Wnt Signaling Is Regulated by Endoplasmic Reticulum Retention

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts

Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli

AbstractSonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. Here, we show that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, we show that the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein

Conserved Role of unc-79 in Ethanol Responses in Lightweight Mutant Mice

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans

Bone morphogenetic protein signaling molecules in facial morphogenesis and dysmorphogenesis

Facial morphogenesis is a dynamic multi-step process involving the formation of the neural crest cell derived facial prominences, their coordinated outgrowth, fusion and differentiation to generate the final facial form. Defects in each of these processes are associated with facial dysmorphogenesis. The identification of genes that are involved in cleft lip is the first aim of this thesis. PCR based differential display was used to compare gene expression profiles in cleft and non-cleft chicken embryos. The second manner to identify candidate genes involved in orofacial clefting was by examining expression patterns of previously cloned genes at the specific time and place where lip fusion occurs. This approach identified Bone morphogenetic proteins (BMPs) as potential genes that are involved with the outgrowth and fusion of facial prominences. Little was known about where BMP receptors and BMP antagonists are expressed in the face or the function of endogenous BMPs in the face. The expression of BMP ligands (Bmp-2, Bmp-4 and Bmp- 7) was correlated with the expression of BMP receptors (BmpR-IA, BmpR-IB and BmpR-II) and noggin, a BMP antagonist, in order to predict where endogenous BMP signaling occurs in the face. Bmp-2, Bmp-4 and Bmp-7 are expressed in the zone of fusion between the frontonasal mass and maxillary prominences. Noggin expression is restricted to the frontonasal mass epithelium and is downregulated at the corners of the frontonasal mass just prior to fusion. Later in development, Bmps are expressed in the perichondrium of cartilage whereas noggin is expressed within differentiating cartilage. The BmpR-IA and BmpR-II are expressed ubiquitously whereas BmpR-IB is expressed in a subset of tissues. This expression data was used to design studies that examined BMP function during primary palate closure and facial cartilage morphogenesis. Noggin protein was applied to regions with high BMP expression in the zone of fusion and subsequently induced clefts. These experiments show for the first time that endogenous BMPs regulate two aspects of lip closure; outgrowth of facial prominences and thinning of the frontonasal epithelium prior to fusion. We also examined the well-established retinoic acid model for cleft lip to see whether BMPs were mediating this defect. Ectopic retinoic acid increases Bmp-2 and Bmp-4 expression prior to the induction of cleft lip in chickens. Mimicking this result by applying BMP-2 to the face also induced clefts. These results are related to non-syndromic human orofacial clefting and demonstrate the utility of studying cleft lip in non-mammalian model systems. Finally, experiments were carried out that would directly test the function of the BMP receptors in craniofacial development. Whereas expression of dominant-negative (dn) and constitutively active (ca) BMP type I receptors seldom affected early facial morphology, there were significant effects on chondrogenesis. These mutant forms of the type IA and IB receptors regulated the size and shape of cartilage elements. The dn form of the BMPRIB inhibited chondrogenesis, whereas ca-BMPRIA and ca-BMPRIB increased cartilage formation. The embryos injected with active viruses also lacked feather germs over much of the head and about 50% of specimens did not form an egg tooth. It appears that type IA and type IB receptors play similar roles in regulating cartilage and feather formation in the skull. Overall, these findings identify BMP signalling molecules as critical regulators of facial morphogenesis and dysmorphogenesis.Dentistry, Faculty ofGraduat

Cortical bone porosity in rabbit models of osteoporosis

Cortical bone porosity is intimately linked with remodeling, is of growing clinical interest, and is increasingly accessible by imaging. Thus, the potential of animal models of osteoporosis (OP) to provide a platform for studying how porosity develops and responds to interventions is tremendous. To date, rabbit models of OP have largely focused on trabecular microarchitecture or bone density; some such as ovariectomy (OVX) have uncertain efficacy and cortical porosity has not been extensively reported. Our primary objective was to characterize tibial cortical porosity in rabbit-based models of OP, including OVX, glucocorticoids (GC), and OVX + GC relative to controls (SHAM). We sought to: (i) test the hypothesis that intracortical remodeling is elevated in these models; (ii) contrast cortical remodeling and porosity in these models with that induced by parathyroid hormone (1–34; PTH); and (iii) contrast trabecular morphology in the proximal tibia across all groups. Evidence that an increase in cortical porosity occurred in all groups was observed, although this was the least robust for GC. Histomorphometric measures supported the hypothesis that remodeling rate was elevated in all groups and also revealed evidence of uncoupling of bone resorption and formation in the GC and OVX + GC groups. For trabecular bone, a pattern of loss was observed for OVX, GC, and OVX + GC groups, whereas the opposite was observed for PTH. Change in trabecular number best explained these patterns. Taken together, the findings indicated rabbit models provide a viable and varied platform for the study of OP and associated changes in cortical remodeling and porosity. Intriguingly, the evidence revealed differing effects on the cortical and trabecular envelopes for the PTH model.</p

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans