Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates

Amphibian holoblastic cleavage in which all blastomeres contribute to any one of the three primary germ layers has been widely thought to be a developmental pattern in the stem lineage of vertebrates, and meroblastic cleavage to have evolved independently in each vertebrate lineage. In extant primitive vertebrates, agnathan lamprey and basal bony fishes also undergo holoblastic cleavage, and their vegetal blastomeres have been generally thought to contribute to embryonic endoderm. However, the present marker analyses in basal ray-finned fish bichir and agnathan lamprey embryos indicated that their mesoderm and endoderm develop in the equatorial marginal zone, and their vegetal cell mass is extraembryonic nutritive yolk cells, having non-cell autonomous meso-endoderm inducing activity. Eomesodermin (eomes), but not VegT, orthologs are expressed maternally in these animals, suggesting that VegT is a maternal factor for endoderm differentiation only in amphibian. The study raises the viewpoint that the lamprey/bichir type holoblastic development would have been ancestral to extant vertebrates and retained in their stem lineage; amphibian-type holoblastic development would have been acquired secondarily, accompanied by the exploitation of new molecular machinery such as maternal VegT.     

Structure and expression of three Emx genes in the dogfish Scyliorhinus canicula: functional and evolutionary implications

We report the characterization of three Emx genes in a chondrichthyan, the dogfish Scyliorhinus canicula. Comparisons of these genes with their osteichthyan counterparts indicate that the gnathostome Emx genes belong to three distinct orthology classes, each containing one of the dogfish genes and either the tetrapod Emx1 genes (Emx1 class), the osteichthyan Emx2 genes (Emx2 class) or the zebrafish Emx1 gene (Emx3 class). While the three classes could be retrieved from the pufferfish genome data, no indication of an Emx3-related gene in tetrapods could be found in the databases, suggesting that this class may have been lost in this taxon. Expression pattern comparisons of the three dogfish Emx genes and their osteichthyan counterparts indicate that not only telencephalic, but also diencephalic Emx expression territories are highly conserved among gnathostomes. In particular, all gnathostomes share an early, dynamic phase of Emx expression, spanning presumptive dorsal diencephalic territories, which involves Emx3 in the dogfish, but another orthology class, Emx2, in tetrapods. In addition, the dogfish Emx2 gene shows a highly specific expression domain in the cephalic paraxial mesoderm from the end of gastrulation and throughout neurulation, which suggests a role in the segmentation of the cephalic mesoderm.     

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.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

Origin of pancreatic precursors in the chick embryo and the mechanism of endoderm regionalization

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.     

Fate and plasticity of the endoderm in the early chick embryo

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.     

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

Background  

The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Retinoic Acid Signaling Organizes Endodermal Organ Specification along the Entire Antero-Posterior Axis

Background

Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.

Methodology/Principal Findings

RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA ⁺ cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.

Conclusions/Significance

Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.     

Characterization of sFRP2‐like in amphioxus: insights into the evolutionary conservation of Wnt antagonizing function

Wnt signaling plays a key role in embryonic patterning and morphogenetic movements. The secreted Frizzled‐related proteins (sFRPs) antagonize Wnt signaling, but their roles in development are poorly understood. To determine whether function of sFRPs is conserved between amphioxus and vertebrates, we characterized sFRP2‐like function in the amphioxus, Branchiostoma belcheri tsingtauense (B. belcheri). As in other species of Branchiostome, in B. belcheri, expression of sFRP2‐like is restricted to the mesendoderm during gastrulation and to the anterior mesoderm and endoderm during neurulation. Functional analyses in frog (Xenopus laevis) indicate that amphioxus sFRP2‐like potently inhibits both canonical and non‐canonical Wnts. Thus, sFRP‐2 probably functions in amphioxus embryos to inhibit Wnt signaling anteriorly. Moreover, dorsal overexpression of amphioxus sFRP2‐like in Xenopus embryos, like inhibition of Wnt11, blocks gastrulation movements. This implies that sFRP2‐like may also modulate Wnt signaling during gastrulation movements in amphioxus.     

Towards a synthetic view of axis specification mechanisms in vertebrates: insights from the dogfish

The genetic mechanisms, which control axis specification, apparently extensively diverge across vertebrates. In amphibians and teleosts, they are tightly linked to the establishment of an early dorso-ventral polarity. This polarity has no equivalent in amniotes, which unlike the former, retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extra-embryonic tissues for the establishment of their early rostro-caudal pattern. In order to better understand the links between these seemingly highly divergent mechanisms, we have used an evo-devo approach, aimed at reconstructing the gnathostome ancestral state and focussed on a chondrichthyan, the dogfish Scyliorhinus canicula. A detailed molecular characterization of the dogfish embryo at blastula and gastrula stages highlights striking similarities with all vertebrate model organisms including amniotes. It suggests the presence in the dogfish of territories homologous to the hypoblast and extra-embryonic ectoderm of the latter, which may therefore reflect the primitive condition of jawed vertebrates. In the ancestral state, these territories are specified at opposite sides of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians and teleosts, and aligned with the later forming embryonic axis, from head to tail. Amniotes have diverged from this pattern through a posterior expansion of extra-embryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages. These data delineate the broad outlines of the gnathostome ancestral pattern of axis specification and highlight an unexpected unity of mechanisms across jawed vertebrates. They illustrate the complementarity of comparative and genetic approaches for a comprehensive view of developmental mechanisms themselves. To cite this article: M. Coolen et al., C. R. Biologies 332 (2009).     

Molecular expression of Ly6k, a putative glycosylphosphatidyl-inositol-anchored membrane protein on the mouse testicular germ cells

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.