Ontogeny of a synaptophysin-mediated GABA transmission mechanism from the ciliary band-associated strand to the ciliary band during the development of the sea urchin Hemicentrotus pulcherrimus

Abstract

Swimming activity of the sea urchin larva depends on ciliary beating primarily in the circumoral ciliary band (CB) and is regulated by several neurotransmitters, such as 5HT, dopamine and γ-aminobutyric acid (GABA). Accordingly, the larval swimming activity is severely inhibited by 3-mercaptopropionic acid [a glutamate decarboxylase (GAD) inhibitor]. Although GABA is detected in the CB, GAD is absent. GAD is expressed in the spatially segregated nearby ciliary band-associated strand (CBAS). Thus, it is assumed that GABA transmission extends from the CBAS to the CB. Here, we examined the synaptic transmission mechanism by focusing on the spatiotemporal expression pattern of synaptophysin (Syp), a synaptic vesicle glycoprotein. The sea urchin has a single copy of the Syp gene, which encodes a 266-amino acid protein with possibly 4 transmembrane domains. We generated an anti-Syp antibody (Ab).Immunoblotting (IB) detected Ab binding to a single band at approximately 38 kDa.Whole-mount immunohistochemistry (WMIHC) detected high intensity Ab binding to the CBAS. Syp was initially detected at the mesenchyme blastula stage (mBL) as a single band by IB. Accordingly, WMIHC detected Syp in the cytoplasm of small patches of several ectodermal cells at the mBL stage. Syp has also been detected in the cytoplasm of blastocoelar cells from the prism stage to the 2-arm pluteus stage (2aPL). By the 4aPL stage, Syp was expressed in the CBAS and was moderately expressed among the blastocoelar cells. Distinctive co-localization of GABA and Syp was not detected until the 2aPL stage. Beginning at the 4aPL stage, GABA was detected in the CB and Syp-positive puncta in the CBAS. In the CB, GABA was co-localized with the GABA-A receptor (GABA A R). Thus, the GABA signal may be transmitted from GAD in the CBAS through a Syp-mediated system to the CB and then, in the CB, to the basal body of the cilia through GABA A R.

Involvement of Huntingtin in Development and Ciliary Beating Regulation of Larvae of the Sea Urchin, Hemicentrotus pulcherrimus

“…At the apical tuft region near the serotonergic apical ganglion, on the other hand, long cilia do not beat as actively as the shorter cilia in the other regions [ 20 ]. However, we did not detect Hp-Htt at or around the basal body of the cilia of the ciliary band; rather, Hp-Htt was located in the CBAS, which contains several signaling proteins, such as encephalopsin [ 19 , 24 ], synaptophysin [ 19 , 22 , 25 ], and 5HThpr [ 19 , 32 ]. Hp-Htt is also located in stomach wall cells ( Figure 3 B inset, H).…”

“…Huntingtin is a large single molecule of around 348 kDa (HD-Human; P42858; [ 15 , 16 , 21 , 22 , 23 ]) to 350 kDa [ 24 , 25 , 26 , 27 , 28 ] in humans, and around 339 kDa in the sea urchin Strongylocentrotus purpuratus [Sp-Hunt (Sp-Htt); XP_030841199.1, XP_030848870.1]. The present immunoblotting of H. plucherrimus (Hp-Htt) detected a single band of around 350 kDa from the swimming blastula stage to the pL stage, indicating that the size is very close to those of above HD-human and Sp-Htt, but larger than that of Ciona intestinalis [ 15 ].…”

“…CBAS formation starts from the 2aPL stage, accompanied by the blastocoel-to-larval body surface egression of the GAD-expressing blastocoelar cells between the oral ectoderm and the ciliary band [ 18 , 22 , 23 , 24 , 25 ]. On the other hand, the other GAD/Hp-Htt-expressing cells left in the blastocoel form the large blastocoelar networks ( Figure 3 E–G, Figure 5 C,G).…”

“…These neurotransmitter mechanisms, such as the GABAA receptor for GABA transmission, involve Huntingtin [ 23 , 45 ]. In sea urchin larvae, since GABA [ 19 , 22 , 23 ] and GABAA receptor are present in the ciliary band [ 23 ] and GAD is present in the CBAS of sea urchin larva [ 19 ], the histological Hp-Htt location may indicate the presence of its regulatory function in ciliary beating through the neurotransmission process.…”