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.Holoprosencephaly (HPE), the most developmental common defect of the forebrain, is best explained by a “mutation with modifier” model. However, HPE modifier genes have not been identified. Here, we report HPE‐associated missense variants within the Hedgehog coreceptor BOC (arrows). Functional analyses of these variants, along with previous work in mouse models, are consistent with the conclusion that these variants act as phenotypic modifiers of a driver mutation or environmental insult.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138902/1/humu23286-sup-0001-text.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138902/2/humu23286_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138902/3/humu23286.pd

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 β-galactosidase (SA-β-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 β1 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 β1 activation (weak or mislocalized integrin β1 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. © 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders1

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

<em>Cdon</em> Mutation and Fetal Ethanol Exposure Synergize to Produce Midline Signaling Defects and Holoprosencephaly Spectrum Disorders in Mice

<div><p>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 (<em>Cdon</em>, which encodes a SHH co-receptor) and a suspected HPE teratogen, ethanol. Loss of <em>Cdon</em> 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.</p> </div

Rescue of Holoprosencephaly in Fetal Alcohol-Exposed <i>Cdon</i> Mutant Mice by Reduced Gene Dosage of <i>Ptch1</i>

<div><p>Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice. <i>Cdon</i> mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator <i>Ptch1</i> from <i>Cdon^−/−</i> embryos and compared their response to ethanol with that of <i>Cdon^−/−;Ptch1^+/+</i> embryos. <i>Ptch1</i> heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced <i>Ptch1</i> gene dosage was on penetrance, as those <i>Cdon^−/−;Ptch1^+/−</i> embryos that displayed HPE did not show major differences in phenotype from <i>Cdon^−/−;Ptch1^+/+</i> embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.</p></div

Microwave Sensor for Non-Destructive, Volume-Independent Liquid Characterization

In this letter, we propose a microwave sensor for nondestructive, volume-independent liquid characterization over a 50 mL volume range. The proposed sensor is based on a 1.08-GHz slot-coupled cylindrical dielectric resonator and liquid-positioning jigs that control the degree of coupling. To ensure a stable resonance over a 50 mL volume range, we introduce a new criterion in which the resonance frequency monotonically shifts with respect to both the volume and permittivity of the liquid under test (LUT). The sensor was validated with ethanol solutions in different concentrations and volumes. The sensor demonstrates a resolution of approximately 5% ethanol concentration regardless of LUT volumes and a sensitivity of 1.04 MHz/% averaged at 25 mL. © 2017 IEEE.11Nscopu

Frequency of HPE spectrum defects in ethanol-treated 129S6.<i>Cdon^−/−</i> mice at different stages of analysis.

*<p>p<0.05, when comparing ethanol-treated <i>Cdon^−/−</i> embryos with saline and ethanol-treated <i>Cdon^+/+</i> embryos and saline-treated <i>Cdon^−/−</i> embryos.</p>**<p>p>0.05, due to the relatively low n (4) for this analysis and the partial penetrance of the phenotype. However, in our experience, lobar HPE and defective palatogenesis are not observed in either <i>Cdon^+/+</i> or <i>Cdon^−/−</i> embryos on the 129S6 background <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002999#pgen.1002999-Zhang1" target="_blank">[27]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002999#pgen.1002999-Zhang2" target="_blank">[28]</a>.</p>1<p>Agnathia spectrum phenotypes include complete lack of mandible (1/14) and fused, shortened mandibular bone (2/14).</p

Defective expression of <i>Foxa2</i> and <i>Gsc</i> at the late streak stage of ethanol-treated <i>Cdon^−/−</i> embryos.

<p>Whole mount in situ hybridization analyses of <i>Foxa2</i> (A–D), <i>Gsc</i> (E–H) and <i>Cer1</i> (I–L) in embryos of the indicated genotype and treatment harvested at E7.25. <i>Foxa2</i> and <i>Gsc</i> expression was analyzed at the late streak stage and was decreased in EtOH-treated <i>Cdon</i> mutants (D and H, arrows). <i>Cer1</i> was analyzed at the early bud stage and was unaffected by loss of <i>Cdon</i> or EtOH treatment. (M) Whole mount in situ hybridization analysis of <i>Cdon</i> expression in a wild-type E7.25 embryo at the late streak stage. <i>Cdon</i> expression is observed in the ectoderm, mesoderm and allantoic bud. <i>Cdon</i> expression was also monitored via ß-galactosidase (ß-gal) activity derived from a <i>LacZ</i> reporter knocked into the <i>Cdon</i> locus in mutant E7.5 embryos (O) at the early bud stage; a wild-type embryo stained for ß-gal activity is shown as a negative control (N). <i>Cdon</i> expression is seen mainly in ectoderm, mesoderm and allantois. a, amniotic cavity; all, allantois; e, ectoderm; m, mesoderm; *, exocoelom. Scale bars, 200 µm.</p

Structures of ethanol and <i>t</i>-butyl alcohol.

<p>Structures of ethanol and <i>t</i>-butyl alcohol.</p

Defective expression of SHH pathway target genes in the ventral forebrains of ethanol-treated <i>Cdon^−/−</i> embryos.

<p>Whole mount in situ hybridization analyses of <i>Ptch1</i> (A–D), <i>Gli1</i> (E–H), <i>Fgf8</i> (I–L) and <i>Nkx2.1</i> (M–Q) expression in E10.0 (30–36 somites) embryos of the indicated genotype and treatment (A–L, lateral views; M–Q, ventral views). <i>Ptch1</i> and <i>Gli1</i> expression in the ventral telencephalon was decreased in EtOH-treated <i>Cdon</i> mutants (<i>D</i> and <i>H</i>, arrows). <i>Fgf8</i> expression in the commissural plate but not at the midbrain-hindbrain boundary, was diminished in EtOH-treated <i>Cdon</i> mutants (L, arrow and arrowhead, respectively). Expression of the ventral forebrain marker <i>Nkx2.1</i> was diminished by ethanol treatment of <i>Cdo</i> mutants (P and Q, arrows; P and Q show moderately and severely affected embryos, respectively). Scale bar, 250 µm.</p