Development of a dopaminergic system in sea urchin embryos and larvae

Abstract

SUMMARYThe mechanisms that regulate the organized swimming movements of sea urchin blastulae are largely unknown. Using immunohistochemistry, we found that dopamine (DA) and the Hemicentrotus pulcherrimus homolog of the dopamine receptor D1 (Hp-DRD1) were strongly co-localized in 1-2m diameter granules (DA/DRD1 granules). Furthermore, these granules were arranged across the entire surface of blastulae as they developed locomotory cilia before hatching, and remained evident until metamorphosis. DA/DRD1 granules were associated with the basal bodies of cilia, and were densely packed in the ciliary band by the eight-arm pluteus stage. The transcription of Hp-DRD1 was detected from the unfertilized egg stage throughout the period of larval development. Treatment with S-(-)-carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, for 20-24h (i) from soon after insemination until the 20h post-fertilization (20hpf) early gastrula stage and (ii) from the 24hpf prism larva stage until the 48hpf pluteus stage, inhibited the formation of DA granules and decreased the swimming activity of blastulae and larvae in a dose-dependent manner. Exogenous DA rescued these deprivations. The formation of DRD1 granules was not affected. However, in 48hpf plutei, the serotonergic nervous system (5HT-NS) developed normally. Morpholino antisense oligonucleotides directed against Hp-DRD1 inhibited the formation of DRD1 granules and the swimming of larvae, but did not disturb the formation of DA granules. Thus, the formation of DRD1 granules and DA granules occurs chronologically closely but mechanically independently and the swimming of blastulae is regulated by the dopaminergic system. In plutei, the 5HT-NS closely surrounded the ciliary bands, suggesting the functional collaboration with the dopaminergic system in larvae. Supplementary material available online at

Development of the γ-amino butyric acid (GABA)-ergic signaling system and its role in larval swimming in sea urchin

“…Antisera against Hp-GabrA and Hp-Gabarap were raised in accordance with a method described previously (Katow, 2008) (Yaguchi and Katow, 2003;Katow et al, 2004;Hara and Katow, 2005;Katow 2008;Katow et al, 2010). The amino acid sequences were chosen on the basis of a Physico-Chemical Profiles analysis within Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_pcprof.html), and are specific to GABA A R protein of invertebrates and GABARAP of both vertebrates and invertebrates according to a BLAST search.…”

“…Embryos and larvae were fixed as described before in 4% paraformaldehyde diluted in FSW for 20min at ambient temperature (Katow et al, 2010) at the 6-7h.p.f. morula stage, 15h.p.f.…”

“…plutei using anti-DRD1 rabbit antibody (diluted 1:500 in PBST) (Katow et al, 2010) and the mouse anti-GabrA antiserum as described above.…”

“…For double-staining with two different primary antibodies, visualization was performed with Alexa Fluor 488-tagged antibody or Alexa Fluor 594-tagged antibody as described previously (Katow et al, 2010 …”

“…The swimming activity of sea urchin larvae depends strongly on the larval serotonergic nervous system (Katow et al, 2004;Katow et al, 2007) and dopaminergic regulatory system (Katow et al, 2010). These serotonergic and dopaminergic regulatory systems are regarded as excitatory (Varju et al, 2001;Li et al, 2006).…”