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|a A netrin homolog of the sea urchin, Hemicentrotus pulcherrimus (HpNetrin) was characterized in terms of its expression behavior. The predicted amino acid sequence was an ortholog of hemichordate netrin-1. Reverse transcriptase-PCR, immunoblotting, and whole mount immunohistochemistry showed that gene transcription and protein expression occurred from 15-hour post-fertilization (hpf) swimming blastula stage to, at least 53-hpf pluteus stage. HpNetrin was detected on the entire basal surface of the ectoderm in swimming and 16-hpf mesenchyme blastulae. However, by 24-hpf prism stage, immunoreaction on the oral ectoderm decreased, whereas it increased in the aboral ectoderm including near the animal plate ectoderm (area-I). By 48-hpf pluteus stage, the HpNetrin-rich area-I comprised a 40mm wide dorsal midline belt (DMB) that stretched from the dorsal posterior edge of the animal plate to the posterior end of the larval body. Serotonergic cells were first detected in the HpNetrin-moderate area between the anterior DMB and the HpNetrin-poor oral ectoderm near the animal plate in 24-hpf prism larvae. By 48-hpf pluteus stage, these cells extended axons toward the middle-ridge to form a neural plexus of the apical ganglion. At 53-hpf pluteus stage, these axons extended further away from the apical ganglion directly or through the DMB toward the HpNetrin-poor contralateral ectoderm. The protein expression and axon extension pattern were reproduced in embryonic cell-aggregates formed from artificially dissociated 20-hpf gastrulae and resembled small blastula. In Hpnetrin morpholino anti-sense oligonucleotide-injected plutei, serotonergic axon extension was severely inhibited. Thus, HpNetrin functions as a serotonergic axon guidance cue in this basal deuterostome.
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