phd in europe vienna

Application | Alumni |  Shop |  Publications |  FAQ

• Mission Statement
• Organisation
• Location & Contact
• News from the DA
• Media Coverage
• Publications
• Partners & Networks
• Diploma Programme (DLG)
• Master of Advanced International Studies (MAIS)
• MSc Environmental Technology & International Affairs (ETIA)
• MSc Digital International Affairs (DIA)
• PhD Interdisciplinary International Studies (IIS)
• Recognised Students
• Lectures & Discussions
• Discussion Papers
• Handbuch Außenpolitik
• Department of International History
• Department of International Economics
• Department of International and EU Law
• Culture in Diplomacy
• International Economic Law and Human Rights
• Recharging Austro-American Relations
• Reinvigorating the United Nations

Application

• Fees & Funding
• Immigration & Health
• International Campus
• Living at the DA
• da.link career services
• Academic Calendar 2024-25
• Inauguration Ceremonies
• Graduation Ceremonies
• DA Students Initiative
• Student Societies
• dasicon24: Digital Diplomacy
• Charity Ball at the DA
• Student Ambassadors
• Study Trips
• German Summer Course
• Online-Intensivkurs Deutsch
• French DFP RI Diploma
• Clients & Partners
• Human Rights Space
• Hammer-Purgstall 250
• Event Highlights 2022
• Event Mailing List

Are you in search of an inspiring research environment? Are you looking to improve your scientific and analytical skills to prepare you for a career in social research or academics? The PhD programme at the Vienna School of International Studies might just be what you are looking for! The four-year doctoral programme ‘Interdisciplinary International Studies’ (IIS) is organised in cooperation with the University of Vienna open to graduates who wish to pursue a research project linking at least two of the four disciplines legal studies, political science, international economics and history.

Throughout the programme, doctoral candidates are guided by internationally acclaimed scholars from all over the world, who are dedicated to support our students' academic journeys exploring scientific skills, data collection and analysis as well as academic publications among others.

In addition to that, IIS students have the opportunity to participate in the academic life at the DA through teaching activities, research assistantships and more. The small but vibrant research community at the DA provides PhD students with opportunities to connect with peers in advanced academic positions.

The doctoral programme is built on three modules: Module 1 at the Vienna School of International Studies (“DA”), Module 2 at the University of Vienna (UW) and Module 3 “Disssertation”.

• For Module 1 (DA), doctoral students are required to complete coursework and sit field examinations in both disciplines. Coursework includes research seminars, workshops, courses relevant for their research topic and extensive reading courses focussing on their research disciplines.
• For Module 2 (UW), students are required to develop their research project. To complete this module students are expected to hold a colloquium (“faculty public presentation”) to discuss their dissertation prospectus with the dissertation committee to gain approval for the project.
• Module 3 focuses on research and writing of the doctoral thesis. The primary purpose of PhD study is the preparation and presentation of a substantial piece of original research in the form of a thesis.

For more detailed information on the PhD IIS programme, please consult the information tabs and documents on this page or contact us. To apply, please visit our application portal at https://application.da-vienna.ac.at .

In cooperation with

Phd iis key facts.

Programme start: 23 September 2024

Application deadline: 20 May 2024

Admission results: Beginning of June 2024

Tuition fee*:  EUR 14,700

Financial assistance: Financial Support Application Form

* applies to Module 1 only

PROGRAMME OVERVIEW

Supervision, faculty list of potential reserach supervisors.

"There really is no other place like the Diplomatische Akademie Wien; not only is the level of commitment by the students incredible, the academic possibilities are also endless. My favourite part of pursuing my PhD at the DA are my weekly meetings with my supervisor, Professor Patrick Müller, where we not only discuss my project in depth, but all stages and processes related to my PhD. This highlights the level of dedication of my incredible supervisor at the DA. I could not imagine a better place to pursue my PhD!"

Charlott Gebauer 3 rd PhD in IIS programme, Vienna School of International Studies

"During the first year of the doctoral programme, classes at both the Vienna School of International Studies and the University of Vienna helped me to broaden and deepen my knowledge of international relations and African studies, and to get to know my fellow students. Furthermore, I was in close contact with my supervisors, who greatly helped me to refine my proposal."

Colin Hendrickx 3 rd PhD in IIS programme, Vienna School of International Studies

"The first year of the programme has allowed me to come away with an excellent understanding of all of the requisite fundamentals of both international relations theory and international law. The strong and vibrant faculty at the DA was of paramount importance throughout the planning process of my research project."

Vanessa Sant’Anna Bonifacio Tavares 2 nd PhD in IIS programme, Vienna School of International Studies

PhD Programme

Training young scientists is one of the core missions of the IMP. In 1993, the IMP co-founded an international PhD programme in molecular life sciences with the University of Vienna. Today, the "Vienna BioCenter PhD Program" is one of the most prestigious international PhD programmes in biology.

"Empowering curious researchers" is the motto of the Vienna BioCenter PhD Program, the programme that enables students to do research for their PhD at an IMP lab. The main priorities of this programme are the effective mentoring of PhD students and ensuring that all candidates have the support and resources they need to develop their projects.

The selection for the Vienna BioCenter PhD Program is very competitive and successful candidates are rewarded with ample resources. PhD students at the IMP enjoy access to outstanding facilities and scientific services, opportunities to do any experiment useful to support their work and a pleasant work atmosphere in a modern building. All this is offered in the frame of a training programme that has become one of Europe’s most distinguished PhD programmes in molecular biology since its establishment in 1993.

The IMP and its neighbouring institutions offer a vibrant scientific atmosphere. Lecture series and weekly seminars provide PhD students with immediate access to many of the world’s most outstanding biologists. The Vienna BioCenter community with 150 research groups is a fertile ground for stimulating encounters and a good spirit – not last socially.

Meet former and current IMP PhD Students

Apply to the Vienna BioCenter PhD Program

The programme has two international calls per year and recruits approximately 15 motivated young scientists in each call (all PhD Students at IMP are recruited through the Vienna BioCenter PhD Program). Access current calls via the official website of the programme:

Vienna BioCenter PhD Program

What are Group Leaders looking for in a PhD applicant? The Vienna BioCenter PhD Program is competitive and highly selective, so passion for science and research experience will be an important pre-condition. However, there is more to a good applicant. One IMP Group Leader stated that she was looking for "PhD candidates that have curious and creative minds, are passionate about science, are easy going and communicative, are highly motivated and have endurance."

And what can you expect from the programme? The best way to convey the essence of the IMP PhD student experience is perhaps to listen to past and present PhD students – for a start, please note our testimonials below, or read the interview with the 2018 Weintraub Award laureate Annika Nichols on her experience as a PhD student at the IMP.

The Vienna BioCenter is highly international with excellent service facilities supporting the scientific research done here. This is really a great place to pursue competitive science.

A flexible curriculum

The programme's curriculum is flexible and was designed for and with PhD students. The Vienna BioCenter PhD students start with a 3-week introductory course – “Prime your PhD” – which is designed to prime and facilitate the implementation and development of their research projects, specifically on this campus. Throughout their training, students (like postdocs ) present their work to the campus community at the Monday Seminars .

This weekly seminar series is a great vehicle to share knowledge and expertise between different research groups and different institutes. At the same time, the seminars are an opportunity to train PhD students in presentation skills.

Numerous seminars and journal clubs take place on a regular basis, alongside several advanced courses such as scientific writing, advanced microscopy, data analysis, and others. In addition, PhD students benefit from career development workshops.

Each student is free to choose which courses he or she wants to participate in – moreover, students are encouraged to suggest and develop new courses. In addition, the VBC PhD students organise activities such as a retreat and a symposium each year. For this, they are provided with funding, support – and a lot of freedom to develop through task planning and implementation.

Alumni: What former PhD students did

Out of 140 Vienna BioCenter PhD Students that graduated after PhD research at the IMP between in the 1998-2008 decade, no less than 50 are now in Group Leader positions.

A recent survey showed very high satisfaction among alumni: virtually all survey participants see their time at the Vienna BioCenter as having been a great asset for their careers.

More information

You will find further information on the Vienna BioCenter PhD Program and the forms to apply during open call at the Vienna BioCenter training website.

https://training.vbc.ac.at/

Cookie Settings

We use cookies that are technically necessary for the functionality of our website, but also cookies for functional and analysis purposes to optimise the user experience on our site. With your consent to the cookies, data is processed both by us and by third-party providers, some of which are based in third countries (e.g. the USA).

You can find more information about the tools and the partners in our data protection declaration, in which we also explain exactly what data transfer to the USA can mean. You can individually adjust or revoke your cookie settings at any time if you wish.

These cookies are necessary for the basic functions of the website. You can block or delete them in your browser settings, but you then run the risk that some parts of the website will not function properly.

PHPSESSID Technically necessary cookie from the web server. storage duration: session

fe_users Used by TYPO3 as function for user administration. storage duration: session

Necessary to obtain consent for certain cookies and thus for the use of certain tools.

cookieUser Used to recognise the browser and its cookie history (consent or rejection of particular cookies with date und time) by an anonymised identification number. storage duration: 2 years

cookieNotice Controlling the cookie consent storage duration: 180 days

cookieNoticeAll Used to check if the user allows all cookies. storage duration: 180 days

cookieNoticegta Used to check if the user allows analytics cookies. storage duration: 180 days

cookieNoticetwitter Used to check if the user allows Twitter. storage duration: 180 days

cookieNoticevideo Used to check if the user allows YouTube. storage duration: 180 days

Google Analytics: Web analysis service for evaluating visitor information in order to optimise the website and its offering.

Google Tag Manager: Tag management system for the central integration of tags via a user interface.

Google Ireland Limited Google Building Gordon House, 4 Barrow St, Dublin, D04 E5W5, Irland

Alphabet Inc. 1600 Amphitheatre Parkway Mountain View, CA 94043, USA

Google LLC 1600 Amphitheatre Parkway Mountain View, CA 94043, USA

This service may transfer the collected data to another country. Please note that this service may transfer data outside the European Union and the European Economic Area and to a country that does not provide an adequate level of data protection. If the data is transferred to the US, there is a risk that your data may be processed by US authorities for control and monitoring purposes, without any possible redress available to you. Below is a list of countries to which the data will be transferred.

USA, Singapore, Taiwan, Chile

_ga Used by Google Analytics to determine users. storage duration: 2 years

_gid Used by Google Analytics to determine users. storage duration: 24 hours

_gat Used by Google Analytics to throttle requests. storage duration: 1 minute

Enables the display of videos.

Alphabet Inc. 1600 Amphitheatre Parkway Mountain View, CA 94043, USA Google Ireland Limited Google Building Gordon House, 4 Barrow St, Dublin, D04 E5W5, Irland Google LLC 1600 Amphitheatre Parkway Mountain View, CA 94043, USA

This service may transfer the collected data to another country. Please note that this service may transfer data outside the European Union and the European Economic Area and to a country that does not provide an adequate level of data protection. If the data is transferred to the US, there is a risk that your data may be processed by US authorities for control and monitoring purposes, without any possible redress available to you. Below is a list of the countries to which the data will be transferred.

__Secure-3PAPISID Builds a profile of website visitor interests to show relevant and personalized ads through retargeting. storage duration: 2 years

__Secure-3PSID Builds a profile of website visitor interests to show relevant and personalized ads through retargeting. storage duration: 2 years

__Secure-3PSIDCC These cookies are used to deliver ads that are more relevant to the user and its interests. They contain an encrypted unique ID to identify the user. storage duration: 2 years

YSC This cookie is set by YouTube to track views of embedded videos. It contains an encrypted unique ID to identify the user. storage duration: session

CONSENT Stores visitors’ preferences and personalizes ads. storage duration: persistent

PREF YouTube uses the ‘PREF’ cookie to store information such as a user’s preferred page configuration and playback preferences like autoplay, shuffle content, and player size. storage duration: 8 months

VISITOR_INFO1_LIVE This cookie is set by YouTube to track views of embedded videos. It contains an encrypted unique ID to identify the user.. storage duration: 3 months

Enables the display of embedded tweets.

Twitter Inc. One Cumberland Place, Fenian Street, Dublin 2 D02 AX07, Irland

This service may transfer the collected data to another country. Please note that this service may transfer data outside the European Union and the European Economic Area and to a country that does not provide an adequate level of data protection. If the data is transferred to the US, there is a risk that your data may be processed by US authorities for control and monitoring purposes, without any possible redress available to you. Below is a list of the countries to which the data will be transferred. USA

guest_id Unique id that identifies the user’s session storage duration: 2 years

personalization_id This cookie is set due to Twitter integration and sharing capabilities for the social media storage duration: 2 years

d_prefs Used by Twitter services, to monitor referral links, and login status. storage duration: 6 months

guest_id_marketing Used to detect whether a user is logged into Twitter storage duration: 2 years

guest_id_ads This cookie is set due to Twitter integration and sharing capabilities for the social media storage duration: 2 years

Our PhD and Doctoral Programs

• Studies & Further Education
• PhD & Doctoral Programs

Are you looking for a Ph.D. position? Our next Ph.D. Call starts on 15.10.2024

Training the next generation of young scientists is a long tradition of ours!

MedUni Vienna has been training doctors and young researchers since 1365 and can therefore look back on a long tradition in research and education. Here you can work closely with respected academics and researchers while taking your first independent steps as a scientist. As a young researcher, you’ll be part of a research team from the very beginning and develop your first original project with guidance from a supervisor. Your work will shape the knowledge base of tomorrow’s doctors and have a positive influence on the lives of many people. Medical research is meaningful and rewarding work for anyone who is enthusiastic about science and research. You’ll acquire a deep understanding of your field and its methods and develop important transferable skills you’ll need to succeed in the future. The university and the Alumni club offer excellent networking and training opportunities. At the end of your studies, you’ll write your dissertation, and after a successful defense, you’ll be rewarded with your Ph.D. and be recognized as a fully trained scientist (R2-R3 researcher profile EU level). The doctoral program is full-time and linked to employment at MedUni Vienna.

A Ph.D. is the basis of a successful career in research, education, medicine, or any research-orientated position in the public or private sector. 

Depending on your educational background, you can focus your studies on basic or clinical research. For more detail on our interdisciplinary Ph.D. and doctoral programs, please click on the links to respective postgraduate training programs. Become a part of our constant endeavour to keep humanity vital and healthy!

Click here for general information on doctoral studies at the Medical University of Vienna.

UN 094 PhD – Doctor of Philosophy (UN094)

Joint phd studies, the doctoral programme of applied medical science (un790), contact us.

We have collected all information about our PhD studies for you in our FAQ section . Please contact us directly by Email for any questions left unanswered and make an appointment with us. We will be pleased to provide you with information on any arising questions from recruiting to details about the complete academic calendar.

Our office is located in the Study Departement Building of the Medical University Vienna at the following address :

Währinger Straße 25a A-1090 Vienna, Austria

Contact for general inquiries

Dieter Breitenbaum Vesna Dominkovic

P: +43 (0)1 40160-21029 P: +43 (0)1 40160-21030

Contact for PhD Call recruiting 

Stephanie Danzinger P: +43 (0)1 40160-21033

We use cookies

We use cookies that are technically necessary for the functionality of our website, but also cookies for functional, marketing and analysis purposes to optimise the user experience on our site. With your consent to the cookies, data is processed both by us and by third-party providers, some of which are based in third countries (e.g. the USA). You can find more information about the tools and the partners in our data protection declaration, in which we also explain exactly what data transfer to the USA can mean. You can individually adjust or revoke your cookie settings at any time if you wish.

These cookies are necessary for the basic functions of the website. You can block or delete them in your browser settings, but you then run the risk that some parts of the website will not function properly.

• PHPSESSID Technically necessary cookie from the web server. storage duration: session
• fe_typo_user_live Used by TYPO3 as function for user administration. storage duration: session

Necessary to obtain consent for certain cookies and thus for the use of certain tools.

• cookieUser Used to recognise the browser and its cookie history (consent or rejection of particular cookies with date und time) by an anonymised identification number. storage duration: 1 years
• fcc_cookie_consent Cookie Consent storage duration: 1 years
• fcc_cookie_consent_minified Cookie Consent storage duration: 1 years
• cookie_matomo Cookie Consent storage duration: 1 years

To track users on your website(s) or app(s), the default Matomo Tracking code in JavaScript uses 1st party cookies, which are set on the domain of your website.

• pk_ses short lived cookies used to temporarily store data for the visit storage duration: 30 minutes
• pk_id used to store a few details about the user such as the unique visitor ID storage duration: 13 months

• Show search form Hide search form
• Quick links
• Staff search
• Search Search --> Websites Staff search Start search

PhD Orientation Week, 02-04.10.2024

Doctoral insights symposium, 24.09.2024.

Two day academic writing workshop, 17.&18.10.2024, 09-17:00

Engaging in academic work, particularly through reading and writing, requires a balance between maintaining a steady writing routine and producing...

Building Social Worlds: How Minds Shapes Societies and Societies Shape Minds, 14.10.2024

Organisational team of five of our doctoral candidates have invited a speaker, Professor Alex Thornto of Cognitive Evolution at the Human Biological...

VBC PhD Symposium, abstract submission deadline: 02.09.2024

The theme of Symposium this year is 'Into the unknown: Exploring Uncharted Territories in Research', with the aim to highlight the exploratory science...

Doctoral Insights Symposium, 24.09.2024, 14:00

This Symposium is organized by Vienna BioCenter, and has the aim to guide individuals interested in pursuing a PhD in Life Sciences!

doc:muv Mentoring Program, registration deadline: 07.07.2024

The career development program doc:muv is intendent for female* prae-doc scientists of all disciplines and faculties of the University of Vienna,...

