Pijuan-Sala B, Griffiths JA, Guibentif C, Hiscock TW, Jawaid W, Calero-Nieto FJ, Mulas C, Ibarra-Soria X, Tyser RCV, Ho DLL, Reik W, Srinivas S, Simons BD, Nichols J, Marioni JC & Göttgens B. (2019). A single-cell molecular map of mouse gastrulation and early organogenesis. Nature , 566, 490-495. PMID: 30787436 DOI.
A single-cell molecular map of mouse gastrulation and early organogenesis.
Abstract Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine. PMID: 30787436 PMCID: PMC6522369 DOI: 10.1038/s41586-019-0933-9
Self-organization of a human organizer by combined Wnt and Nodal signalling
Nature. 2018 Jun;558(7708):132-135. doi: 10.1038/s41586-018-0150-y. Epub 2018 May 23.
Martyn I1,2, Kanno TY1, Ruzo A1, Siggia ED3, Brivanlou AH4.
In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology. PMID: 29795348 DOI: 10.1038/s41586-018-0150-y
Molecular regulation of Nodal signaling during mesendoderm formation
Acta Biochim Biophys Sin (Shanghai). 2018 Jan 1;50(1):74-81. doi: 10.1093/abbs/gmx128.
Wei S1, Wang Q2.
Abstract One of the most important events during vertebrate embryogenesis is the formation or specification of the three germ layers, endoderm, mesoderm, and ectoderm. After a series of rapid cleavages, embryos form the mesendoderm and ectoderm during late blastulation and early gastrulation. The mesendoderm then further differentiates into the mesoderm and endoderm. Nodal, a member of the transforming growth factor β (TGF-β) superfamily, plays a pivotal role in mesendoderm formation by regulating the expression of a number of critical transcription factors, including Mix-like, GATA, Sox, and Fox. Because the Nodal signal transduction pathway is well-characterized, increasing effort has been made to delineate the spatiotemporal modulation of Nodal signaling during embryonic development. In this review, we summarize the recent progress delineating molecular regulation of Nodal signal intensity and duration during mesendoderm formation. KEYWORDS: Nodal signal; Smad2; mesendoderm; vertebrate embryo PMID: 29206913 DOI: 10.1093/abbs/gmx128
BMP and FGF signaling interact to pattern mesoderm by controlling basic helix-loop-helix transcription factor activity
Elife. 2018 Jun 7;7. pii: e31018. doi: 10.7554/eLife.31018. [Epub ahead of print]
Row RH1, Pegg A2, Kinney B1, Farr GH3, Maves L3, Lowell S2, Wilson V4, Martin BL1.
The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer. KEYWORDS: developmental biology; mouse; stem cells; zebrafish PMID: 29877796 DOI: 10.7554/eLife.31018
Establishment of the Vertebrate Germ Layers
Adv Exp Med Biol. 2017;953:307-381.
Tseng WC1, Munisha M1, Gutierrez JB2,3, Dougan ST4. Author information Abstract The process of germ layer formation is a universal feature of animal development. The germ layers separate the cells that produce the internal organs and tissues from those that produce the nervous system and outer tissues. Their discovery in the early nineteenth century transformed embryology from a purely descriptive field into a rigorous scientific discipline, in which hypotheses could be tested by observation and experimentation. By systematically addressing the questions of how the germ layers are formed and how they generate overall body plan, scientists have made fundamental contributions to the fields of evolution, cell signaling, morphogenesis, and stem cell biology. At each step, this work was advanced by the development of innovative methods of observing cell behavior in vivo and in culture. Here, we take an historical approach to describe our current understanding of vertebrate germ layer formation as it relates to the long-standing questions of developmental biology. By comparing how germ layers form in distantly related vertebrate species, we find that highly conserved molecular pathways can be adapted to perform the same function in dramatically different embryonic environments. KEYWORDS: Amniote; Amphibian; Ectoderm; Endoderm; Extraembryonic tissues; Fgf; Mesoderm; Morphogen; Nodal; Pander; TGF-beta; Teleost; Temporal gradient PMID: 27975275 DOI: 10.1007/978-3-319-46095-6_7
Review - Molecular specification of germ layers in vertebrate embryos
Cell Mol Life Sci. 2016 Mar;73(5):923-47. doi: 10.1007/s00018-015-2092-y. Epub 2015 Dec 14.
