Delta-Notch signaling induces hypochord development in zebrafish

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. We show that hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with no tail-expressing midline precursors and paraxial mesoderm, which expresses deltaC and deltaD. Loss-of-function experiments revealed that deltaC and deltaD were required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocked no tail expression and promoted her4 expression. We propose that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord.     

Endoderm patterning by the notochord: development of the hypochord in Xenopus

The patterning and differentiation of the vertebrate endoderm requires signaling from adjacent tissues. In this report, we demonstrate that signals from the notochord are critical for the development of the hypochord, which is a transient, endodermally derived structure that lies immediately ventral to the notochord in the amphibian and fish embryo. It appears likely that the hypochord is required for the formation of the dorsal aorta in these organisms. We show that removal of the notochord during early neurulation leads to the complete failure of hypochord development and to the elimination of expression of the hypochord marker, VEGF. Removal of the notochord during late neurulation, however, does not interfere with hypochord formation. These results suggest that signals arising in the notochord instruct cells in the underlying endoderm to take on a hypochord fate during early neural stages, and that the hypochord does not depend on further notochord signals for maintenance. In reciprocal experiments, when the endoderm receives excess notochord signaling, a significantly enlarged hypochord develops. Overall, these results demonstrate that, in addition to patterning neural and mesodermal tissues, the notochord plays an important role in patterning of the endoderm.     

T-Box genes and developmental decisions that cells make

During gastrulation in vertebrate embryos, three definitive germ layers (ectoderm, mesoderm, and endoderm) are formed by organized and coordinated cell movements. In zebrafish, further subdivision of the mesoderm gives rise to the axial, adaxial and paraxial mesoderm. The axial mesoderm contributes to the prechordal plate and notochord whereas the adaxial and paraxial cells give rise to slow and fast muscles, respectively (Devoto et al., 1996; Blagden et al., 1997; Currie and Ingham, 1998). An inductive interaction in which the notochord plays an essential role will also provide an input in forming other specialized types of tissue contributing to the axial structures: the floor plate located dorsally to the notochord in the ventral spinal cord and the hypochord located ventrally of the notochord and deriving probably from the endoerm. It is known that despite the difference in developmental roles (Str?hle et al., 1993; Krauss et al., 1993), the floor plate and hypochord co-express a number of common molecular markers (Jan et al., 1995; our unpublished results) that may illustrate a certain similarity of their origin. Their close proximity to the notochord determines specialized features of these structures that differ substantially from the rest of the neural tube and endoderm, correspondingly. Once formed under the influence of the notochordal signaling, the floor plate will acquire an ability, similar to the notochord, to express genes of the Hedgehog family and several other groups of genes and to induce specification of ventral cell types in the neural tube during later development (for review, see Korzh, 1998). The biology of the hypochord is much less understood. It seems that the hypochord develops slightly later than the floor plate. It may be required for proper positioning of the dorsal aorta as well as induction of some other endoderm derivatives.     

A revised model of Xenopus dorsal midline development: differential and separable requirements for Notch and Shh signaling

The development of the vertebrate dorsal midline (floor plate, notochord, and hypochord) has been an area of classical research and debate. Previous studies in vertebrates have led to contrasting models for the roles of Shh and Notch signaling in specification of the floor plate, by late inductive or early allocation mechanisms, respectively. Here, we show that Notch signaling plays an integral role in cell fate decisions in the dorsal midline of Xenopus laevis, similar to that observed in zebrafish and chick. Notch signaling promotes floor plate and hypochord fates over notochord, but has variable effects on Shh expression in the midline. In contrast to previous reports in frog, we find that Shh signaling is not required for floor plate vs. notochord decisions and plays a minor role in floor plate specification, where it acts in parallel to Notch signaling. As in zebrafish, Shh signaling is required for specification of the lateral floor plate in the frog. We also find that the medial floor plate in Xenopus comprises two distinct populations of cells, each dependent upon different signals for its specification. Using expression analysis of several midline markers, and dissection of functional relationships, we propose a revised allocation mechanism of dorsal midline specification in Xenopus. Our model is distinct from those proposed to date, and may serve as a guide for future studies in frog and other vertebrate organisms.     

A family of Xenopus BTB-Kelch repeat proteins related to ENC-1: new markers for early events in floorplate and placode development

We report the expression of a family of Xenopus genes encoding BTB-Kelch repeat proteins related to mouse ENC-1. Xenopus ENC-related-1 (Xencr-1) is expressed throughout the dorsal midline in dorsal endoderm, notochord, and deep neuroectoderm cells during early neurula stages. Later, Xencr-1 expression is downregulated in the notochord in an anterior-to-posterior progression but is maintained in the floorplate and dorsal endoderm. Xencr-3 is expressed strongly in several cranial placodes during the early neurula stages, placing it among the earliest molecular markers of placode precursor populations.     

Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio)

The hypochord of the zebrafish embryo (Danio rerio) emerges at the 9-somite stage as a single row of cells in the dorsomedial endoderm immediately ventral to the notochord. It is recognizable from the 2(nd) or 3(rd) somite and extends along the trunk to the same extent as the somites. A basal lamina surrounds the hypochord and its cells are slightly larger than the nearby endoderm cells. TEM studies have shown that the hypochord cells contain, in addition to mitochondria, well-developed rough endoplasmic reticula and Golgi networks, indicating synthetic activity. Once formed, the hypochord will stay in close association with the notochord, and this axial complex gradually moves dorsally, separating the hypochord from the endoderm as a one-cell-wide, rod-like structure that is bean-shaped in transverse section. This is the situation in the 15-somite embryo, at the level of the 4-5(th) somites. In the gap between the hypochord and the endoderm, angioblast cells aggregate and start to form the dorsal aorta, which becomes intimately associated with the hypochord. In the 17-somite embryo the aortic rudiment is established just ventral to the hypochord as a tube with a lumen. As development proceeds, the size of the hypochord decreases. In the pec fin embryo the hypochord is still recognizable in the posterior trunk, but has apparently vanished in anterior regions. The temporal correlation between the appearance of the hypochord and the formation of the dorsal aorta, coupled with the intimate relationship between these structures, suggest that the hypochord may play a role in the positioning of the dorsal aorta.     

Hypochord,an enigmatic embryonic structure: Study of the axolotl embryo

The hypochord of the axolotl embryo is first visible at an early tailbud stage, forming a rod-like structure, situated immediately under the notochord. A profusion of extracellular matrix fibrils is attached to the dorsolateral regions of the hypochord, linking it with the somites. A basal lamina develops around the hypochord, indicating an epithelial type of cell differentiation. Abundant rough endoplasmic reticula in the hypochord cells suggest lively synthetic activity. Prospective endoderm cells were vitally labeled with the lipophilic dye 1,1-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) at the gastrula stage. Cells labeled with the dye were later found in the hypochord as well as in the gut endoderm. This shows that the hypochord is of endodermal origin, contrary to recent suggestions that the hypochord is of mesodermal origin, but consistent with histological data. After about 8 days of existence, the hypochord disappears. Experimental results, using an apoptosis detection kit, indicate that the hypochord cells may disintegrate by a type of apoptotic cell death. The close association between the hypochord and developing dorsal aorta suggests that the hypochord could be involved in the positioning of the dorsal aorta, which forms under it. J Morphol 232:57–66, 1997. © 1997 Wiley-Liss, Inc.     

A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor

The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2-4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.     

Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.     

Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death

BACKGROUND: Sonic hedgehog (Shh) is well known for its role in patterning tissues, including structures of the head. Haploinsufficiency for SHH in humans results in holoprosencephaly, a syndrome characterized by facial and forebrain abnormalities. Shh null mice have cyclopia and loss of branchial arch structures. It is unclear, however, whether these phenotypes arise solely from the early function of Shh in patterning midline structures, or whether Shh plays other roles in head development. RESULTS: To address the role of Shh after floorplate induction, we inhibited Shh signaling by injecting hybridoma cells that secrete a function-blocking anti-Shh antibody into the chick cranial mesenchyme. The antibody subsequently bound to Shh in the floorplate, notochord, and the pharyngeal endoderm. Perturbation of Shh signaling at this stage resulted in a significant reduction in head size after 1 day, loss of branchial arch structures after 2 days, and embryos with smaller heads after 7 days. Cell death was significantly increased in the neural tube and neural crest after 1 day, and neural crest cell death was not secondary to the loss of neural tube cells. CONCLUSIONS: Reduction of Shh signaling after neural tube closure resulted in a transient decrease in neural tube cell proliferation and an extensive increase in cell death in the neural tube and neural crest, which in turn resulted in decreased head size. The phenotypes observed after reduction of Shh are similar to those observed after cranial neural crest ablation. Thus, our results demonstrate a role for Shh in coordinating the proliferation and survival of cells of the neural tube and cranial neural crest.     

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.     

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.     

The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling

Interactions between niche cells and stem cells are vital for proper control over stem cell self-renewal and differentiation. However, there are few tissues where the initial establishment of a niche has been studied. The Drosophila testis houses two stem cell populations, which each lie adjacent to somatic niche cells. Although these niche cells sustain spermatogenesis throughout life, it is not understood how their fate is established. Here, we show that Notch signaling is necessary to specify niche cell fate in the developing gonad. Surprisingly, our results indicate that adjacent endoderm is the source of the Notch-activating ligand Delta. We also find that niche cell specification occurs earlier than anticipated, well before the expression of extant markers for niche cell fate. This work further suggests that endoderm plays a dual role in germline development. The endoderm assists both in delivering germ cells to the somatic gonadal mesoderm, and in specifying the niche where these cells will subsequently develop as stem cells. Because in mammals primordial germ cells also track through endoderm on their way to the genital ridge, our work raises the possibility that conserved mechanisms are employed to regulate germline niche formation.     

Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues

The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.     

Early posterior/ventral fate specification in the vertebrate embryo

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.