Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.     

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.     

Expression of estrogen induced gene 121-like (EIG121L) during early Xenopus development

Estrogen induced gene 121 (EIG121) and EIG121-like (EIG121L) are evolutionarily conserved genes. But, their function is still unknown. Here, we report the expression pattern of Xenopus EIG121-like (xEIG121L) during early development. Its expression was first detected at stage 9 after mid-blastula transition, attained its maximal level at the gastrula stage, and remained constant until the tadpole stage. Whole-mount in situ hybridization revealed that xEIG121L was expressed strongly in the ventral ectoderm at the gastrula stage, and in the anterior ectoderm surrounding the neural plate at the neurula stage. xEIG121L expression was especially high in the presumptive hatching gland and cement gland regions in the neurula. At the tailbud stage, xEIG121L expression was limited to the hatching gland; an inverted Y type staining, characteristic of the hatching gland, was observed. However, at the tadpole stage, xEIG121L was expressed broadly in the head, heart and fin.     

Xenopus Tetraspanin-1 regulates gastrulation movements and neural differentiation in the early Xenopus embryo

The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.     

Ndst1, a heparan sulfate modification enzyme, regulates neuroectodermal patterning by enhancing Wnt signaling in Xenopus

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.     

Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo

We identified three novel genes that were expressed within the anterior non-neural ectoderm of Xenopus early neurula embryos. The expression of these genes was observed in the different areas complementary to the expression zone of a homeodomain gene Xanf-1 in the anterior neural plate. One of these genes, a Ras-like GTP-ase Ras-dva, marked the anterior placodal ectoderm area; a second, an Agr family homologous gene, XAgr2, was expressed in the anterior-most ectoderm in the cement gland primordium, and a third, novel gene Nlo was expressed in the lateral neural folds. The genes were transiently expressed in the developing cement and hatching gland primordia, and repressed in the mature cement and hatching glands. XAgr2 and Nlo were also expressed in the otic vesicles, and Ras-dva was expressed in the dorso-lateral column of the neural tube.     

Long- and short-range signals control the dynamic expression of an animal hemisphere-specific gene in Xenopus

Little is known of the control of gene expression in the animal hemisphere of the Xenopus embryo. Here we show that expression of FoxI1e, a gene essential for normal ectoderm formation, is expressed regionally within the animal hemisphere, in a highly dynamic fashion. In situ hybridization shows that FoxI1e is expressed in a wave-like fashion that is initiated on the dorsal side of the animal hemisphere, extends across to the ventral side by the mid-gastrula stage, and is then turned off in the dorsal ectoderm, the neural plate, at the neurula stage. It is confined to the inner layers of cells in the animal cap, and is expressed in a mosaic fashion throughout. We show that this dynamic pattern of expression is controlled by both short- and long-range signals. Notch signaling controls both the mosaic, and dorsal/ventral changes in expression, and is controlled, in turn, by Vg1 signaling from the vegetal mass. FoxI1e expression is also regulated by nodal signaling downstream of VegT. Canonical Wnt signaling contributes only to late changes in the FoxI1e expression pattern.These results provide new insights into the roles of vegetally localized mRNAs in controlling zygotic genes expressed in the animal hemisphere by long-range signaling. They also provide novel insights into the role of Notch signaling at the earliest stages of vertebrate 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.     

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.     

The genesis and transmission of epidermal potentials in an amphibian embryo

The genesis and transmission of action potentials in epidermal cells of the newt (Cynops pyrrhogaster) embryo were investigated with special reference to cellular differentiation during development. Typical action potentials can be recorded from any of the epidermal cells at Stage 31. These potentials consist of a fast spike (18 msec) followed by a slow component (164 msec). The potential is graded with current intensity, and only the slow component initiates action potentials in adjacent cells and induces a transmission to other cells. The fast spike was found in all epidermal cells throughout the embryonic stages examined (Stages 26–47). The slow potential, however, appears at Stage 28, persists until Stage $\text{36}\text{37}$ just before hatching and then disappears at Stage $\text{38}\text{42}$. Electrical recordings from traumatic embryos (embryos without neural crest cells) or from cultured epidermal cell masses isolated from the pregastrula or the ventral region of the neurula, were compared with the intact embryo. No differences were observed in either the form of the action potential or its transmission. Thus these action potentials appear to be derived from epidermal cells, and are not of nervous origin. Evidence suggests that the transient establishment of excitable membranes in epidermal cells during differentiation is closely related to neural cell differentiation.     

