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Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer's vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon-/- embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry. [ABSTRACT FROM AUTHOR]

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer’s vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon−/− embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry.

Abstract Background The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging‐related muscle wasting. Motor neuron diseases or aging‐related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule‐downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin‐mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown. Methods Hb9‐Cre recombinase‐driven motor neuron‐specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed. Results Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging‐related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P

Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.

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. [Display omitted] • CDON contributes to proper digit specification, growth and patterning of the limb. • HH co-receptors are required for proximo-distal axis patterning of the limb. • HH co-receptors play partially redundant roles in HH-dependent digit specification. [ABSTRACT FROM AUTHOR]

Background: N6-methyladenosine (m6A) is the most common RNA modification, which plays a pivotal role in tumor development and progression. In this study, we assessed the role of m6A methyltransferase METTL3 in FRAS1-involved cell proliferation and colony formation of non-small cell lung cancer (NSCLC) cell lines.Methods: Cell viability was analyzed by Cell Counting Kit (CCK-8) and colony formation. M6A RNA immunoprecipitation (IP), Ribosomal immunoprecipitation, RNA immunoprecipitation (RIP) were performed to verify the relationship between METTL3, FRAS1 and YTHDF1. Rescue experiments to confirm the regulatory mechanism of METTL3-FRAS1 promoted NSCLC cell proliferation through CDON by cooperating YTHDF1.Results: We found that FRAS1 was correlated with poor prognosis in NSCLC patients, of which the transcript undergoes m6A modification regulated by METTL3. METTL3 silence reduced cell viability of NSCLC cells HCC827 and NCI-H1975, which could be restored by FRAS1 overexpression. The m6A modification of FRAS1 could be recognized by YTHDF1. FRAS1 silence or YTHDF1 silence could rescue the elevated NSCLC cell proliferation, colony formation, and tumor growth induced by METTL3 overexpression in vitro and in vivo.Conclusions: Our study reveals that METTL3-FRAS1 plays a crucial role in NSCLC cell proliferation, colony formation, and tumor growth through the regulation of CDON by cooperating YTHDF1. [ABSTRACT FROM AUTHOR]

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon , but not boc , is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer's vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon -/- embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Deng, Ran, Chang, Li, Li, Huang, Huang, Zhang, Li, Liu, Liang, Guo and Huang.)

Abstract 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.

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-offunction 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. [ABSTRACT FROM AUTHOR]

Ocular coloboma is caused by failure of optic fissure closure during development and recognized as part of the microphthalmia, anophthalmia, and coloboma (MAC) spectrum. While many genes are known to cause colobomatous microphthalmia, relatively few have been reported in coloboma with normal eye size. Genetic analysis including trio exome sequencing and Sanger sequencing was undertaken in a family with two siblings affected with bilateral coloboma of the iris, retina, and choroid. Pathogenic variants in MAC genes were excluded. Trio analysis identified compound heterozygous donor splice site variants in CDON, a cell‐surface receptor known to function in the Sonic Hedgehog pathway, c.928 + 1G > A and c.2650 + 1G > T, in both affected individuals. Heterozygous missense and truncating CDON variants are associated with dominant holoprosencephaly (HPE) with incomplete penetrance and Cdon−/− mice display variable HPE and coloboma. A homozygous nonsense allele of uncertain significance was recently identified in a consanguineous patient with coloboma and a second molecular diagnosis. We report the first compound heterozygous variants in CDON as a cause of isolated coloboma. CDON is the first HPE gene identified to cause recessive coloboma. Given the phenotypic overlap, further examination of HPE genes in coloboma is indicated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cases with multiple molecular diagnoses are challenging to diagnose clinically, yet may be resolved by unbiased exome sequencing analysis. We report an infant with developmental delay, severe growth delay, dysmorphic features, and multiple congenital anomalies including retinal coloboma, congenital pyloric stenosis, and circumferential skin creases. Exome sequencing identified a homozygous missense variant in MAPRE2 and a homozygous stopgain (nonsense) variant in CDON. Variants in MAPRE2, encoding a regulator of microtubule dynamics, lead to congenital symmetric circumferential skin creases type 2, with associated dysmorphism, small growth parameters, and congenital cardiac and genital anomalies. Monoallelic variants in CDON, encoding a coreceptor for sonic hedgehog, have been associated with autosomal dominant pituitary stalk interruption syndrome and holoprosencephaly. Cdon−/− mice have multiple eye defects including coloboma, consistent with the observed human phenotype. Thus, the complex phenotypic presentation of the infant may potentially be attributed to a dual molecular diagnosis. Furthermore, we present CDON as a candidate gene for coloboma formation in addition to the known holoprosencephaly phenotype, and propose to expand the allelic spectrum of CDON to variants associated with autosomal recessive inheritance in addition to dominant inheritance. [ABSTRACT FROM AUTHOR]

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.

