Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation

Background

The notochord is a signaling center required for the patterning of the vertebrate embryic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood.

Methodology/Principal Findings

Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete.

Conclusions/Significance

Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.     

Notch destabilises maternal beta-catenin and restricts dorsal-anterior development in Xenopus

The blastula chordin- and noggin-expressing centre (BCNE) is the predecessor of the Spemann-Mangold's organiser and also contains the precursors of the brain. This signalling centre comprises animal-dorsal and marginal-dorsal cells and appears as a consequence of the nuclear accumulation of β-catenin on the dorsal side. Here, we propose a role for Notch that was not previously explored during early development in vertebrates. Notch initially destabilises β-catenin in a process that does not depend on its phosphorylation by GSK3. This is important to restrict the BCNE to its normal extent and to control the size of the brain.     

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.     

Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, β-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early β-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator β-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

A Bmp Reporter with Ultrasensitive Characteristics Reveals That High Bmp Signaling Is Not Required for Cortical Hem Fate

Insights into Bone morphogenetic protein (Bmp) functions during forebrain development have been limited by a lack of Bmp signaling readouts. Here we used a novel Bmp signaling reporter (“BRE-gal” mice) to study Bmp signaling in the dorsal telencephalon. At early stages, BRE-gal expression was restricted to the dorsal telencephalic midline. At later stages, strong BRE-gal expression occurred in neurons of the marginal zone and dentate gyrus. Comparisons to nuclear phospho-Smad1/5/8 (pSmad) and Msx1 indicated that BRE-gal expression occurred exclusively in neural cells with high-level Bmp signaling. BRE-gal responsiveness to Bmps was confirmed in reporter-negative cortical cells cultured with Bmp4, and both in vivo and in vitro, BRE-gal expression was switch-like, or ultrasensitive. In the early dorsal telencephalon, BRE-gal expression negatively correlated with the cortical selector gene Lhx2, indicating a BRE-gal expression border that coincides with the cortex-hem boundary. However, in Lhx2 null chimeras, neither BRE-gal nor nuclear pSmad increases were observed in ectopic hem cells. These findings establish BRE-gal as an ultrasensitive reporter of Bmp signaling in the dorsal telencephalon, imply that hem fate can be specified at different Bmp signaling intensities, and suggest that Lhx2 primarily regulates the responses to – rather than the intensity of – Bmp signaling in dorsal telencephalic cells.     

Induction of notochord by the organizer inXenopus

Summary One important step in understanding early development is to define the cell interactions involved in establishing tissue types. In amphibian embryos, one such interaction is the induction by the organizer region after the late blastula stage of lateral and ventral regions of the marginal zone (MZ) to form dorsal tissue types such as muscle. It is not known whether the organizer can also induce lateral MZ to form notochord after the late blastula stage. We find that this induction occurs under experimental conditions and plays a role in normalXenopus development. The ability to induce notochord is strongest at the center of the organizer along the dorsal midline and weaker at the lateral edges of the organizer. Organizer tissue along the dorsal midline, which would differentiate as notochord in normal development, can exhibit organizer functions such as the induction of the dorsolateral MZ to form notochord without later differentiating as notochord itself. Thus organizer activity can be dissociated from subsequent notochord formation.     

Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain

The division of the mammalian forebrain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic proteins (BMPs) have been implicated in dorsal midline development, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline development in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signaling plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression.     

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.     

Structural Requirements for PACSIN/Syndapin Operation during Zebrafish Embryonic Notochord Development

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.     

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.     

Zebrafish IGF Genes: Gene Duplication,Conservation and Divergence,and Novel Roles in Midline and Notochord Development

Insulin-like growth factors (IGFs) are key regulators of development, growth, and longevity. In most vertebrate species including humans, there is one IGF-1 gene and one IGF-2 gene. Here we report the identification and functional characterization of 4 distinct IGF genes (termed as igf-1a, -1b, -2a, and -2b) in zebrafish. These genes encode 4 structurally distinct and functional IGF peptides. IGF-1a and IGF-2a mRNAs were detected in multiple tissues in adult fish. IGF-1b mRNA was detected only in the gonad and IGF-2b mRNA only in the liver. Functional analysis showed that all 4 IGFs caused similar developmental defects but with different potencies. Many of these embryos had fully or partially duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. IGF-2a, the most potent IGF, was analyzed in depth. IGF-2a expression caused defects in the midline formation and expansion of the notochord but it did not alter the anterior neural patterning. These results not only provide new insights into the functional conservation and divergence of the multiple igf genes but also reveal a novel role of IGF signaling in midline formation and notochord development in a vertebrate model.     

Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, β-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early β-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator β-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.     

Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, β-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early β-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator β-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Delta-mediated specification of midline cell fates in zebrafish embryos

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.     

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.     

Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis

In humans, holoprosencephaly (HPE) is a common birth defect characterized by the absence of midline cells from brain, facial, and oral structures. To understand the pathoetiology of HPE, we investigated the involvement of mammalian prechordal plate (PrCP) cells in HPE pathogenesis and the requirement of the secreted protein sonic hedgehog (Shh) in PrCP development. We show using rat PrCP lesion experiments and DiI labeling that PrCP cells are essential for midline development of the forebrain, foregut endoderm, and ventral cranial mesoderm in mammals. We demonstrate that PrCP cells do not develop into ventral cranial mesoderm in Shh^−/− embryos. Using Shh^−/− and chimeric embryos we show that Shh signal is required for the maintenance of PrCP cells in a non-cell autonomous manner. In addition, the hedgehog (HH)-responding cells that normally appear during PrCP development to contribute to midline tissues, do not develop in the absence of Shh signaling. This suggests that Shh protein secreted from PrCP cells induces the differentiation of HH-responding cells into midline cells. In the present study, we show that the maintenance of a viable population of PrCP cells by Shh signal is an essential process in development of the midline of the brain and craniofacial structures. These findings provide new insight into the mechanism underlying HPE pathoetiology during dynamic brain and craniofacial morphogenesis.