Hensen's node gives rise to the ventral midline of the foregut: implications for organizing head and heart development

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

Head-organizing activities of endodermal tissues in vertebrates.

The embryonic organizer represents the major regulatory centre for the establishment of the body axes during gastrulation. Here, we discuss the endodermal contributions to the organizer of amphibia, birds and mammals. We differentiate between the definitive, prospective liver endoderm, and the primitive, prospective extraembryonic endoderm, the latter addressed as the hypoblast in birds and the visceral endoderm in mammals. We further discuss the role of the prechordal plate, a mesendodermal tissue underlying the prospective forebrain. Our conclusion points out the similarity of the amphibian and the avian organizer, with a concentration of inductive potentials in time and space. On the other hand, we discuss the unique feature of mammals, that have shifted certain aspects of the head organizer into the anterior visceral endoderm.     

BMP signalling regulates anteroposterior endoderm patterning in zebrafish

In vertebrates, the embryonic dorsoventral asymmetry is regulated by the bone morphogenetic proteins (Bmp) activity gradient. In the present study, we have used dorsalized swirl (bmp2b) and ventralized chordino (chordin) zebrafish mutants to investigate the effects of dorsoventral signalling on endoderm patterning and on the differentiation and positioning of its derivatives. Alterations of dorsoventral Bmp signalling do not perturb the induction of endodermal precursors, as shown by normal amounts of cells expressing cas and sox17 in swirl and chordino gastrulae, but affect dramatically the expression pattern of her5, a regulator of endoderm anteroposterior patterning in zebrafish. In particular, increased levels of Bmp signalling in chordino gastrulae are associated with a markedly reduced her5 expression domain, that may be abolished by injecting bmp2b mRNA. Conversely, in swirl mutants, lacking Bmp2b, the her5 expression domain is expanded. Thus, a gradient of Bmp2b signalling defines the extension of the her5 expression domain at gastrulation and the allocation of anterior endodermal precursors. A balanced Bmp2b signalling is also required for the normal development of the pancreas, as shown by the sharp reduction of the pancreatic primordium in swirl embryos and its expansion in chordino mutants. In the latter, at 3 days post-fertilization, the increased Bmp signalling does not compromise the endocrine/exocrine pancreas compartmentalization, but the right/left positioning of the pancreas and liver is randomized. Our results suggest that by regulating the expression of her5, the Bmp2b/Chordin gradient directs the anteroposterior patterning of endoderm in zebrafish embryos.     

Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling

Although retinoic acid (RA) has been implicated as one of the diffusible signals regulating forebrain development, patterning of the forebrain has not been analyzed in detail in knockout mouse mutants deficient in embryonic RA synthesis. We show that the retinaldehyde dehydrogenase 2 (RALDH2) enzyme is responsible for RA synthesis in the mouse craniofacial region and forebrain between the 8- and 15-somite stages. Raldh2-/- knockout embryos exhibit defective morphogenesis of various forebrain derivatives, including the ventral diencephalon, the optic and telencephalic vesicles. These defects are preceded by regionally decreased cell proliferation in the neuroepithelium, correlating with abnormally low D-cyclin gene expression. Increases in cell death also contribute to the morphological deficiencies at later stages. Molecular analyses reveal abnormally low levels of FGF signaling in the craniofacial region, and impaired sonic hedgehog signaling in the ventral diencephalon. Expression levels of several regulators of diencephalic, telencephalic and optic development therefore cannot be maintained. These results unveil crucial roles of RA during early mouse forebrain development, which may involve the regulation of the expansion of neural progenitor cells through a crosstalk with FGF and sonic hedgehog signaling pathways.     

Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish

Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-β signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis.     

The role of prechordal mesendoderm in neural patterning

Prechordal mesendoderm is formed in response to Nodal and maternal beta-Catenin signaling and is regulated by signals from anterior endoderm and chordamesoderm. Prechordal mesendodermal cells are involved in neural induction and in anteroposterior and dorsoventral neural patterning. Inhibitors of Wnt and BMP growth factors secreted by prechordal mesendoderm mediate neural induction and anteroposterior and dorsoventral patterning, whereas SHH and TGF betas mediate dorsoventral patterning.     

The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann's organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain- and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes- and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.     

The zinc-finger protein CNBP is required for forebrain formation in the mouse

Mouse mutants have allowed us to gain significant insight into axis development. However, much remains to be learned about the cellular and molecular basis of early forebrain patterning. We describe a lethal mutation mouse strain generated using promoter-trap mutagenesis. The mutants exhibit severe forebrain truncation in homozygous mouse embryos and various craniofacial defects in heterozygotes. We show that the defects are caused by disruption of the gene encoding cellular nucleic acid binding protein (CNBP); Cnbp transgenic mice were able to rescue fully the mutant phenotype. Cnbp is first expressed in the anterior visceral endoderm (AVE) and, subsequently, in the anterior definitive endoderm (ADE), anterior neuroectoderm (ANE), anterior mesendoderm (AME), headfolds and forebrain. In Cnbp(-/-) embryos, the visceral endoderm remains in the distal tip of the conceptus and the ADE fails to form, whereas the node and notochord form normally. A substantial reduction in cell proliferation was observed in the anterior regions of Cnbp(-/-) embryos at gastrulation and neural-fold stages. In these regions, Myc expression was absent, indicating CNBP targets Myc in rostral head formation. Our findings demonstrate that Cnbp is essential for the forebrain induction and specification.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.     

Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.     

Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.     

Development of chick axial mesoderm: specification of prechordal mesoderm by anterior endoderm-derived TGFbeta family signalling

Two populations of axial mesoderm cells can be recognised in the chick embryo, posterior notochord and anterior prechordal mesoderm. We have examined the cellular and molecular events that govern the specification of prechordal mesoderm. We report that notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen's node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Together these experiments suggest that, in vivo, signalling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor (beta) (TGFbeta) superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together our results suggest that anterior endoderm-derived TGFbetas may specify prechordal mesoderm character in chick axial mesoderm.     

Induction and patterning of the telencephalon in Xenopus laevis

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.     

Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation

We identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial mesendoderm such as prechordal plate and notochord. dkk1 expression in the dorso-anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm. dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish.     

The pitx2 homeobox protein is required early for endoderm formation and nodal signaling.

Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.     

The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embryo

The anterior midline tissue (AML) of the late gastrula mouse embryo comprises the axial mesendoderm and the ventral neuroectoderm of the prospective forebrain, midbrain and rostral hindbrain. In this study, we have investigated the morphogenetic role of defined segments of the AML by testing their inductive and patterning activity and by assessing the impact of their ablation on the patterning of the neural tube at the early-somite-stage. Both rostral and caudal segments of the AML were found to induce neural gene activity in the host tissue; however, the de novo gene activity did not show any regional characteristic that might be correlated with the segmental origin of the AML. Removal of the rostral AML that contains the prechordal plate resulted in a truncation of the head accompanied by the loss of several forebrain markers. However, the remaining tissues reconstituted Gsc and Shh activity and expressed the ventral forebrain marker Nkx2.1. Furthermore, analysis of Gsc-deficient embryos reveals that the morphogenetic function of the rostral AML requires Gsc activity. Removal of the caudal AML led to a complete loss of midline molecular markers anterior to the 4th somite. In addition, Nkx2.1 expression was not detected in the ventral neural tube. The maintenance and function of the rostral AML therefore require inductive signals emanating from the caudal AML. Our results point to a role for AML in the refinement of the anteroposterior patterning and morphogenesis of the brain.