CDON contributes to Hedgehog-dependent patterning and growth of the developing limb.

Hedgehog (HH) signaling is a major driver of tissue patterning during embryonic development through the regulation of a multitude of cell behaviors including cell fate specification, proliferation, migration, and survival. HH ligands signal through the canonical receptor PTCH1 and three co-receptors, GAS1, CDON and BOC. While previous studies demonstrated an overlapping and collective requirement for these co-receptors in early HH-dependent processes, the early embryonic lethality of Gas1;Cdon;Boc mutants precluded an assessment of their collective contribution to later HH-dependent signaling events. Specifically, a collective role for these co-receptors during limb development has yet to be explored. Here, we investigate the combined contribution of these co-receptors to digit specification, limb patterning and long bone growth through limb-specific conditional deletion of Cdon in a Gas1;Boc null background. Combined deletion of Gas1, Cdon and Boc in the limb results in digit loss as well as defects in limb outgrowth and long bone patterning. Taken together, these data demonstrate that GAS1, CDON and BOC are collectively required for HH-dependent patterning and growth of the developing limb.

Δ9-Tetrahydrocannabinol inhibits Hedgehog-dependent patterning during development.

Many developmental disorders are thought to arise from an interaction between genetic and environmental risk factors. The Hedgehog (HH) signaling pathway regulates myriad developmental processes, and pathway inhibition is associated with birth defects, including holoprosencephaly (HPE). Cannabinoids are HH pathway inhibitors, but little is known of their effects on HH-dependent processes in mammalian embryos, and their mechanism of action is unclear. We report that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) induces two hallmark HH loss-of-function phenotypes (HPE and ventral neural tube patterning defects) in Cdon mutant mice, which have a subthreshold deficit in HH signaling. THC therefore acts as a 'conditional teratogen', dependent on a complementary but insufficient genetic insult. In vitro findings indicate that THC is a direct inhibitor of the essential HH signal transducer smoothened. The canonical THC receptor, cannabinoid receptor-type 1, is not required for THC to inhibit HH signaling. Cannabis consumption during pregnancy may contribute to a combination of risk factors underlying specific developmental disorders. These findings therefore have significant public health relevance.

Lamina-associated polypeptide 1 is dispensable for embryonic myogenesis but required for postnatal skeletal muscle growth.

Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that has been implicated in striated muscle maintenance. Mutations in its gene have been linked to muscular dystrophy and cardiomyopathy. As germline deletion of the gene encoding LAP1 is perinatal lethal, we explored its potential role in myogenic differentiation and development by generating a conditional knockout mouse in which the protein is depleted from muscle progenitors at embryonic day 8.5 (Myf5-Lap1CKO mice). Although cultured myoblasts lacking LAP1 demonstrated defective terminal differentiation and altered expression of muscle regulatory factors, embryonic myogenesis and formation of skeletal muscle occurred in both mice with a Lap1 germline deletion and Myf5-Lap1CKO mice. However, skeletal muscle fibres were hypotrophic and their nuclei were morphologically abnormal with a wider perinuclear space than normal myonuclei. Myf5-Lap1CKO mouse skeletal muscle contained fewer satellite cells than normal and these cells had evidence of reduced myogenic potential. Abnormalities in signalling pathways required for postnatal hypertrophic growth were also observed in skeletal muscles of these mice. Our results demonstrate that early embryonic depletion of LAP1 does not impair myogenesis but that it is necessary for postnatal skeletal muscle growth.

Modeling the complex etiology of holoprosencephaly in mice.

Holoprosencephaly (HPE) is a common developmental defect caused by failure to define the midline of the forebrain and/or midface. HPE is associated with heterozygous mutations in Nodal and Sonic hedgehog (SHH) pathway components, but clinical presentation is highly variable, and many mutation carriers are unaffected. It is therefore thought that such mutations interact with more common modifiers, genetic and/or environmental, to produce severe patterning defects. Modifiers are difficult to identify, as their effects are context-dependent and occur within the complex genetic and environmental landscapes that characterize human populations. This has made a full understanding of HPE etiology challenging. We discuss here the use of mice, a genetically tractable model sensitive to teratogens, as a system to address this challenge. Mice carrying mutations in human HPE genes often display wide variations in phenotypic penetrance and expressivity when placed on different genetic backgrounds, demonstrating the existence of silent HPE modifier genes. Studies with mouse lines carrying SHH pathway mutations on appropriate genetic backgrounds have led to identification of both genetic and environmental modifiers that synergize with the mutations to produce a spectrum of HPE phenotypes. These models favor a scenario in which multiple modifying influences-both genetic and environmental, sensitizing and protective-interact with bona fide HPE mutations to grade phenotypic outcomes. Despite the complex interplay of HPE risk factors, mouse models have helped establish some clear concepts in HPE etiology. A combination of mouse and human cohort studies should improve our understanding of this fascinating and medically important issue.

