Background The buccopharyngeal membrane is a thin layer of cells covering the embryonic mouth. The perforation of this structure creates an opening connecting the external and the digestive tube which is essential for oral cavity formation. In humans, persistence of the buccopharyngeal membrane can lead to orofacial defects such as choanal atresia, oral synechiaes and cleft palate. Little is known about the causes of a persistent buccopharyngeal membrane and importantly how this structure ruptures. Results We have determined that Xenopus embryos deficient JNK signaling, using antisense and pharmacological approaches, have a persistent buccopharyngeal membrane. JNK deficient embryos have decreased cell division and increased cellular stress and apoptosis. However, altering these processes independently of JNK did not affect buccopharyngeal membrane perforation. JNK deficient embryos also have increased intercellular adhesion and defects in e-cadherin localization. Conversely, embryos with overactive JNK have epidermal fragility, increased E-cadherin internalization and increased membrane localized clathrin. In the buccopharyngeal membrane, clathrin is colocalized with active JNK. Further, inhibition of endocytosis results in a persistent buccopharyngeal membrane, mimicking the JNK deficient phenotype. Conclusions The results of this study suggest that JNK has a role in the disassembly adherens junctions via endocytosis that is required during buccopharyngeal membrane perforation.

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The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signaling, and lipid transfer. Using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometer proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization and mobility. These findings indicate that desmosomes and the keratin cytoskeleton pattern the distribution of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

Statement: Critical desmosomal protein, desmoplakin, is required for proper distribution and levels of cytoskeletal elements and e-cadherin. Thus embryos with decreased desmoplakin have defects in epidermal integrity and morphogenesis. AbstractDesmoplakin (Dsp) is a unique and critical desmosomal protein, however, it is unclear whether this protein and desmosomes themselves are required for epidermal morphogenesis. Using morpholinos or Crispr/Cas9 mutagenesis we decreased the function of Dsp in frog embryos to better understand its role during epidermal development. Dsp morphant and mutant embryos had developmental defects that mimicked what has been reported in mammals. Such defects included epidermal fragility which correlated with reduction in cortical keratin and junctional e-cadherin in the developing epidermis. Dsp protein sequence and expression are also highly similar with mammals and suggest shared function across vertebrates. Most importantly, we also uncovered a novel function for Dsp in the morphogenesis of the epidermis in X. laevis. Specifically, Dsp is required during the process of radial intercalation where basally located cells move into the outer epidermal layer. Once inserted these newly intercalated cells expand their apical surface and then they differentiate into specific epidermal cell types. Decreased levels of Dsp resulted in the failure of the radially intercalating cells to expand their apical surface, thereby reducing the number of differentiated multiciliated and secretory cells.Dsp is also required in the development of other ectodermally derived structures such as the mouth, eye and fin that utilize intercalating-like cell movements. We have developed a novel system, in the frog, to demonstrate for the first time that desmosomes not only protect against mechanical stress but are also critical for epidermal morphogenesis.

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