Background: The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown., Methods: Hb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1 G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed., Results: Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 μm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment., Conclusions: Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration., (© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development. [ABSTRACT FROM AUTHOR]

On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon-/- hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon-/- hearts. In agreement with altered Cx43 expression, functional analysis both using Cdon-/- cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon-/- hearts exhibit hyperactive Wnt signaling as evident by ß-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/ß-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy. [ABSTRACT FROM AUTHOR]

During central nervous system development, oligodendrocyte progenitors elaborate multiple branched processes to contact axons and initiate myelination. Using cultured primary rat oligodendrocytes ( OLGs), we have recently demonstrated that a cell surface protein belonging to the immunoglobulin superfamily, cell adhesion molecule-related, down-regulated by oncogenes (Cdon), is important in initiating OLG differentiation and axon myelination by promoting the formation of branched cellular processes; however, the molecular mechanism by which Cdon regulates OLG differentiation is not known. Here, using Cdon immunoprecipitation ( IP) and liquid chromatography-tandem mass spectrometry analysis, we identified serine/threonine kinase TANK-binding kinase 1 ( TBK1) as a candidate novel target of Cdon. We confirmed this interaction using co- IP and immunofluorescence with TBK1 antibodies, showing that TBK1 partly co-localizes with Cdon along cellular processes in puncta-like structures. We show that TBK1 is expressed throughout OLG differentiation, and surprisingly, that levels of phosphorylated TBK1 (ser172) increase during OLG maturation, while total levels of TBK1 protein decrease. To investigate function, TBK1 expression was knocked down using si RNA in OLG primary cultures, reducing protein levels by 69%. Two myelin-specific proteins, myelin basic protein and myelin-associated glycoprotein, were similarly reduced when examined at day 2 and day 4 of OLG differentiation. Reduced Cdon or TBK1 expression also decreased Akt phosphorylation at Threonine 308 in OLG. Our findings provide evidence that a Cdon- TBK1 complex is associated with Akt phosphorylation and early OLG differentiation. [ABSTRACT FROM AUTHOR]

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations. [ABSTRACT FROM AUTHOR]

Steinfeld syndrome (MIM #184705) was first reported in 1982. It is characterised by holoprosencephaly and limb defects, however other anomalies may also be present. Following the initial description, three further cases have been reported in the literature. We report on a 23-year-old girl, with features of microform holoprosencephaly and bilateral congenital elbow dislocation in association with hypoplastic radial heads. She was identified to have a variant in the CDON gene inherited from her father who had ocular hypotelorism, but no other clinical features. We discuss the clinical features of Steinfeld syndrome, and broaden the phenotypic spectrum of this condition. Structural analysis suggests that this variant could lead to destabilisation of binding of CDON with hedgehog proteins. Further work needs to be done to confirm whether mutations in the CDON gene are the cause of Steinfeld syndrome. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Proper levels of Hedgehog (HH) signaling are essential during embryonic development and adult tissue homeostasis. A central mechanism to control HH pathway activity is through the regulation of secreted HH ligands at the plasma membrane. Recent studies have revealed a collective requirement for the cell surface co-receptors GAS1, CDON and BOC in HH signal transduction. Despite their requirement in HH pathway function, the mechanisms by which these proteins act to promote HH signaling remain poorly understood. Here we focus on the function of the two structurally related co-receptors, CDON and BOC. We utilized an in vivo gain-of-function approach in the developing chicken spinal cord to dissect the structural requirements for CDON and BOC function in HH signal transduction. Notably, we find that although CDON and BOC display functional redundancy during HH-dependent ventral neural patterning, these molecules utilize distinct molecular mechanisms to execute their HH-promoting effects. Specifically, we define distinct membrane attachment requirements for CDON and BOC function in HH signal transduction. Further, we identify novel and separate extracellular motifs in CDON and BOC that are required to promote HH signaling. Together, these data suggest that HH co-receptors employ distinct mechanisms to mediate HH pathway activity. [ABSTRACT FROM AUTHOR]