Kiecker C1, Bates T1,2, Bell E3.
In order to generate the tissues and organs of a multicellular organism, different cell types have to be generated during embryonic development. The first step in this process of cellular diversification is the formation of the three germ layers: ectoderm, endoderm and mesoderm. The ectoderm gives rise to the nervous system, epidermis and various neural crest-derived tissues, the endoderm goes on to form the gastrointestinal, respiratory and urinary systems as well as many endocrine glands, and the mesoderm will form the notochord, axial skeleton, cartilage, connective tissue, trunk muscles, kidneys and blood. Classic experiments in amphibian embryos revealed the tissue interactions involved in germ layer formation and provided the groundwork for the identification of secreted and intracellular factors involved in this process. We will begin this review by summarising the key findings of those studies. We will then evaluate them in the light of more recent genetic studies that helped clarify which of the previously identified factors are required for germ layer formation in vivo, and to what extent the mechanisms identified in amphibians are conserved across other vertebrate species. Collectively, these studies have started to reveal the gene regulatory network (GRN) underlying vertebrate germ layer specification and we will conclude our review by providing examples how our understanding of this GRN can be employed to differentiate stem cells in a targeted fashion for therapeutic purposes. KEYWORDS: Activin; FGF; Induction; Mesendoderm; Nieuwkoop Centre; Nodal; Spemann organiser; TGFβ; Vg1; Wnt
PMID 26667903 PMCID: PMC4744249 DOI: 10.1007/s00018-015-2092-y
Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo
Development. 2015 Jan 1;142(1):92-8. doi: 10.1242/dev.111583.
Stankova V1, Tsikolia N1, Viebahn C2.
During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms. © 2015. Published by The Company of Biologists Ltd. KEYWORDS: Amniote evolution; Cell migration; Mammalian gastrulation; Mesoderm formation; Planar cell polarity; Primitive streak
PLoS One. 2014 Feb 18;9(2):e88811. doi: 10.1371/journal.pone.0088811. eCollection 2014.
Meteorin regulates mesendoderm development by enhancing nodal expression
Kim YY1, Moon JS2, Kwon MC2, Shin J1, Im SK1, Kim HA3, Han JK2, Kong YY3. Author information Abstract During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin-null embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin-null embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.
PMID: 24558432 PMCID: PMC3928293 DOI: 10.1371/journal.pone.0088811
Early left-right asymmetries during axial morphogenesis in the chick embryo
Genesis. 2014 Jun;52(6):614-25. doi: 10.1002/dvg.22773. Epub 2014 Apr 5.
Otto A1, Pieper T, Viebahn C, Tsikolia N.
The primitive node is the "hub" of early left-right patterning in the chick embryo: (1) it undergoes asymmetrical morphogenesis immediately after its appearance at Stage 4; (2) it is closely linked to the emerging asymmetrical expression of nodal and shh at Stage 5; and (3) its asymmetry is spatiotemporally related to the emerging notochord, the midline barrier maintaining molecular left-right patterning from Stage 6 onward. Here, we study the correlation of node asymmetry to notochord marker expression using high-resolution histology, and we test pharmacological inhibition of shh signaling using cyclopamine at Stages 4 and 5. Just as noggin expression mirrors an intriguing structural continuity between the right node shoulder and the notochord, shh expression in the left node shoulder confirms a similar continuity with the future floor plate. Shh inhibition at Stage 4 or 5 suppressed nodal in both its paraxial or lateral plate mesoderm domains, respectively, and resulted in randomized heart looping. Thus, the "primordial" paraxial nodal asymmetry at Stage 4/5 (1) appears to be dependent on, but not instructed by, shh signaling and (2) may be fixed by asymmetrical roots of the notochord and the floor plate, thereby adding further twists to the node's pivotal role during left-right patterning. © 2014 Wiley Periodicals, Inc. KEYWORDS: avian; ectoderm; gastrulation; mesoderm; patterning PMID 24648137
Generation of organized germ layers from a single mouse embryonic stem cell
Nat Commun. 2014 May 30;5:4000. doi: 10.1038/ncomms5000.
Poh YC1, Chen J2, Hong Y2, Yi H2, Zhang S2, Chen J2, Wu DC3, Wang L2, Jia Q2, Singh R3, Yao W2, Tan Y1, Tajik A3, Tanaka TS4, Wang N1.
Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.
PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation
Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development.
Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors
PLoS One. 2013;8(2):e57698. doi: 10.1371/journal.pone.0057698. Epub 2013 Feb 28.
Kuo CL, Lam CM, Hewitt JE, Scotting PJ. Source Centre for Genetics and Genomics, School of Biology, University of Nottingham, QMC, Nottingham, United Kingdom.
The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.
Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo
Proc Natl Acad Sci U S A. 2013 Nov 27. [Epub ahead of print]
Plouhinec JL, Zakin L, Moriyama Y, De Robertis EM. Source Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662.
The vertebrate body plan follows stereotypical dorsal-ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers-ectoderm, mesoderm, and endoderm-are affected coordinately by the Chordin-BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called "Brachet's cleft" in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet's cleft.
Planar cell movements and oriented cell division during early primitive streak formation in the mammalian embryo
Dev Dyn. 2011 Aug;240(8):1905-16. doi: 10.1002/dvdy.22687.
Halacheva V, Fuchs M, Dönitz J, Reupke T, Püschel B, Viebahn C. Source Department of Anatomy and Embryology, Centre of Anatomy, University of Göttingen, Germany.
Formation of the mammalian primitive streak appears to rely on cell proliferation to a minor extent only, but compensating cell movements have not yet been directly observed. This study analyses individual cell migration and proliferation simultaneously, using multiphoton and differential interference contrast time-lapse microscopy of late pregastrulation rabbit blastocysts. Epiblast cells in the posterior gastrula extension area accumulated medially and displayed complex planar movements including U-turns and a novel type of processional cell movement. In the same area metaphase plates tended to be aligned parallel to the anterior-posterior axis, and statistical analysis showed that rotations of metaphase plates causing preferred orientation were near-complete 8 min before anaphase onset; in some cases, rotations were strikingly rapid, achieving up to 45° per min. The mammalian primitive streak appears to be formed initially with its typically minimal anteroposterior elongation by a combination of oriented cell divisions with dedicated planar cell movements. Copyright © 2011 Wiley-Liss, Inc. PMID 21761476
Deficiency in crumbs homolog 2 (Crb2) affects gastrulation and results in embryonic lethality in mice
Dev Dyn. 2011 Dec;240(12):2646-56. doi: 10.1002/dvdy.22778.
Xiao Z, Patrakka J, Nukui M, Chi L, Niu D, Betsholtz C, Pikkarainan T, Vainio S, Tryggvason K. Source Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institutet, Stockholm, Sweden.
The Crumbs family of transmembrane proteins has an important role in the differentiation of the apical membrane domain in various cell types, regulating such processes as epithelial cell polarization. The mammalian Crumbs protein family is composed of three members. Here, we inactivated the mouse Crb2 gene with gene-targeting techniques and found that the protein is crucial for early embryonic development with severe abnormalities appearing in Crb2-deficient embryos at late-gastrulation. Our findings indicate that the primary defect in the mutant embryos is disturbed polarity of the epiblast cells at the primitive streak, which affects epithelial to mesenchymal transition (EMT) during gastrulation, resulting in impaired mesoderm and endoderm formation, and embryonic lethality by embryonic day 12.5. These findings therefore indicate a novel role for the Crumbs family of proteins. Developmental Dynamics 240:2646-2656, 2011. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Rethinking embryonic germ layers
Dev Cell. 2011 Apr 19;20(4):e2.
Kimelman D. Source University of Washington, Department of Biochemistry, Seattle, WA 98195-7350, USA.
One of the central concepts we teach in developmental biology is that a major early decision made by the embryo is how to allocate cells to the three germ layers: ectoderm (epidermis, neural), mesoderm (muscle, cardiovascular), and endoderm (gut, liver). Using an in vivo lineage-labeling strategy based on random activation of a nonfunctional lacZ (β-galactosidase) gene in the mouse embryo, Tzouanacou et al. show that while the surface ectoderm (epidermis) is set aside early, a bipotential neuromesodermal cell population produces both neural and muscle cells as the body continues to grow out during the somitogenesis stages. This nicely challenges our simple assumptions about how the germ layers are established in early development while providing the basis for a mechanism that coordinates allocation of cells to the spinal cord and the muscles as the embryonic body elongates. This PaperPick refers to "Redefining the Progression of Lineage Segregations during Mammalian Embryogenesis by Clonal Analysis" by E. Tzouanacou, A. Wegener, F.J. Wymeersch, V. Wilson, and J.F. Nicolas, published in September 2009. VIDEO ABSTRACT:
Copyright © 2011 Elsevier Inc. All rights reserved.
J Biol Chem. 2010 Oct 26
KZP controls canonical WNT8 signaling to modulate dorsoventral patterning during zebrafish gastrulation. Yao S, Qian M, Deng S, Xie L, Yang H, Xiao C, Zhang T, Xu H, Zhao X, Wei YQ, Mo X.
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University,, China. Abstract During vertebrate embryonic development, the body axis formation requires action of wnt signals and their antagonists. Zygotic canonical wnt8 expression appears exclusively at the ventrolateral margin and mediates Wnt/beta-catenin activities to promote posterior and ventral cell fate. However, the mechanisms involved in the initiation of zygotic wnt8 signals are completely unknown. Here we identify a novel maternal derived transcriptional factor, Kaiso zinc finger containing protein (KZP), is an important determinant for the initiation of zygotic wnt signal in zebrafish. We demonstrated that KZP recognized specific consensus sequences and bound directly to wnt8 promoter to control the initiation of wnt8 expression. Depletion of KZP strongly dorsalized embryos characterized by the expansion of dorsal genes expression while overexpression caused posteriorization, which phenocopied wnt8 depletion or overexpression phenotypes and can be rescued by alternation of wnt8 activity. Thus, our results provided the first insight into the mechanism involved in the initiation of zygotic canonical wnt signal by maternal derived factor.
A requirement for FGF signalling in the formation of primitive streak-like intermediates from primitive ectoderm in culture
PLoS One. 2010 Sep 3;5(9):e12555.
Zheng Z, de Iongh RU, Rathjen PD, Rathjen J.
Department of Zoology, University of Melbourne, Parkville, Australia.
BACKGROUND: Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.
METHODOLOGY/PRINCIPAL FINDINGS: Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.
CONCLUSIONS/SIGNIFICANCE: FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.
Modeling gastrulation in the chick embryo: formation of the primitive streak
PLoS One. 2010 May 11;5(5):e10571.
Vasiev B, Balter A, Chaplain M, Glazier JA, Weijer CJ.
Division of Mathematics, University of Dundee, Dundee, United Kingdom. email@example.com
The body plan of all higher organisms develops during gastrulation. Gastrulation results from the integration of cell proliferation, differentiation and migration of thousands of cells. In the chick embryo gastrulation starts with the formation of the primitive streak, the site of invagination of mesoderm and endoderm cells, from cells overlaying Koller's Sickle. Streak formation is associated with large-scale cell flows that carry the mesoderm cells overlying Koller's sickle into the central midline region of the embryo. We use multi-cell computer simulations to investigate possible mechanisms underlying the formation of the primitive streak in the chick embryo. Our simulations suggest that the formation of the primitive streak employs chemotactic movement of a subpopulation of streak cells, as well as differential adhesion between the mesoderm cells and the other cells in the epiblast. Both chemo-attraction and chemo-repulsion between various combinations of cell types can create a streak. However, only one combination successfully reproduces experimental observations of the manner in which two streaks in the same embryo interact. This finding supports a mechanism in which streak tip cells produce a diffusible morphogen which repels cells in the surrounding epiblast. On the other hand, chemotactic interaction alone does not reproduce the experimental observation that the large-scale vortical cell flows develop simultaneously with streak initiation. In our model the formation of large scale cell flows requires an additional mechanism that coordinates and aligns the motion of neighboring cells.
- Non-redundant roles for Proﬁlin2 and Proﬁlin1 during vertebrate gastrulation Deepak K. Khadka a, Wei Liu a, Raymond Habas
Wnt signaling pathway has emerged as a key regulator of gastrulation.