BOC is a modifier gene in holoprosencephaly.

Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, has a complex etiology. Heterozygous, loss-of-function mutations in the sonic hedgehog (SHH) pathway are associated with HPE. However, mutation carriers display highly variable clinical presentation, leading to an "autosomal dominant with modifier" model, in which the penetrance and expressivity of a predisposing mutation is graded by genetic or environmental modifiers. Such modifiers have not been identified. Boc encodes a SHH coreceptor and is a silent HPE modifier gene in mice. Here, we report the identification of missense BOC variants in HPE patients. Consistent with these alleles functioning as HPE modifiers, individual variant BOC proteins had either loss- or gain-of-function properties in cell-based SHH signaling assays. Therefore, in addition to heterozygous loss-of-function mutations in specific SHH pathway genes and an ill-defined environmental component, our findings identify a third variable in HPE: low-frequency modifier genes, BOC being the first identified.

Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice.

Holoprosencephaly (HPE) is a remarkably common congenital anomaly characterized by failure to define the midline of the forebrain and midface. HPE is associated with heterozygous mutations in Sonic hedgehog (SHH) pathway components, but clinical presentation is extremely variable and many mutation carriers are unaffected. It has been proposed that these observations are best explained by a multiple-hit model, in which the penetrance and expressivity of an HPE mutation is enhanced by a second mutation or the presence of cooperating, but otherwise silent, modifier genes. Non-genetic risk factors are also implicated in HPE, and gene-environment interactions may provide an alternative multiple-hit model to purely genetic multiple-hit models; however, there is little evidence for this contention. We report here a mouse model in which there is dramatic synergy between mutation of a bona fide HPE gene (Cdon, which encodes a SHH co-receptor) and a suspected HPE teratogen, ethanol. Loss of Cdon and in utero ethanol exposure in 129S6 mice give little or no phenotype individually, but together produce defects in early midline patterning, inhibition of SHH signaling in the developing forebrain, and a broad spectrum of HPE phenotypes. Our findings argue that ethanol is indeed a risk factor for HPE, but genetically predisposed individuals, such as those with SHH pathway mutations, may be particularly susceptible. Furthermore, gene-environment interactions are likely to be important in the multifactorial etiology of HPE.

Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development.

Id2 is a helix-loop-helix transcription factor essential for normal development, and its expression is dysregulated in many human neurological conditions. Although it is speculated that elevated Id2 levels contribute to the pathogenesis of these disorders, it is unknown whether dysregulated Id2 expression is sufficient to perturb normal brain development or function. Here, we show that mice with elevated Id2 expression during embryonic stages develop microcephaly, and that females in particular are prone to generalized tonic-clonic seizures. Analyses of Id2 transgenic brains indicate that Id2 activity is highly cell context specific: elevated Id2 expression in naive neural stem cells (NSCs) in early neuroepithelium induces apoptosis and loss of NSCs and intermediate progenitors. Activation of Id2 in maturing neuroepithelium results in less severe phenotypes and is accompanied by elevation of G1 cyclin expression and p53 target gene expression. In contrast, activation of Id2 in committed intermediate progenitors has no significant phenotype. Functional analysis with Id2-overexpressing and Id2-null NSCs shows that Id2 negatively regulates NSC self-renewal in vivo, in contrast to previous cell culture experiments. Deletion of p53 function from Id2-transgenic brains rescues apoptosis and results in increased incidence of brain tumors. Furthermore, Id2 overexpression normalizes the increased self-renewal of p53-null NSCs, suggesting that Id2 activates and modulates the p53 pathway in NSCs. Together, these data suggest that elevated Id2 expression in embryonic brains can cause deregulated NSC self-renewal, differentiation, and survival that manifest in multiple neurological outcomes in mature brains, including microcephaly, seizures, and brain tumors.

Neogenin regulates Sonic Hedgehog pathway activity during digit patterning.

BACKGROUND: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. RESULTS: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. CONCLUSIONS: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning.

Concepts in Multifactorial Etiology of Developmental Disorders: Gene-Gene and Gene-Environment Interactions in Holoprosencephaly.

Many common developmental disorders are thought to arise from a complex set of genetic and environmental risk factors. These factors interact with each other to affect the strength and duration of key developmental signaling pathways, thereby increasing the possibility that they fail to achieve the thresholds required for normal embryonic patterning. One such disorder, holoprosencephaly (HPE), serves as a useful model system in understanding various forms of multifactorial etiology. Genomic analysis of HPE cases, epidemiology, and mechanistic studies of animal models have illuminated multiple potential ways that risk factors interact to produce adverse developmental outcomes. Among these are: 1) interactions between driver and modifier genes; 2) oligogenic inheritance, wherein each parent provides predisposing variants in one or multiple distinct loci; 3) interactions between genetic susceptibilities and environmental risk factors that may be insufficient on their own; and 4) interactions of multiple genetic variants with multiple non-genetic risk factors. These studies combine to provide concepts that illuminate HPE and are also applicable to additional disorders with complex etiology, including neural tube defects, congenital heart defects, and oro-facial clefting.

Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly.

Holoprosencephaly (HPE), a defect in midline patterning of the forebrain and midface, arises ~1 in 250 conceptions. It is associated with predisposing mutations in the Nodal and Hedgehog (HH) pathways, with penetrance and expressivity graded by genetic and environmental modifiers, via poorly understood mechanisms. CDON is a multifunctional co-receptor, including for the HH pathway. In mice, Cdon mutation synergizes with fetal alcohol exposure, producing HPE phenotypes closely resembling those seen in humans. We report here that, unexpectedly, Nodal signaling is a major point of synergistic interaction between Cdon mutation and fetal alcohol. Window-of-sensitivity, genetic, and in vitro findings are consistent with a model whereby brief exposure of Cdon mutant embryos to ethanol during gastrulation transiently and partially inhibits Nodal pathway activity, with consequent effects on midline patterning. These results illuminate mechanisms of gene-environment interaction in a multifactorial model of a common birth defect.

A common birth defect known as holoprosencephaly affects how the brain and face of a fetus develop in the womb. In many cases, the condition is so severe that the fetus dies before, or shortly after, birth. Mutations in certain genes that control how the fetus develops are associated with holoprosencephaly. For example, mutations in components of the Hedgehog and Nodal signaling pathways, which transmit information that help cells to become specialized, increase the risk that a fetus will develop holoprosencephaly. Environmental factors, such as exposure to alcohol in the womb, are also thought to contribute to this condition. A gene known as Cdon is a component of the Hedgehog signaling pathway. In 2012, a team of researchers reported that mice with a mutation in the Cdon gene exposed to alcohol in the womb develop symptoms similar to holoprosencephaly in humans. Here, Hong et al. ­ including some of the researchers involved in the previous work ­ set out to understand how Cdon and alcohol work together to cause holoprosencephaly in the mutant mice. First, the team exposed pregnant mice to alcohol at different times during gestation to find out when their young were sensitive to developing holoprosencephaly. This showed that the young mice were most sensitive in early pregnancy when the Nodal pathway was active in their growing bodies. Further experiments found that alcohol and mutations in Cdon change Nodal signaling in cells. Together, these findings demonstrate that exposure to alcohol in the womb works together with the mutant form of Cdon via the Nodal signaling pathway, rather than the Hedgehog pathway, to cause holoprosencephaly in mice. The causes of many common birth defects are complex and difficult to distinguish at the level of individual cases. The work of Hong et al. illuminates how multiple risk factors during pregnancy, which may not create any problems on their own, may work together to produce birth defects in the fetus. The findings also offer new ways to understand how exposure to alcohol in the womb affects the fetus. Ultimately, understanding how birth defects form could lead to new strategies to prevent them in the future.

Effect of Body Mass Index on Postoperative Nausea and Vomiting: Propensity Analysis.

