The vertebrate skull is composed of bones derived from neural crest cells and mesoderm. The evolutionary capacity of the skull has been linked, in part, to the emergence of neural crest cells; however, this increased capacity for evolutionary change requires that variation within neural crest- and mesoderm-derived bones remains partly autonomous. One way to assess whether tissue origin leads to discrete patterns of variation is through measures of morphological integration and modularity. In this study, we use a neural crest-specific gap junction alpha-1 (Gja1) knockout mouse model (Cx43cKO) to determine the effect of tissue origin on skull integration and modularity. Micro-computed tomography images obtained from embryonic, newborn, and 2-month Cx43cKO and wildtype (Cx43WT) mice were used to measure and compare skull shape, size, integration, and modularity between genotypes. To determine if the phenotypic differences observed between genotypes reflect Cx43 function, mRNA expression data for markers of bone differentiation were measured from the neural crest-derived frontal bones and mesoderm-derived parietal and occipital bones. We found that patterns of integration and modularity change over development and these changes correspond with differences in Cx43 expression throughout the lifespan. Most interestingly, the patterns of developmental integration and modularity we observed at birth were influenced most greatly by tissue interactions, rather than Cx43 expression in the bones. Ultimately, our findings highlight the power of experimental models for investigating integration and modularity and the importance of tissue interactions in skull development.

Pannexin 1 (PANX1) is upregulated in many cancers, where its channel activity and signalling promote tumorigenic properties. Here, we report that potential internal translation start sites exist in mouse and human PANX1 which have implications in trafficking and protein interaction. Using mouse PANX1 constructs for each internal methionine (M) we saw that the shorter PANX1 isoforms were glycosylated, able to traffic to the cell surface and PANX1-M37 formed channels which could be activated by C-terminus cleavage or α1-adrenoceptor stimulation. Furthermore, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer, and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, and using Expasy PeptideCutter we did not find any evidence of hPANX1 cleavage sites which would produce a 25 kDa fragment, suggesting a potential alternative translation initiation site as the source of hPANX1-25K. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at multiple sites including the canonical N255 and novel N338 and N394 residues, and can interact with both β-catenin and full length hPANX1. Using cell surface biotinylation and immunocytochemistry, we also determined hPANX1-25K exhibits a predominantly intracellular localization. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and patient-matched normal skin, indicating that it may be differentially regulated in normal and cancer cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

Immunotherapies for malignant melanoma seek to boost the anti‐tumoral response of CD8 + T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel‐forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo . Here, we crossed Panx1 knockout ( Panx1 −/− ) mice with the inducible melanoma model Braf CA , Pten loxP , Tyr::CreER T2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten‐driven primary tumor formation or improve survival. However, tumors in BPC‐ Panx1 −/− mice exhibited a significant increase in the infiltration of CD8 + T lymphocytes, with no changes in the expression of early T‐cell activation marker CD69, lymphocyte activation gene 3 protein (LAG‐3) checkpoint receptor, or programmed cell death ligand‐1 (PD‐L1) in tumors when compared to the BPC‐ Panx1 +/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF / Pten ‐driven melanoma progression in vivo , it does increase the infiltration of effector immune T‐cell populations in the tumor microenvironment. We propose that PANX1‐targeted therapy could be explored as a strategy to increase tumor‐infiltrating lymphocytes to boost anti‐tumor immunity.

The channel-forming glycoprotein PANX3 functions in cutaneous wound healing and keratinocyte differentiation, but its role in maintaining skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3-knockout (KO) mice and found that KO dorsal skin showed sex differences at different ages but generally had reduced dermal and hypodermal areas compared with age-matched controls. Transcriptomic analysis of the KO epidermis revealed reduced E-cadherin stabilization and Wnt signaling compared with that of wild-type, consistent with the inability of primary KO keratinocytes to adhere in culture and diminished epidermal barrier function in KO mice. We also observed increased inflammatory signaling in the KO epidermis and a higher incidence of dermatitis in aged KO mice compared with that in wild-type controls. These findings suggest that during skin aging, PANX3 is critical in the maintenance of dorsal skin architecture, keratinocyte cell-cell and cell-matrix adhesion, and inflammatory skin responses.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

