Identification of neural transcription factors required for the differentiation of three neuronal subtypes in the sea urchin embryo

Abstract

Correct patterning of the nervous system is essential for an organism's survival and complex behavior. Embryologists have used the sea urchin as a model for decades, but our understanding of sea urchin nervous system patterning is incomplete. Previous histochemical studies identified multiple neurotransmitters in the pluteus larvae of several sea urchin species. However, little is known about how, where and when neural subtypes are differentially specified during development. Here, we examine the molecular mechanisms of neuronal subtype specification in 3 distinct neural subtypes in the Lytechinus variegatus larva. We show that these subtypes are specified through Delta/Notch signaling and identify a different transcription factor required for the development of each neural subtype. Our results show achaete-scute and neurogenin are proneural for the serotonergic neurons of the apical organ and cholinergic neurons of the ciliary band, respectively. We also show that orthopedia is not proneural but is necessary for the differentiation of the cholinergic/catecholaminergic postoral neurons. Interestingly, these transcription factors are used similarly during vertebrate neurogenesis. We believe this study is a starting point for building a neural gene regulatory network in the sea urchin and for finding conserved deuterostome neurogenic mechanisms.

Neurogenesis in the sea urchin embryo is initiated uniquely in three domains

“…1A), is characterized by specification of four to five serotonergic neurons. A number of neurons differentiate in the CB and in the post oral region just outside the CB (Garner et al, 2016;Slota and McClay, 2018) (Fig. 1A).…”

“…Neurogenin (Ngn), for example, is not expressed until mid-gastrula in the CB proneural progenitors ( Fig. S1; Slota and McClay, 2018). Thus, there is not a single time that proneural specification is initiated.…”

“…Lv_INSM was cloned using PCR primers (forward, 5′-ACGTCTGCA-ATATGCCTCGAAACTTTCT-3′; reverse, 5′-AGAGTAGGCATTTGTA-GGAGGATCACCTGT-3′) for a partial 1740 bp fragment. All other genes used in this study have been published (Angerer et al, 2000;Bradham et al, 2009;Croce et al, 2011;Rottinger et al, 2008;Slota and McClay, 2018;Sweet et al, 2002;Yuh et al, 2005).…”

“…The bilaterally symmetrical larva of the sea urchin develops a nervous system that begins functioning when the larva begins to feed, which in Lytechinus variegatus occurs between 24 and 48 hours post fertilization (hpf ). By that time, 40-50 neurons constitute the nervous system composed of about four serotonergic, 30-35 cholinergic and five to ten dopaminergic neurons (Garner et al, 2016;Hinman and Burke, 2018;Slota and McClay, 2018). These neurons differentiate in three locations in the larva.…”

“…Most of the cholinergic neurons are distributed within the ciliary band (CB), which is located between the dorsal and ventral ectoderm (Nakajima et al, 2004;Burke et al, 2006b). A population of neurons that are dopaminergic and cholinergic resides just outside the CB (Slota and McClay, 2018). Cholinergic and dopaminergic neurons also differentiate just outside the gut tube in the regions of the sphincters separating the three gut compartments (Wei et al, 2011;L.…”