Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics

Suzan Stelloo; Maria Teresa Alejo-Vinogradova; Charlotte A. G. H. van Gelder; Dick W. Zijlmans; Marek J. van Oostrom; Juan Manuel Valverde; Lieke A. Lamers; Teja Rus; Paula Sobrevals Alcaraz; Tilman Schafers; Cristina Furlan; Pascal W. T. C. Jansen; Marijke P. A. Baltissen; Katharina F. Sonnen; Boudewijn Burgering; Maarten A. F. M. Altelaar; Harmjan R. Vos; Michiel Vermeulen

doi:10.1016/j.stem.2024.04.017

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics

Suzan Stelloo^*, Maria Teresa Alejo-Vinogradova, Charlotte A. G. H. van Gelder, Dick W. Zijlmans, Marek J. van Oostrom, Juan Manuel Valverde, Lieke A. Lamers, Teja Rus, Paula Sobrevals Alcaraz, Tilman Schafers, Cristina Furlan, Pascal W. T. C. Jansen, Marijke P. A. Baltissen, Katharina F. Sonnen, Boudewijn Burgering, Maarten A. F. M. Altelaar, Harmjan R. Vos, Michiel Vermeulen^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometrybased proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Original language	English
Pages (from-to)	1072-1090.e8
Number of pages	28
Journal	Cell Stem Cell
Volume	31
Issue number	7
DOIs	https://doi.org/10.1016/j.stem.2024.04.017
Publication status	Published - 5 Jul 2024

Bibliographical note

Publisher Copyright:
© 2024 The Authors

Funding

We would like to thank Rob Woestenenk, Paul Ruijs, Laura Wingens, Tom van Oorschot, Nick C.M. Pelzers, Susan Zwakenberg, and Marjolein Vliem for their technical support with cell sorting. We thank Marieke Willemse and Gert-Jan Bakker from the Radboud Technology Center for Microscopy for training and assistance with confocal microscopy and Okolab, respectively. We thank Maarten van der Sande and Siebren Fr\u00F6lich for seq2science support and Sybren Rinzema for sequencing data management. We would also like to thank Susanne van den Brink and Sandra de Vries for their valuable advice on gastruloid culturing and St\u00E9fane Nedelec for advice on HOX proteins. Additionally, we thank all members of the Vermeulen lab for their input and suggestions. We thank the laboratory of Iftach Nachman (Tel Aviv University) for sharing the dual BRA/SOX17 reporter cell line. This work was supported by a VENI grant from the Netherlands Organisation for Scientific Research (NWO, VI.Veni.212.076); Pluripotent Stem cells for Inherited Diseases and Embryonic Research (PSIDER) grant from ZonMw (10250042110004); EPIC-XS (project number 823839), funded by the Horizon 2020 programme of the European Union; and the NWO-funded Netherlands Proteomics Centre through the National Road Map for Large-scale Infrastructures program X-Omics (project 184.034.019). J.M.V. is supported by scholarships from the Ministry of Science and Technology of Costa Rica (MICITT) and the University of Costa Rica (UCR). The Vermeulen and Burgering labs are part of the Oncode Institute, which is partly funded by the Dutch Cancer Society (KWF). The research reported in this publication was supported by Oncode Accelerator, a Dutch National Growth Fund project under grant number NGFOP2201. Conceptualization, M.V. and S.S.; methodology, S.S. K.F.S. H.R.V. and C.A.G.H.v.G.; formal analysis, S.S. M.T.A.-V. C.A.G.H.v.G. P.S.A. and J.M.V.; investigation, S.S. M.T.A.-V. L.A.L. D.W.Z. M.J.v.O. J.M.V. C.A.G.H.v.G. P.S.A. T.R. and M.P.A.B.; data curation, S.S. P.S.A. and H.R.V.; resources, P.W.T.C.J. and C.F.; writing\u2014original draft, S.S. M.T.A.-V. and M.V.; writing\u2014review and editing, all authors; visualization, S.S. M.T.A.-V. C.A.G.H.v.G. P.S.A. and T.S.; supervision, S.S. M.A.F.M.A. K.F.S. B.B. H.R.V. and M.V.; funding acquisition, S.S. M.V. M.A.F.M.A. J.M.V. and B.B. The authors declare no competing interests. We would like to thank Rob Woestenenk, Paul Ruijs, Laura Wingens, Tom van Oorschot, Nick C.M. Pelzers, Susan Zwakenberg, and Marjolein Vliem for their technical support with cell sorting. We thank Marieke Willemse and Gert-Jan Bakker from the Radboud Technology Center for Microscopy for training and assistance with confocal microscopy and Okolab, respectively. We thank Maarten van der Sande and Siebren Fr\u00F6lich for seq2science support and Sybren Rinzema for sequencing data management. We would also like to thank Susanne van den Brink and Sandra de Vries for their valuable advice on gastruloid culturing and St\u00E9fane Nedelec for advice on HOX proteins. Additionally, we thank all members of the Vermeulen lab for their input and suggestions. We thank the laboratory of Iftach Nachman (Tel Aviv University) for sharing the dual BRA/SOX17 reporter cell line. This work was supported by a VENI grant from the Netherlands Organisation for Scientific Research (NWO, VI.Veni.212.076 ); Pluripotent Stem cells for Inherited Diseases and Embryonic Research (PSIDER) grant from ZonMw ( 10250042110004 ); EPIC-XS (project number 823839 ), funded by the Horizon 2020 programme of the European Union ; and the NWO -funded Netherlands Proteomics Centre through the National Road Map for Large-scale Infrastructures program X-Omics (project 184.034.019 ). J.M.V. is supported by scholarships from the Ministry of Science and Technology of Costa Rica (MICITT) and the University of Costa Rica (UCR) . The Vermeulen and Burgering labs are part of the Oncode Institute, which is partly funded by the Dutch Cancer Society (KWF) . The research reported in this publication was supported by Oncode Accelerator , a Dutch National Growth Fund project under grant number NGFOP2201 .

