Germ layer formation at gastrulation

Stable Identifier
Homo sapiens
Primitive streak formation
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Due to ethical considerations, most research on mammalian gastrulation has been performed on mouse embryos. Therefore most of the reactions described in this section are the results of research in mouse embryos. Significant research has also been performed on non-human primates such as cynomolgus monkeys (Macaca fascicularis) (Nakamura et al. 2016, Sasaki et al. 2016). More recently, human gastrula-like cell assemblages ("gastruloids") generated from pluripotent stem cells have been developed (Moris et al. 2020) and are now being compared with mouse embryos (reviewed in Rossant and Tam 2021, Ghimire et al. 2021).
At the beginning of gastrulation in the mouse, the primitive streak forms in a region of BMP, WNT, FGF, and NODAL signaling. In the mouse embryo, NODAL is expressed throughout the epiblast before anterior-posterior axis induction and is required for pluripotency (reviewed in Robertson 2014). NODAL signaling is restricted to the posterior side of the embryo by the secretion of NODAL and WNT antagonists (CER1, LEFTY1) from the anterior visceral endoderm (AVE) (reviewed in Stower and Srinivas 2014). In human embryonic stem cells (hESCs) NODAL is also crucial for maintenance of pluripotency (James et al. 2005, Vallier et al. 2004). In mouse embryos, NODAL and WNT3 are required for formation of the primitive streak (Conlon et al. 1994, Brennan et al. 2001, Liu et al. 1999) and NODAL expression subsequently becomes restricted to the node at the anterior end of the primitive streak (Zhou et al. 1993). Pro-NODAL secreted by the epiblast in response to BMP4 signalling from the extraembryonic ectoderm is converted to mature NODAL by furin (PCSK3) secreted from the extraembryonic ectoderm. NODAL maintains BMP4 expression in the extraembryonic ectoderm which then activates WNT3 in the posterior epiblast. WNT signaling, in turn, amplifies NODAL expression (Brennan et al. 2001). The order of events in this signaling cascade may be different in human embryos due to differences in early embryo architecture.
NODAL, BMP, and WNT show similar effects on human 2D gastruloids (Martyn et al. 2019). Mesoderm and definitive endoderm progenitors appear to be already separate and distinct in the primitive streak, therefore bipotential mesendoderm progenitors may be transitory if they exist (Probst et al. 2021). This is an area of ongoing research.
Mesoderm is formed by an epithelial-mesenchymal transition that produces an ingression of cells through the primitive streak. Endoderm does not show a complete epithelial-mesenchymal transition and instead forms by cell plasticity (a partial epithelial-mesenchymal transition in which both E-cadherin and N-Cadherin are expressed) (inferred from mouse embryos in Scheibner et al. 2021). However, in mouse embryos endoderm progenitors still ingress through the anterior region of the primitive streak, migrate with mesoderm cells, and eventually integrate into the visceral endoderm layer to give rise to the definitive endoderm (Viotti et al. 2014).
Specific types of mesoderm are formed sequentially according to the time and position of ingression of cells through the primitive streak. This patterning is caused by gradients of NODAL, WNT, and BMP signaling that activate transcriptional programs in the mesoderm progenitors.
T-box transcription factor T (TBXT, T, Brachyury) and Eomesodermin (EOMES) are two of the first transcription factors expressed in mesoderm and endoderm progenitors in the primitive streak (reviewed in Probst and Arnold 2016). The two factors combined are required for formation of all mesoderm and endoderm (Arnold et al. 2008, Tosic et al. 2019).
TBXT is activated by WNT signaling (via beta-catenin acting with LEF1 or TCF1) and BMP4 and is expressed in mesodermal and axial mesodermal progenitors and in the primitive streak during gastrulation, later becoming localized to the notochord and tailbud. TBXT is an early marker of mesodermal differentiation and is often used in studies of embryonic stem cells. In hESCs TBXT is expressed in both mesodermal and endodermal progenitors, it regulates different sets of target genes depending on the signaling environment (Faial et al. 2015).
Expression of EOMES is activated by NODAL via SMAD2 and SMAD3 and is observed in the posterior epiblast prior to formation of the primitive streak and in mesoderm and endoderm progenitors during the first day of gastrulation. EOMES in combination with SMAD2,3 is crucial for the activation of definitive endoderm genes (Teo et al. 2011). TBXT and EOMES generally activate expression of mesoderm genes and repress expression of genes associated with pluripotency such as SOX2 and NANOG.
Some transcription factors are particularly important for regulating gastrulation and are also used as markers for particular stages and morphological features. For example, Goosecoid (GSC) expression marks the onset of gastrulation, is first observed in the primitive streak, and becomes localized to the anterior end of the primitive streak and then the axial mesoderm (Blum et al. 1992). SMAD2 and SMAD3 activated by NODAL are recruited to the GSC promoter by FOXH1, which is already located at the promoter. MIXL1 also binds the GSC promoter and activates expression. In mice, GSC is a regulator of head development.
MIXL1 is required for formation of both mesoderm and definitive endoderm (Hart et al. 2002) and is expressed early throughout the primitive streak and in nascent mesoderm cells exiting the streak. Expression of MIXL1 is mediated downstream by NODAL through SMAD2 and SMAD3 binding to the promoter of MIXL1. EOMES also plays a direct role in activating MIXL1 and GSC expression in hESCs (Teo et al. 2011) and in mouse embryos (Tosic et al. 2019)..
Developing mesoderm becomes specified by expression of transcription factors such as MESP1, a marker of cardiac progenitors. (See the Reactome pathway "Cardiogenesis".)

