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.     

The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development. At embryonic day (E) 5.5, Sfrp5 is expressed in the visceral endoderm at the distal tip region of the embryo and at E6.5 in the AVE opposite the primitive streak. In Lim1 embryos, which lack anterior neural tissue and sometimes form a secondary body axis, Sfrp5-expressing cells fail to move towards the anterior and remain at the distal tip of E6.5 embryos. When compared with Dkk1, which encodes another secreted Wnt antagonist molecule present in the visceral endoderm, Sfrp5 and Dkk1 expression overlap but Sfrp5 is expressed more broadly in the AVE. Between E7.5 and 8, Sfrp5 is expressed in the foregut endoderm underlying the cardiac mesoderm. At E8.5, Sfrp5 is expressed in the ventral foregut endoderm that gives rise to the liver. Additional domains of Sfrp5 expression occur in the dorsal neural tube and in the forebrain anterior to the optic placode. These findings identify a gene encoding a secreted Wnt antagonist that is expressed in the extraembryonic visceral endoderm and anterior definitive endoderm during axis formation and organogenesis in the mouse.     

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps

The development of the anterior foregut of the mammalian embryo involves changes in the behavior of both the epithelial endoderm and the adjacent mesoderm. Morphogenetic processes that occur include the extrusion of midline notochord cells from the epithelial definitive endoderm, the folding of the endoderm into a foregut tube, and the subsequent separation of the foregut tube into trachea and esophagus. Defects in foregut morphogenesis underlie the constellation of human birth defects known as esophageal atresia (EA) and tracheoesophageal fistula (TEF). Here, we review what is known about the cellular events in foregut morphogenesis and the gene mutations associated with EA and TEF in mice and humans. We present new evidence that about 70% of mouse embryos homozygous null for Nog, the gene encoding noggin, a bone morphogenetic protein (Bmp) antagonist, have EA/TEF as well as defects in lung branching. This phenotype appears to correlate with abnormal morphogenesis of the notochord and defects in its separation from the definitive endoderm. The abnormalities in foregut and lung morphogenesis of Nog null mutant can be rescued by reducing the gene dose of Bmp4 by 50%. This suggests that normal foregut morphogenesis requires that the level of Bmp4 activity is carefully controlled by means of antagonists such as noggin. Several mechanisms are suggested for how Bmps normally function, including by regulating the intercellular adhesion and behavior of notochord and foregut endoderm cells. Future research must determine how Noggin/Bmp antagonism fits into the network of other factors known to regulate tracheal and esophagus development, both in mouse or humans.     

Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation

Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1^−/− mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1^−/− esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1^−/− foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.     

Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of midstreak to neural plate stages of the mouse embryo was studied by microinjecting horseradish peroxidase (HRP) into single axial endoderm cells in situ, and tracing the labeled descendants to early somite stages in vitro. Axial endoderm cells along the anterior fifth of the late streak/neural plate stage embryo contributed descendants either to the yolk sac endoderm or to the anterior intestinal portal. Cells of the exposed head process contributed to the trunk endoderm and notochord; neighboring endoderm cells contributed to the dorsal foregut. Contributions to the ventral foregut came from endoderm at, and anterior to, the distal tip of the younger, midstreak embryo (in which the head process was not yet exposed). Endoderm over the primitive streak contributed to the postsomite endoderm. We argue from these results and those in the literature that during gastrulation the axial embryonic endoderm is of mixed lineage: (1) an anterior population of cells is derived from primitive endoderm and contributes to the yolk sac endoderm; (2) a population at, and anterior to, the distal tip of the midstreak embryo, extending more anteriorly at late streak/neural plate stages, is presumed to emerge from primitive ectoderm at the beginning of gastrulation and contributes to the foregut and anterior intestinal portal; (3) the axial portion of the head process that begins to incorporate into the ventral surface at the late streak stage contributes to notochord and trunk endoderm. Cells or their descendants that were destined to die within 24 hr were evident at the midstreak stage. There was a linear trend in the incidence of cell death among labeled cells at the late streak/neural plate stages, ranging from 27% caudal to the node to 57% in the anterior fifth of the embryo. The surviving axial endoderm cells divided sufficiently fast to double the population in 24 hr.     

Expression patterns of fork head and goosecoid homologues in the mollusc Patella vulgata supports the ancestry of the anterior mesendoderm across Bilateria

We have characterised orthologues of the genes fork head and goosecoid in the gastropod Patella vulgata. In this species, the anterior-posterior (AP) axis is determined just before gastrulation, and leads to the specification of two mesodermal components on each side of the presumptive endoderm, one anterior (ectomesoderm), and one posterior (endomesoderm). Both fork head and goosecoid are expressed from the time the AP axis is specified, up to the end of gastrulation. fork head mRNA is detected in the whole endoderm, as well as in the anterior mesoderm, whereas goosecoid is only expressed anteriorly, in the three germ layers. The two genes are thus coexpressed in the anterior mesoderm, suggesting the latter's homology with vertebrate prechordal mesoderm. In addition, since prechordal plate is known to belong to an anterior, so called "head organiser", and since its inductive role is dependent on the function of the vertebrate fork head and goosecoid orthologues, we further suggest that the anterior mesoderm may also have a role in anterior inductive patterning in Spiralia. Finally, we propose that a mode of axial development involving two organisers, one anterior and one posterior, is ancestral to the Bilateria, and that both organisers evolved from the single head organiser of a putative hydra-like ancestor.     

