Il ruolo di efrina A.d nello sviluppo del sistema nervoso della larva di ascidia

Vertebrates, including humans, possess a highly complicated dorsal tubular central nervous system. Many studies have been conducted to investigate the mechanism of neural circuit formation in the brain of vertebrates. Due to its complexity, however, full understanding is still elusive. The closest surviving relatives of vertebrates are tunicates, including ascidians, which are also known as sea squirts, very simple organisms that share basic body plans, including the dorsal tubular central nervous system with vertebrates. Thus, they provide a superb model to investigate cellular and molecular mechanisms in chordate development. The central nervous system of the sea squirt larva consists of three regions from the front: the brain vesicle, motor ganglion, and nerve cord. The nerve cord, which corresponds to the spinal cord of vertebrates, mostly consists of ependymal cells, a type of glial cell. The axons of motor ganglion neurons extend along the ependymal cells of the nerve cord and control the tail movement. A recent study identified a set of genes that are predominantly or specifically expressed in the nerve cord ependymal cells of the sea squirt Ciona intestinalis type A. Among these genes, ephrinA.d, which codes for an extracellular signaling molecule ephrin, is specifically expressed in the middle to the posterior half of the lateral nerve cord but not in the anterior part. Ephrin-Eph (ephrin receptor) signaling is known for acting as repellent signals in axonal guidance during the nervous system development in vertebrates. Therefore, ephrinA.d may play an important role in restricting neuromuscular junction to the anterior tail region of the sea squirt larvae. However, their roles in axon guidance during Ciona’s development have not been elucidated. In this study, I attempted to experimentally verify the possibility that ephrinA.d is involved in controlling the axon guidance and investigate its roles in neural circuit formation. The cDNAs encoding ephrin and nervous system-specific gene promoters have been cloned and stored in the laboratory of developmental biology -Prof. Kusakabe’s lab- at Konan University. I prepared a plasmid DNA, pSP-gnrh2>ephrinA.d:mCherry, in which the DNA sequence encoding a fusion protein of ephrinA.d and a red fluorescence protein mCherry was connected to the upstream cis-regulatory region of the gnrh2 gene, which is expressed in both the anterior and posterior nerve cord. The plasmid DNA construct was transfected into Ciona embryos to ectopically express ephrinA.d in the anterior nerve cord. Transfected larvae were fixed and then subjected to immunofluorescence staining to visualize the localization of the ephrinA.d:mCherry fusion protein and axons of various neurons. The fluorescence signals were observed using conventional as well as confocal fluorescence microscopes. When ephrinA.d:mCherry was overexpressed in the anterior nerve cord, axon growth of the GABAergic/glycinergic interneurons located in the anterior nerve cord was observed to be abnormal, suggesting that ephrinA.d acts as a repellent signal for axon development of these neurons. Research is still ongoing to explore the effect of ephrinA.d on other types of neurons such as glutamatergic and cholinergic neurons, and to investigate the importance of the ephrinA.d signal for the generation of the rhythmic movements of the Ciona larval tail that is coordinated by these neurons.