The impact of body mass index (BMI) on postoperative nausea and vomiting (PONV) is controversial, and few studies have focused on their relationship. We investigated the effects of BMI on PONV, taking into account other PONV risk factors. We analyzed adults over the age of 18 years who received general anesthesia between 2015 and 2019, using propensity score matching. Before propensity score matching, odds ratios (ORs) for PONV were lower for overweight (OR, 0.91; 95% confidence interval (CI), 0.87-0.96; p < 0.0001) or obese patients (OR, 0.77; 95% CI, 0.71-0.84; p < 0.0001) than for normal-BMI patients. After matching, the ORs for PONV of overweight (OR, 0.89; 95% CI, 0.80-0.98; p = 0.016) and obese patients (OR, 0.71; 95% CI, 0.63-0.79; p < 0.0001) were low. However, the ORs of underweight patients did not differ from those of normal-BMI patients, irrespective of matching. Therefore, the incidence of PONV may be lower among adults with a higher­than­normal BMI.

Satellite cell-specific ablation of Cdon impairs integrin activation, FGF signalling, and muscle regeneration.

BACKGROUND: Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. METHODS: We generated inducible satellite cell-specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2 . To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin-induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. RESULTS: Satellite cell-specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon-depleted myofibers exhibited 32 ± 9.6% of EdU-positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon-ablated myoblasts were positive for senescence-associated ß-galactosidase (SA-ß-gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post-injury Day 4 revealed alterations in genes related to mitogen-activated protein kinase signalling (P < 8.29 e-5 ) and extracellular matrix (P < 2.65 e-24 ). Consistent with this, Cdon-depleted tibialis anterior muscles had reduced phosphorylated extracellular signal-regulated kinase (p-ERK) protein levels and expression of ERK targets, such as Fos (0.23-fold) and Egr1 (0.31-fold), relative to mock-treated control muscles (P < 0.001). Cdon-depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin ß1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24-/- myofibers, showed decreased Cdon levels (Cdon-positive cells in Zmpste24-/- = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin ß1 activation (weak or mislocalized integrin ß1 in Zmpste24-/- = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). CONCLUSIONS: Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.

Late-onset megaconial myopathy in mice lacking group I Paks.

BACKGROUND: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase ß (Chk ß) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKß are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. METHODS: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase ß (Chk ß) were analyzed for Pak1/2 phosphorylation. RESULTS: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkß and are a model for human diseases associated with CHKß deficiency. Pak1/2 and Chkß activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. CONCLUSIONS: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKß deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

The anesthetic action of ethanol analyzed by genetics in Caenorhabditis elegans.

Acute exposure to ethanol causes paralysis at high concentrations in the nematode Caenorhabditis elegans. We set out to elucidate the mechanism of the anesthetic action of ethanol by genetic approaches. We identified nine mutations that conferred reduced sensitivity to ethanol after chemical, irradiation, or transposon insertion mutagenesis. Of these nine, we further characterized five mutations that defined four genes, jud-1-jud-4. Analysis of the phenotypes of the animals heterozygous for two unlinked genes revealed that jud-1 and jud-3 act synergistically in a gene dose-dependent manner. We cloned jud-4 and found that it encodes a protein with limited homology to human Homer proteins. jud-4 was expressed in the hypodermis and vulva muscles, suggesting that this gene acts in tissues directly exposed to the external environment. Characterization of the other mutations identified in this study will facilitate the elucidation of the molecular mechanism for the anesthetic action of ethanol.

Ethanol itself is a holoprosencephaly-inducing teratogen.

Ethanol is a teratogen, inducing a variety of structural defects in developing humans and animals that are exposed in utero. Mechanisms of ethanol teratogenicity in specific defects are not well understood. Oxidative metabolism of ethanol by alcohol dehydrogenase or cytochrome P450 2E1 has been implicated in some of ethanol's teratogenic effects, either via production of acetaldehyde or competitive inhibition of retinoic acid synthesis. Generalized oxidative stress in response to ethanol may also play a role in its teratogenicity. Among the developmental defects that ethanol has been implicated in is holoprosencephaly, a failure to define the midline of the forebrain and midface that is associated with a deficiency in Sonic hedgehog pathway function. Etiologically, holoprosencephaly is thought to arise from a complex combination of genetic and environmental factors. We have developed a gene-environment interaction model of holoprosencephaly in mice, in which mutation of the Sonic hedgehog coreceptor, Cdon, synergizes with transient in utero exposure to ethanol. This system was used to address whether oxidative metabolism is required for ethanol's teratogenic activity in holoprosencephaly. We report here that t-butyl alcohol, which is neither a substrate nor an inhibitor of alcohol dehydrogenases or Cyp2E1, is a potent inducer of holoprosencephaly in Cdon mutant mice. Additionally, antioxidant treatment did not prevent ethanol- or t-butyl alcohol-induced HPE in these mice. These findings are consistent with the conclusion that ethanol itself, rather than a consequence of its metabolism, is a holoprosencephaly-inducing teratogen.