Nuclear Factor One (NFI) family of transcription factors regulate proliferation and multiple aspects of differentiation, playing analogous roles in embryonic development and various types of cancer. While all NFI family members are expressed in the developing brain and are involved in progression of brain cancers, their role in neuroblastoma has not been studied. Here we show that NFIB is required for the survival and proliferation of SH-SY5Y neuroblastoma cells, assessed by viability and colony formation assays. Cdon, an Ig superfamily member, is a SHH dependence receptor that acts as a tumor suppressor in neuroblastoma. In the absence of NFI, Cdon is upregulated in the developing mouse brain, however the mechanisms by which its transcription is regulated remains unknown. We report CDON as a downstream target of NFIs in SH-SY5Y cells. There are three putative NFI binding sites within the one kb CDON promoter, two of which are occupied by NFIs in SH-SY5Y cells and human neural stem cells. In dual-luciferase assays, Nfib directly represses CDON proximal promoter activity. Moreover, silencing NFIB leads to upregulation of CDON in SH-SY5Y cells, however, decreased cell proliferation in NFIB silenced cells could not be rescued by concomitantly silencing CDON , suggesting other molecular players are involved. For instance, p21, an NFI target in glioblastoma and breast cancer cells, is also upregulated upon NFIB knock-down. We propose that NFIB is indispensable for SH-SY5Y cells which may involve regulation of apoptosis inducer proteins CDON and p21. • CDON is a downstream target of NFIB in SH-SY5Y neuroblastoma cells. • NFIs occupy two of identified NFI binding sites on CDON promoter in SH-SY5Y. • NFIB directly represses CDON proximal promoter activity. • NFIB is required for the survival and proliferation of SH-SY5Y cells. [ABSTRACT FROM AUTHOR]

Objective: Screen the known craniosynostotic related gene, FGFR1 (exon 7), and two new identified potential candidates, CER1 and COON, in patients with syndromic and nonsyndromic metopic craniosynostosis to determine if they might be causative genes. Design: Using single-strand conformational polymorphisms (SSCPs), denaturing high-performance liquid chromatography, and/or direct sequencing, we analyzed a total of 81 patients for FGFR1 (exon 7), 70 for CER1, and 44 for COON. Patients: Patients were ascertained in the Centro de Estudos do Genoma Humano in São Paulo, Brazil (n = 39), the Craniofacial Unit, Oxford, U.K. (n = 23), and the Johns Hopkins University, Baltimore, Maryland (n = 31). Clinical inclusion criteria included a triangular head and/or forehead, with or without a metopic ridge, and a radiographic documentation of metopic synostosis. Both syndromic and nonsyndromic patients were studied. Results: No sequence alterations were found for FGFR1 (exon 7). Different patterns of SSCP migration for CER1 compatible with the segregation of single nucleotide polymorphisms reported in the region were identified. Seventeen sequence alterations were detected in the coding region of CDON, seven of which are new, but segregation analysis in parents and homology studies did not indicate a pathological role. Conclusions: FGFR1 (exon 7), CER1, and COON are not related to trigonocephaly in our sample and should not be considered as causative genes for metopic synostosis. Screening of FGFR1 (exon 7) for diagnostic purposes should not be performed in trigonocephalic patients. [ABSTRACT FROM AUTHOR]