1st Summer School of the International Max Planck Research School on Cognitive NeuroImaging (IMPRS CoNI), registration deadline: 20.06.2024

We are excited to invite you to the 1st Summer School of the International Max Planck Research School on Cognitive NeuroImaging (IMPRS CoNI), as a...

 PhD Stories

Lange Nacht der Forschung

The doors were wied open from four different locations, from the main University building, to Biology and Chemistry building as well as University...

Rojan Amini-Nejad has been awarded a dissertation price

Congratulations to Rojan Amini-Nejad for winning the ÖAW Dissertation Prize for Migration Research!

Julia Reiter has been awarded the City of Vienna Impact Award 2023

Congratulations to Julia Reiter for winning the Impact Award 2023!

 Defenses

PhD defense of Lisa Stempfer, 23.09.2024, 9:00

Public Defense in the Doctoral Research Field: Psychology

“Meta-Analytical Investigations of Boredom: Associations With Arousal and Performance”

PhD defense of Hanna Mües, 20.09.2024, 10:00

“Too stressed for sex? Associations between sexual experiences, sexual behavior, stress,...

PhD defense of Courtney Pike, 18.09.2024, 17:00

Public Defense in the Doctoral Research Field: Biology

''Investigating the behavior and larval development of the invasive parasitic fly, Philornis...

PhD defense of Franziska Laaber, 02.09.2024, 11:00

“A Psychological Perspective on Self-Determined Digital Technology Use: Conceptualizing,...

PhD defense of Sarah Emser, 02.09.2024, 13:30

''Mitochondrial impact in hibernation: insights into conservation and translation of...

PhD defense of Marlene Forstinger, 26.08.2024, 11:30

“Contingent Suppression of Visual Attention: Novel Evidence for Proactive Top-Down Control...

 Books

Raben - Das Geheimnis ihrer erstaunlichen Intelligenz und sozialen Fähigkeiten

Thomas Bugnyar, 2022, Brandstätter Verlag. Sie sind bekannt für ihre verblüffende Intelligenz, für das clevere Benutzen von Werkzeugen und für ihr...

Buchtipp: Wie klug sind wir wirklich?

Jakob Pietschnig, 2021, Ecowin. Was Intelligenz ist, wie man sie messen kann und warum das so spannend ist, erklärt der österreichische...

Buchtipp: Von singenden Mäusen und quietschenden Elefanten

Angela Stöger, Brandtstätter Verlag, 2021. Wie Tiere kommunizieren und was wir lernen, wenn wir ihnen wirklich zuhören

Doctoral (PhD) programmes

Description.

The doctorate programme has a standard duration of 6 semesters. Aside from the completion of a doctoral thesis (dissertation), elective courses must be passed for a total credit of at least 18 ECTS from the current doctoral curriculum (standardised for all disciplines). Detailed information about this course selection can found in the doctoral curriculum. Credits earned and previously applied to attaining a master degree cannot be credited to doctoral studies.

The doctorate programme is concluded with a research presentation and thesis defense (Rigorosum) in front of an examination committee. According to the degree programme, the graduate is awarded the academic degree  Doctor of Social and Economic Sciences , abbreviated  Dr.rer.soc.oec.  or  Doctor of Engineering Sciences , abbreviated  Dr.techn .

Starting out from the outcomes of the 2017 Research Day, the framework conditions of the procedure for doctorate studies in the Faculty of Architecture and Planning was discussed by a working group of experts from a wide range of disciplines, and concrete proposals for a further development were developed. On the basis of the current regulations for study matters for the doctoral programme, the formats and processes documented in the  Guidelines Doctoral Programme 2020  focused on an improved networking of the candidates with one another, on more transparency both internally and externally, and stepped-up support of candidates. In practice this means:

• In a  Colloquium  taking place twice a year, active candidates are invited to present an interim exposé and to follow this up by sharing their intermediate results in an informal talk afterwards. The occasion is intended as a networking encounter. This has the aim of encouraging knowledge-sharing between the candidates – beginners as well as advanced – also to create synergies and to enhance the visibility of the individual research projects on Faculty level.
• The  Review  introduces a feedback option for the candidates. One year after inscription the candidate has the opportunity of presenting to a review jury consisting of Faculty members and external experts the developed research plan and thus gain support through a tangible resonance to their thesis project.
• The  option of specific units of instruction and courses  for candidates aims to be adapted continually to current needs and followed up according to requirement. The corresponding resources will be made available to cover these.

The complete version of the Guidelines is available online.

Requirements

At least one of the following requirements must be fulfilled before enrolling a doctorate programme at TU Wien:

• Completion of university master or teaching certification degree in a related discipline
• Completion of equivalent master studies at an accredited Austrian or foreign institution of higher education
• Completion of a master programme at a technical college accredited in accordance with the Austrian law governing such institutions  Fachhochschulgesetz § 5 Abs. 3

In cases not meeting the first stated condition, the applicant may be individually required to take supplemental courses in the doctorate programme.

In the latter two cases, the certificate of admission may include individual requirements for taking supplemental courses. The requirements for graduates of several (Austrian) college programmes are stipulated in  regulations  (available only in German) issued by the Federal Ministry of Science, Research and Economy. The extent of additionally required course credits can be up to 44 semester hours, equivalent to 60 ECTS points. For more information about application requirements, see the  Admission Office ; foreign students may also consult to the  Student Exchange Bureau  of the university’s student union (HTU).

Students must enroll in the doctoral programme at the  Admissions Office .

A  TU Wien account  is registered upon enrollment, which is maintained for the duration of doctoral studies. Since account authorisation takes a certain amount of time, early registration is recommended to ensure a problem-free programme start.

Graduation Ceremony

For consultation hours, please register in advance via e-mail.

Dean's Office of Faculty for Architecture and Planning Mail: [email protected] Tel.:  +43 (1) 58801 25006

• Industry Insights
• How to apply
• Talents for Future
• Program Description
• How to Apply
• Participating Labs
• Housing/Benefits
• Award Winners
• Testimonials/Alumni
• Steering Committee
• Partner Universities
• Open Positions
• Roles and expectations
• Program Steering Committee
• Core Training
• Elective Courses
• Scientific Speakers
• Workshop Speakers
• Registration/Travel Grants
• FAQs/Contact
• Graduations & Awards
• Guidelines/Procedures
• PhD Representatives
• Post Doc Mentoring
• Applications
• Two-Mentor Scheme
• Career Guidance
• Training Program
• VIP² Advisory Board (VAB)
• Post Doc Representatives
• Career Development Plan

Autumn Call Now Open

Application Submission Deadline

15 October 2024

Each year our students organize a symposium highlighting various disciplines present across our campus

Empowering Curious Researchers

The Vienna BioCenter is a leading life science location in Europe: a thriving campus that combines basic research institutes, universities and biotech companies.

Four research institutes collaborate in creating an exceptional training environment, where students and junior researchers have all the necessary resources to conduct meaningful projects: there is virtually no experiment that cannot be done at the VBC. More on Research at VBC

Our training programs are designed to prime and/or advance learning of critical skills, while supporting students and postdocs to become independent, critical and innovative thinkers, establish professional networks and ultimately move on to their next position.

What sets us apart? Two main factors contribute to our vibrant scientific community:

• An extremely collaborative faculty, people that thrive from the success of their colleagues on campus and are extremely supportive.
• A supreme research infrastructure: state-of-the-art equipment and facilities managed by top expert.

If you are passionate about science and have a pioneering mindset, then join us in Vienna!

Other News, Events

For more news check out our news page here

Forthcoming Defensio

For information on forthcoming defences please check our graduation page here

This website uses cookies to ensure you get the best experience on our website.

Technically required

Purpose: technical enabling for the use of analysis tools Processing operations: no collection of personal data Storage period: - Joint controller: Google Ireland Limited, Gordon House, Barrow Street, Dublin 4, Ireland Legal basis for data processing: voluntary, consent that can be revoked at any time Consequences of non-consent: No direct impact on the functionality of the website; however, limited opportunities for further development and error analysis

Analysis / statistics

Anonymous evaluation for troubleshooting and further development

Doctoral Programmes at TU Wien

E784 - Doctoral Programme in Social and Economic Sciences E786 - Doctoral Programme in Technical Sciences E791 - Doctoral Programme in Natural Sciences

Curricula , opens an external URL in a new window

Admission and prerequisites

The prerequisite for admission to a doctoral programme at TU Wien is either

• successful completion of a relevant master's, teacher training or other appropriate degree programme,
• successful completion of an equivalent degree programme at an accredited tertiary educational institution in or out of Austria or
• successful completion of a relevant degree or master's programme from a technical college in accordance with § 6 Para. 4 Fachhochschulstudiengesetz [Technical Colleges Act],
• Supervision assurance of a full or associate Professor of TU Wien.

The doctoral programme usually lasts for six semesters.  In addition to the dissertation, the current standardised programme for doctoral students stipulates that a total of 180 ECTS of modules (162 ECTS of which are the dissertation) must be completed.  More detailed information regarding this selection can be found in the programme. Examinations taken as part of a master's or other degree programme cannot be recognised for this. The doctoral programme is assessed in the viva voce, a general examination by a committee involving defence of the dissertation by the candidate.  Graduates of a doctorate in the technical sciences are awarded the title of Doktor/in der Technischen Wissenschaften (Dr. techn.), graduates of a doctorate in the natural sciences are awarded the title of Doktor/in der Naturwissenschaften (Dr. rer. nat.) and graduates of a doctorate in the social and economic sciences are awarded the title of Doktor/in der Sozial- und Wirtschaftswissenschaften (Dr.rer.soc.oec.).

Further Information

Admission office.

If you need information on how to apply at the TU Wien regardless of academic degree, this is your go to Admission .

Fachschaft Doktorat:

Fachschaft Doktorat is the representation of interests of doctoral candidates at the TU Wien. Their members provide information and give advice and support concerning all aspects of the doctoral programs.

http://fsdr.at , opens an external URL in a new window

Further information

Admission office

Karlsplatz 13, Stiege 2/Halbstock 1040 Wien

Phone: +43 1 58801 41188 Email:  [email protected]

Fachschaft Doktorat

Karlsplatz 13 1040 Wien Main building, ground floor, next to Fachschaft Raumplanung and Fachschaft Architektur Room number: AF EG 32

Email:  [email protected] Website: fsdr.at , opens an external URL in a new window

• Top menu level Studies
• Back: list subpages of parent page "Studies" Back to: Studies
• Doctoral Programmes
• Study Application , opens an external URL in a new window

About Cookies and other techniques

Our website uses cookies and integrates content from third-party providers to ensure you get the best experience on our website, for analytical purposes, to provide social media features, and for targeted advertising. This it is necessary in order to pass information on to respective service providers. If you would like additional information about cookies and content from third-party providers on this website, please see our Data protection declaration .

These cookies are required to help our website run smoothly.

These cookies help us to continuously improve our services and adapt our website to your needs. We statistically evaluate the pseudonymized data collected from our website.

With the help of these cookies and third-party content we strive to improve our offer for our users. By means of anonymized data of website users we can optimize the user flow. This enables us to improve ads and website content.

• Research at Vienna BioCenter
• Research Entities
• Anthropogenic Evolution
• Biochemistry, Biophysics, Structure
• Cell and Chromosome Biology
• Cognitive and Behavioral Biology
• Computational Biology, Genomes and Evolution
• Disease Mechanisms, Immunology, Pathogens
• Gene Regulation, RNA, Epigenetics
• Microbiology and Environmental Systems Science
• Neurobiology
• Plant Sciences
• Stem Cells, Development, Regeneration
• Research Groups
• CRISPR/Cas9
• MECHANISMS OF CHROMOSOME SEGREGATION
• HUMAN BRAIN ORGANOIDS
• EPIGENETICS
• 1001 GENOMES
• Recent Research Highlights
• Austrian BioImaging/CMI
• Computational Biology Training
• Electron Microscopy
• Light Microscopy
• Metabolomics
• Next Generation Sequencing
• Preclinical Phenotyping
• Protein Technologies
• Vienna Drosophila Resource Center
• Child Care Center
• VBCF Landing Page
• Projects & Grants
• My VBCF Info-Hub

• Companies at Vienna BioCenter
• Ablevia Biotech
• ADvantage Therapeutics
• Affiris CVD GmbH
• aitiologic GmbH
• BioNTech R&D Austria
• bit.bio discovery GmbH
• CALYXHA Biotechnologies
• G.ST Antivirals
• HeartBeat.bio AG
• HOOKIPA Pharma Inc.
• Miti Biosystems
• MyeloPro Diagnostics and Research GmbH
• Myllia Biotechnology
• Pregenerate
• proxygen GmbH
• QUANTRO Therapeutics
• Thermo Fisher Scientific
• THT Biomaterials GmbH
• Tridem Bioscience
• X4 Pharmaceuticals
• YGION Biomedical GmbH
• LabConsulting
• IG StartUP Solutions GmbH

• Open Positions (current)
• Welcome Office
• Campus Life
• Life in Vienna

• About Vienna BioCenter
• Who is Here (Members)
• Management Team
• Virtual Tour
• Events at the Vienna BioCenter
• Sustainability

Open Positions

The Campus. The Vienna BioCenter is a top biology research center in Europe conducting innovative research in all areas of the life sciences.

The Areas. The wide range of expertise, from biophysics and structural biology to cell biology and evolution, combined with a plethora of model systems, ensures diversity and interdisciplinarity.

The Research. Purely curiosity-driven research is highly valued and encouraged. A focus on basic biomedical research and mechanisms of disease, laying the groundwork for future solutions to global health problems.

The Infrastructure. Cutting-edge research infrastructure enables innovative research and provides a competitive locational advantage to all researchers on campus.

The City. Located in the city consistently ranked #1 in the Mercer Quality of Living Ranking, the Vienna BioCenter offers some of the best research opportunities in Europe and has become a magnet for inquisitive and ambitious scientists from all over the world.

The different entities at Vienna BioCenter offer career opportunities at various stages, both in science, science support and administration. See the full list of job opportunities on campus below.

The website uses cookies to ensure you get the best experience on our website.

Analysis / statistics

Anonymous evaluation for troubleshooting and further development

Purpose: error analysis, statistical evaluation of our website accesses, campaign analysis, conversion tracking, retargeting Processing operations: Collection of access data, data from your browser and data about the content accessed; Execution of analysis software and storage of data on your terminal device, anonymization of the data collected; Evaluation of the anonymous data in the form of statistics Storage period: data on your device for up to two years. Joint controller: Google Ireland Limited, Gordon House, Barrow Street, Dublin 4, Ireland Legal basis for data processing: voluntary, consent that can be revoked at any time Consequences of non-consent: No direct impact on the functionality of the website; however limited opportunities for further development and error analysis Data transfer to the USA: Your data is processed by the provider Google in the USA, which involves corresponding risks, e.g. B. a secret data access by US authorities. With your consent, you also consent to the processing of your data in the USA.

Fully-funded PhD Positions in the Life Sciences/Molecular Biosciences

Empowering Curious Researchers at the Vienna BioCenter PhD Program

Are you a curious and motivated student looking to launch a successful scientific career? Look no further than the Vienna BioCenter PhD Program , one of the best PhD programs in Europe. Our program empowers curious researchers to develop their own PhD projects and make important scientific discoveries in a dynamic and multidisciplinary research environment.

At the Vienna BioCenter Campus, you will have access to state-of-the-art facilities and be supervised by top scientists in the field, providing you with the resources and support you need to succeed. Our campus is highly multidisciplinary, with research areas ranging from molecules to populations, ensuring that you have the opportunity to explore a wide range of topics and make interdisciplinary connections.

Our Mission: Promoting Interdisciplinary Research at the Highest Level

Our mission is to promote interdisciplinary research in the Life Sciences at the highest level and to help PhD students develop into tomorrow's leading scientists through a comprehensive training program. We believe in the power of interdisciplinary research to drive innovation and tackle complex scientific challenges, and we strive to create an environment that fosters collaboration and innovation.

The Vienna BioCenter is a world-class scientific research environment where students and group leaders from different backgrounds interact in an inter-disciplinary, open, and creative environment. By promoting the exchange of ideas and bringing together different and often complementary disciplines, we provide outstanding opportunities for ambitious and motivated students to advance their scientific careers.

Our training program is designed to educate students in all areas relevant for a career in science, providing additional opportunities to acquire skills relevant for other career paths. We believe that scientific training should be comprehensive and prepare students for a wide range of future opportunities, whether in academia, industry, or beyond.

Vienna: The Best City for Life Quality in the World

Not only is the Vienna BioCenter PhD Program one of the best in Europe, but it is located in the number one city for life quality in the world. Vienna consistently ranks at the top of global quality of life indexes, with a high standard of living, excellent public transportation, and a rich cultural heritage.

Vienna is a city that values innovation and creativity, with a thriving arts and music scene and a long history of scientific discovery. As a student at the Vienna BioCenter PhD Program, you will have the opportunity to immerse yourself in this vibrant and dynamic city, exploring its many museums, galleries, and cultural institutions.

In addition, Vienna is located at the heart of Europe, providing easy access to other major cities and research institutions. This makes it an ideal location for students who are looking to network and collaborate with other researchers across the continent.

An Outstanding Opportunity for Ambitious and Motivated Students

The Vienna BioCenter PhD Program offers an outstanding opportunity for ambitious and motivated students looking to launch a successful scientific career. With access to state-of-the-art facilities, top scientists, and a dynamic and multidisciplinary research environment, our program provides the resources and support you need to make important scientific discoveries and advance your career; located in one of the best cities for life quality in the world.

If you are looking for a comprehensive training program that prepares you for a wide range of future opportunities, look no further than the Vienna BioCenter PhD Program.

We have two calls per year with approx. 30 fully-funded positions per call.