Involvement of an inner nuclear membrane protein, Nemp1, in Xenopus neural development through an interaction with the chromatin protein BAF

To clarify the molecular mechanisms of neural development in vertebrates, we analyzed a novel gene, termed nemp1 (nuclear envelope integral membrane protein 1), which is expressed in the Xenopus anterior neuroectoderm at the neurula stage. Nemp1 has a putative signal peptide and five transmembrane domains, but does not have any other known domains. We show that Nemp1 is localized to the inner nuclear membrane (INM) with its evolutionarily conserved C-terminal region facing the nucleoplasm. Both overexpression and knockdown of Nemp1 in Xenopus embryos reduced the expression of early eye marker genes, rax, tbx3, and pax6, and later resulted mainly in severe eye defects at the tailbud stage. In contrast, the expression of a forebrain/midbrain marker, otx2, and a pan-neural marker, sox2, was largely unaffected. Deletion analysis of Nemp1 showed that nuclear envelope-localization of the C-terminal region is necessary for its eye-reducing activity. Furthermore, nemp1 is coexpressed with baf (barrier-to-autointegration factor) in the eye anlagen, and that Nemp1 interacts with BAF through the BAF-binding site in the C-terminal region and this site is required for Nemp1 activity. These data suggest that Nemp1 is involved in the expression of eye marker genes by functioning at the INM at least partly through BAF.     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Xenopus radial spoke protein 3 gene is expressed in the multiciliated cells of epidermis and otic vesicles and sequentially in the nephrostomes

We describe the phylogenetic analysis and expression pattern of the Xenopus radial spoke protein 3 (RSP3) gene during early development. The Xenopus RSP3 protein presents characteristic features of the RSP3 family. It contains a radial spoke domain, which is 75 and 72 % identical to the corresponding region of human and Chlamydomonas RSP3 proteins, respectively. Examination of the phylogenetic relationship between the Xenopus RSP3 protein and its known homologues from different deuterostomes indicates that the RSP3 proteins are highly conserved among deuterostomes. Whole-mount in situ hybridization analyses show that Xenopus RSP3 is a maternal mRNA enriched in the animal hemisphere during cleavage stages. The expression is detected in the dorsal region of the embryo during gastrulation, then in the presumptive neuroectoderm at the end of gastrulation. During neurulation and at the subsequent stages, the expression of RSP3 mRNA is detected in the entire multiciliated cells of epidermis. At tail-bud stages, it is progressively expressed in the otic vesicles and sequentially expressed in the nephrostomes. Expression could be also detected in the floor plate of the neural tube. This expression pattern persists until at least late tail-bud stages.     

The space-time distribution of the mRNA of the nuclear proteins c-myc and P-53 in the development of the clawed toad studied by hybridization in situ]

We studied distribution of mRNA for nuclear protooncogene c-myc and nuclear protein P-53 in mature oocytes and embryos of Xenopus laevis from the stage of fertilization up to the stage of hatching by in situ hybridization with histological sections. mRNA for c-myc was present in all cells of the embryo at all studied developmental stages. Between the stage of fertilization and up to the late blastula, mRNA concentration for c-myc decreased progressively in all embryonic cells. During gastrulation a local increase in the concentration of this messenger was found in dorsal mesoderm and ectoderm. At the stage of neurula increased concentration of mRNA for c-myc was observed in all cells of the embryo but the hybridization signal increased particularly distinctly in cells of the neural tube. In studies of P-53 mRNA distribution hybridization signal was detected only in brain cells after stage 20 of development (after closure of the neural folds) and up to the stage of hatching.