Literature References
PubMed ID Title Journal Year
28057273 Eomesodermin-At Dawn of Cell Fate Decisions During Early Embryogenesis

Probst, S, Arnold, SJ

Curr Top Dev Biol 2017
7924997 A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse

Robertson, EJ, Kispert, A, Barth, KS, Herrmann, B, Takaesu, N, Lyons, KM, Conlon, FL

Development 1994
26015544 Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells

Smith, JC, Diamanti, E, Pedersen, RA, Trotter, MW, Mascetti, VL, Faial, T, Gentsch, GE, Ortmann, D, Bernardo, AS, Mendjan, S

Development 2015
29795348 Self-organization of a human organizer by combined Wnt and Nodal signalling

Siggia, ED, Martyn, I, Brivanlou, AH, Kanno, TY, Ruzo, A

Nature 2018
33484705 Human gastrulation: The embryo and its models

Moris, N, Martinez Arias, A, Ghimire, S, Mantziou, V

Dev Biol 2021
11418863 Nodal signalling in the epiblast patterns the early mouse embryo

Robertson, EJ, Lu, CC, Brennan, J, Norris, DP, Beddington, RS, Rodriguez, TA

Nature 2001
19129791 Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo

Arnold, SJ, Robertson, EJ

Nat Rev Mol Cell Biol 2009
8429908 Nodal is a novel TGF-beta-like gene expressed in the mouse node during gastrulation

Lowe, L, Sasaki, H, Zhou, X, Hogan, BL, Kuehn, MR

Nature 1993
34417090 Human embryonic development: from peri-implantation to gastrulation

Xiao, Z, Wang, Y, Wang, H, Zhai, J

Trends Cell Biol 2021
25349455 Gutsy moves in mice: cellular and molecular dynamics of endoderm morphogenesis

Hadjantonakis, AK, Viotti, M, Foley, AC

Philos Trans R Soc Lond B Biol Sci 2014
18171685 Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse

Bikoff, EK, Hofmann, UK, Arnold, SJ, Robertson, EJ

Development 2008
33199445 Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation

Grün, D, Schwan, C, Probst, S, Tosic, J, Arnold, SJ, Sagar, S

Development 2021
12117810 Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo

Hart, AH, Stadler, ES, Li, R, Stanley, EG, Robb, L, Elefanty, AG, Sourris, K, Tam, PPL, Hartley, L

Development 2002
15501227 Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Pedersen, RA, Vallier, L, Reynolds, D

Dev Biol 2004
31792383 Eomes and Brachyury control pluripotency exit and germ-layer segregation by changing the chromatin state

Köttgen, M, Barg, M, Kim, GJ, Pavlovic, M, Hein, L, Hofherr, A, Mersiowsky, SL, Probst, S, Tosic, J, Arnold, SJ, Schröder, CM

Nat Cell Biol 2019
27556940 A developmental coordinate of pluripotency among mice, monkeys and humans

Saitou, M, Tsuchiya, H, Okamoto, I, Yamamoto, T, Sasaki, K, Iwatani, C, Seita, Y, Nakamura, S, Yabuta, Y, Nakamura, T

Nature 2016
15703277 TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells

Hemmati-Brivanlou, A, James, D, Levine, AJ, Besser, D

Development 2005
32528178 An in vitro model of early anteroposterior organization during human development

Moris, N, Martinez Arias, A, Anlas, K, Alemany, A, Ghimire, S, van Oudenaarden, A, Balayo, T, van den Brink, SC, Schröder, J

Nature 2020
33667412 Opportunities and challenges with stem cell-based embryo models

Rossant, J, Tam, PPL

Stem Cell Reports 2021
34168324 Epithelial cell plasticity drives endoderm formation during gastrulation

Böttcher, A, Ansarullah, A, Lickert, H, Cernilogar, FM, Beckers, J, Sterr, M, Burtscher, I, Büttner, M, Schotta, G, Scheibner, K, Theis, FJ, Schirge, S, Irmler, M, Yang, D

Nat Cell Biol 2021
24704361 Dose-dependent Nodal/Smad signals pattern the early mouse embryo

Robertson, EJ

Semin Cell Dev Biol 2014
27720607 The Germ Cell Fate of Cynomolgus Monkeys Is Specified in the Nascent Amnion

Saitou, M, Tsuchiya, H, Shiraki, N, Okamoto, I, Yamamoto, T, Sasaki, K, Iwatani, C, Takakuwa, T, Seita, Y, Nakamura, S, Yabuta, Y, Nakamura, T

Dev Cell 2016
25349454 Heading forwards: anterior visceral endoderm migration in patterning the mouse embryo

Stower, MJ, Srinivas, S

Philos Trans R Soc Lond B Biol Sci 2014
10431240 Requirement for Wnt3 in vertebrate axis formation

Bradley, A, Albrecht, U, Behringer, RR, Wakamiya, M, Shea, MJ, Liu, P

Nat Genet 1999
21245162 Pluripotency factors regulate definitive endoderm specification through eomesodermin

Dunn, NR, Chng, Z, Trotter, MW, Vallier, L, Teo, AK, Ang, LT, Brown, S, Arnold, SJ, Robertson, EJ

Genes Dev. 2011
1352187 Gastrulation in the mouse: the role of the homeobox gene goosecoid

Blumberg, B, Steinbeisser, H, De Robertis, EM, Gaunt, SJ, Blum, M, Cho, KW, Bittner, D

Cell 1992
31476530 Mesoderm specification and diversification: from single cells to emergent tissues

Hadjantonakis, AK, Ferretti, E

Curr Opin Cell Biol 2019
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