Autumn call is open from 1 September to 15 October

Spring call is open for applications from 1 March to 15 April

Check out our website for further information

Register your interest for this PhD

The university will respond to you directly. You will have a FindAPhD account to view your sent enquiries and receive email alerts with new PhD opportunities and guidance to help you choose the right programme.

It looks like you alredy have a FindAPhD Account

Log in to save time sending your enquiry and view previously sent enquiries

Which subjects are you interested in studying?

What would you like more information about *, which age group are you optional.

The information you submit to this university will only be used by them to deal with your enquiry. For more information on how we use your data, please read our privacy statement

Your enquiry has been emailed successfully

You can view your previously sent enquiries in MyFindAPhD

Vienna Biocenter

FindAPhD. Copyright 2005-2024 All rights reserved.

Unknown    ( change )

Have you got time to answer some quick questions about PhD study?

Select your nearest city

You haven’t completed your profile yet. To get the most out of FindAPhD, finish your profile and receive these benefits:

• Monthly chance to win one of ten £10 Amazon vouchers ; winners will be notified every month.*
• The latest PhD projects delivered straight to your inbox
• Access to our £6,000 scholarship competition
• Weekly newsletter with funding opportunities, research proposal tips and much more
• Early access to our physical and virtual postgraduate study fairs

Or begin browsing FindAPhD.com

or begin browsing FindAPhD.com

*Offer only available for the duration of your active subscription, and subject to change. You MUST claim your prize within 72 hours, if not we will redraw.

Create your account

Looking to list your PhD opportunities? Log in here .

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Open access
• Published: 09 September 2024

FoxO transcription factors actuate the formative pluripotency specific gene expression programme

• Laura Santini   ORCID: orcid.org/0000-0001-9968-2459 1 , 2 ,
• Saskia Kowald 1 ,
• Luis Miguel Cerron-Alvan 1 , 2 ,
• Michelle Huth   ORCID: orcid.org/0000-0002-1152-9140 1 , 2 ,
• Anna Philina Fabing 1 ,
• Giovanni Sestini   ORCID: orcid.org/0000-0002-6643-7585 2 , 3 ,
• Nicolas Rivron   ORCID: orcid.org/0000-0003-1590-5964 3 &
• Martin Leeb   ORCID: orcid.org/0000-0001-5114-4782 1  

Nature Communications volume  15 , Article number:  7879 ( 2024 ) Cite this article

1 Altmetric

Metrics details

• Differentiation
• Embryonic stem cells
• Growth factor signalling
• Stem-cell differentiation

Naïve pluripotency is sustained by a self-reinforcing gene regulatory network (GRN) comprising core and naïve pluripotency-specific transcription factors (TFs). Upon exiting naïve pluripotency, embryonic stem cells (ESCs) transition through a formative post-implantation-like pluripotent state, where they acquire competence for lineage choice. However, the mechanisms underlying disengagement from the naïve GRN and initiation of the formative GRN are unclear. Here, we demonstrate that phosphorylated AKT acts as a gatekeeper that prevents nuclear localisation of FoxO TFs in naïve ESCs. PTEN-mediated reduction of AKT activity upon exit from naïve pluripotency allows nuclear entry of FoxO TFs, enforcing a cell fate transition by binding and activating formative pluripotency-specific enhancers. Indeed, FoxO TFs are necessary and sufficient for the activation of the formative pluripotency-specific GRN. Our work uncovers a pivotal role for FoxO TFs in establishing formative post-implantation pluripotency, a critical early embryonic cell fate transition.

Similar content being viewed by others

The transcription factor OCT6 promotes the dissolution of the naïve pluripotent state by repressing Nanog and activating a formative state gene regulatory network

Cell fate roadmap of human primed-to-naive transition reveals preimplantation cell lineage signatures

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Introduction.

Pluripotent cells can give rise to all specialised cells that form an adult organism. During mouse embryonic development, a population of naïve pluripotent cells arises in the pre-implantation epiblast around embryonic day E3.5–E4.5 1 . During the transition from pre- to post-implantation development, epiblast cells navigate through a continuum of pluripotency states. Starting from a naïve state with unrestricted potential, cells transit through a formative state to acquire competence for somatic and germ cell lineage specification, ultimately entering a primed state where they initiate expression of lineage markers 2 , 3 , 4 , 5 .

The naïve pluripotent state of pre-implantation epiblast cells can be captured in vitro using mouse embryonic stem cells (mESCs) 6 , 7 . Maintenance of a homogeneous ground state of pluripotency requires the addition of two small molecule inhibitors to the culture media: PD0325901 (MEK1/2 inhibitor) and CHIRON (GSK3ɑ/β inhibitor), collectively referred to as 2i 8 . mESCs cultured in 2i resemble the E4.5 pre-implantation epiblast in terms of epigenetic and transcriptional status 1 , 9 , 10 , 11 . Naïve identity is defined by the expression of a self-reinforcing gene regulatory network (GRN) that comprises the core pluripotency transcription factors (TFs) Oct4 (gene name: Pou5f1 ) and Sox2 , and naïve-specific TFs including Nanog , Esrrb , Tbx3, Klf4, Klf5 and others 12 , 13 , 14 , 15 .

A balanced interplay of several signalling inputs is responsible for the maintenance of the naïve-specific GRN 16 . The cytokine LIF (Leukaemia Inhibitory Factor) plays a key role in maintaining pluripotency and was the first identified exogenous factor that can support mouse ESC culture along with serum supplementation 17 , 18 . LIF mainly activates the JAK/STAT3 and PI3K/AKT pathways which are crucial to sustain naïve pluripotency 15 . Although the role of the JAK/STAT pathway has been intensively studied, the function of PI3K/AKT signalling in pluripotency has received much less attention 19 . Overexpression of a constitutively active form of AKT is sufficient to maintain mESCs in an undifferentiated state, even in the absence of LIF 20 . PI3K/AKT signalling is thought to support naïve pluripotency through inhibition of both the MEK/ERK and the GSK3 pathways 21 , 22 , 23 , although the underlying mechanisms are unclear. Furthermore, AKT signalling feeds directly into the naïve GRN by activating the expression of Tbx3 and Nanog 15 , 24 .

Exit from the naïve pluripotent state and initiation of formative pluripotency can be recapitulated in vitro by releasing cells from 2i inhibition into basal N2B27 medium. This change in conditions leads to loss of self-renewal in the naïve state and an irreversible commitment to differentiate approximately 48 h after 2i withdrawal 22 . The exit from naïve pluripotency results in the dismantling of the naïve-specific GRN and the establishment of a new formative state-specific GRN. This is accompanied by a profound shift in the signalling landscape: LIF and AKT signalling are reduced, concomitant with an increase in FGF/ERK activity 15 , 25 , 26 . We and others found that Pten , a negative regulator of AKT, is among the top hits in genetic screens for drivers of ESC differentiation, highlighting the importance of downregulating the PI3K/AKT pathway to ensure timely exit from the naïve state 27 , 28 , 29 , 30 , 31 . However, how exactly Pten regulates the exit from naïve pluripotency remains elusive.

In this study, we find that FoxO transcription factors are regulated by AKT and play a previously unrecognised but critical role in the transition from naïve to formative pluripotency. Our findings indicate that AKT acts as a gatekeeper by maintaining FoxO TFs in the cytoplasm in the naïve state. At the initiation of differentiation, elevated PTEN levels lead to a reduction in AKT signalling, allowing FoxO TFs to localise to the nucleus where they play a pivotal role in facilitating the transition from naïve to formative pluripotency by regulating a switch in operative GRNs. Our findings uncover an intricate mechanism that regulates the orderly transition between gene regulatory networks that determine distinct pluripotent states.

PTEN controls pAKT for timely exit from naïve pluripotency

We previously found that mESCs lacking Pten exhibit a pronounced defect in the exit from naïve pluripotency 28 . Indeed, 24 h after 2i-removal in N2B27 medium (N24), Pten KO mESCs displayed higher Rex1-GFPd2 (Rex1-GFP) reporter activity than wild-type (WT) cells (Fig.  1a, b ). Rex1 is specifically expressed in the naïve state, and its downregulation coincides with irreversible commitment to differentiation 22 , 27 . This defect in exit from the naïve state in Pten KO ESCs was rescued by expressing Pten through transfection of a plasmid encoding 3xFLAG-PTEN (Supplementary Fig.  1a, b ).

a Schematic representation of our experimental setup. Nx indicates differentiation time (x) after 2i withdrawal in N2B27 medium. b Flow cytometry analysis of Rex1-GFP levels in WT and two independent Pten KO clones in naïve pluripotency supporting conditions (2i, green) and 24 h after 2i removal (N24, blue). One representative of n  = 11 independent experiments is shown. c Pten expression levels (transcript per million, TPM) from a 2 h-resolved RNA-seq differentiation time course in WT 28 . d Western blot analysis for indicated proteins in WT and Pten KO in 2i/LIF, at N24 and N48. TUBULIN served as a loading control. e Quantification of pAKT (S473) levels by flow cytometry in indicated cells and conditions. Mean and standard deviation (SD) of mean fluorescence intensity (MFI) from n  = 3 independent experiments (distinct shades of grey) are shown. p values result from paired, two-tailed t -tests. f Heatmap showing row-normalised expression values of indicated naïve and formative genes in indicated genotypes and conditions. g Scatter plots showing correlation between differentially expressed genes (DEGs, p adj. ≤ 0.05, |log2foldchange (log2FC)| ≤ 0.5) in Pten or Tsc2 KO in 2i and N24. Trend lines with 95% confidence intervals, and Pearson’s correlation coefficients ( R ) are shown. h Flow cytometry analysis of Rex1-GFP levels in indicated cell lines at N24, after indicated treatments. Rapamycin-treated WT cells are shown as dashed grey line. One representative of n  = 5 independent experiments is shown. i Expression levels of indicated genes in indicated cell lines at N24 after indicated treatments measured by RNA-seq. p adj. values resulting from DESeq analysis are indicated in the plot. j Box plot showing expression of 377 naïve genes (naïve early and naïve late genes 33 ) in indicated KOs and conditions measured by RNA-seq. p values from two-tailed Wilcoxon signed rank tests are indicated. k Box plot showing the expression of 377 naïve genes in indicated KOs and conditions measured by RNA-seq. p value from two-tailed Wilcoxon signed rank test is indicated. All box plots in this paper show the 25th and 75th percentiles and median. Whiskers indicate minimum and maximum values.

Pten mRNA and protein levels increase during exit from naïve pluripotency, while phospho-AKT (pAKT) levels are concomitantly reduced (Fig.  1c–e ), suggesting that PTEN may promote the transition to formative pluripotency by decreasing AKT activity. In Pten KOs, phospho-AKT levels were significantly higher than in WT cells (Fig.  1d ). Thus, we set out to delineate the molecular mechanism by which PTEN-mediated AKT inhibition might drive exit from naïve pluripotency.

When active, AKT phosphorylates several targets that play crucial roles in distinct cellular processes. Among AKT targets, TSC2 (Tuberous Sclerosis 2), GSK3 (Glycogen Synthase Kinase 3), and the FOXO (Forkhead box O) class of transcription factors are strong candidates for mediating changes in mESC differentiation states. Indeed, these factors have been identified as hits in genetic screens for differentiation drivers 27 , 28 , 29 , 30 , 31 , suggesting that the AKT/mTORC1, AKT/GSK3 and AKT/FOXO signalling axes could all participate in the regulation of the transition from naïve to formative pluripotency.

Therefore, we started by investigating the involvement of AKT/mTORC1. AKT-mediated phosphorylation leads to TSC2 inhibition and consequent mTORC1 activation. mTORC1 is one of two distinct complexes containing the serine/threonine protein kinase mTOR and regulates essential cellular processes, including cell growth, protein synthesis and autophagy via phosphorylation of S6K, 4EBP-1 and ULK1, respectively 32 . ESCs lacking Tsc2 retained higher Rex1-GFP levels at N24 compared to WT cells, similar to Pten KO cells 28 (Supplementary Fig.  1c, d ).

To evaluate whether Pten acts through the AKT/mTORC1 axis to regulate the exit from naïve pluripotency, we inspected RNA sequencing (RNA-seq) data from Pten and Tsc2 KO mESCs 28 (Supplementary Fig.  1e ). Both KOs showed delayed downregulation of naïve and delayed upregulation of formative marker genes at N24, with more pronounced effects observed in Tsc2 KOs (Fig.  1f ). Both in 2i and at N24, Pten and Tsc2 KOs deregulated a similar set of genes (differentially expressed genes, DEGs; Fig.  1g ). In 2i, DEGs were enriched for terms associated with lysosomal and metabolic regulation, in line with the known role of mTORC1 in regulating those processes 27 , 31 , 32 (Supplementary Fig.  1f ). Consistent with the observed naïve exit defect, genes upregulated at N24 were enriched for terms related to pluripotency (Supplementary Fig.  1g ).

Next, we inspected the phosphorylation level of direct targets of mTORC1 in WT, Pten KO and Tsc2 KO ESCs. Phospho-4EBP1 (p4EBP1) and phospho-S6K (pS6K) were similarly increased in both KOs, confirming that Pten or Tsc2 deletion increases mTORC1 pathway activity. Addition of the mTORC1 inhibitor Rapamycin reduced mTORC1 activity in both KOs (Supplementary Fig.  1h, i ).

We then assessed whether this reduction restored normal differentiation potential. Rapamycin treatment promoted faster downregulation of Rex1-GFP in WT ESCs, and resulted in a complete rescue of the differentiation defect of Tsc2 KO cells (Fig.  1h ), in line with previously published data 27 . In contrast, in Pten KO cells Rapamycin achieved only a partial rescue. The restoration of differentiation potential was specific for ESCs depleted for members of the PI3K/AKT/mTOR pathway. Naïve exit-defective ESCs lacking the WNT/GSK3 pathway effector Tcf7l1 22 , 28 did not restore Rex1-GFP downregulation kinetics upon Rapamycin treatment.

To evaluate the extent of phenotypic rescue of Pten KO cells by Rapamycin, we performed RNA-seq using WT, Pten KO and Tsc2 KO mESCs, differentiated in presence of DMSO or Rapamycin (Supplementary Fig.  1j ). Changes in naïve pluripotency expression upon Rapamycin treatment were TF-specific: While addition of Rapamycin restored Nanog expression to WT levels in both Pten and Tsc2 KOs, Esrrb and Klf5 expression remained elevated in Pten KOs compared to Tsc2 KOs (Fig.  1i ). Further corroborating an incomplete rescue of Pten KOs by mTORC1 inhibition, a set of 377 previously identified naïve pluripotency-specific genes 33 showed a significantly stronger reduction in expression levels upon Rapamycin treatment in Tsc2 compared to Pten KOs (Fig.  1j, k ). Together, these results highlight that the Pten KO phenotype is not exclusively determined by hyperactivity of mTORC1.

This prompted us to next investigate the role of AKT-mediated phosphorylation of GSK3 on Serine 9, which tags it for degradation and thereby stabilises β-catenin 22 . GSK3 phosphorylation was recently proposed to be crucial for maintaining pluripotency in Pten KO mESCs 23 . Consistently, phospho-GSK3 (pGSK3) levels are increased in Pten KO cells (Supplementary Fig.  1k ). We hypothesised that if indeed deactivation of the GSK3-TCF7L1 axis of the WNT pathway leads to the differentiation defect in Pten KO cells, then the resulting WNT pathway hyperactivity should be epistatic to the pharmacological inhibition of GSK3 by CHIRON. Such an epistatic interaction was observed in Tcf7l1 KOs, where the activity of the β-catenin destruction complex is rendered obsolete and, hence, the addition of CHIRON had no additional effect (Supplementary Fig.  1l ). In contrast, treatment of Pten KOs with CHIRON resulted in delayed differentiation speeds akin to WT cells. Furthermore, we did not previously observe a transcriptional signature typical of increased WNT activity in Pten KO ESCs 28 . This suggests that AKT-hyperactivity-dependent phosphorylation of GSK3 in Pten mutants has little causative impact on the exit from naïve pluripotency in our cellular system.

Collectively, these data show that Pten regulates pathways in addition to mTORC1 that are relevant for proper exit from naïve pluripotency. As the role of GSK3 appeared minor, we focussed our attention on FoxO TFs, the third signalling axis downstream of AKT.

FoxO nuclear translocation promotes naïve pluripotency exit

FoxO TFs regulate several crucial cellular processes, including cell cycle, apoptosis and DNA repair 34 . AKT-mediated phosphorylation retains FoxO TFs in the cytoplasm. In both WT and Pten KO ESCs cultured in 2i, immunofluorescence (IF) analysis revealed a largely cytoplasmic localisation of FOXO1. Upon exit from the naïve state in WT cells, we observed that FOXO1 translocated to the nucleus, concomitant with reduced ESRRB levels (Fig.  2a, b ) and a downregulation of AKT activity (Fig.  1d, e ) 35 . Increased nuclear FOXO1 levels could be observed as early as 8 h after 2i withdrawal (N8) and increased further until N24. This nuclear translocation of FOXO1 at the onset of ESC differentiation was severely impaired in Pten KO cells, which maintained a clear cytoplasmic FOXO1 localisation at N24 (Fig.  2a, c ). Nucleo-cytoplasmic fractionation experiments showed similar results for FOXO3 (Supplementary Fig.  2a ). Sequestration of FOXO1 in the cytoplasm is regulated through pAKT-mediated FOXO1 phosphorylation (pFOXO1). Consistently, we observed a decrease in pFOXO1 concomitant with an increase in total FOXO1 during the exit from naïve pluripotency in WT. In Pten mutant cells pFOXO1 levels are elevated in 2i and at N24 (Supplementary Fig.  2b ).

a Confocal microscopy images after IF, showing FOXO1 (purple) and ESRRB (green) in WT and Pten KOs in 2i and at N24. DAPI staining is shown in blue. One representative image from n  = 3 independent experiments is shown. Scale bar = 20 μM. b Quantification of FOXO1 and ESRRB nuclear intensity (in arbitrary unit, arb. units) measured from confocal images of WT cells in 2i ( n  = 135) and 8 h (N8, n  = 136), 16 h (N16, n  = 88) and 24 h (N24, n  = 127) after 2i withdrawal. Data were obtained from n  = 2 independent experiments. p values are derived from a two-tailed Wilcoxon rank sum test. c Quantification of FOXO1 nuclear intensity (arb. units) based on images of WT ( n  = 189 [2i] and n  = 164 [N24]) and Pten KO ( n  = 100 [2i] and n  = 153 [N24]), as in ( a ). Data were obtained from n  = 2 independent experiments. p values were calculated as in ( b ). d Confocal microscopy images after IF showing FOXO1 (white), SOX2 (green), GATA4 (red), or OTX2 (red) in WT E4.5, E4.75 and E5.5 embryos. Hoechst staining is shown in cyan. One representative image of n  = 3 independent experiments is shown. Scale bar = 100 μM. e Quantification of FOXO1 nuclear intensity (arb. units) measured from confocal images of WT epiblast from E4.5 ( n  = 12), E4.75 ( n  = 12) and E5.5 ( n  = 8) embryos, as in ( d ). Data were obtained from n  = 3 independent experiments. Indicated p values are derived from a two-tailed Wilcoxon rank sum test.

Supporting a functional relevance of nuclear shuttling of FoxO TFs in vivo, we detected transient nuclear FOXO1 in the OTX2-positive epiblast of E4.75 embryos. This was not detectable in E4.5 or in E5.5 epiblast cells (Fig.  2d, e and Supplementary Fig.  2c ). This is consistent with the hypothesis that FOXO1 nuclear shuttling is involved in the initiation of formative pluripotency in in vivo peri-implantation development.

To test whether nuclear translocation of FOXO1 was indeed AKT-dependent, we analysed the nuclear vs. cytoplasmic localisation of FOXO1 after treatment with the specific allosteric AKT inhibitor MK-2206 36 . MK-2206 treatment elevated levels of nuclear FOXO1, in both 2i and at N24, and resulted in expedited downregulation of Rex1-GFP in WT cells (Supplementary Fig.  2d–f ), consistent with a requirement for reducing AKT activity to enable and possibly trigger the exit from naïve pluripotency.

Our results support the hypothesis that nuclear translocation of FoxO1 is crucial to induce proper exit from naïve pluripotency. To test this hypothesis, we performed doxycycline-induced expression of a constitutively nuclear version of FoxO1 (3xFLAG-FoxO1 nuc ) 37 . This treatment was sufficient to extinguish naïve pluripotency in WT cells cultured in 2i within 8 h in a dose-dependent manner (Supplementary Fig.  2g–j ). Underscoring a causal link between aberrant localisation of FOXO1 in Pten mutant ESCs and defects in naïve exit, 3xFLAG-FoxO1 nuc expression in Pten KO ESCs rescued their differentiation defect (Supplementary Fig.  2k ). Interestingly, overexpression of nuclear FoxO1 in Pten KO cells had distinct results on different naïve markers: While it reduced the expression of Nanog and Esrrb , in line with the global rescue of the differentiation defect of Pten mutants, it upregulated the expression of Klf5 in a dose-dependent manner (Supplementary Fig.  2l ). This indicates that FoxO1 can exert target-specific activator or silencer function. We also noted that prolonged exposure to high levels of nuclear FoxO1 (>24 h) was cytotoxic, probably due to induction of the pro-apoptotic programme by FoxO1 38 .

In summary, our data show that AKT-mediated nuclear translocation of FoxO1, and possibly other FoxO TFs, is essential for the timely exit from naïve pluripotency. We conclude that lack of nuclear translocation of FoxO TFs underlies, at least in part, the differentiation defect observed in Pten KO mESCs.

FoxO TFs bind enhancers activated in formative pluripotency

To explore the molecular role of FoxO TFs in the transition from naïve to formative pluripotency, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis for FOXO1 and FOXO3 in WT and Pten KO cells. This analysis was performed in 2i and at N24, that is, 24 h into the formative transition (Fig.  3a and Supplementary Fig.  3a–c ). In WT cells, we observed a strong increase in the number of genomic loci bound by FOXO1 and FOXO3 at N24 compared to 2i. We detected 1314 and 391 peaks in 2i and 2840 and 623 peaks at N24 for FOXO1 and FOXO3, respectively. This is consistent with increased nuclear localisation of FoxO TFs at the exit from naïve pluripotency. In agreement with the largely cytoplasmic localisation in the absence of Pten , FOXO TF ChIP-seq signals were barely detectable in Pten KO cells (Supplementary Fig.  3b, c ). Of note, due to low amounts of precipitated DNA in 2i, ChIP-seq library-prep will most likely have amplified signal in 2i relative to N24. Hence, a direct quantitative comparison between 2i and N24 FOXO TF signal is not possible.

a Heatmap displaying FoxO1 signal within a 1.5 kb window around FoxO1 peak-centres in WT cells at 2i and N24, categorised into 2i-only ( n  = 612, green), N24-only ( n  = 2138, blue) and shared peaks ( n  = 702, purple). b Heatmaps showing H3K27ac (left) and p300 (right) signal in ESCs and EpiLCs 39 in a 1.5 kb window around FoxO1 peak-centres. c Venn diagrams showing overlaps between FoxO1 peaks in 2i and N24 with ESC and EpiLC enhancers (top). Bar plots display the percentage of FoxO1 peaks overlapping with ESC-specific (ESCe), EpiLC-specific (EpiLCe) or shared (ESCe&EpiLCe) enhancers (bottom).  p values are derived from hypergeometric tests. d Heatmap showing ATAC-seq signal within a 1.5 kb window around FoxO1 peak-centres in WT and Pten KOs in 2i or at N24. e Heatmaps showing Oct4 signal in ESCs and EpiLCs 39 in a 1.5 kb window around FoxO1 peak-centres. f Venn diagrams showing the overlap between FoxO1 peaks in 2i or at N24 with Oct4 peaks in ESCs or EpiLCs (top). Bar plots showing the percentage of OCT4-bound enhancers overlapping with FoxO1 peaks (bottom). p values are derived from hypergeometric tests. g Heatmap showing Otx2 signal in EpiLCs 39 in a 1.5 kb window around FoxO1 peak-centres. h Venn diagram showing the overlap between FoxO1 peaks at N24 with Otx2 peaks in EpiLCs (top). Bar plots showing the percentage of OTX2-bound enhancers overlapping with FoxO1 peaks (bottom).  p values are derived from hypergeometric tests. i Heatmap showing Esrrb signal in WT cells in 2iL, N48 and N96 33 in a 1.5 kb window around FoxO1 peak-centres. j Venn diagrams showing the overlap between FoxO1 peaks in 2i and at N24 with Esrrb peaks in 2iL, N48 or N96 (top). Bar plots showing the percentage of FoxO1 peaks (2i-only, N24-only or shared) that overlap with Esrrb peaks (bottom).  p values are derived from hypergeometric tests. k Venn diagrams showing the overlap between FoxO1 peaks in 2i and at N24 with β-catenin peaks (top). Barplots showing the percentage of FoxO1 peaks in indicated categories overlapping with β-catenin peaks (bottom).  p values are derived from hypergeometric tests.

FOXO1 and FOXO3 ChIP-seq data showed a significant overlap. Almost 50% of FOXO3 peaks overlapped with FOXO1 peaks, and FOXO1 signal was detected at virtually all FOXO3 peaks (Supplementary Fig.  3c, d ). Based on this finding, we performed all downstream analysis with the group of FOXO1-bound loci, if not otherwise stated. We then divided FOXO1-bound regions into three groups depending on peak-calling results: 2i-only peaks (612), N24-only peaks (2138) and shared peaks (702) (Fig.  3a ). KEGG and REACTOME pathway enrichment analyses (EA) showed an enrichment for ubiquitin mediated proteolysis in the 2i-specific peaks and for PI3K/AKT signalling, focal adhesion and actin cytoskeleton related terms in the N24 specific peaks. Shared peaks were enriched for terms related to general pluripotency and TGF-β signalling (Supplementary Fig.  3e and Supplementary Data  1 ). All peak categories were enriched for FoxO-motifs (Supplementary Data  1 ).

FoxO TF peaks were located mainly outside of promoter regions (Supplementary Fig.  3f ), indicating a potential contribution of FoxO TFs to enhancer regulation. To test this, we utilised published ChIP-seq datasets 39 for the enhancer marks H3K27ac and p300, obtained in ESCs and in epiblast-like cells (EpiLCs). EpiLCs represent the in vitro counterpart of formative epiblast cells of the E5.5 blastocyst 40 and correspond to a developmental state akin to mESCs at N24. We found that strong H3K27ac and p300 signals in EpiLCs overlapped with the regions we identified as bound by FoxO TFs at N24 (Fig.  3b ). Moreover, regions bound by FoxO TFs in 2i were significantly enriched in ESC-specific enhancers and enhancers active in ESCs and EpiLCs 41 (Fig.  3c ), whereas EpiLC-specific enhancers were not significantly bound by FOXO1 in 2i. In contrast, N24 FoxO1-peaks significantly overlapped with shared and EpiLC-specific enhancers while showing no significant overlap with ESC-specific enhancers. A striking total of 74% of FoxO1 peaks at N24 are found on EpiLC enhancers. Consistently, we detected a significant enrichment of FoxO1 and FoxO3 motifs in EpiLC-specific enhancers (Supplementary Fig.  3g ). This is consistent with a role for FoxO TFs in activating formative-specific regulatory regions upon exit from the naïve state.

To assess a potential role of AKT signalling in mediating chromatin accessibility, we performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) in WT and Pten KO cells, in 2i and at N24 (Fig.  3d and Supplementary Fig.  3h ). Regions that showed FoxO TF binding exclusively in 2i were open only in the naïve state and showed weak ATAC-seq signal at N24. In contrast, regions exhibiting FoxO TF binding at N24 showed near equal ATAC-seq signal in both 2i and at N24. At the resolution of our analysis, ATAC-seq signal was indistinguishable between WT and Pten KO cells, in which FoxO TF translocation to the nucleus is largely abolished. This suggests that FoxO TFs are unlikely to act as pioneer factors that open chromatin during the naïve to formative transition. Instead, our data is consistent with a role of FoxO TFs in activating already open poised chromatin as suggested before 42 also during the exit from naïve pluripotency.

OCT4 and OTX2 are key regulators of general and formative pluripotency, respectively. OTX2 causes relocation of OCT4 from ESC- to EpiLC-specific enhancers upon the exit from the naïve state 39 . FoxO1 2i peaks showed a significant but relatively weak overlap with OCT4-bound ESC enhancers, and 4% of all OCT4-bound ESC enhancers were FoxO1 targets (Fig.  3e, f ). In contrast, OCT4-bound EpiLC enhancers showed a stronger and highly significant overlap with FOXO1 at N24, and 12% of OCT4-bound EpiLC-enhancers were FoxO1 targets at N24 (Fig.  3f ). A similarly large portion of 14% of OTX2-bound EpiLC enhancers were bound by FOXO1 at N24 (Fig.  3g, h ). Moreover, our analysis clearly showed that colocalization of FOXO1 with OCT4 and OTX2 occurs nearly exclusively on enhancers, suggesting that FoxO TFs regulate enhancer activation in cooperation with pluripotency-state specific TFs.

Recently it was shown that Esrrb , originally identified as a naïve pluripotency-specific TF, performs additional functions in the initiation of the formative transcription programme 33 . To investigate whether FoxO TFs cooperate with ESRRB, we examined the overlap on chromatin between FOXO1 and ESRRB throughout the pluripotency continuum (Fig.  3i, j ). These analyses showed that a significant 35% of 2i-specific and 38% of shared FoxO1 peaks are also bound by ESRRB in 2i (Fig.  3j ). Regions bound by ESRRB at N48 still showed a highly significant overlap with FOXO1 binding, with 28% of shared FoxO1 peaks also decorated by ESRRB at N48. These FOXO1-ESRRB co-bound loci were in close proximity to, and thus potentially regulate, known formative ( Otx2 , Dnmt3a , Lef1 ) and naïve marker genes ( Nanog , Esrrb , Zfp42 , Klf2 , Klf4 , Klf5 , Tfcp2l1 , Nr5a2 , Prdm14 ) (Supplementary Data  2 ). Co-bound regions consistently show an overall stronger binding intensity of FOXO1 and ESRRB and also exhibit higher overall H3K27ac and p300 levels compared to loci bound by only FOXO1 or only ESRRB (Supplementary Fig.  3i ) . We also noted that regions only bound by ESRRB exhibited weaker ATAC-seq signal, indicating that ESRRB requires additional factors such as FOXO1 to achieve full chromatin activation. FOXO1-only bound regions, in turn, showed clear enhancer activity in 2i and at N24, suggesting that FOXO1 can work independently of ESRRB.

FoxO TFs have been shown to interact with the WNT pathway effector β-catenin to enhance transcriptional output 43 . In line with this, a highly significant 50% of all FOXO1-bound regions were also occupied by β-CATENIN (Fig.  3k ), suggesting a functional interaction of FoxO TFs and β-catenin during the transition to formative pluripotency.

In sum, our findings reveal that FoxO TFs bind to a large set of enhancers that are activated upon the exit from naïve pluripotency. FoxO1 cooperates with core components of the naïve and formative TF-repertoire. We hypothesise that this is to ensure faithful firing of the formative GRN.

FoxO TFs instruct rewiring of the naïve to the formative GRN

We next sought to identify the transcriptional consequences of FoxO TF induced changes to chromatin at the exit from naïve pluripotency. We specifically wanted to know which components of the formative state-specific GRN might be functionally dependent on regulation by FoxO TFs. To this end, we compared the changes in transcript levels upon exit from naïve pluripotency 28 between distinct sets of FoxO1-bound genes as defined above. Overall, FoxO1 targets were highly enriched in genes differentially expressed within the first 24 h of ESC differentiation (Supplementary Fig.  4a ). 2i-specific FoxO1 targets were overall downregulated at N24, while genes bound by FOXO1 at N24 showed an overall upregulation during naïve exit (Fig.  4a and Supplementary Fig.  4b ). The expression of targets bound in both conditions (2i&N24) did not show a strong and consistent directional change in gene expression during the exit from naïve pluripotency. To further investigate the expression kinetics of FoxO1 target genes during the exit from naïve pluripotency, we followed their transcript levels across a 32 h differentiation time course at a 2 h-resolution 28 . We found that 2i-specific FoxO1 targets showed a largely continuous downregulation, N24-specific targets a continuous upregulation, and 2i&N24 targets a lack of clear directionality (Supplementary Fig.  4c ).

a Boxplot illustrating expression of 2i-specific ( n  = 249, green), N24-specific ( n  = 889, blue), and 2i&N24 ( n  = 486, purple) FoxO1 targets in WT cells at N24, measured by RNA-seq. Data show log2FC relative to 2i and p values from Wilcoxon rank sum tests. Non-targets are shown in grey. b Bootstrapping analysis (30,000 draws) for gene-sets of the same size as the sample gene-sets (2i-specific, 249 genes; N24-specific, 889 genes; 2i&N24, 486 genes). The distribution of average log2FC is plotted, with p values indicating the probability of the predicted exceeding the measured effect-size. c Upset plot showing the overlap of FoxO1 targets with core naïve and formative markers. d Genome browser snapshots displaying FoxO1 ChIP-signal in WT on selected naïve markers in 2i (green) and N24 (blue). Called peaks are indicated (2i-only: green, N24-only: blue, shared: purple). e Genome browser snapshots showing FoxO1 ChIP-signal in WT cells on selected formative markers in 2i (green) and N24 (blue). Called peaks are indicated (2i-only: green, N24-only: blue, shared: purple). f Box plot depicting expression of formative genes ( n  = 832) in WT cells at N24, divided into FoxO1 N24 targets (blue) or non-targets (grey), as measured by RNA-seq. Data are shown as log2FC relative to 2i. p value from two-tailed Wilcoxon rank sum tests is indicated in the plot. g Box plot showing the expression of core formative genes ( n  = 12) in WT cells at N24, divided into FoxO1 N24 targets (blue) or non-targets (grey), measured by RNA-seq. Data are shown as log2FC relative to 2i. p value from two-tailed Wilcoxon rank sum test is indicated. h Box plot showing the expression of FoxO1 targets ( n  = 726, blue) and non-targets ( n  = 5061, grey) in E5.5 and E6.5 embryos 28 , 68 . Absolute log2FC relative to E4.5 are shown. p values from two-tailed Wilcoxon rank sum test are indicated. i Similar to ( b ), bootstrapping analysis (30,000 draws) for gene-sets of the same size as the sample gene-sets ( n  = group size). The distribution of average log2FC is plotted, with p values indicating the probability of the predicted exceeding the measured effect-size.

To further determine whether the observed gene expression changes were stronger than expected by chance, we computed the distribution of average log2 fold changes (log2FC) across 30,000 randomly sampled gene groups. Sampled gene groups were matched in size to different FoxO1 target groups (Fig.  4b ) and individual genes were drawn from RNA-seq data comparing expression between 2i and N24. The results showed that 2i- and N24-specific FoxO1 targets by far exceed the levels of gene expression changes observed in any randomly sampled gene group. Specifically, FoxO1 2i targets are highly significantly more downregulated, and N24 FoxO1 targets are highly significantly more upregulated during the exit from naïve pluripotency than randomly selected gene sets. These findings suggest that FoxO1 binding is a major determinant of gene expression changes during differentiation.

