Yukio Ohtsuka

The Ascidian Dihydropyridine-Resistant Calcium Channel as the Prototype of Chordate L-Type Calcium Channel

This review describes recent findings on voltage-gated Ca channel (Cav channel) cloned from ascidians, the most primitive chordates. Ascidian L-type like Cav channel has several unusual features: (1) it is closely related to the prototype of chordate L-type Cav channels by sequence alignment; (2) it is resistant to dihydropyridine due to single amino acid change in the pore region, and (3) maternally provided RNA putatively encodes a truncated protein which has remarkable suppressive effect on Cav channel expression during development. Ascidian Cav channel will provide a useful molecular clue in the future to understand Ca2+-regulated cell differentiation and physiology with the background of recently defined ascidian genome and molecular biological tools.

Nodal signaling regulates specification of ascidian peripheral neurons through control of the BMP signal

The neural crest and neurogenic placodes are thought to be a vertebrate innovation that gives rise to much of the peripheral nervous system (PNS). Despite their importance for understanding chordate evolution and vertebrate origins, little is known about the evolutionary origin of these structures. Here, we investigated the mechanisms underlying the development of ascidian trunk epidermal sensory neurons (ESNs), which are thought to function as mechanosensory neurons in the rostral-dorsal trunk epidermis. We found that trunk ESNs are derived from the anterior and lateral neural plate border, as is the case in the vertebrate PNS. Pharmacological experiments indicated that intermediate levels of bone morphogenetic protein (BMP) signal induce formation of ESNs from anterior ectodermal cells. Gene knockdown experiments demonstrated that HrBMPa (60A-subclass BMP) and HrBMPb (dpp-subclass BMP) act to induce trunk ESNs at the tailbud stage and that anterior trunk ESN specification requires Chordin-mediated antagonism of the BMP signal, but posterior trunk ESN specification does not. We also found that Nodal functions as a neural plate border inducer in ascidians. Nodal signaling regulates expression of HrBMPs and HrChordin in the lateral neural plate, and consequently specifies trunk ESNs. Collectively, these findings show that BMP signaling that is regulated spatiotemporally by Nodal signaling is required for trunk ESN specification, which clearly differs from the BMP gradient model proposed for vertebrate neural induction.

DHP-insensitive L-type-like Ca channel of ascidian acquires sensitivity to DHP with single amino acid change in domain III P-region

TuCa1, an ascidian homolog of L‐type Ca channel α1‐subunit, has many critical sites required for binding 1,4‐dihydropyridines (DHPs), but is insensitive to DHPs and methyl 2,5‐dimethyl‐4‐[2‐(phenylmethyl)benzoyl]‐1H‐pyrrole‐3‐carboxylate (FPL‐64176). We have substituted Ser for Ala1016 at the P‐region of domain III in TuCa1 (TuCa1/A1016S) and functionally expressed the channel in Xenopus oocyte along with rabbit α2/δ and β2b. TuCa1/A1016S has gained DHP sensitivity as high as that of a mammalian neuronal L‐type Ca channel (rbCII), but remained resistant to FPL‐64176. These results reinforce the view that Ser1016 in TuCa1/A1016S participates in DHP binding, but there exist other novel sites that fully acquire sensitivity to FPL‐64176.

Primary structure, functional characterization and developmental expression of the ascidian Kv4-class potassium channel

Ascidians belong to the primitive chordates and their larvae show symmetrical beating of the tail, which is reminiscent of the swimming pattern in primitive vertebrates. Since ascidian larva contains only a small number of neurons in their entire larval nervous system, they will potentially provide a simple model for the study of animal locomotion. In a step towards the goal of establishing the molecular basis underlying ascidian larval neurophysiology, we describe here a Kv4 class of voltage-gated potassium channel, TuKv4, from Halocynthia roretzi. Whole mount in situ hybridization indicates that TuKv4 is expressed in most of larval neurons including motor neurons. TuKv4-currents reconstituted in Xenopus oocytes show currents with similar properties to the lower-threshold A-type currents from cleavage-arrested ascidian blastomeres of neural lineage. However, the voltage-dependency of the steady-state inactivation and activation was shifted leftward by 20 mV, as compared with native A-type currents, suggesting that other components may be required to restore full function of the Kv4 channel. Unexpectedly, another isoform lacking C-terminal cytoplasmic region was also isolated. This truncated isoform did not lead to a functional current in Xenopus oocytes. RT-PCR analysis showed that the truncated form is transiently expressed during larval development, suggesting some developmental role for potassium channel expression.

Functional analysis of synaptotagmin gene regulatory regions in two distantly related ascidian species.

We have studied the structure and function of a promoter region of the Halocynthia synaptotagmin (Hr‐Syt) gene, which is abundantly expressed in neuronal cells. Our previous analysis suggested that the expression of Hr‐Syt is regulated by at least one epidermal and two neuronal regulatory regions. In this study, the regulatory regions of Hr‐Syt promoter were further characterized by using two species of ascidians, Halocynthia roretzi and Ciona intestinalis embryos. A putative GATA transcription factor binding site in the epidermal regulatory region has ectodermal enhancer activity in the Halocynthia embryo. Neuronal expression of Hr‐Syt was regulated by multiple redundant enhancer regions. Among these enhancer regions, a 200‐bp (–2900/–2700) region drove the reporter expression in neurons in both species of ascidian. Although the synaptotagmin promoter sequences did not show overall similarity between Hr‐Syt and Ciona synaptotagmin (Ci‐Syt), 5′‐upsteream two short sequences of Ci‐Syt have similarity to the –2766/–2732 region of the Hr‐Syt promoter. The homeodomain binding sites in this region are required for the neuronal enhancer activity. These results suggest that GATA and homeodomain transcription factors regulate the expression of synaptotagmin.