Establishment of a developmental atlas and transgenetic tools in the ascidian Styela clava

The ascidian Styela clava is an ecologically important species that is distributed along coastal regions worldwide.It has a long history as a model animal for evolutionary and developmental biology research owing to its phylogenetic position between vertebrates and invertebrates,and its classical mosaic expression patterns.However,the standard developmental atlas and protocols and tools for molecular manipulation of this organism are inadequate.In this study,we established a standard developmental table and provided a web-based digital image resource for S.clava embryogenesis at each developmental stage from fertilized eggs to hatching larvae by utilizing confocal laser microscopy and 3D reconstruction images.It takes around 10 h for fertilized eggs to develop into swimming larvae and 20–30 min to complete the tail regression processes at the metamorphic stage.We observed that the notochord cells in S.clava embryos did not produce an extracellular lumen like Ciona robusta,but showed polarized elongation behaviors,providing us an ideal comparative model to study tissue morphogenesis.In addition,we established a chemical-washing procedure to remove the chorion easily from the fertilized eggs.Based on the dechorionation technique,we further realized transgenic manipulation by electroporation and successfully applied tissue-specific fluorescent labeling in S.clava embryos.Our work provides a standard imaging atlas and powerful genetic tools for investigating embryogenesis and evolution using S.clava as a model organism.

Injury-induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori

Previous study showed that diapause in Bombyx mori eggs can be ter- minated by dechorionation and that activation in the mitogen-activated protein kinase （MAPK）/extracellular signal-regulated kinase （ERK） in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that me- chanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho-ERK levels and that injury increased the phospho-ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HC1. Effects of anaerobiosis on dechorionation-stimulated phospho-ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is in- volved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation-stimulated phospho-ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphory- lation of c-jun N-terminal kinases （JNKs） and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in .INK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho-ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury-induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs.