Elevated NO signals correlated with altered cellular morphologies in ectodermal periderm cells suggestive of localized signaling in chicken embryo development

The constitutive nitric oxide synthases (cNOS) are calcium activated, membrane raft associated enzymes that make nitric oxide (NO), a short-lived signaling gas molecule with multiple cellular targets regulating many physiological processes. In chicken embryos, dynamic NO signals initiate in the outermost ectoderm layer, called periderm, and signal to underlying mesodermal tissues. The periderm has a pattern of elevated NO that we propose to form round/fusiform cells distinctive from its conventional, squamous barrier role. To investigate, 2.5 day-old chicken embryos were incubated with 25 uM MBC, a membrane raft disruptor blocking NO signaling, followed by SEM imaging. Control embryos, untreated with MBC, showed an abundance of altered periderm cells above the neural tube with high NO levels at the embryo dorsal midline. In contrast, MBC (>6 hours) caused reduced NO signaling in periderm cells with loss of the NO pattern. Instead, NO was elevated in newly made fusiform cells that were widely distributed in the ectoderm. MBC treatment overnight, reduced round/fusiform cells returning to a flattened morphology. Additionally, cytochalasin B (5 mg/mL) treatment of ectoderm for 2 hours caused periderm cells to round suggesting stabilization of the flattened cells by microfilaments. Lastly, ectoderm microinjections with diI and diO revealed fluorescent cells away from injection site because of active cellular movements. In conclusion, the periderm layer is under high cellular tension that activates cNOS, resulting in NO induced microfilament disassembly. We propose a novel signaling connection between rapid embryo growth, ectoderm stretch and concomitant activation of cNOS and NO signaling in development.