Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator.

Holoprosencephaly (HPE) is a clinically heterogeneous developmental anomaly affecting the CNS and face, in which the embryonic forebrain fails to divide into distinct halves. Numerous genetic loci and environmental factors are implicated in HPE, but mutation in the sonic hedgehog (Shh) gene is an established cause in both humans and mice. As growth arrest-specific 1 (Gas1) encodes a membrane glycoprotein previously identified as a Shh antagonist in the somite, we analyzed the craniofacial. phenotype of mice harboring a targeted Gas1 deletion. Gas1(-/-) mice exhibited microform HPE, including midfacial hypoplasia, premaxillary incisor fusion, and cleft palate, in addition to severe ear defects; however, gross integrity of the forebrain remained intact. These defects were associated with partial loss of Shh signaling in cells at a distance from the source of transcription, suggesting that Gas1 can potentiate hedgehog signaling in the early face. Loss of a single Shh allele in a Gas1(-/-) background significantly exacerbated the midline craniofacial phenotype, providing genetic evidence that Sbb and Gasl interact. As human GAS1 maps to chromosome 9q21.3-q22, a region previously associated with nonsyndromic cleft palate and congenital deafness, our results establish GAS1 as a potential locus for several human craniofacial malformations.

Growth arrest specific gene 1 (Gas1) has long been regarded as a cell cycle inhibitor of the G(0) to S phase transition. How GAS1, a GPI-anchored plasma membrane protein, directs intracellular changes without an extracellular ligand or a transmembrane protein partner has been puzzling. A recent series of biochemical and molecular genetic studies assigned the mammalian Hedgehog (HH) growth factor to be a ligand for GAS1 in vitro and in vivo. HH has enjoyed considerable attention for its profound role in embryonic patterning as a classic morphogen, i.e. inducing various cell types in a concentration-dependent manner. GAS1 appears to help transform the HH concentration gradient into its morphogenic activity gradient by acting cooperatively with the HH receptor, the 12-transmembrane protein Patched 1 (PTC1). These findings provoke intriguing thoughts on how HH and GAS1 may coordinate cell proliferation and differentiation to create biological patterns. The role of HH extends to human genetic diseases, stem cell renewal, and cancer growth, and we consider the possibility of GAS1's involvement in these processes as well.

PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in postmitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1 alpha. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1 alpha. The N-terminal 115 residues of NDN are sufficient for interaction with the bHLH domains of ARNT2 or HIF1 alpha but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1 alpha dimers, respectively. (C) 2007 Elsevier Inc. All rights reserved.

The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) contain neuroendocrine cells that modulate pituitary secretion to maintain homeostasis. These two nuclei have a common developmental origin but they eventually form at locations distant from each other. Little is known about the molecular cues that direct the segregation of PVN and SON. As a means to identify potential factors, we have documented expression patterns of genes with known guidance roles in neural migration. Here, we focus on two groups of ligand/receptor families classified to mediate chemo-repulsion of neurons and their axons: the Slit/Robo and the Semaphorin/Plexin/Neuropilin families. Their dynamic expression patterns within and around the common PVN/SON progenitor as well as the mature PVN and SON may provide a framework for understanding the formation of these two important nuclei. (C) 2008 Elsevier B.V. All Fights reserved.

A robust and well-organized rhythm is a key feature of many neuronal networks, including those that regulate essential behaviors such as circadian rhythmogenesis, breathing, and locomotion. Here we show that excitatory V3-derived neurons are necessary for a robust and organized locomotor rhythm during walking. When V3-mediated neurotransmission is selectively blocked by the expression of the tetanus toxin light chain subunit (TeNT), the regularity and robustness of the locomotor rhythm is severely perturbed. A similar degeneration in the locomotor rhythm occurs when the excitability of V3-derived neurons is reduced acutely by ligand-induced activation of the allatostatin receptor. The V3-derived neurons additionally function to balance the locomotor output between both halves of the spinal cord, thereby ensuring a symmetrical pattern of locomotor activity during walking. We propose that the V3 neurons establish a regular and balanced motor rhythm by distributing excitatory drive between both halves of the spinal cord.

Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.

Holoprosencephaly (HPE) is a common birth defect predominantly affecting the forebrain and face and has been linked to mutations in the sonic hedgehog (SHH) gene. HPE is genetically heterogeneous, and clinical presentation represents a spectrum of phenotypes. We have previously shown that Gas1 encodes a cell-autonomous Hedgehog signaling enhancer. Combining cell surface binding, in vitro activity, and explant culture assays, we provide evidence that SHH contains a previously unknown unique binding surface for its interaction with GAS1 and that this surface is also important for maximal signaling activity. Within this surface, the Asn-115 residue of human SHH has been documented to associate with HPE when mutated to lysine (N115K). We provide evidence that HPE associated with this mutation can be mechanistically explained by a severely reduced binding of SHH to GAS1, and we predict a similar result if a mutation were to occur at Tyr-80. Our data should encourage future searches for mutations in GAS1 as possible modifiers contributing to the wide spectrum of HPE.