Recent work reported transcriptome analysis of differentiating mESCs up to 96 h after 2i withdrawal and defined 6 distinct groups based on gene expression kinetics: naïve early and naïve late (downregulated early or late upon naïve exit), formative early and formative late (upregulated during naïve exit), committed early and committed late (upregulated late during naïve exit) 33 . We found that 2i-specific FoxO1 targets were enriched for naïve and formative early genes, whereas N24-specific and 2i&N24 FoxO1 targets were enriched for naïve early, formative (early and late) and committed early genes (Supplementary Fig.  4d ).

As these results indicated a link between FoxO TFs and central components of the naïve and formative GRNs, we further tested whether FoxO1 binds and potentially regulates core members of the naïve or formative GRN. We found that 9 out of 13 core naïve marker genes (modified from ref. 44 ) were bound by FoxO1 in 2i ( Tfcp2l1 , Nr5a2 ), in both 2i and at N24 ( Nanog , Esrrb , Zfp42 , Klf2 , Klf4, Prdm14 ) or only at N24 ( Klf5 ) (Fig.  4c, d and Supplementary Data  2 ). N24-specific binding of Klf5 might explain why this naïve marker gene is further up- instead of down-regulated, when nuclear FOXO1 is expressed in Pten KOs during differentiation (Supplementary Fig.  2l ) . Conversely, 8 out of 12 key formative marker genes 44 (hereafter referred to as core formative genes) were bound by FoxO1 at N24 ( Dnmt3b , Pou3f1 , Fgf5 , Sall2 , Pim2 ) or under both conditions ( Dnmt3a , Otx2 , Lef1 ) (Fig.  4c , e and Supplementary Fig.  4e ). Notably, formative genes (early and late, as described above) and core formative genes that are direct FoxO1 targets showed a significantly stronger upregulation during the exit from naïve pluripotency than non FoxO1-targets (Fig.  4f, g ).

Notably, in vivo FoxO1 targets are upregulated more strongly than non-targets between E4.5 and E5.5 (Fig.  4h ). This is consistent with our previous results showing high nuclear FoxO1 levels in E4.75 blastocysts (Fig.  2d, e ), and we hypothesise that transient nuclear localisation of FoxO1 in rosette-stage blastocysts at E4.75 facilitates the establishment of the formative post-implantation like pluripotency programme.

Among the FoxO1 targets, we identified a significant number of genes that were also found in a genetic screen for genes controlling the exit from naïve pluripotency, here referred to as ‘exit factors’ (Supplementary Fig.  4f ) 28 , 29 . Among exit factors that are upregulated during naïve exit, FoxO1 targets showed significantly stronger regulation (Supplementary Fig.  4g ). This suggests that FoxO TFs might function by facilitating the activity of multiple processes required for proper differentiation.

In total, 22% of genes differentially expressed between WT and Pten KOs at N24 were FoxO1 target genes (Supplementary Fig.  4h ). Furthermore, those components of the core formative GRN that are FoxO1 targets exhibited a stronger deficiency in upregulation compared to non-targets in Pten mutant cells at N24 (Supplementary Fig.  4i ). Further highlighting a causal link between inactivation of the FoxO-signalling axis in Pten KOs and the molecular defects observed in these mutants, FoxO1 2i target genes showed a significantly stronger downregulation compared to non-targets in Pten KO ESCs (Supplementary Fig.  4j, k ).

To finally assay the impact of FoxO1 relative to other known master regulators of pluripotency, we again performed bootstrapping experiments, akin to the ones described above (Fig.  4b ). We generated the distribution of average log2FC during naïve exit across 30,000 random sampled groups of genes, each matched to the size of the respective TF-target group (Fig.  4i ). Our analyses show that FoxO1 targets exhibit both a stronger positive and negative transcriptional response during naïve exit than targets of well-established pluripotency TFs, such as OCT4, SOX2, ESRRB or NANOG. Only OTX2-bound genes exhibited a slightly stronger upregulation and TCF7L1- and β-CATENIN bound genes a stronger downregulation than FoxO1 target genes. Notably, even within OTX2-bound genes, genes co-bound by OTX2 and FOXO1 showed the strongest transcriptional upregulation, suggesting an interaction between OTX2 and FOXO1 at the initiation of the formative GRN (Supplementary Fig.  4l ). Genes co-bound by FOXO1 and TCF7L1 in ESCs showed a significantly stronger transcriptional downregulation during differentiation compared to single factor targets and non-targets (Supplementary Fig.  4m ), suggesting a cooperative role of FOXO1 and TCF7L1 in silencing the naïve pluripotency specific GRN. Together, these data support a dual role for FoxO1 in activation of the formative and silencing of naïve specific genes, depending on the chromatin context.

In sum, our data suggest that FoxO1 acts as a key regulator of the naïve to formative transition by binding to and regulating major components of the naïve and formative-specific GRNs. This function is potentially performed in cooperation with core pluripotency TFs such as OCT4, ESRRB, NANOG, TCF7L1 and OTX2 which are known to regulate multiple pluripotency states and transitions. Our analysis further indicates that the effect size of FoxO1 regulation surpasses that of most other core pluripotency TFs. Together, this positions FoxO1 as central TF to regulate the transition from naïve to formative pluripotency.

FoxO TFs are required for proper exit from naïve pluripotency

To investigate a potential requirement for FoxO1 for the transition from naïve to formative pluripotency, we analysed the differentiation capacity of ESCs after FoxO1 depletion. To this end, we turned to knockdown (KD) experiments using short-interfering RNAs (siRNAs). Treatment with siRNAs against FoxO1 resulted in the downregulation of transcript and protein levels (Supplementary Fig.  5a, b ). Cells with reduced levels of FoxO1 (siFoxO1) retained higher expression of Rex1-GFP at N24 compared to cells treated with scrambled siRNAs (siScr), suggesting that FoxO1 is required for proper exit from naïve pluripotency (Fig.  5a ).

a Flow cytometry analysis of Rex1-GFP levels in WT cells transfected with control (siGFP and siScr, grey) or siRNAs targeting FoxO1 (siFoxO1, red). One representative of n  = 3 independent experiments is shown. b Volcano plot showing RNA-seq data of WT cells at N24, treated with siRNA against FoxO1 . DEGs ( p adj. ≤ 0.2) that are bound by FoxO1 are colour coded depending on whether they are bound only in 2i (green), only at N24 (blue) or in both conditions (purple). Selected naïve and formative genes are indicated in the plot. For each quadrant, percentages (%) of FoxO1 2i-only, N24-only and 2i&N24 targets are indicated. c CUT&RUN analysis of H3K27ac levels on indicated enhancers after FoxO1 siRNA treatment. Signal was normalised to a genomic background region that did not exhibit any H3K27ac signal in WT cells. Data were further normalised to a H3K27ac peak found in Drosophila spike-in cells. Data are shown as relative to siScr control. n  = 2 biological replicates. An independent experiment with further n  = 2 biological replicates shown in Supplementary Fig.  5g .

KEGG pathway enrichment analysis after RNA-seq analysis of FoxO1-siRNA treated cells at N24 showed that upregulated genes were enriched for signalling pathways associated with pluripotency maintenance (Supplementary Data  1 ), confirming the differentiation defect. Further consistent with the differentiation delay upon FoxO1 depletion, we observed that naïve pluripotency markers failed to be down- and formative markers upregulated upon FoxO1 KD (Supplementary Fig.  5c ). Both genes that were up- and downregulated upon FoxO1 depletion were highly enriched in FoxO1 ChIP-seq targets (Fig.  5b and Supplementary Fig.  5d ), with 30% and 25%, respectively, being bound by FOXO1.

Overall, genes up- or downregulated in Pten KOs at N24 were also up- or downregulated in siFoxO1-treated cells at N24 (Supplementary Fig.  5e ) and FoxO1 target genes showed a stronger deregulation in the absence of either Pten or FoxO1 compared to non-targets (Supplementary Fig.  5f ). This further supports the proposition that the Pten KO phenotype is caused, in part, by misregulated FoxO TF localisation.

To evaluate whether activation of formative-specific enhancers depends on FoxO1 action, we performed CUT&RUN experiments. We assayed H3K27ac levels as a proxy for enhancer activity on a set of known formative gene enhancers in presence and after depletion of FoxO1 . These experiments showed that indeed activity of enhancers of Dnmt3b , Pou3f1 and Sall2 and the enhancer of the known FoxO1 target Enpp2 are dependent on normal FoxO1 levels (Fig.  5c and Supplementary Fig.  5g ).

Together, our results show that proper levels of FoxO1 are required for the effective exit from naïve pluripotency and that loss of FoxO1 abrogates proper formative pluripotency-specific enhancer activation.

Formative GRN activation by forced nuclear FoxO1 shuttling

If nuclear shuttling of FoxO TFs is indeed involved in initiating the formative GRN, then artificial inactivation of AKT in the naïve pluripotent state and consequent nuclear accumulation of FoxO TFs should trigger expression of formative-specific genes. To test this hypothesis, we performed RNA-seq after treatment with the AKT inhibitor MK-2206 in 2i and at N24. Consistent with data shown before (Supplementary Fig.  2f ), PCA analysis indicated that MK-2206 treatment propelled cells towards a more differentiated state, both when added to naïve ESCs and to cells exiting naïve pluripotency. (Supplementary Fig.  6a ). Importantly, FoxO1 targets identified by ChIP-seq represented 27% and 30% of all deregulated genes upon MK-2206 treatment in 2i cells or at N24, respectively (Fig.  6a ). Moreover, 44 out of the 105 DEGs in 2i that were not direct FoxO1 targets were differentially expressed during naïve exit in WT cells. MK-2206 treatment in the naïve state led to a highly significant upregulation of FoxO1 N24 targets but had less of an impact on genes that are already FoxO1 targets in 2i (Fig.  6b ).

a Venn diagrams showing the overlap between FoxO1 targets (purple) and genes differentially expressed upon MK-2206 treatment in 2i (top) or at N24 (bottom) (DEGs, p value ≤ 0.05, grey). p values derived from hypergeometric tests of the overlaps are indicated. b Bootstrapping analysis (30,000 draws) of gene-sets of the same size as the sample gene sets (2i-specific, 200 genes; N24-specific, 731 genes; 2i&N24, 420 genes). The distribution of average log2FC is plotted. Empirical p values for the observed log2FC changes in FoxO1 target gene groups are shown. c Box plot showing the expression of core formative genes ( n  = 12) in WT cells after MK-2206 treatment in 2i, divided into FoxO1 N24 targets (blue) and non-targets (grey). Data are shown as log2FC relative to NT cells. p value from two-tailed Wilcoxon rank sum test is indicated. d Box plot showing the expression of core formative genes ( n  = 12) in WT cells after MK-2206 treatment at N24, divided into FoxO1 N24 targets (blue) or non-targets (grey). Data are shown as log2FC relative to NT cells. p value from two-tailed Wilcoxon rank sum test is indicated. e Box plot showing the expression of formative genes ( n  = 814) in WT cells treated with MK-2206 in 2i, divided into FoxO1 N24 targets (blue) or non-targets (grey). Data are shown as log2FC relative to NT cells. p value from two-tailed Wilcoxon rank sum test is shown. f Box plot showing the expression of naïve genes ( n  = 411) in WT cells treated with MK-2206 in 2i, divided into FoxO1 N24 targets (blue) or non-targets (grey). Data are shown as log2FC relative to NT cells. p value from two-tailed Wilcoxon rank sum test is indicated.

Core formative FoxO1 targets Pou3f1 , Pim2 , Dnmt3b , Lef1 , Fgf5 , Otx2 and Sall2 showed a stronger response to MK-2206 treatment in both 2i and at N24 than non-targets ( Hes6 , Sox12 , Sox3 , Tead2 ) (Fig.  6c, d ). Consistent results were obtained using a larger set of formative-specific genes (Fig.  6e ). Interestingly, FoxO1-bound components of the naïve GRN also showed a positive response to MK-2206 treatment in 2i medium (Fig.  6f ). A specific response to FoxO signalling rather than mTORC1 deregulation is shown by the fact that treatment with Rapamycin had no specific effect on FoxO1 targets (Supplementary Fig.  6b ). Together, this suggests that AKT inhibition and subsequent nuclear FOXO1 translocation is sufficient to initiate major components of the formative GRN, even in ground state culture conditions in 2i medium.

In sum, our work uncovers a role for FoxO TFs downstream of AKT signalling in facilitating the transition from a naïve to a formative pluripotency specific GRN. Our data shows that this is achieved by FoxO1 targeting and regulating large parts of the formative and naïve specific GRNs. Hence, FoxO TFs are pivotal factors in mediating the transition from naïve to formative pluripotency.

In this work, we uncovered a mechanism through which PTEN-mediated AKT regulation controls the transition from naïve to formative pluripotency by regulating nuclear FoxO TF localisation. We demonstrate that FoxO TFs play a fundamental role in orchestrating the exit from naïve pluripotency. Their activity is precisely gated by the activity of PTEN and released once the exit from naïve pluripotency commences (Fig.  7 ). We propose a mechanism for FoxO1 akin to an actuator in mechanical systems that converts control signals into physical action. Accordingly, FoxO1 acts as an actuator by converting signalling inputs into biological outcomes. By facilitating coordinated dismantling of the naïve and ordered establishment of the formative GRNs, FoxO1 ensures timely and faithful differentiation.

Schematic illustration of the proposed model of FoxO TF action at the exit from naïve pluripotency.

FoxO TFs are well-established regulators of multiple fundamental cellular processes including stress response, DNA repair, cell cycle and apoptosis, metabolism and ageing 45 . Although mostly studied for their role in apoptosis, longevity, and cancer, previous studies have reported a function for FoxO TFs in the regulation of cell fate 38 , 46 , 47 , 48 , 49 , 50 , 51 . A recent report has shown an important role for FoxO1 in regulating diapause in mouse embryos 52 . Moreover, consistent with a role in establishing formative pluripotent identity, FoxO TFs are reported inhibitors of reprogramming to a naïve pluripotent ESC state 53 , 54 .

FoxO4 was proposed to be the only FoxO TF family member necessary for human ESC differentiation into the neuronal lineage 49 , whereas FoxO1 depletion resulted in loss of ESC pluripotency in human and mouse ESCs 51 . Consistent with the latter findings, we could not establish FoxO1 KO ES cells. We cannot exclude that this was due to technical rather than biological hurdles, but note that exclusively in 2i, FoxO1 binds to a number of E3-ligases including Mdm2 , Fbx17 and Huwe1 and REACTOME pathway analysis of 2i-only bound genes shows an enrichment for the term ‘Regulation of TP53 Expression and Degradation’. This could indicate a possible role for FoxO1 in maintenance of proper protein homoeostasis or cell cycle control in ESCs.

Relocating transcription factors from the cytoplasm to the nucleus or vice versa allows cells to rapidly respond to changes in signalling inputs by providing a cell state switch with rapid on-off kinetics. A relevant example is the regulation of TFE3, a bHLH transcription factor that is relocated from the nucleus to the cytoplasm. TFE3 is found in the nucleus of naïve ESCs, where it sustains the naïve GRN via transcriptional control of Esrrb 27 . Induced by a metabolic shift, mTORC1-dependent and mTORC1-independent nutrient-sensing pathways converge to cause TFE3 export from the nucleus, thus contributing to the extinction of the naïve GRN 27 , 31 . Whether the export of TFE3 and the import of FoxO TFs are coordinated remains an interesting open question.

Once translocated to the nucleus, FoxO TFs play key roles in the establishment of the formative GRN and contribute to the expression of many genes that are themselves required for the exit from naïve pluripotency. Our ChIP-seq experiment revealed that FoxO1 binds to multiple genomic locations even in naïve conditions and that FoxO1 2i targets are part of the core naïve TF-network. Hence, FoxO1 could be necessary for the maintenance of naïve identity by directly regulating the naïve TF-network. However, FoxO TFs remain bound to multiple core naïve genes even 24 h after the onset of differentiation, when most of them have been transcriptionally inactivated. These binding dynamics pose the question of whether FoxO TFs can act not only as activators, but also as repressors depending on cellular context. Such a role is consistent with the large cohort of naïve TFs bound by FoxO TFs during differentiation, the observed downregulation of several naïve genes upon FoxO1 nuclear overexpression and the increased levels of FoxO TF-bound core naïve genes after FoxO1 siRNA treatment.

But how can such a silencing function be reconciled with the fact that FoxO TFs are mainly known as activators of gene expression? In a recent study on human transcriptional effector domains it was shown that FoxO1 can display activator and repressor activity 55 . Hence, whether FoxO TFs act as activators or repressors might depend on cellular environment, post-translational modifications, or on the co-binding of other TFs and co-factors. This potential dual role of FoxO TFs in mediating both activation and silencing will be an exciting question for future research.

Our data show that many FoxO TF-bound genomic regions are enhancers that are also bound by ESRRB, OCT4, NANOG, OTX2, β-CATENIN and TCF7L1. This places FoxO TFs as a core component of both naïve and formative GRNs. β-CATENIN was reported to physically interact with FOXO4 56 , 57 to increase its activity 43 . Interaction between FoxO TFs and TCF transcription factor derived peptides, in turn, was reported to disrupt β-CATENIN condensate formation, thereby interfering with β-catenin-driven gene expression 58 . Consistent with a repression enhancing role for FoxO1 in cooperation with Tcf7l1 , FOXO1-TCF7L1 co-bound genes show a significantly stronger downregulation compared to single factor targets. These results are exciting, because they suggest that AKT regulates Wnt signalling not only through phosphorylation of GSK3 but achieves both positive and negative regulation of Wnt signalling target genes through interaction of FOXO1 with β-CATENIN and TCF7L1, respectively.