Nuclear Factor One (NFI) family of transcription factors regulate proliferation and multiple aspects of differentiation, playing analogous roles in embryonic development and various types of cancer. While all NFI family members are expressed in the developing brain and are involved in progression of brain cancers, their role in neuroblastoma has not been studied. Here we show that NFIB is required for the survival and proliferation of SH-SY5Y neuroblastoma cells, assessed by viability and colony formation assays. Cdon, an Ig superfamily member, is a SHH dependence receptor that acts as a tumor suppressor in neuroblastoma. In the absence of NFI, Cdon is upregulated in the developing mouse brain, however the mechanisms by which its transcription is regulated remains unknown. We report CDON as a downstream target of NFIs in SH-SY5Y cells. There are three putative NFI binding sites within the one kb CDON promoter, two of which are occupied by NFIs in SH-SY5Y cells and human neural stem cells. In dual-luciferase assays, Nfib directly represses CDON proximal promoter activity. Moreover, silencing NFIB leads to upregulation of CDON in SH-SY5Y cells, however, decreased cell proliferation in NFIB silenced cells could not be rescued by concomitantly silencing CDON, suggesting other molecular players are involved. For instance, p21, an NFI target in glioblastoma and breast cancer cells, is also upregulated upon NFIB knock-down. We propose that NFIB is indispensable for SH-SY5Y cells which may involve regulation of apoptosis inducer proteins CDON and p21., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: Sonic Hedgehog (Shh) is a tightly regulated membrane-associated morphogen and a known driver of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). After processing, Shh remains at the plasma membrane of Shh producing cells, thereby limiting its distribution and signal strength. In PDAC, the release of Shh from tumor cells is necessary to promote a tumor-permissive microenvironment. Mechanisms regulating Shh sequestration and/or release from tumor cells to signal distant stromal cells are not well known. Previously, our laboratory demonstrated that the Drosophila transmembrane protein Boi, sequesters Hh at the membrane of Hh-producing cells. In response to dietary cholesterol or in the absence of boi, Hh is constitutively released to promote proliferation in distant cells. In this study, we investigated the conservation of this mechanism in mammals by exploring the role of the human boi homolog, CDON, in PDAC. Methods: Using PDAC cell-lines BxPC-3, Capan-2, and MIA PaCa-2, along with normal pancreatic epithelial cells (PDEC), we investigated Shh expression via Immunoblot and real-time, quantitative polymerase chain reaction in addition to Shh release via enzyme-linked immunoassay following cholesterol treatment and/or transfection with either RNA interference to reduce CDON expression or with human CDON to increase expression. Results: Consistent with our Boi model, CDON suppresses Shh release, which is alleviated in response to dietary cholesterol. However, over-expressing CDON suppresses cholesterol-mediated Shh release in some PDAC contexts, which may be relative to the mutational burden of the cells. Conclusion: Identifying mechanisms that either sequester or stimulate Shh release from the tumor cell membrane may provide new avenues to reduce signaling between the tumor and its surrounding environment, which may restrain tumor development., Competing Interests: No competing financial interests exist., (© Jennifer I. Alexander et al., 2021; Published by Mary Ann Liebert, Inc.)

The Hedgehog (HH) pathway controls multiple aspects of craniofacial development. HH ligands signal through the canonical receptor PTCH1, and three co-receptors: GAS1, CDON and BOC. Together, these co-receptors are required during embryogenesis to mediate proper HH signaling. Here, we investigated the individual and combined contributions of GAS1, CDON and BOC to HH-dependent mammalian craniofacial development. Notably, individual deletion of either Gas1 or Cdon results in variable holoprosencephaly phenotypes in mice, even on a congenic background. In contrast, we find that Boc deletion results in facial widening that correlates with increased HH target gene expression. In addition, Boc deletion in a Gas1 null background partially ameliorates the craniofacial defects observed in Gas1 single mutants; a phenotype that persists over developmental time, resulting in significant improvements to a subset of craniofacial structures. This contrasts with HH-dependent phenotypes in other tissues that significantly worsen following combined deletion of Gas1 and Boc Together, these data indicate that BOC acts as a multi-functional regulator of HH signaling during craniofacial development, alternately promoting or restraining HH pathway activity in a tissue-specific fashion., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon -/- hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon -/- hearts exhibit hyperactive Wnt signaling as evident by β-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/β-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.Cdon -/- cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon -/- hearts exhibit hyperactive Wnt signaling as evident by β-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/β-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.

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., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon(-/-) embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon(-/-) mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon(-/-) mice at either age. Moreover, adult Cdon(-/-) mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon(-/-) mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.

During central nervous system development, oligodendrocyte progenitors (OLPs) establish multiple branched processes and axonal contacts to initiate myelination. A complete understanding of the molecular signals implicated in cell surface interaction to initiate myelination/remyelination is currently lacking. The objective of our study was to assess whether Cdon, a cell surface protein that was shown to participate in muscle and neuron cell development, is involved in oligodendrocyte (OLG) differentiation and myelination. Here, we demonstrate that endogenous Cdon protein is expressed in OLPs, increasing in the early differentiation stages and decreasing in mature OLGs. Immunocytochemistry of endogenous Cdon showed localization on both OLG cell membranes and cellular processes exhibiting puncta- or varicosity-like structures. Cdon knockdown with siRNA decreased protein levels by 62% as well as two myelin-specific proteins, MBP and MAG. Conversely, overexpression of full-length rat Cdon increased myelin proteins in OLGs. The complexity of OLGs branching and contact point numbers with axons were also increased in Cdon overexpressing cells growing alone or in coculture with dorsal root ganglion neurons (DRGNs). Furthermore, myelination of DRGNs was decreased when OLPs were transfected with Cdon siRNA. Altogether, our results suggest that Cdon participates in OLG differentiation and myelination, most likely in the initial stages of development., (© 2016 Wiley Periodicals, Inc.)