Funders	Funder number
Horizon 2020 Framework Programme
Oncode Institute
European Commission
KWF Kankerbestrijding
Universidad de Costa Rica
Tel Aviv University
Pluripotent Stem cells for Inherited Diseases and Embryonic Research
NWO-funded Netherlands Proteomics Centre
Ministry of Science and Technology, Croatia
Netherlands Proteomics Centre	184.034.019
European Proteomics Infrastructure Consortium providing access	823839
Dutch National Growth Fund	NGFOP2201
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	VI.Veni.212.076
ZonMw	10250042110004

Keywords

ZEB2
dTAG system
developmental biology
enhancers
gastruloids
germ layers
interaction proteomics
proteomics
somitoids
transcription factors

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1-s2.0-S1934590924001449-mainFinal published version, 7.39 MBLicence: CC BY

Cite this

Stelloo, S., Alejo-Vinogradova, M. T., Gelder, C. A. G. H. V., Zijlmans, D. W., Oostrom, M. J. V., Valverde, J. M., Lamers, L. A., Rus, T., Alcaraz, P. S., Schafers, T., Furlan, C., Jansen, P. W. T. C., Baltissen, M. P. A., Sonnen, K. F., Burgering, B., Altelaar, M. A. F. M., Vos, H. R., & Vermeulen, M. (2024). Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics. Cell Stem Cell, 31(7), 1072-1090.e8. https://doi.org/10.1016/j.stem.2024.04.017

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title = "Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics",

abstract = "Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometrybased proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.",

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author = "Suzan Stelloo and Alejo-Vinogradova, {Maria Teresa} and Gelder, {Charlotte A. G. H. van} and Zijlmans, {Dick W.} and Oostrom, {Marek J. van} and Valverde, {Juan Manuel} and Lamers, {Lieke A.} and Teja Rus and Alcaraz, {Paula Sobrevals} and Tilman Schafers and Cristina Furlan and Jansen, {Pascal W. T. C.} and Baltissen, {Marijke P. A.} and Sonnen, {Katharina F.} and Boudewijn Burgering and Altelaar, {Maarten A. F. M.} and Vos, {Harmjan R.} and Michiel Vermeulen",

note = "Publisher Copyright: {\textcopyright} 2024 The Authors",

year = "2024",

month = jul,

day = "5",

doi = "10.1016/j.stem.2024.04.017",

language = "English",

volume = "31",

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Stelloo, S, Alejo-Vinogradova, MT, Gelder, CAGHV, Zijlmans, DW, Oostrom, MJV, Valverde, JM, Lamers, LA, Rus, T, Alcaraz, PS, Schafers, T, Furlan, C, Jansen, PWTC, Baltissen, MPA, Sonnen, KF, Burgering, B, Altelaar, MAFM, Vos, HR & Vermeulen, M 2024, 'Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics', Cell Stem Cell, vol. 31, no. 7, pp. 1072-1090.e8. https://doi.org/10.1016/j.stem.2024.04.017

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T1 - Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics

AU - Stelloo, Suzan

AU - Alejo-Vinogradova, Maria Teresa

AU - Gelder, Charlotte A. G. H. van

AU - Zijlmans, Dick W.

AU - Oostrom, Marek J. van

AU - Valverde, Juan Manuel

AU - Lamers, Lieke A.

AU - Rus, Teja

AU - Alcaraz, Paula Sobrevals

AU - Schafers, Tilman

AU - Furlan, Cristina

AU - Jansen, Pascal W. T. C.

AU - Baltissen, Marijke P. A.

AU - Sonnen, Katharina F.

AU - Burgering, Boudewijn

AU - Altelaar, Maarten A. F. M.

AU - Vos, Harmjan R.

AU - Vermeulen, Michiel

PY - 2024/7/5

Y1 - 2024/7/5

N2 - Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometrybased proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

AB - Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometrybased proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

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KW - somitoids

KW - transcription factors

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Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics

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Keywords

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