It is also tempting to speculate about a function for cytoplasmic phosphorylated FoxO TFs, which are abundant in the naïve state. Cytoplasmic phosphorylated FoxO TFs can bind to the scaffold protein IQGAP1, and this interaction prevents IQGAP1-mediated ERK-activation 59 . Such an interaction would enable cytoplasmic phospho-FoxO to stabilise naïve identity by inhibiting ERK signalling. Nuclear translocation of FoxO TFs at the onset of differentiation would release ERK inhibition and facilitate the exit from naïve pluripotency. Hence, FoxO nuclear shuttling could execute a dual role to firstly transcriptionally control the naïve and formative core GRNs, and secondly to allow ERK to exert its crucial function at the exit from naïve pluripotency. This would position FoxO TFs as a central cell fate switch that can both shield naïve identity and disrupt it under differentiation-permissive conditions but remains pure speculation.

FoxO TFs are classically seen as tumour suppressors, but a pro-tumorigenic role has been proposed 60 , 61 . Whether the control of cell fate specific programmes in response to a shift in signalling states, as reported here, contributes to the role of FoxO TFs in tumour biology remains to be investigated.

Our work shows that AKT functionally controls at least two major regulatory axes that regulate the transition to post-implantation-like pluripotency, mTORC1 and FoxO TFs. Our epistasis experiments suggest only a minor role for GSK3, which was reported before 23 . However, in the absence of experimental evidence, we cannot formally exclude a contribution of other targets apart from mTORC1 and FoxO TFs that might contribute to proper cell fate transition to formative identity.

Interestingly, FoxO1 is not only regulating embryonic peri-implantation development, but also acts on the maternal side of the pregnancy as critical regulator of endometrial receptivity in vivo 62 , 63 . This suggests a concomitant pivotal dual role for FoxO1 at the time of implantation: to initiate post-implantation epiblast fate in the embryo and to prepare the uterus for proper implantation of the same.

In sum, our work highlights a previously unappreciated role of AKT in safeguarding pluripotency by ensuring cytoplasmic sequestration of FoxO TFs. Upon nuclear translocation, FoxO TFs play a key role in the shutdown of the naïve pluripotent identity and the initiation of the formative gene expression programme. Our findings place FoxO transcription factors, and specifically FoxO1, as a crucial component of the pluripotency TF-network with a pivotal role to instruct formative fate. Signalling-instructed translocation of lineage determining TFs such as FoxO1 provides a paradigm that allows the precise and effective control of delicately balanced GRNs that govern stem cell transitions.

Cell culture

Mouse embryonic stem cells (mESCs) were cultured on gelatin-coated (Sigma-Aldrich, G1890) plates in DMEM high-glucose medium (Sigma-Aldrich, D5671) supplemented with 10% FBS (Gibco, 10270-106), 2 mM L-Glutamine (Sigma-Aldrich, G7513), 0.1 mM NEAA (Sigma-Aldrich, M7145), 1 mM Sodium Pyruvate (Sigma-Aldrich, S8636), 10 µg/ml penicillin-streptomycin (Sigma-Aldrich, P4333), 55 µM β-mercaptoethanol (Fisher Scientific, 21985-023), 10 ng/ml LIF (batch tested, in-house) and 2i (1.5 μM PD0325901 and 3 μM CHIR99021), referred to here as ES DMEM-2i medium. mESCs were passaged every second day and routinely tested negative for mycoplasma infection 28 , 64 .

All cell lines used in this study were derived from a parental cell line carrying a Rex1-GFPd2 reporter (destabilised version of the GFP transgene (GFPd2) under control of the endogenous Rex1 promoter 22 ) and a Cas9 transgene (EF1alpha-Cas9 cassette targeted to the Rosa26 locus (RC9 cells) 30 . Cells lacking Pten , Tsc2 or Tcf7l1 ( Pten KO, Tsc2 KO and Tcf7l1 KO) had been generated previously 28 . Pten-rescue cell lines were generated by cloning the Pten coding sequence (amplified by PCR from RC9 genomic DNA) into a pCAG-3xFLAG-empty-pgk-hph vector 27 .

All work reported here complies with all relevant ethical regulations applicable to research conducted at the Max Perutz Laboratories and the University of Vienna. All animal experiments were approved by the IMP/IMBA animal house and performed in accordance with institutional guidelines.

Monolayer differentiation

mESCs were plated on gelatin-coated plates at a final density of 1 × 10 4 cells/cm 2 in N2B27 medium (1:1 mix of DMEM/F12 (Gibco, 21331020) and Neurobasal medium (Gibco, 21103049) supplemented with N2 (homemade), B27 Serum-Free Supplement (Gibco, 17504-044), 2 mM L-Glutamine (Sigma-Aldrich, G7513), 0.1 mM NEAA (Sigma-Aldrich, M7145), 10 µg/ml penicillin-streptomycin (Sigma-Aldrich, P4333), 55 µM β-mercaptoethanol (Fisher Scientific, 21985-023)) and 2i (1 μM PD0325901 and 3 μM CHIR99021), here referred to as N2B27-2i medium. The following day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation for the indicated time, or to fresh N2B27-2i for undifferentiated controls.

Rapamycin treatment

mESCs were plated on gelatin-coated plates in N2B27-2i + DMSO (Sigma-Aldrich, D2650) or N2B27-2i + 20 nM Rapamycin (Enzo Life Sciences, BML-A275-0005). The following day, cells were washed with PBS and medium was exchanged to N2B27 + DMSO or N2B27 + 20 nM Rapamycin to induce differentiation for the indicated time.

MK-2206 treatment

mESCs were plated on gelatin-coated plates in N2B27-2i or N2B27-2i + 1 μM MK-2206 (Cayman Chemical, 11593). The following day, cells were washed with PBS and medium was exchanged to either N2B27 or N2B27 + 1 μM MK-2206 to induce differentiation for the indicated time, or to N2B27-2i or N2B27-2i + 1 μM MK-2206 for the undifferentiated controls.

mESCs were plated on gelatin-coated plates in N2B27-2i and transfected with FlexiTube siRNAs (Qiagen) using DharmaFECT 1 (Fisher Scientific, T-2001). The next day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or fresh N2B27-2i for the undifferentiated controls. Two siRNAs targeting FoxO1 (SI01005200, SI02694153) were used at a final concentration of 20 nM. As controls, siRNAs targeting GFP (siGFP) or scrambled siRNAs (siScr) were used.

Expression of nuclear FoxO1

A FoxO1 coding sequence carrying three mutations (T24A, S253D and S316A) was cloned from an Addgene vector (Plasmid #12149) 65 into a pB-TetOn-3xFLAG-Empty-PolyA-Puro vector. The resulting plasmid, here referred to as 3xFLAG-FoxO1 nuc , was transfected into RC9 and Pten KO cells. Single cell derived, independent clones were selected and expanded for further experiments. For experiments in differentiation-permissive conditions, RC9-based and Pten KO-based clones were plated on gelatin-coated plates in N2B27-2i. The following day, cells were washed with PBS and medium was exchanged to N2B27 with or without 500 ng/ml of doxycycline (Sigma-Aldrich, D9891). After 8 h, medium was changed to N2B27, and cells were differentiated for further 16 h. For the experiments in naïve conditions, RC9-based clones were plated in N2B27-2i and cultured for 48 h. The last 8 h before harvesting, 500 ng/ml of doxycycline was added to the medium.

Flow cytometry analysis

After the indicated amount of time in differentiation (N2B27-based) or control (N2B27-2i-based) medium conditions, cells were harvested using 0.25% trypsin/EDTA and resuspended in ES-DMEM medium to neutralise trypsin. Rex1-GFPd2 levels were measured using the LSR Fortessa flow cytometer (BD bioscience) and then analysed with the FlowJo software (v10, BD bioscience).

Real-time PCR analysis

After the indicated amount of time in differentiation (N2B27-based) or control (N2B27-2i-based) medium conditions, cells were washed with PBS and harvested in RNA Lysis buffer containing 1% (v/v) 2-mercaptoethanol and stored at −80 °C before isolation of RNA. RNA was extracted using the ExtractMe kit (Blirt, EM15) following the manufacturer’s instructions. 0.25–1 µg of RNA was reverse-transcribed into cDNA using the SensiFAST cDNA Synthesis Kit (Bioline, BIO-65054). Real-time PCR was performed on the CFX384 Touch real-time PCR detection system (Bio-Rad) using the Sensifast SYBR no Rox kit (Bioline, BIO-98020). Data analysis and visualisation was performed using Microsoft Excel (Office 365) and R (v4.2.2). Used primers are listed in Supplementary Data  3 .

Immunoblotting analysis

After the indicated amount of time in differentiation (N2B27-based) or control (N2B27-2i-based) medium conditions, cells were washed with PBS and harvested in 1x RIPA buffer (Sigma-Aldrich, 20-188) supplemented with Complete Mini EDTA-free protease inhibitor cocktail (Roche, 04693159001) and PhosSTOP (phosphatase inhibitor cocktail (Roche, 04906845001). Protein extraction was performed by incubating the samples on ice for 15 min and then collecting the supernatant after centrifugation at 14,000 ×  g at 4 °C for 30 min. Protein concentration was determined using a Bradford Assay (Bio-Rad). Eight to twenty µg whole cell lysates were separated on 8–12% SDS–PAGE gels (depending on the molecular weight of the target proteins) and subsequently blotted on 0.2 µm nitrocellulose membranes (Amersham). Membranes were blocked at RT for 1 h with 5% milk diluted in PBS (Sigma-Aldrich, P4417) containing 0.1% Tween-20 (PBS-T). Primary antibodies were incubated overnight at 4 °C or for 1 h at room temperature (RT). Secondary antibodies were incubated for 1 h at RT. The following primary antibodies were diluted in PBS-T containing 5% BSA and used 1:1000 for anti-phospho-Akt(Ser473) (rabbit; Cell Signalling, 4058), 1:1000 for anti-phospho-Akt(Thr308) (rabbit; Cell Signalling, 13038), 1:1000 for anti-pan-Akt (rabbit; Cell Signalling, 4691), 1:1000 for anti-phospho-GSK3β(Ser9) (rabbit; Cell Signalling, 9336), 1:1000 for anti-GSK3β (rabbit; Cell Signalling, 12456), 1:1000 for anti-phospho-4E-BP1(Ser65) (rabbit; Cell Signalling, 9451), 1:1000 for anti-4E-BP1 (rabbit; Cell Signalling, 9644), 1:1000 for anti-phospho-p70 S6 kinase(Thr389) (rabbit; Cell Signalling, 9234), 1:1000 for anti-p70 S6 kinase (rabbit; Cell Signalling, 2708), 1:1000 for anti-PTEN (rabbit; Cell Signalling, 9559), 1:1000 for anti-TSC2 (rabbit; Cell Signalling, 4308), 1:1000 for anti-FoxO1 (rabbit; Cell Signalling, 2880), 1:1000 for anti-phospho-FoxO1(Ser256) (rabbit; Cell Signalling, 9461) and 1:1000 for anti-FoxO3a (rabbit; Cell Signalling, 2497). The following primary antibodies were diluted in PBS-T containing 5% milk and used 1:1000 for anti-Oct4 (rabbit; Abcam, ab19857), 1:5000 for anti-Tubulin (mouse; Sigma-Aldrich, T8203), 1:10,000 for anti-GAPDH(G-9) (mouse; Santa Cruz Biotechnology, sc-365062) and 1:10,000 for anti-Vinculin(H-10) (mouse; Santa Cruz Biotechnology, sc-25336). Secondary antibodies were diluted in 5% milk and used 1:10,000 for anti-rabbit IgG (Amersham, NA934) and 1:15,000 for anti-mouse IgG (goat; Santa Cruz Biotechnology, sc-2064). Chemiluminescence signal was detected using the ECL Select detection kit (GE Healthcare, GERPN2235) with a ChemiDoc system (Bio-Rad). Data analysis was performed using ImageLab (v5.2.1). Uncropped images are available in the Source Data File.

Immunofluorescence analysis

mESCs were plated at a final density of 1 × 10 4 cells/cm 2 on fibronectin-coated (MM Merck Millipore, FC010) µ-Slide 8 Well Glass Bottom Chamber Slides (Ibidi, 80827) in N2B27-2i medium. The following day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or to fresh N2B27-2i for the undifferentiated controls. After the indicated amount of time, cells were washed with PBS and fixed for 15 min at RT with freshly diluted 4% PFA (16% paraformaldehyde diluted in 1:4 in PBS) (SCI Science Services, E15710). Cells were washed in PBS and subsequently permeabilized with PBS containing 0.1% Triton-X for 10 min at RT. Cells were washed 3x in PBS-T and then blocked using PBS-T containing 5% BSA (blocking buffer) for 30 min at 4 °C. Primary antibodies were incubated overnight at 4 °C. Cells were washed 3x with PBS-T. Secondary antibodies were incubated for 1 h at RT. Cells were washed 2x with PBS-T. Nuclei were stained with 1 µg/ml DAPI (Sigma-Aldrich, D9542) for 10 min at RT. Cells were washed 3x with PBS and stored in PBS at 4 °C until the image acquisition procedure. Primary and secondary antibodies were diluted in blocking buffer and used 1:100 for anti-FoxO1 (rabbit; Cell Signalling, 2880), 1:100 for anti-ESRRB (mouse; R&D Systems, PP-H6705-00), 1:250 for anti-FLAG M2 (mouse; Sigma-Aldrich F1804), 1:200 for anti-NANOG (rabbit; NovusBio, NB100-58842), 1:500 for anti-mouse Alexa-555 (donkey; Cell Signalling, 4409), 1:500 for anti-rabbit Alexa-647 (goat; Cell Signalling, 4414). Images were acquired using a Zeiss LSM 980 confocal microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 (WD 0.19 mm) objective. Images were analysed using Fiji/ImageJ (v2.9.0/1.54f). For quantifying the nuclear intensity of the stained proteins, segmentation was performed on the DAPI channel with cellpose with the following settings: cell diameter (in pixels) = 170, flow_threshold = 0.4, cellprob_threshold = 0.0, stitch_threshold = 0.0, model = cyto2. The obtained nuclei outlines were imported into Fiji/ImageJ and used to create mask objects. The latter were used to measure the mean fluorescence intensity of each nucleus in all recorded channels. Nuclei out of focus were manually removed from the analysis. Data analysis and visualisation was subsequently performed in R.

Intracellular staining

After the indicated amount of time in differentiation (N2B27-based) or control (N2B27-2i-based) medium conditions, cells were harvested using 0.25% trypsin/EDTA and resuspended in ES-DMEM medium to neutralise trypsin. Cells were centrifuged, and cell pellets were washed twice with PBS before fixation with 2% PFA for 15 min at RT. Cells were washed with FACS buffer (PBS containing 5% BSA) and subsequently permeabilized with ice-cold MeOH for 10 min on ice. After 3 washes in FACS buffer, cells were incubated in FACS buffer for 10 min in the dark. Subsequently, cells were incubated with primary antibodies for 1 h at RT. Cells were then washed 3x with FACS buffer and incubated with secondary antibodies for 15 min on ice. Cells were washed 3x with FACS buffer and stored in FACS buffer until flow cytometry analysis at the LSR Fortessa flow cytometer. Primary and secondary antibodies were diluted in FACS buffer and used 1:100 for anti-phospho-Akt(Ser473) (rabbit; Cell Signalling, 4058) and 1:500 for anti-rabbit Alexa-647 (goat; Cell Signalling, 4414). Flow cytometry data was analysed with the FlowJo software. Mean fluorescence intensities (MFI) for stained samples were calculated by subtracting the MFI of their relative controls (cells stained only with the secondary antibody). Data analysis and visualisation was performed in R.

Embryo staining

Blastocysts from B6CBAF1 mice were isolated at embryonic stages E3.5, E4.5, E4.75 and E5.5. The samples were fixed with 4% paraformaldehyde for 30 min and stored in 1X PBS at 4 °C. To prepare samples for IF, they were permeabilized with 0.3% PBST (PBS + Triton) for 60 min at room temperature before incubating with Blocking buffer (10% Donkey Serum, in 0.3% PBST) for 3 h. Primary antibody incubation was performed overnight at 4 °C in blocking buffer. All primary antibodies were used at a dilution of 1:200. Prior adding the secondary antibody, samples were washed three times with 0.3% PBST.

The embryos were incubated with the secondary antibody for 1 h at room temperature before washing three times with 0.3 % PBST. All the secondary antibodies and Hoechst staining were used at a dilution of 1:300. The samples were then stored at 4 °C in PBS prior imaging. The following antibodies were used: Gata4 (Monoclonal, Mouse, Santa Cruz Biotechnology, sc-25310) + Secondary Ab (Anti-Mouse, Donkey, AF 647, Invitrogen A31571), Cdx2 (Monoclonal, Mouse, Emergo Europe/BioGenex MU392A-5UC) + Secondary Ab (Anti-Mouse, Donkey, AF 647, Invitrogen A31571), FoxO1 (rabbit C29H4) + Secondary Ab (Anti-Rabbit IgG, Donkey, AF 568, Invitrogen A10042), Sox2 (Monoclonal, Rat Invitrogen 14-9811-82) + Secondary Ab (Anti-Rat, Donkey, AF 488, Invitrogen A21208), Hoechst (Life Technology Corporation, 33342). Images were obtained on an Olympus spinning disk confocal (inverted) with 40x/0.75 (Air) WD 0.5 mm objective. Images were analysed using Fiji/ImageJ (v2.0.0/1.53t). For each embryonic stage, at least 3 embryos were analysed. Single nuclei were manually segmented at multiple z-sections, and the mean intensity was calculated using the Fiji Measure tool.

