Xenopus Meis3 protein forms a hindbrain-inducing center by activating FGF/MAP kinase and PCP pathways

Knockdown studies in Xenopus demonstrated that the XMeis3 gene is required for proper hindbrain formation. An explant assay was developed to distinguish between autonomous and inductive activities of XMeis3 protein. Animal cap explants caudalized by XMeis3 were recombined with explants neuralized by the BMP dominant-negative receptor protein. XMeis3-expressing cells induced convergent extension cell elongations in juxtaposed neuralized explants. Elongated explants expressed hindbrain and primary neuron markers, and anterior neural marker expression was extinguished. Cell elongation was dependent on FGF/MAP-kinase and Wnt-PCP activities. XMeis3 activates FGF/MAP-kinase signaling, which then modulates the PCP pathway. In this manner, XMeis3 protein establishes a hindbrain-inducing center that determines anteroposterior patterning in the brain.     

Ras-mediated FGF signaling is required for the formation of posterior but not anterior neural tissue in Xenopus laevis

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, we have employed the dominant negative Ras mutant, N17Ras, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction.     

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.     

Expression of the Xhox3 Homeobox Protein in Xenopus Embryos: Blocking Its Early Function Suggests the Requirement of Xhox3 for Normal Posterior Development

Antibodies directed against the product of the Xenopus homeobox gene Xhox3 were raised and used to localize the expression of Xhox3 in the embryo at different stages of development. These studies suggest that endogenous Xhox3 protein is distributed in a graded fashion in the nuclei of mesodermal cells along the anterior-posterior (A-P) and dorso-ventral (D-V) axes in the postgastrula embryo with low levels in anterior and ventral regions and higher levels in posterior and dorsal regions. Xhox3 protein is also detected at different times in the midbrain, spinal cord and hindbrain. In the hindbrain, Xhox3 displays different metameric expression patterns in dorsal and ventral regions during early embryogenesis and metamorphosis. We have tested for the early function of Xhox3 by injecting antibodies against the Xhox3 protein into the cytoplasm of developing embryos. A significant number of embryos injected with Xhox3 antibodies show posterior (trunk and tail) deficiencies. This posterior deficient phenotype constitutes the opposite of the anterior (head) deficient phenotype obtained after overexpresson of Xhox3 reported previously. These results suggest that expression of Xhox3 in the posterior mesoderm is necessary for posterior development and that the graded distribution of Xhox3 in the embryonic mesoderm is required for the development of normal embryonic axial pattern.     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation.

We previously showed that otx2 regulates Xenopus cement gland formation in the ectoderm. Here, we show that otx2 is sufficient to direct anterior neural gene expression, and that its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning.     

Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development

In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.     

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

Xenopus XsalF: anterior neuroectodermal specification by attenuating cellular responsiveness to Wnt signaling

Here we show that XsalF, a frog homolog of the Drosophila homeotic selector spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3beta and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3beta and Tcf3 mimic those of XsalF while injections of GSK3beta and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3beta and Tcf3 expression.     

Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos.

Neural cell markers have been used to examine the effect of retinoic acid (RA) on the development of the central nervous system (CNS) of Xenopus embryos. RA treatment of neurula stage embryos resulted in a concentration-dependent perturbation of anterior CNS development leading to a reduction in the size of the forebrain, midbrain and hindbrain. In addition the overt segmental organization of the hindbrain was abolished by high concentrations of RA. The regional expression of two cell-specific markers, the homeobox protein Xhox3 and the neurotransmitter serotonin was also examined in embryos exposed to RA. Treatment with RA caused a concentration-dependent change in the pattern of expression of Xhox3 and serotonin and resulted in the ectopic appearance of immunoreactive neurons in anterior regions of the CNS, including the forebrain. Collectively, our results extend previous studies by showing that RA treatment of embryos at the neurula stage inhibits the development of anterior regions of the CNS while promoting the differentiation of more posterior cell types. The relevance of these findings to the possible role of endogenous retinoids in the determination of neural cell fate and axial patterning is discussed.     

A novel member of the Xenopus Zic family, Zic5, mediates neural crest development

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.     

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Misexpression of Polycomb-group proteins in Xenopus alters anterior neural development and represses neural target genes.

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, we have cloned a Xenopus homolog of Drosophila Polycomblike, XPcl1. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33, XBmi-1, and mPh2. These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons

XIHbox 6 is an early spatially restricted marker for molecular studies of neural induction. The sequence of the full-length XIHbox 6 protein is reported. An antibody raised against a beta-galactosidase/XIHbox 6 fusion protein was used to analyze the expression of XIHbox 6 proteins during frog embryogenesis. The anterior border of XIHbox 6 expression lies just posterior of the hindbrain/spinal cord junction. Immunostaining extends the entire length of the spinal cord. A much weaker transient expression with a similar anterior border is observed in mesoderm. Almost all nuclei in the newly closed spinal cord contain XIHbox 6. The number of positive nuclei decreases over the next stages of development, until in later embryos XIHbox 6 is restricted to nuclei of the dividing neuroepithelium, and not the mantle or marginal zones of the spinal cord. When the limb buds begin to grow, there is a second burst of XIHbox 6 expression in proliferating neurons of the cervical and lumbar enlargements, where nerves arise that supply the limbs. The data suggest that XIHbox 6 expression is spatially and temporally restricted to immature neurons of the spinal cord, before their differentiation into mature neurons.