Select members of the Wnt family of secreted glycoproteins have been implicated in inducing the myogenic determinant genes Pax3, MyoD and Myf5 during mammalian embryogenesis(1,2), but the mechanism of induction has not been defined. We describe an unexpected role for protein kinase A (PKA) signalling via CREB in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer(3).

We report here a novel approach to direct gene expression in the mouse somite based on the combined application of adenovirus-mediated gene delivery and whole embryo ex vivo culture. As proof of principle, we show functional analysis of somites microinjected with an engineered virus expressing an activated form of Smoothened, the signaling receptor for Sonic Hedgehog (SHH). As adenovirus can infect many embryonic tissues in the mouse, this method may provide an effective alternative to conventional transgenesis for targeted spatial and temporal gene expression. genesis (c) 2005 Wiley-Liss, Inc.

SIM1 is a transcription factor essential for the developmental expression of the endocrine hormone genes, e. g. vasopressin (Vp) and oxytocin (Ot), in the paraventricular nucleus (PVN) and supraoptic nucleus ( SON) of the hypothalamus. Mice mutant for Sim1 lack structural PVN and SON, attributed in previous studies to the death of the PVN/SON progenitor cells. Here, we use a tau-LacZ knock-in allele at the Sim1 locus to trace Sim1 mutant cells and show that they are generated normally and survive to birth, contrasting to the previous proposal. Mutant cells adopt neuronal characteristics and maintain their PVN/SON identity as they continue to express PVN/SON progenitor markers. However, they occupy an ectopic position between the normal PVN and SON, indicating a defect in neuronal migration. To explore candidate molecular cues that contribute to PVN/SON neuronal migration, we focused on the Plexin family of genes. We found that PlexinA1 is expressed in regions surrounding the PVN and SON, whereas PlexinC1 is expressed within the PVN and SON. PlexinA1 expression becomes up-regulated in Sim1 mutant cells, whereas PlexinC1 expression is down-regulated. Finally, the PlexinC1 mutant has a selective defect in partitioning the VP and OT neurons coherently into the PVN and SON. Together, our results uncover a transcriptional regulation of neuronal migration cues initiated by Sim1 that contribute to the organization of the PVN and SON.

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.