Nucleo-cytoplasmic fractionation

Subcellular fractionation experiments were performed following a protocol adapted from Rockland ( https://www.rockland.com/resources/nuclear-and-cytoplasmatic-extract-protocol/ ). A total of 1 × 10 4 cells/cm 2 of WT and Pten KO cells were plated in 10 cm gelatine-coated plates. The next day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or to fresh N2B27-2i for the undifferentiated controls. After 24 h cells were harvested. 1/20 of cells were processed as described above for total protein extraction. The rest was used for the nucleo-cytoplasmic fractionation. Cell pellets were resuspended in 5 pellet volumes of CE buffer adjusted to pH 7.6 (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% NP-40, 1 mM DTT, 1 mM PMSF) and incubated on ice for 3 min. Samples were pelleted by centrifugation at 300 ×  g for 4 min at 4 °C and the supernatant (the cytoplasmic fraction) was transferred to clean tubes. The nuclei were washed carefully with 5 pellet volumes of CE buffer (without NP-40), and then pelleted at 300 ×  g for 4 min at 4 °C. The supernatant was discarded, and the nuclei were resuspended in 1 pellet volume of NE buffer adjusted to pH 8.0 (20 mM Tris Cl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol) and the salt concentration was then adjusted to 400 mM with 5 M NaCl. An additional pellet volume of NE buffer was added to the extracts before incubating them for 10 min on ice. The extracts were vortexed every 3 min during the incubation time. Both cytoplasmic and nuclear fractions were spun at maximum speed to pellet any remaining nuclei. Cytoplasmic and nuclear fractions were transferred to clean tubes; 20% glycerol was added to the cytoplasmic fraction. Both fractions were stored at −80 °C.

RNA-sequencing analysis

For differentiation experiments using RC9, Pten KO and Tsc2 KO cells, and for the 2h-resolved time course, count tables generated in a previous study were used 28 . QuantSeq analysis was performed for all the other RNA-seq experiments described in the manuscript. For the Rapamycin experiment, WT, Pten KO and Tsc2 KO cells treated with DMSO or Rapamycin from two independent differentiation assays were sequenced (duplicates). For the FoxO1 knockdown experiment, WT cells were treated either with an individual siRNA sequence targeting FoxO1 transcript, or with a combination of both siRNAs (triplicates, siRNA #1, siRNA#2, siRNA#1 + siRNA#2) in a reverse transfection approach 64 . For the control sample (siCtrl), two siGFP samples combined with 1 siScr sample were considered a triplicate. For the MK-2206 experiment, WT cells left untreated or treated with MK-2206 from two independent differentiation assays were sequenced (duplicates). Library preparation (according to the Lexogen 3’ mRNA Seq Library Prep Kit), multiplexing (by qPCRs) and sequencing on an Illumina NextSeq2000 P3 platform was carried out at the VBCF NGS facility. Five to ten million of single-end reads at 50 bp read length were generated per sample. The resulting fastq files were analysed with a Nextflow 23.04.1.5866/nf-core/rnaseq v3.10.1 pipeline. Quality control was performed using fastQC (v0.11.9), and transcripts were mapped to the mm10 assembly mouse reference genome using Salmon (v1.9.0) as a pseudoaligner and STAR (v2.7.10a) as an aligner. DESeq2 (v1.38.3) was used to generate normalised count tables and to perform differential expression analyses (FDR-adjusted p value ≤ 0.05; H0: log2FC = 0). pheatmap (v1.0.12), EnhancedVolcano (v1.16.0), UpsetR (v1.4.0), eulerr (v7.0.0) and ggplot2 (v3.4.0) were used for data visualisation in R. Combined lists of upregulated and downregulated genes in Pten KO and Tsc2 KO were generated by selecting genes differentially expressed (DEGs) in both KOs (log2FC ≥ 0.5 for the upregulated genes, and log2FC ≤ −0.5 for the downregulated genes). Whenever we compared gene groups from published transcriptomics datasets, we only considered genes that are present also in our datasets.

Chromatin immunoprecipitation (ChIP)

FoxO1 and FoxO3 ChIP were performed as described 41 . 1.5 × 10 4 cells/cm 2 of WT and Pten KO cells were plated in duplicate (for Pten KO cells, each replicate corresponded to a different clone) on 15 cm gelatine-coated plates. The next day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or to fresh N2B27-2i for the undifferentiated controls. After 24 h, cells were harvested. Cells were washed with PBS, and then cross-linked directly on the plate with 1% formaldehyde in PBS for 10 min. Subsequently, 0.125 M glycine was added to the plates for 10 min to quench cross-linking. The plates were washed 2x with PBS, and then cells were scraped off in ice-cold PBS containing 0.01% Triton-X. Cells were pelleted by centrifugation at 500 ×  g for 5 min and flash-frozen in liquid nitrogen. Cell pellets were resuspended in 5 ml LB1 (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100, 1 mM PMSF, 1 × Complete Mini EDTA-free protease inhibitor cocktail) to extract nuclei and rotated vertically for 10 min at 4 °C. Nuclei were pelleted by centrifugation at 1350 ×  g for 5 min at 4 °C, and then resuspended in 5 ml LB2 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA1, mM PMSF, 1xComplete Mini EDTA-free protease inhibitor cocktail), and rotated vertically for 10 min at RT. Samples were pelleted by centrifugation at 1350 ×  g for 5 min at 4 °C and then resuspended in 1.5 ml LB3 (10 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1 mM PMSF, 1xComplete Mini EDTA-free protease inhibitor cocktail) and 200 μl sonication beads (diagenode) in Bioruptor® Pico Tubes (diagenode). Chromatin was sonicated for 13 cycles with 30 s on, 45 s off parameters. Sonicated samples were transferred to fresh tubes and centrifuged at 16,000 ×  g at 4 °C to pellet cellular debris. 1.1 ml of supernatant were collected and transferred to fresh tubes. A total of 110 μl of 10% Triton-X were added to a final concentration of 1%. For each sample, 50 μl were collected as input and 1 ml was used for immunoprecipitation. Chromatin was incubated with 20 μl (1:50 dilution) of anti-FoxO1 (rabbit; Cell Signalling, 2880) or anti-FoxO3a (rabbit; Cell Signalling, 2497) antibodies overnight at 4 °C with vertical rotation. Samples were collected after 14 h. A total of 100 μl of Dynabeads protein G (Thermo Fisher Scientific, 10765583) per sample were washed in ice-cold blocking solution (PBS containing 0.5% BSA) and then incubated with the antibody-bound chromatin solutions for 4 h. Beads were washed 5x in ice-cold RIPA wash buffer (50 mM HEPES pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate), and then 3x with TE + 50 mM NaCl. Samples were eluted in 210 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) for 15 min at 65 °C. Supernatant containing the antibody-bound chromatin fraction was separated from the beads. Three volumes of elution buffer were added to the input samples. Decrosslinking was performed by incubating both input and ChIP samples at 65 °C overnight. The next day, one volume of TE and RNaseA (to a 0.2 mg/ml final concentration) were added to the input and ChIP samples, followed by incubation for 2 h at 37 °C. Final salt concentration was adjusted to 5.25 mM CaCl 2 with 300 mM CaCl 2 in 10 mM Tris pH 8.0. Samples were then incubated with 0.2 mg/ml Proteinase K for 30 min at 55 °C. DNA was extracted with phenol-chloroform using Phase Lock GelTM tubes (Quantabio, 733-2478) and then precipitated in EtOH. DNA pellets were dissolved in H 2 O.

ChIP-sequencing analysis

Libraries were prepared at the VBCF NGS facility and sequenced on an Illumina NovaSeq platform. Within the FoxO1 ChIP experiment, 20–40 million paired-end reads at 150 bp read length were generated for the input samples, and 80–130 million reads for the ChIP samples. Within the FoxO3 ChIP experiment, ~ 25 million single-end reads at 100 bp read length were generated for both input and ChIP samples. RC9-based FoxO3 ChIP samples were re-sequenced to increase sequencing depth, and an additional ~ 60 million of paired-end reads at 150 bp read length per sample were generated. R1 reads from the two sequencing runs were concatenated before data processing. FoxO1 ChIP and input reads were processed using a paired-end mode, while FoxO3 ChIP and input samples following a single-end mode. Quality control of fastq files was performed using fastQC (v0.11.9) before and after trimming sequencing adaptor fragments with trim-galore (v0.6.7) and cutadapt (v3.5). Additional 2 bp at the 3’ end were removed with the parameters --three_prime_clip_R1 2 ( --three_prime_clip_R2 2 in case of paired-end reads). The trimmed reads were aligned to the mm10 assembly mouse reference genome with bowtie2 (v2.4.4), with an alignment rate of 80–98%. The obtained sam files were converted to bam files with samtools (v1.13), and uniquely mapping reads were extracted by removing duplicate reads (as potential PCR artefacts) with samtools markdup . Peak calling was performed on bam files with macs2 (v2.2.7.1) on combined ChIP duplicates, using all input samples as control files. Potential artefactual regions listed in the mm10 blacklist ( http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm10-mouse/mm10.blacklist.bed.gz ) were removed from the obtained bed files using bedtools (v2.31.0). Peaks assigned to unidentified regions (chrUn) were manually removed.

ATAC-sequencing

A total of 1 × 10 4 cells/cm 2 cells were plated on gelatine-coated plates. The next day, cells were washed with PBS and medium was exchanged to either N2B27 without 2i to induce differentiation, or to fresh N2B27-2i for the undifferentiated controls. After 24 h cells were harvested and counted. 250,000 cells per sample were submitted to the VBCF NGS facility for further processing and ATAC-seq library preparation (Bulk ATAC-seq Illumina). In brief, cells were lysed with 0.5x lysis buffer (0.01 M Tris-HCl pH 7.5, 0.01 M NaCl, 0.003 M MgCl 2 , 1% BSA, 0.1% Tween-20, 0.05% NP-40, 0.005% Digitonin, 0.001 M DTT, 1 U/μl RNAse inhibitor), and tagmentation reaction (Tn5 Illumina) was performed on 50,000 isolated nuclei. Libraries were prepared with the Nextera DNA Library Preparation kit and sequenced on an Illumina NovaSeq platform. A total of 80–170 million of paired-end reads at 150 bp read length were generated per sample. The resulting fastq files were analysed with a Nextflow 23.04.1.5866/nf-core/atacseq v2.0 pipeline. Quality control was performed using fastQC (v0.11.9), and reads were mapped to the mm10 assembly mouse reference genome using bowtie2 (v2.4.4). Peak calling was performed on the bam files generated by the nfcore pipeline with Genrich (v0.6.1). Consensus peaks were defined as peaks with at least a 50% overlap between replicates and generated with samtools intersect . Bed files were filtered for the mm10 blacklist using bedtools. Peaks assigned to unidentified regions (chrUn) were manually removed.

CUT&RUN analysis

The ChIC/CUT&RUN Assay Kit (Active Motif, catalogue No. 53180) was used for the CUT&RUN experiment. FoxO1 siRNA treatment was performed as described before using siRNAs directed against FoxO1 and scrambled siRNAs as controls. A total of 125,000 to 500,000 cells were collected per condition in biological duplicates from two independent experiments ( n  = 4). As spike-in, 25,000 Drosophila melanogaster S2 cells were added to each sample. CUT&RUN was performed according to the manufacturer’s protocol, but ethanol precipitation performed to purify DNA instead of column purification. One µg of Histone H3K27ac antibody (rabbit pAb, Thermo Fisher Scientific, 39133) was used per sample. IgG antibody supplied with the CUT&RUN kit was used as a control. DNA was eluted in 100 µL and used as qPCR template. FoxO1 bound H3K27ac positive enhancer sequences near formative genes and control regions not bound by either FOXO1 or H3K27ac were amplified to assay H3K27ac levels. A known H3K27ac decorated region in Drosophila S2 cells (chr3L:4104063-4104149, Aug. 2014- BDGP Release 6 + ISO1 MT/dm6), was used to normalise signal between samples. The qPCR primers used are listed in the Supplementary Data  3 .

Motif enrichment analyses

Motif enrichment analysis was performed with Homer (v4.11). For finding motifs enriched in FoxO1 or FoxO3-bound regions, findMotifsGenome.pl was used with default settings. For finding enriched motifs in ESCs or EpiLCs enhancers, enhancer lists from the Bücker lab were used (filtered to exclude TSS 41 ). A list of overlapping enhancers between ESCs and EpiLCs was generated with bedtools and used as background to identify ESC-specific and EpiLC-specific enriched motifs with findMotifsGenome.pl using default settings. According to the Homer documentation, a motif can be considered robustly enriched when its associated p value is lower than 1 × 10 −50 .

Data integration analyses

Downstream analyses for ChIP-seq and ATAC-seq were performed with Deeptools (v3.5.1). BigWigs were generated from single bam files with bamCoverage using a binsize of 10 bp and a normalisation coverage to 1x mouse genome size (RPGC) excluding the X chromosome. Principal component analysis (PCA) was performed with multiBigwigSummary and plotPCA . Bigwig replicates were merged using bigWigMerge , and heatmaps were plotted on merged BigWigs using computeMatrix and plotHeatmap . Heatmaps were sorted based on FoxO1 or FoxO3 signals. Three lists of peaks (separately for FoxO1 and FoxO3 ChIP) were generated with bedtools by intersecting WT (RC9) bed files: 2i-only peaks, N24-only peaks and shared peaks. Peak to gene association was performed on these peak lists in R with ChIPSeeker (v1.34.1). Oct4 and Otx2 ChIP BigWigs and peak lists were obtained from ref. 39 . Esrrb ChIP BigWigs and bed files were obtained from published data 33 . β-catenin ChIP bed files 66 were downloaded from CODEX ( https://codex.stemcells.cam.ac.uk/ ), and mm9 coordinates were converted into mm10 coordinates using the liftOver tool from UCSC ( https://genome.ucsc.edu/cgi-bin/hgLiftOver ). Overlap between genomic regions was performed with ChIPPeakAnno (v3.32.0). For the FoxO1-FoxO3 ChIP overlap, the background for the hypergeometric test was set to the total number of detected open chromatin regions (generated by merging ATAC-seq peaks in 2i with ATAC-seq peaks at N24 with ESC). For all other overlaps, the background used was obtained by merging all ATAC-seq peaks with the lists of ESC and EpiLC enhancers 41 . Genome tracks shown in Fig.  4 were generated using karyoploteR (v1.24.0) in R. Lists of NANOG, OCT4, SOX2 and TCF7L1 targets were obtained from ref. 67 . Gene expression changes during peri-implantation development in vivo were assessed using previously described and processed data 68 , 28 .

Enrichment analyses

All enrichment analyses presented in this work were performed in R with hypeR (v2.0.1). For KEGG pathway enrichment analysis, mouse KEGG database was downloaded from http://rest.kegg.jp/link/mmu/pathway and transformed into a hypeR-compatible gene-set using the gsets function. The mouse REACTOME dataset was downloaded using the msigdb_gsets function within hypeR. As background, for FoxO1 target enrichment analyses a list of genes associated to any accessible region as detected by ATAC-seq was used, while for the other enrichment analyses the list of all detected genes in the relative RNA-seq experiment was used. Custom gene-sets were also generated with the gsets function. As background for FoxO TF-based enrichments, a list of genes associated with all open chromatin regions was used. Enrichment results were visualised with ggplot2.

Statistical analysis and data representation

All statistical analyses were performed in R with the ggpubr (v0.6.0) package. Information on statistical tests and replicate numbers are provided in the figure legends. Wherever necessary, correction for multiple testing was performed. All box plots show the 25th percentile (Q1), median (Q2) and 75th percentile (Q3); whiskers indicate minimum (Q1 − 1.5 * inter quartile range [IQR]) and maximum (Q3 + 1.5*IQR) values.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The NGS data generated in this study have been deposited in the GEO database under accession code GSE253480 . Data from the following accession numbers were used in this study: GSE56138 ; GSE184137 ; GSE43565 . All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files.  Source data are provided with this paper.

Boroviak, T., Loos, R., Bertone, P., Smith, A. & Nichols, J. The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification. Nat. Cell Biol. 16 , 513–525 (2014).

Article   Google Scholar  

Kinoshita, M. & Smith, A. Pluripotency deconstructed. Dev. Growth Differ. 60 , 44–52 (2018).

Article   PubMed   Google Scholar  

Kinoshita, M. et al. Capture of mouse and human stem cells with features of formative pluripotency. Cell Stem Cell 28 , 453–471.e8 (2021).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4 , 487–492 (2009).

Article   CAS   PubMed   Google Scholar  

Smith, A. Formative pluripotency: the executive phase in a developmental continuum. Development 144 , 365–373 (2017).

Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292 , 154–156 (1981).

Article   ADS   CAS   PubMed   Google Scholar  

Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78 , 7634–7638 (1981).

Article   ADS   CAS   PubMed   PubMed Central   Google Scholar  

Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453 , 519–523 (2008).

Boroviak, T. et al. Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev. Cell 35 , 366–382 (2015).

Ficz, G. et al. FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13 , 351–359 (2013).

Lee, H. J., Hore, T. A. & Reik, W. Reprogramming the methylome: erasing memory and creating diversity. Cell Stem Cell 14 , 710–719 (2014).

Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133 , 1106–1117 (2008).

Dunn, S. J., Martello, G., Yordanov, B., Emmott, S. & Smith, A. G. Defining an essential transcription factor program for naïve pluripotency. Science 344 , 1156–1160 (2014).

