Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

During mouse gastrulation, cells in the primitive streak undergo epithelial–mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8–Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.     

FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak.

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 -/- embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic beta-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.     

Back and forth between cell fate specification and movement during vertebrate gastrulation

Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal, and Wnt have well-recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal, and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.     

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.     

Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation

The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin--gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.     

Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells

Wnt proteins play important roles in many developmental events. Wnts are divided into two groups according to biological function. The Wnt-5a class proteins function in morphogenetic movement during embryogenesis. Previously, a Wnt-5 homolog has been isolated from the ascidian, Halocynthia roretzi. HrWnt-5 is expressed in the notochord until the tail-bud stage, implying a role in the notochord. In this study, the function of HrWnt-5 was investigated. When HrWnt-5 mRNA was injected into fertilized eggs, the embryos showed morphologic defects at around the neurula stage. The anterior-posterior axis was shorter than in control embryos. These defects were caused by the abnormal movement of notochord cells. However, the overexpression of HrWnt-5 mRNA did not affect the differentiation of tissues, suggesting that HrWnt-5 solely regulates the morphogenetic movement. Although endogenous HrWnt-5 is expressed in the notochord, the overexpression of HrWnt-5 mRNA caused the defects, suggesting that the amount of HrWnt-5 mRNA in the notochord is strictly regulated. These results suggest that HrWnt-5 regulates the morphogenetic movement of notochord cells during ascidian embryogenesis.     

Subdivision and developmental fate of the head mesoderm in Drosophila melanogaster

In this paper, we define temporal and spatial subdivisions of the embryonic head mesoderm and describe the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the “T-bar” of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. We distinguish between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that we interpret as a rudimentary cephalic aorta (“cephalic vascular rudiment”). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells.     

PDGF-A controls mesoderm cell orientation and radial intercalation during Xenopus gastrulation

Radial intercalation is a common, yet poorly understood, morphogenetic process in the developing embryo. By analyzing cell rearrangement in the prechordal mesoderm during Xenopus gastrulation, we have identified a mechanism for radial intercalation. It involves cell orientation in response to a long-range signal mediated by platelet-derived growth factor (PDGF-A) and directional intercellular migration. When PDGF-A signaling is inhibited, prechordal mesoderm cells fail to orient towards the ectoderm, the endogenous source of PDGF-A, and no longer migrate towards it. Consequently, the prechordal mesoderm fails to spread during gastrulation. Orientation and directional migration can be rescued specifically by the expression of a short splicing isoform of PDGF-A, but not by a long matrix-binding isoform, consistent with a requirement for long-range signaling.     

Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.     

The Spemann-Mangold organizer: the control of fate specification and morphogenetic rearrangements during gastrulation in Xenopus

Vertebrate embryonic development is controlled by sequentially operating signalling centres that organize spatial pattern by inductive interactions. The embryonic body plan is established during gastrulation through the action of the Spemann-Mangold or gastrula organizer, a signalling source discovered 75 years ago by Hans Spemann and Hilde Mangold. Transplantation of the organizer to a heterotopic location in a recipient embryo results in the formation of a secondary embryonic body axis, in which several tissue types, most notably somites and the neural tube, are derived from ventral host cells. Because of these non-cell autonomous recruiting or inducing activities the organizer has become a paradigm for studying intercellular communication in the vertebrate embryo. Here, I review some of the recent advances in understanding 1) the initiation of the Spemann-Mangold organizer, 2) its function in pattern formation along the dorsal-ventral and anterior-posterior axes and 3) the integration of cell fate specification events and downstream execution of morphogenetic movements during gastrulation in Xenopus laevis.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation

During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas Dishevelled is enriched posteriorly. Asymmetrical localization of Prickle and Dishevelled to the opposite cell edges in zebrafish gastrula parallels their distribution in fly, and suggests that noncanonical Wnt signaling defines distinct anterior and posterior cell properties to bias cell intercalations.     

Heat induced developmental uncoupling of mesoderm from ectoderm and endoderm germ layer derivatives during Artemia postembryonic segmentation

Taking advantage of the fact than segmentation inArtemia is largely a postembryonic process making it more susceptible to environmental influences, heat treatments ofArtemia newly-hatched nauplii were shown to induce a severe inhibition of mesodermal structures, without apparently affecting the corresponding ectodermal and endodermal derivaives. This inhibition was reversible and with enough time the missing mesodermal structures developed. These results indicate that ectoderm and endoderm development can proceed without neccessarily a concomitant mesodermal differentiation, which in turn can be largely uncoupled from that of the rest of the germ layer derivatives.     

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.     

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.