BACKGROUND:We previously determined a nonlinear relationship between connexin 43 (Cx43) function and craniofacial phenotypic variation in the mutant mouse model G60S/+, and that this variation was driven by nasal bone deviation. While nonlinearities in the genotype-phenotype map appear to be common, few studies have looked at the developmental processes that underlie this nonlinearity. Here, we investigated the potential tissue-level developmental determinants of the variation in nasal bone phenotype in G60S/+ mice through postnatal development.RESULTS:The deviated nasal bone phenotype arises by postnatal day 21 and becomes more severe by 3 months in G60S/+ mice. Measures of nasal bone remodeling including the number of osteoclasts, mineralizing surface, mineral apposition rate, and bone formation rate are significantly greater in G60S/+ mice compared to wild-type mice at 2 months, but these differences do not correspond with nasal bone deviation. The degree of nasal bone deviation does significantly and negatively correlate with the ratio between nasal bone and cartilaginous nasal septum length.CONCLUSIONS:Our findings indicate that the mean phenotypic changes observed between G60S/+ and wild-type mice are due to reduced bone growth, but the increased phenotypic variation found within mutant mice is due to discordant growth between nasal cartilage and bone.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Most features of the craniofacial complex that are necessary for feeding derive from cranial neural crest cells (cNCCs). These pluripotent stem cells originate from the dorsal neural folds and undergo a series of coordinated processes such as induction, epithelial‐to‐mesenchymal transition (EMT), migration, and differentiation. While most of these processes have been well‐studied for their roles in development, much remains to be known about the final step cNCCs must undergo, differentiation. Connexin‐43 (Cx43) is a gap junctional protein that is widely expressed, evolutionarily conserved, and has been well‐studied for its impacts on neural crest and bone. Previous research has shown that loss of Cx43 function impacts neural crest EMT and migration and can delay early osteoblast and chondrocyte differentiation; however, it is unknown how Cx43 deficiency in cNCCs impacts cNCC differentiation into osteochondrogenic lineages and the resultant impacts on craniofacial morphology. Therefore, the purpose of this project is to determine the effects of a neural crest‐specific loss of Cx43 on osteogenic and chondrogenic differentiation, and how these effects may alter skull phenotype.

Pannexin channel isoforms (Panx1‐3) are thought to release nucleotides into the extracellular milieu and have been shown to effect vascular hemodynamics. For this reason, we examined their mRNA and protein expression in hypertensive humans and genetically‐inbred hypertensive mice. In both mouse and humans, we found a significant reduction in Panx3 expression in resistance artery endothelium. Thus, we hypothesized Panx3 may be a regulator of vascular function. In en face endothelial preparations from 3rd order mesenteric arteries, we localized Panx3 to the Golgi Apparatus as opposed to Panx1 which localized to the plasma membrane. Next, we generated an inducible, endothelial cell Panx3 knockout mouse (Panx3ECKO). Radiotelemetry revealed a renin‐independent spontaneous hypertension, with unremarkable immune infiltration in the kidney. There was no change in cytoplasmic or released ATP. To understand how Panx3 may regulate blood pressure, we examined whether Panx3 interacted with B Cell Lymphoma 6 (BCL6), a potential binding partner. En face proximity ligation assays demonstrated an interaction between Panx3 and BCL6 in the Golgi. Panx3ECKO mice exhibited significantly decreased BCL6 protein, hinting that Panx3 may stabilize BCL6 by binding at the BCL6 ubiquitin sites. In silico “threading” of the Panx3 sequence onto the cryo‐EM structure of Panx1 confirmed this site of interaction. BCL6 is a NFκB repressor, thus its degradation in Panx3ECKO mice caused an increase in NFκB activity with IκBα and p100 significantly upregulated. A novel mimetic peptide designed to block Panx3‐BCL6 interactions was administered into C57Bl/6J mice, which recapitulated these results. In addition, Panx3ECKO mice had increased endothelial NOX4 (but not NOX1 or NOX2), likely due to increased NFκB activity—this correlated with a significant increase in plasma H2O2, nitrotyrosine (NT3), and 4‐hydroxynonenal (4‐HNE). In line with this observation, 3rd order mesenteric arteries from Panx3ECKO mice constricted (not dilated) to acetylcholine, which was rescued with the H2O2‐scavenger catalase (1000U/mL), suggesting that vascular oxidative stress drives hypertension in Panx3ECKO mice. Interestingly, Panx3ECKO mice also exhibit a significant increase in IL‐4 receptors on endothelium, and increased IL‐4 cytokines in bone marrow lysates. Because IL‐4 can drive BCL6 expression in other cell types, we suggest a possible homeostatic immune‐endothelial signaling axis. These data elucidate a novel Golgi‐localized oxidative signaling pathway in endothelium with a potential immune‐derived negative feedback loop.

BACKGROUND:Prenatal alcohol exposure (PAE) can result in developmental defects that include growth restriction, craniofacial anomalies, and cognitive behavioral deficits, though the presence and severity of these adverse outcomes can vary dramatically among exposed individuals. Preclinical animal models have demonstrated that the dose and timing of PAE account for much, but not all, of this phenotypic variation, suggesting that additional factors mitigate the effects of PAE. Here, we used a mouse model to investigate whether maternal age modulates the effects of PAE on the severity and variation in offspring growth and craniofacial outcomes.METHODS:Nulliparous C57BL/6N dams received either an intraperitoneal injection of ethanol (EtOH) or vehicle solution on gestational day 7.5. Dams were divided into four groups: (1) EtOH-treated young dams (6 to 10 weeks); (2) control young dams; (3) EtOH-treated old dams (6 to 7 months); and (4) old control dams. Neonate offspring growth restriction was measured through body mass and organ-to-body mass ratios, while skeletal craniofacial features were imaged using micro-CT and analyzed for size, shape, and variation.RESULTS:PAE and advanced maternal age each increased the risk of low birthweight and growth restriction in offspring, but these factors in combination changed the nature of the growth restriction. Similarly, both PAE and advanced maternal age individually caused changes to craniofacial morphology such as smaller skull size, dysmorphic skull shape, and greater skull shape variation and asymmetry. Interestingly, while the combination of PAE and advanced maternal age did not affect mean skull shape or size, it significantly increased the variation and asymmetry of those measures.CONCLUSION:Our results indicate that maternal age modulates the effects of PAE, but that the effects of this combination on offspring outcomes are more complex than simply scaling the effects of either factor.

Background We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively. Results Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower. Conclusions Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

BACKGROUND:Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD).METHODS:C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression.RESULTS:In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs.CONCLUSION:PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

Pannexin 3 (Panx3) expression is generally thought to be restricted to skin, cartilage and bone where it is routinely observed on intracellular compartments. However, multiple RNAseq studies have demonstrated consistent expression of Panx3 in endothelium throughout the vasculature. A physiological role for endothelial Panx3 has yet to be described. To assess the expression of Panx3 in intact endothelium, we first performed immunogold labelling on high pressure frozen third‐order mesenteric arteries. Panx3 preferentially localized to internal membranes in transverse sections of the endothelium. Next, we used multiple Panx3 antibodies raised against different topographical locations on third‐order mesenteric arteries prepared en face. We found that endothelial Panx3 was polarized downstream of blood flow, co‐localizing with a subset of calnexin‐positive perinuclear membranes, indicating association with the endoplasmic reticulum (ER). To test whether Panx3 was flow‐sensitive, primary cultures of human endothelial cells were seeded in parallel plate flow chambers and exposed to laminar shear stress (10 dynes/cm2 for 18 hours). Panx3 was polarized in the direction of flow similar to that seen in intact resistance arteries. To interrogate the physiological role of endothelial Panx3, we generated an inducible, cell‐specific Panx3 knockout mouse (Panx3fl/fl/Cdh5‐CreERT2+), which reduces both Panx3 mRNA and protein following tamoxifen injection. Panx3fl/fl/Cdh5‐CreERT2+ mice had normal blood cell parameters, heart function, body length, body weight, and fat content. Because we identified Panx3 in the ER perinuclear region, we began by assessing intracellular calcium on intact third‐order mesenteric arteries. Loss of endothelial Panx3 reduced intracellular calcium activity in endothelium under unstimulated conditions. Because calcium is vital to arterial dilation, we performed pressure myography. We found that resistance arteries from Panx3fl/fl/Cdh5‐CreERT2+ mice exhibit significantly enhanced vasoconstriction in response to phenylephrine, indicating impaired endothelial negative feedback on the smooth muscle cells. Due to the role of resistance arteries in blood pressure regulation, we hypothesized there may be a change in the blood pressure of these mice. We used radiotelemetry and found Panx3fl/fl/Cdh5‐CreERT2+ mice were hypertensive compared to tamoxifen‐injected littermate controls. The sum of this data suggests that endothelial Panx3 is polarized on the ER where it can regulate calcium activity in the endothelium, independent of ATP release, and may be important for vascular function.Support or Funding InformationHL149221 (AGW) and HL120840 (BEI)

Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood. Integrin-linked kinase (ILK) is a scaffold protein that regulates microtubule stability and F-actin dynamics. Here we show that ILK is necessary for normal trafficking of melanosomes along microtubule tracks. In the absence of ILK, immature melanosomes are not retained in perinuclear regions, and mature melanosome trafficking along microtubule tracks is impaired. These deficits can be attenuated by microtubule stabilization. Microtubules are also necessary for the formation of dendrites in melanocytes, and Ilk inactivation reduces melanocyte dendricity. Activation of glycogen synthase kinase-3 (GSK-3) interferes with microtubule assembly. Significantly, inhibition of GSK-3 activity or exogenous expression of the GSK-3 substrate collapsin response mediator protein 2 (CRMP2) in ILK-deficient melanocytes restored dendricity. ILK is also required for normal melanin transfer, and GSK-3 inhibition in melanocytes partially restored melanin transfer to neighboring keratinocytes. Thus, our work shows that ILK is a central modulator of melanosome movements in primary epidermal melanocytes and identifies ILK and GSK-3 as important modulators of melanin transfer to keratinocytes, a key process for epidermal UV photoprotection.

Oculodentodigital dysplasia (ODDD) is a disease caused by mutations in the GJA1 gene that encodes the gap-junctional protein connexin43 (Cx43). ODDD affects multiple organs, but craniofacial anomalies are typical. However, details on the timing of phenotypic presentation of these abnormalities and their correspondence with potential cellular changes are incomplete. Here, we perform the first assessment of the development of the ODDD craniofacial phenotype in the Cx43I130T/+ mouse model and show that the phenotypic features commonly found in patients with the disorder arise in mice between E17.5 and birth and become more profound with age. Using mice heterozygous for the I130T mutation of Gja1, we provide a detailed analysis of the craniofacial phenotype in this ODDD model using shape analyses based on micro-CT images. Results show that in addition to differences in facial bone morphology, there are significant shape differences in the cranial base. Mutant mice display delayed ossification at E17.5 and birth, particularly in bones of the face and cranial vault but ossification is normal at three months. Our immunohistochemical analyses of the palatine bone indicate that osteoblast differentiation is delayed in Cx43I130T/+ mice compared to their wildtype littermates, which likely contributes to the phenotypic variations observed in the facial bones. Our histological and immunohistochemical analyses of the synchondroses of the cranial base show no differences in molecular indicators of chondrocyte differentiation in mutant mice, suggesting that the differences to cranial base morphology displayed by Cx43I130T/+ mice are not due to differences in chondrocyte proliferation or differentiation. Together, our findings suggest that Cx43I130T/+ mice represent a surrogate model to not only inform about the craniofacial anomalies found in ODDD patients but also to show that reduced Cx43 function leads to phenotypic changes that are largely due to osteoblast defects.