Martello, G. & Smith, A. The nature of embryonic stem cells. Annu. Rev. Cell Dev. Biol. 30 , 647–675 (2014).

Niwa, H., Ogawa, K., Shimosato, D. & Adachi, K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460 , 118–122 (2009).

Huang, G., Ye, S., Zhou, X., Liu, D. & Ying, Q. L. Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network. Cell. Mol. Life Sci. 72 , 1741–1757 (2015).

Smith, A. G. et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336 , 688–690 (1988).

Williams, R. L. et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336 , 684–687 (1988).

Ohtsuka, S., Nakai-Futatsugi, Y. & Niwa, H. LIF signal in mouse embryonic stem cells. Jak. Stat. 4 , 1–9 (2015).

Watanabe, S. et al. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 25 , 2697–2707 (2006).

Paling, N. R. D., Wheadon, H., Bone, H. K. & Welham, M. J. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J. Biol. Chem. 279 , 48063–48070 (2004).

Wray, J. et al. Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat. Cell Biol. 13 , 838–845 (2011).

Wang, W. et al. Pten-mediated Gsk3β modulates the naïve pluripotency maintenance in embryonic stem cells. Cell Death Dis . 11 , 107 (2020).

Storm, M. P. et al. Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells. J. Biol. Chem. 282 , 6265–6273 (2007).

Kunath, T. et al. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134 , 2895–2902 (2007).

Stavridis, M. P., Simon Lunn, J., Collins, B. J. & Storey, K. G. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development 134 , 2889–2894 (2007).

Betschinger, J. et al. Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell 153 , 335–347 (2013).

Lackner, A. et al. Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency. EMBO J. 40 , 1–23 (2021).

Leeb, M., Dietmann, S., Paramor, M., Niwa, H. & Smith, A. Genetic exploration of the exit from self-renewal using haploid embryonic stem cells. Cell Stem Cell 14 , 385–393 (2014).

Li, M. et al. Genome-wide CRISPR-KO screen uncovers mTORC1-mediated Gsk3 regulation in naive pluripotency maintenance and dissolution. Cell Rep. 24 , 489–502 (2018).

Villegas, F. et al. Lysosomal signaling licenses embryonic stem cell differentiation via inactivation of Tfe3. Cell Stem Cell 24 , 257–270.e8 (2019).

Yu, J. S. L. & Cui, W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development 143 , 3050–3060 (2016).

Carbognin, E. et al. Esrrb guides naive pluripotent cells through the formative transcriptional programme. Nat. Cell Biol. 25 , 643–657 (2023).

Herman, L., Todeschini, A. L. & Veitia, R. A. Forkhead transcription factors in health and disease. Trends Genet . 37 , 460–475 (2020).

Yang, P. et al. Multi-omic profiling reveals dynamics of the phased progression of pluripotency. Cell Syst. 8 , 427–445.e10 (2019).

Hirai, H. et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol. Cancer Ther. 9 , 1956–1967 (2010).

Nakae, J., Kitamura, T., Silver, D. L. & Accili, D. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J. Clin. Invest. 108 , 1359–1367 (2001).

Greer, E. L. & Brunet, A. FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24 , 7410–7425 (2005).

Buecker, C. et al. Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 14 , 838–853 (2014).

Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146 , 519–532 (2011).

Thomas, H. F. et al. Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements. Mol. Cell 81 , 969–982.e13 (2021).

Eijkelenboom, A., Mokry, M., Smits, L. M., Nieuwenhuis, E. E. & Burgering, B. M. T. FOXO3 selectively amplifies enhancer activity to establish target gene regulation. Cell Rep. 5 , 1664–1678 (2013).

Essers, M. A. G. et al. Functional interaction between β-catenin and FOXO in oxidative stress signaling. Science 308 , 1181–1184 (2005).

Kalkan, T. et al. Complementary activity of ETV5, RBPJ, and TCF3 drives formative transition from naive pluripotency. Cell Stem Cell 24 , 785–801.e7 (2019).

Carter, M. E. & Brunet, A. Quick guide FOXO transcription factors. Curr. Biol. 17 , 113–114 (2007).

Paik et al. FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis. Cell Stem Cell 5 , 540–553 (2009).

Renault, V. M. et al. FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell 5 , 527–539 (2009).

Vilchez, D. et al. Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature 489 , 304–308 (2012).

Vilchez, D. et al. FOXO4 is necessary for neural differentiation of human embryonic stem cells. Aging Cell 12 , 518–522 (2013).

Webb, A. E. et al. FOXO3 shares common targets with ASCL1 genome-wide and inhibits ASCL1-dependent neurogenesis. Cell Rep. 4 , 477–491 (2013).

Zhang, X. et al. FOXO1 is an essential regulator of pluripotency in human embryonic stem cells. Nat. Cell Biol. 13 , 1092–1101 (2011).

van der Weijden, V. A. et al. FOXO1-mediated lipid metabolism maintains mammalian embryos in dormancy. Nat. Cell Biol . 26 , 181–193 (2024).

Fu, M. et al. Forkhead box family transcription factors as versatile regulators for cellular reprogramming to pluripotency. Cell Regen. 10 , 1–11 (2021).

Yu, Y. et al. Stimulation of somatic cell reprogramming by ERas-Akt-FoxO1 signaling axis. Stem Cells 32 , 349–363 (2014).

DelRosso, N. et al. Large-scale mapping and mutagenesis of human transcriptional effector domains. Nature 616 , 365–372 (2023).

Doumpas, N. et al. TCF/LEF dependent and independent transcriptional regulation of Wnt/β‐catenin target genes. EMBO J. 38 , 1–14 (2019).

Bourgeois, B. et al. Multiple regulatory intrinsically disordered motifs control FOXO4 transcription factor binding and function. Cell Rep. 36 , 109446 (2021).

Gui, T. et al. Targeted perturbation of signaling-driven condensates. Mol. Cell 83 , 4141–4157.e11 (2023).

Pan, C. et al. AKT‐phosphorylated FOXO 1 suppresses ERK activation and chemoresistance by disrupting IQGAP 1‐MAPK interaction. EMBO J. 36 , 995–1010 (2017).

Hornsveld, M., Dansen, T. B., Derksen, P. W. & Burgering, B. M. T. Re-evaluating the role of FOXOs in cancer. Semin. Cancer Biol. 50 , 90–100 (2018).

Jiramongkol, Y. & Lam, E. W. F. FOXO transcription factor family in cancer and metastasis. Cancer Metastasis Rev. 39 , 681–709 (2020).

Adiguzel, D. & Celik-Ozenci, C. FoxO1 is a cell-specific core transcription factor for endometrial remodeling and homeostasis during menstrual cycle and early pregnancy. Hum. Reprod. Update 27 , 570–583 (2021).

CAS   PubMed   Google Scholar  

Vasquez, Y. M. et al. FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation. PLoS Genet. 14 , 1–27 (2018).

Huth, M. et al. NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation. Genes Dev. 36 , 348–367 (2022).

Kitamura, Y. I. et al. FoxO1 protects against pancreatic β cell failure through NeuroD and MafA induction. Cell Metab. 2 , 153–163 (2005).

Zhang, X., Peterson, K. A., Liu, X. S., Mcmahon, A. P. & Ohba, S. Gene regulatory networks mediating canonical wnt signal-directed control of pluripotency and differentiation in embryo stem cells. Stem Cells 31 , 2667–2679 (2013).

Martello, G. et al. Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal. Cell Stem Cell 11 , 491–504 (2012).

Mohammed, H. et al. Single-cell landscape of transcriptional heterogeneity and cell fate decisions during mouse early gastrulation. Cell Rep. 20 , 1215–1228 (2017).

Download references

Acknowledgements

We thank Kitti Dóra Csályi and Thomas Sauer at the Max Perutz Laboratories FACS Facility for expert support. Next generation sequencing was performed at the Vienna Biocenter Core Facilities (VBCF), and we thank Thomas Grentzinger (VBCF) for ATAC-seq sample processing. We thank Niklas Urbanek, Michael Bindl, Mattia Pitasi, Julia Ramesmayer, Ana Maria Sandru and Stefania Bucconi for experimental support and Ivan Yudushkin for sharing reagents. We thank all members of the Leeb and Buecker labs for helpful discussions and Christa Buecker, Manuela Baccarini, Alexander Stark and Thomas Leonard for support and helpful suggestions. This research was funded in whole, or in part, by the Austrian Science Fund (FWF; dois: 10.55776/I5958 and 10.55776/P35637). L.S. is an OEAW doc.fellowship awardee (DOC/25622) and a member of the FWF-funded doctoral programme ‘Signalling Molecules in Cellular Homeostasis’ (SMICH; W1261). M.L. is a faculty member and speaker of SMICH and a ‘Wiener Wissenschafts- Forschungs- und Technologiefonds (WWTF)’ Vienna Research Group Leader (VRG14-006).

Author information

Authors and affiliations.

Max Perutz Laboratories Vienna, University of Vienna, Vienna BioCenter, 1030, Vienna, Austria

Laura Santini, Saskia Kowald, Luis Miguel Cerron-Alvan, Michelle Huth, Anna Philina Fabing & Martin Leeb

Vienna BioCenter PhD Program, Doctoral School of the University of Vienna, Medical University of Vienna, 1030, Vienna, Austria

Laura Santini, Luis Miguel Cerron-Alvan, Michelle Huth & Giovanni Sestini

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, 1030, Vienna, Austria

Giovanni Sestini & Nicolas Rivron

You can also search for this author in PubMed   Google Scholar

Contributions

L.S. designed and performed wet-lab and computational experiments and wrote the manuscript. L.M.C.A. and M.H. performed experiments and CUT&RUN analyses. S.K. performed siRNA experiments. A.P.F. performed some image analysis. G.S. and N.R. performed embryo IF stainings and analysis. M.L. designed and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Martin Leeb .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Peer review

Peer review information.

Nature Communications thanks Nissim Hay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information, peer review file, description of additional supplementary files, supplementary data 1, supplementary data 2, supplementary data 3, reporting summary, source data, source data, rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Santini, L., Kowald, S., Cerron-Alvan, L.M. et al. FoxO transcription factors actuate the formative pluripotency specific gene expression programme. Nat Commun 15 , 7879 (2024). https://doi.org/10.1038/s41467-024-51794-9

Download citation

Received : 24 January 2024

Accepted : 16 August 2024

Published : 09 September 2024

DOI : https://doi.org/10.1038/s41467-024-51794-9

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

By submitting a comment you agree to abide by our Terms and Community Guidelines . If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

• Aalto University
• Posted on: 9 September 2024

Fully-funded PhD project under Horizon Europe MSCA Doctoral Networks "FOCAL": Network slicing and AI-based resource allocation for optical multi-layered non-terrestrial networks

Job Information

Offer description.

This fully funded PhD position is available under the recently launched Horizon Europe Marie Skłodowska-Curie Action (MSCA) Doctoral Networks (DN) project "FOCAL" (Free-space Optical Communications for Aerial-sateLlite networks). 

• PhD position title: Network slicing and AI-based resource allocation for optical multi-layered non-terrestrial networks
• Objectives: The aim of this PhD thesis will be to design: (i) novel resource management algorithms for the optimization of non-terrestrial networks using model-based and artificial intelligence (AI)-based techniques such as supervised and reinforcement learning; and (ii) network slicing solutions to enable the coexistence of multiple virtual logical networks for different services and to maximize end-to-end energy efficiency, network coverage, and reliability based on QoS requirement of the different application scenarios.
• PhD supervision:  Aalto University, Finland; Nokia Bell Labs, Denmark; Fraunhofer Heinrich Hertz Institute, Germany  

General description of the "FOCAL" MSCA-DN Project: With currently 3 billion people lacking access to broadband internet worldwide, European operators are struggling to fill the white areas with insufficient or nonexistent connectivity. In light of the early promises of 6G wireless networks, it is tempting to consider non-terrestrial networks as complementary to the ground-based radio and fiber infrastructure. However, radio frequency-based satellite systems are nearing their capacity limits and cannot be scaled further. Free-space optical technology is a natural alternative to deliver greater capacity and to better address data security needs by leveraging quantum key distribution. The first optical intersatellite links are being deployed in commercial networks at relatively low data rate (10 Gbps). However, once the most important remaining research challenges are resolved, they could rapidly expand and serve as a catalyst for the development of a new communication backbone in the sky, consisting of aerial and space optical nodes at different altitudes.

The overarching aim of the FOCAL project is to train a new generation of highly qualified doctoral candidates in developing innovative aerial and space optical wireless technologies that will provide future telecommunication networks with ubiquitous connectivity, resilience, and quantum-proof security. In addition to contributing to the fundamental understanding and technical know-how of such networks through co-supervised individual research projects, FOCAL will train talented and innovative researchers with multidisciplinary expertise and skills that are desirable in this European industrial sector. This will be achieved through a combination of theoretical and hands-on research training provided by a unique consortium of 16 academic and 10 industrial partners from 8 countries, resulting in collaborative research including co-supervision and secondments, as well as transferable training skills, which is necessary for prosperous careers in this prominent R&I area.

More information on the project can be found on the following URL:  https://cordis.europa.eu/project/id/101169042  

Where to apply

Requirements.

The candidates must have a MSc (or an equivalent) degree in physics, electrical, electronics, telecommunications or photonics engineering. A solid background in signal processing is an important asset.  The candidate must have a very good English language proficiency (oral and written expression) and be keen for short-term stays ("secondments") in partner institutions. Knowledge of the Finnish language is not required.

In addition, the following qualifications will be highly appreciated:

• Experience in wireless and/or optical communication systems (e.g. from participating in R&D projects)
• Knowledge of simulation tools for wireless communication systems.
• Publications in scientific journals or conferences in areas related to the title of the position.

Additional Information

The enrollments of the Doctoral Candidates (DCs) are under very attractive employment conditions  and competitive salaries offered in Marie-Curie Doctoral Networks.

Candidates must also meet the following criteria: 

• Be of any nationality, but must not have resided or carried out main activity in Finland for more than 12 months in the last 36 months; 
• Have not yet been awarded a PhD degree.

All interested candidates irrespective of age, nationality, gender, race, disability, religion or ethnic background are encouraged to apply. 

The applications must be received from 1-30 September 2024 (the deadline may be extended if the position is not filled by then) by email to the supervisor with CC to [email protected] including the following documents (all strictly in PDF format):

• Curriculum vitae
• Master [alternatively, the expected graduation date of Master degree] and Bachelor diplomas (in a single PDF file)
• Master and Bachelor grades transcripts (in a single PDF file)
• Motivation letter (limited to 500 words)
• The name and contact information of two references or two recommendation letters (in a single PDF file).

Please note, the starting date of the position is flexible.

Also, the applicants must fill the application form following the URL below,  https://docs.google.com/forms/d/1AhWWi3rNseEpJ8LNbWJmDj89cQfwrl5-Vhn0cAdGTS8/  

Work Location(s)

Share this page.

• Show search form Hide search form
• Quick links
• Staff search
• Search Search --> Websites Staff search Start search

Visiting-PhDs

The University of Vienna welcomes Visiting-PhD students from all over the world. The Visiting PhD Programme is a "non-degree-seeking" programme for guest PhDs who would like to spend a research period at the University of Vienna, lasting a maximum of one year. Following the stay, PhD candidates return to their respective home-universities. Visiting-PhDs are enrolled at the university to run their research projects, participate in courses and pass exams as well as to benefit from university's services (libraries, Center for Doctoral Studies, computer-services, etc.).

How can I apply for a Visiting-PhD research stay?

In order to enroll you as a Visiting PhD, we need written confirmation of your mentoring professor at the University of Vienna. It is within the scope of your responsibility to contact a professor and negotiate the mentoring during your research stay at the University of Vienna. As soon as an agreement is reached, we kindly ask you and your mentor to get in touch with us. For the subsequent steps please prepare the following:

• copy of your master diploma in German or English
• passport photo 35x45mm
• copy of your identity card or passport

Please be aware that the University of Vienna does not provide particular funding for Visiting-PhDs. Funding possibilities can be found here.

Visiting PhDs do not pay tuition fees (only the student union's fee).

As a Visiting PhD at the University of Vienna you will for sure spend an exciting time here by getting to know a new research group and exploring a new city.

You have access to the following services of the University of Vienna:

Courses at the faculty.

The faculties are offering course for PhD candidates. These you will find in the course directory . Online registration is possible for almost all courses. In the course catalogue you will find the application periods and the link to the registration via u:space.

Events organized by the doctoral schools

The doctoral schools are very active in bringing doctoral candidates and researchers from the faculty together by different events. Please check the website of the doctoral school you are affiliated to. An overview of the schools you will find here .

Workshops in transferable skills and events from the Center for Doctoral Studies

Every semester, workshops on topics such as literature research and management, academic writing and publishing, time and project management etc. are offered by the Center for Doctoral Studies. Additionally, there are events on topics like well-being or postdoc funding programmes. Check out the website .

Vienna University Library

Of course, you have access to all services our library is offering. With the u:card you already have a valid library card. Inform yourself about the different research tools and services on the website of the Vienna University Library . 

Vienna University Computer Center

The u:account is your personal access to the IT services of the University of Vienna. Students receive a personal e-mail address including a mailbox free of charge as part of their u:account. It is essential to activate and check these e-mails regularly, because important information for example concerning the courses you registered for are sent to this e-mail address. How to activate the account and what services you can profit from can be found on the website .

University Sports Institute

From aqua fitness to ball sports, gymnastics, or popular new sports such as Zumba – the University Sports Institute Vienna offers more than 1,200 courses (from over 120 different types of sport) in modern sports facilities all over Vienna. Check them out on the website . 

Language Centre of the University of Vienna

The Language Center of the University of Vienna offers language courses for German and more than 30 foreign languages.