Non-cell-autonomous role for Cripto in axial midline formation during vertebrate embryogenesis

Several membrane-associated proteins are known to modulate the activity and range of potent morphogenetic signals during development. In particular, members of the EGF-CFC family encode glycosyl-phosphatidylinositol (GPI)-linked proteins that are essential for activity of the transforming growth factor beta (TGFbeta) ligand Nodal, a factor that plays a central role in establishing the vertebrate body plan. Genetic and biochemical studies have indicated that EGF-CFC proteins function as cell-autonomous co-receptors for Nodal; by contrast, cell culture data have suggested that the mammalian EGF-CFC protein Cripto can act as a secreted signaling factor. Here we show that Cripto acts non-cell-autonomously during axial mesendoderm formation in the mouse embryo and may possess intercellular signaling activity in vivo. Phenotypic analysis of hypomorphic mutants demonstrates that Cripto is essential for formation of the notochordal plate, prechordal mesoderm and foregut endoderm during gastrulation. Remarkably, Cripto null mutant cells readily contribute to these tissues in chimeras, indicating non-cell-autonomy. Consistent with these loss-of-function analyses, gain-of-function experiments in chick embryos show that exposure of node/head process mesoderm to soluble Cripto protein results in alterations in cell fates toward anterior mesendoderm, in a manner that is dependent on Nodal signaling. Taken together, our findings support a model in which Cripto can function in trans as an intercellular mediator of Nodal signaling activity.     

Interplay between Wnt2 and Wnt2bb controls multiple steps of early foregut-derived organ development

The vertebrate liver, pancreas and lung arise in close proximity from the multipotent foregut endoderm. Tissue-explant experiments uncovered instructive signals emanating from the neighbouring lateral plate mesoderm, directing the endoderm towards specific organ fates. This suggested that an intricate network of signals is required to control the specification and differentiation of each organ. Here, we show that sequential functions of Wnt2bb and Wnt2 control liver specification and proliferation in zebrafish. Their combined specific activities are essential for liver specification, as their loss of function causes liver agenesis. Conversely, excess wnt2bb or wnt2 induces ectopic liver tissue at the expense of pancreatic and anterior intestinal tissues, revealing the competence of intestinal endoderm to respond to hepatogenic signals. Epistasis experiments revealed that the receptor frizzled homolog 5 (fzd5) mediates part of the broader hepatic competence of the alimentary canal. fzd5 is required for early liver formation and interacts genetically with wnt2 as well as wnt2bb. In addition, lack of both ligands causes agenesis of the swim bladder, the structural homolog of the mammalian lung. Thus, tightly regulated spatiotemporal expression of wnt2bb, wnt2 and fzd5 is central to coordinating early liver, pancreas and swim bladder development from a multipotent foregut endoderm.     

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.     

Regional specification of the endoderm in the early chick embryo

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA , a posterior endoderm marker, but not cSox2 , an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA . At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2 , but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.     

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.     

The Effect of Adriamycin Exposure on the Notochord of Mouse Embryos

The notochord has important structural and signaling properties during vertebrate development with key roles in patterning surrounding tissues, including the foregut. The adriamycin mouse model is an established model of foregut anomalies where exposure of embryos in utero to the drug adriamycin leads to malformations including oesophageal atresia and tracheoesophageal fistula. In addition to foregut abnormalities, treatment also causes branching, displacement, and hypertrophy of the notochord. Here, we explore the hypothesis that the notochord may be a primary target of disruption leading to abnormal patterning of the foregut by examining notochord position and structure in early embryos following adriamycin exposure. Treated (n = 46) and control (n = 30) embryos were examined during the crucial period when the notochord normally delaminates away from the foregut endoderm (6–28 somite pairs). Transverse sections were derived from the anterior foregut and analyzed by confocal microscopy following immunodetection of extracellular matrix markers E‐cadherin and Laminin. In adriamycin‐treated embryos across all stages, the notochord was abnormally displaced ventrally with prolonged attachment to the foregut endoderm. While E‐cadherin was normally detected in the foregut endoderm with no expression in the notochord of control embryos, treated embryos up to 24 somites showed ectopic notochordal expression indicating a change in characteristics of the tissue; specifically an increase in intracellular adhesiveness, which may be instrumental in structural changes, affecting mechanical and signaling properties. This is consistent with disruption of the notochord leading to altered signaling to the foregut causing abnormal patterning and congenital foregut malformations.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.