Role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation

Abstract

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.

Tissue linkage through adjoining basement membranes: The long and the short term of it

“…Examining these complex and transient tissue fusions is experimentally challenging, and thus very little is known about molecular aspects of the BM-BM interactions. JNK signaling promotes both primary mouth opening and wing disc eversion, however, the mechanisms regulating BM-BM associations and attachment are unknown [39–41],…”

Desmoplakin is required for epidermal integrity and morphogenesis in theXenopus laevisembryo

“…Histology was performed as described (Dickinson and Sive, 2006;Houssin et al, 2017) with some modifications. Briefly, embryos were fixed in 2% PFA and 2% glutaraldehyde in PBT buffer for 24 hr and then embedded in plastic resin (JB-4 Plus) and sectioned at 5-7 μm using a tungsten carbide knife.…”

Mouth development

“…These layers intercalate to form a one or two cell thick ‘buccopharyngeal membrane,’ which perforates to open the mouth (Figure (b), st. 40) . Hedgehog signaling regulates buccopharyngeal membrane perforation (st. 39) and recent, elegant data point to c‐Jun N‐terminal kinase (JNK) signaling as a key player in this process, promoting disassembly of adherens junctions via endocytosis . Perforation also requires adjacent NC that may provide tension to pull the mouth open .…”

“…Electron micrographs in Rana japonica, hamster, and chick show that cellular processes between ectoderm and endoderm germ layers increase the surface area to bring the germ layers immediately adjacent . In X. laevis endocytosis of E‐cadherin is required for membrane perforation . One difference among species is whether cell death preceeds perforation: this has been observed in X. laevis, Rana japonica, and mouse but not in zebrafish, chick, or hamster .…”

“…Consistent with this hypothesis, mouse buccopharyngeal membrane has almost no dividing cells, however, cell division in adjacent areas has not been quantified . Inhibition of cell proliferation in X. laevis did not affect mouth opening suggesting that differential cell proliferation is not required for perforation in frog . In addition to cell division, movement of surrounding tissues such as the forebrain or facial prominences may generate tension.…”