Keywords: Wnt, Non-canonical signaling, Proﬁlin1, Proﬁlin2, Daam1, Gastrulation, Morphogenesis, Xenopus
Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly deﬁned. We had previously identiﬁed the Formin protein Daam1 and an effector molecule XProﬁlin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identi- ﬁcation of XProﬁlin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProﬁlin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProﬁlin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProﬁlin1 regulates blastopore closure, overexpression or depletion of XProﬁlin2 speciﬁcally affects convergent exten- sion movement independent of mesodermal speciﬁcation. Speciﬁcally, we show that XProﬁlin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProﬁlin1. Together, our studies demonstrate that XProﬁlin2 and XProﬁlin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.
Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation
Reproduction. 2008 Feb;135(2):181-95. doi: 10.1530/REP-07-0355.
Blomberg L, Hashizume K, Viebahn C. Source Animal Biosciences and Biotechnology Laboratory, USDA Agricultural Research Service, Beltsville, Maryland 20705, USA.
Abstract The molecular basis of ungulate and non-rodent conceptus elongation and gastrulation remains poorly understood; however, use of state-of-the-art genomic technologies is beginning to elucidate the mechanisms regulating these complicated processes. For instance, transcriptome analysis of elongating porcine concepti indicates that protein synthesis and trafficking, cell growth and proliferation, and cellular morphology are major regulated processes. Furthermore, potential autocrine roles of estrogen and interleukin-1-beta in regulating porcine conceptus growth and remodeling and metabolism have become evident. The importance of estrogen in pig is emphasized by the altered expression of essential steroidogenic and trophoblast factors in lagging ovoid concepti. In ruminants, the characteristic mononucleate trophoblast cells differentiate into a second lineage important for implantation, the binucleate trophoblast, and transcriptome profiling of bovine concepti has revealed a gene cluster associated with rapid trophoblast proliferation and differentiation. Gene cluster analysis has also provided evidence of correlated spatiotemporal expression and emphasized the significance of the bovine trophoblast cell lineage and the regulatory mechanism of trophoblast function. As a part of the gastrulation process in the mammalian conceptus, specification of the germ layers and hence definitive body axes occur in advance of primitive streak formation. Processing of the transforming growth factor-beta-signaling molecules nodal and BMP4 by specific proteases is emerging as a decisive step in the initial patterning of the pre-gastrulation embryo. The topography of expression of these and other secreted molecules with reference to embryonic and extraembryonic tissues determines their local interaction potential. Their ensuing signaling leads to the specification of axial epiblast and hypoblast compartments through cellular migration and differentiation and, in particular, the specification of the early germ layer tissues in the epiblast via gene expression characteristic of endoderm and mesoderm precursor cells.
Curr Biol. 2006 Oct 24;16(20):1986-97. Wnt/Frizzled signaling controls C. elegans gastrulation by activating actomyosin contractility. Lee JY, Marston DJ, Walston T, Hardin J, Halberstadt A, Goldstein B. Source Department of Biology, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA. Abstract BACKGROUND: Embryonic patterning mechanisms regulate the cytoskeletal machinery that drives morphogenesis, but there are few cases where links between patterning mechanisms and morphogenesis are well understood. We have used a combination of genetics, in vivo imaging, and cell manipulations to identify such links in C. elegans gastrulation. Gastrulation in C. elegans begins with the internalization of endodermal precursor cells in a process that depends on apical constriction of ingressing cells. RESULTS: We show that ingression of the endodermal precursor cells is regulated by pathways, including a Wnt-Frizzled signaling pathway, that specify endodermal cell fate. We find that Wnt signaling has a role in gastrulation in addition to its earlier roles in regulating endodermal cell fate and cell-cycle timing. In the absence of Wnt signaling, endodermal precursor cells polarize and enrich myosin II apically but fail to contract their apical surfaces. We show that a regulatory myosin light chain normally becomes phosphorylated on the apical side of ingressing cells at a conserved site that can lead to myosin-filament formation and contraction of actomyosin networks and that this phosphorylation depends on Wnt signaling. CONCLUSIONS: We conclude that Wnt signaling regulates C. elegans gastrulation through regulatory myosin light-chain phosphorylation, which results in the contraction of the apical surface of ingressing cells. These findings forge new links between cell-fate specification and morphogenesis, and they represent a novel mechanism by which Wnt signaling can regulate morphogenesis. Comment in The following popper user interface control may not be accessible. Tab to the next button to revert the control to an accessible version.Destroy user interface controlGastrulation: Wnts signal constriction. [Curr Biol. 2006] PMID 17055977