Prenatal ethanol exposure in mice phenocopies Cdon mutation by impeding Shh function in the etiology of optic nerve hypoplasia.

Septo-optic dysplasia (SOD) is a congenital disorder characterized by optic nerve, pituitary and midline brain malformations. The clinical presentation of SOD is highly variable with a poorly understood etiology. The majority of SOD cases are sporadic, but in rare instances inherited mutations have been identified in a small number of transcription factors, some of which regulate the expression of Sonic hedgehog (Shh) during mouse forebrain development. SOD is also associated with young maternal age, suggesting that environmental factors, including alcohol consumption at early stages of pregnancy, might increase the risk of developing this condition. Here, we address the hypothesis that SOD is a multifactorial disorder stemming from interactions between mutations in Shh pathway genes and prenatal ethanol exposure. Mouse embryos with mutations in the Shh co-receptor, Cdon, were treated in utero with ethanol or saline at embryonic day 8 (E8.0) and evaluated for optic nerve hypoplasia (ONH), a prominent feature of SOD. We show that both Cdon-/- mutation and prenatal ethanol exposure independently cause ONH through a similar pathogenic mechanism that involves selective inhibition of Shh signaling in retinal progenitor cells, resulting in their premature cell-cycle arrest, precocious differentiation and failure to properly extend axons to the optic nerve. The ONH phenotype was not exacerbated in Cdon-/- embryos treated with ethanol, suggesting that an intact Shh signaling pathway is required for ethanol to exert its teratogenic effects. These results support a model whereby mutations in Cdon and prenatal ethanol exposure increase SOD risk through spatiotemporal perturbations in Shh signaling activity.

Gene-Environment Interactions and the Etiology of Birth Defects.

It is thought that most structural birth defects are caused by a complex combination of genetic and environmental factors that interact to interfere with morphogenetic processes. It is important not only to identify individual genetic and environmental risk factors for particular defects but also to identify which environmental factors interact specifically with which genetic variants that predispose to the same defect. Genomic and epidemiological studies are critical to this end. Development and analysis of model systems will also be essential for this goal, as well as for understanding the mechanisms that underlie specific gene-environment interactions.

Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans.

Membrane integrity is critical for cell survival, defects of which cause pathological symptoms such as metabolic diseases. In this study, we used ethanol sensitivity of the nematode Caenorhabditis elegans to identify genetic factors involved in membrane integrity. InC. elegans, acute exposure to a high concentration (7% v/v) of ethanol changes membrane permeability, as measured by propidium iodide staining, and causes paralysis. We used the timing of complete paralysis as an indicator for alteration of membrane integrity in our genetic screen, and identified ptr-6 as a gene that confers ethanol resistance when mutated. PTR-6 is a patched-related protein and contains a sterol sensing domain. Inhibition of two PTR-encoding genes,ptr-15 and ptr-23, and mboa-1, encoding an Acyl Co-A: cholesterol acyltransferase homolog, restored ethanol sensitivity of the ptr-6 mutant, suggesting that these ptr genes and mboa-1 are involved in the maintenance of membrane integrity and permeability. Our results suggest that C. elegans can be used as a model system to identify factors involved in metabolic diseases and to screen for therapeutic drugs.

Differential hypoxia response of hsp-16 genes in the nematode.

Small heat shock proteins are induced by various stresses. We here report the differential hypoxia responses of the hsp-16 genes in the nematode. The hsp-16.1 and hsp-16.2 genes in Caenorhabditis elegans responded to hypoxia, while hsp-16.41 and hsp-16.48, which share the promoter regions with hsp-16.1 and hsp-16.2, respectively, did not. For comparative genomic analysis, we identified ten hsp-16 genes in the nematode C.briggsae from the genome database. The comparison of the promoter sequences revealed a new conserved sequence block, CAC(A/T)CT, that was required for the orientation-dependent hypoxia response, but not for other stress responses such as heat or ethanol. We propose a working model for the orientation-dependent promoter usage between two genes sharing the promoter region. We also discuss a possible application of the hypoxia-inducible promoter for conditional gene expression.

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system.

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.