A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo

Sonic hedgehog (Shh) is a key signal in the specification of ventral cell identities along the length of the developing vertebrate neural tube. In the presumptive hindbrain and spinal cord, dorsal development is largely Shh independent. By contrast, we show that Shh is required for cyclin D1 expression and the subsequent growth of both ventral and dorsal regions of the diencephalon and midbrain in early somite-stage mouse embryos. We propose that a Shh-dependent signaling relay regulates proliferation and survival of dorsal cell populations in the diencephalon and midbrain. We present evidence that Fgf15 shows Shh-dependent expression in the diencephalon and may participate in this interaction, at least in part, by regulating the ability of dorsal neural precursors to respond to dorsally secreted Wnt mitogens.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene

The processes that specify early regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The mouse open brain (opb) gene plays an essential role in dorsal neural patterning: in the caudal spinal cord of opb mutants, dorsal cell types are absent and markers of ventral fates, including Shh, expand into dorsal regions. Analysis of the opb mutant phenotype and of opb/opb <--> wild-type chimeric embryos reveals that early in neural development, the wild-type opb gene (opb(+)) is required cell autonomously for the expression of Pax7 in dorsal cells and Pax6 in lateral cells. Thus the opb(+) gene product acts intracellularly in the reception or interpretation of signals that determine cell types in the dorsal 80% of the neural tube. At later stages, the lack of opb(+) causes a non-cell-autonomous expansion of ventral cell types into dorsal regions of the neural tube, revealing that opb(+) controls the production of a diffusible molecule that defines the domain of Shh expression. The data indicate that opb(+) could act as either a novel component of a dorsalizing pathway or a novel intracellular negative regulator of the Shh signal transduction pathway.     

EphB receptor tyrosine kinases control morphological development of the ventral midbrain

EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compound homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants displayed a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which formed an abnormal protrusion into the cephalic flexure. The affected area was comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor caused a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells were unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. Our data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube.     

Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis

Genetic studies have established that heparan sulphate proteoglycans (HSPGs) are required for signalling by key developmental regulators, including Hedgehog, Wnt/Wg, FGF, and BMP/Dpp. Post-synthetic remodelling of heparan sulphate (HS) by Sulf1 has been shown to modulate these same signalling pathways. Sulf1 codes for an N-acetylglucosamine 6-O-endosulfatase, an enzyme that specifically removes the 6-O sulphate group from glucosamine in highly sulfated regions of HS chains. One striking aspect of Sulf1 expression in all vertebrates is its co-localisation with that of Sonic hedgehog in the floor plate of the neural tube. We show here that Sulf1 is required for normal specification of neural progenitors in the ventral neural tube, a process known to require a gradient of Shh activity. We use single-cell injection of mRNA coding for GFP-tagged Shh in early Xenopus embryos and find that Sulf1 restricts ligand diffusion. Moreover, we find that the endogenous distribution of Shh protein in Sulf1 knockdown embryos is altered, where a less steep ventral to dorsal gradient forms in the absence of Sulf1, resulting in more a diffuse distribution of Shh. These data point to an important role for Sulf1 in the ventral neural tube, and suggests a mechanism whereby Sulf1 activity shapes the Shh morphogen gradient by promoting ventral accumulation of high levels of Shh protein.     

Brain, eye, and face defects as a result of ectopic localization of Sonic hedgehog protein in the developing rostral neural tube.

BACKGROUND: Normal development of the face, eyes, and brain requires the coordinated expression of many genes. One gene that has been implicated in the development of each of these structures encodes the secreted protein, Sonic hedgehog (Shh). During central nervous system development, Shh is required for ventral specification along the entire neural axis. To further explore the role of Shh in chick brain and craniofacial development, we overexpressed Shh in the developing rostral neural tube METHODS: In order to determine if Shh is sufficient to ventralize the forebrain, we localized ectopically recombinant Shh protein to the rostral neural tube of chick embryos. The resulting embryos were evaluated morphologically and by assaying gene expression. RESULTS: Disruption in normal gene expression patterns was observed with a reduction or loss in expression of genes normally expressed in the dorsal forebrain (wnt-3a, wnt-4, and Pax-6) and expansion of ventrally expressed genes dorsally (HNF-3beta, Ptc). In addition to the genetic alterations observed in the neural tube, a craniofacial phenotype characterized by a reduction in many cranial neural crest-derived structures was observed. The eyes of Shh-treated embryos were also malformed. They were small with expansion of the retinal pigmented epithelium, enlarged optic stalks, and a reduction of neural retina. DISCUSSION: The ectopic localization of recombinant Shh protein in the rostral neural tube resulted in severe craniofacial anomalies and alterations of gene expression predicted by other studies. The system employed appears to be a model for studying the embryogenesis of malformations that involve the brain, eyes, and face.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.