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.

Keywords: Achaete-Scute; Neural progenitor; Neurogenesis; Neurogenin; Orthopedia; Sea urchin.

Figure 1. Overview of the L. variegatus embryonic nervous system

(A–C) Fluorescent whole mount in situ hybridization shows 3 different neural subtypes: serotonergic neurons express Lv-tph (A), postoral neurons express Lv-chat (B) and ciliary band neurons also express Lv-chat (C). (D) Schematic showing overview of neurons present in 48 hour pluteus larva of L. variegatus. (E) Double fluorescent in situ hybridization shows co-expression of Lv-chat with Lv-th in the postoral neurons in the maximum intensity Z projection. Area in square shown in panels as composite or single channel of a single confocal section. (F) Double fluorescent in situ (maximum intensity Z projection) shows the serotonergic neurons and the ciliary band cholinergic neurons are different cell types and do not co-express Lv-chat and Lv-tph. Area in rectangle shown in panels as composite or single channel of maximum intensity projection. Nuclei (blue) in fluorescent images stained with Hoechst. hr- hour post fertilization, PL-Pluteus, Oral-oral view, Aboral- aboral view. Scale bars: 50 um.

Figure 2. Delta-notch signaling regulates the correct number and location of neurons

(A–H) Whole mount in situ hybridization of delta expression. (A) At 10 hpf (hour post fertilization), delta expression is strictly endomesodermal. (B)At 12 hpf, cells in the oral ectoderm begin to express delta. (C) By 14 hpf expression extends to the apical organ. Delta-expressing cells are continually added to the ectoderm through 24 hpf and beyond. (I–P) Perturbations using a delta morpholino result in an increased number neurons belonging to all three neural subtypes. (I–J) Delta knockdown results in more serotonergic neurons in the apical organ, marked by Lv-tph expression (n=84, 76%). (K–N) Delta knockdown results in more postoral neurons marked by Lv-chat (n=78, 78%) and Lv-th (n=52, 69%). (O–P) Embryos injected with a delta MO have an increased number of ciliary band neurons at 48 hpf marked by Lv-chat (n= 75, 89%). ‘n’ represents the total number of embryos scored, and the percentage indicates the percent of embryos scored with the shown effect. See Fig. S2 for explanation of focal plane shown in (O). Scale bars: 50 um. Embryos cultured at 22° C. Endo- endomesoderm expression, Oral ecto- oral ectoderm, Ap. Organ- apical organ. Nuclei (blue) in fluorescent images stained with Hoechst.

Figure 3. Expression of three neurogenic transcription factors in L. variegatus

(A–D) Expression of Lv-achaete-scute begins at 14 hours post fertilization (hpf) in the apical organ and remains there through pluteus stage. (E–H) Expression of Lv-neurogenin begins in two bilaterally symmetric patches in the oral ectoderm at 18 hpf and then appears in the ciliary band beginning at prism stage. (G–H) Lv-ngn expression remains restricted to cells in the ciliary band in pluteus larva. (I–L) Expression of Lv-orthopedia begins in 2–4 cells in the post oral ectoderm at mid-gastrula stage and expression is found in the line of postoral neurons through pluteus larval stage. (M) Confocal maximum intensity projection shows expression of Lv-ngn does not overlap with Lv-chat expression in the oral ectoderm at late gastrula stage. Cells that express Lv-ngn are anterior to the position of the postoral neurons, marked here by Lv-chat expression. (N) Confocal maximum intensity projection shows at pluteus stage Lv-ngn and Lv-chat are expressed in different neural subtypes with no overlap of expression. (O) Confocal maximum intensity projection shows Lv-ngn expressing neural cells are not the serotonergic neurons of the apical organ because expression does not overlap with expression of Lv-tph. To confirm that the two genes are not expressed in the same cells, combined and split channels of the maximum intensity projection are provided (the region of the embryo shown in the right insets is highlighted by white box). Nuclei (blue) in fluorescent images stained with Hoechst. Scale bars: 50 um. MB-mesenchyme blastula, MG-mid gastrula, LG-late gastrula, PR-prism, PL-pluteus.

Figure 4. Perturbations show Lv-achaete-scute is proneural for serotonergic neurons

(A–D) Perturbations using an Lv-achaete-scute translation-blocking morpholino (MO) shows Lv-ac/sc is upstream of delta (n=197, 94%) and tph (n=130, 72%) in the apical organ which suggests that Lv-achaete-scute is proneural for the serotonergic neurons. A′ and B′ are oral view images the same embryo shown in either A or B. Arrowheads in B′ show delta expressing cells present in the oral ectoderm. Dotted line indicates apical organ. (E–F) Injection of full length achaete-scute RNA results in increased number of serotonergic neurons in the apical organ. ‘n’ represents the total number of embryos scored, and the percentage indicates the percent of embryos scored with the shown effect, μ equals the average number of tph-expressing cells per embryo. (G) A single Apotome section shows that Lv-ac/sc expressing cells in the apical organ also express delta at 14 hpf. (H) A single Apotome section shows that Lv-ac/sc expressing cells in the apical organ also express Lv-tph, confirming Lv-ac/sc is expressed in the serotonergic neurons of the apical organ. Arrowhead shows an example of adjacent cells where one cell expresses Lv-ac/sc and the other expresses Lv-tph. To confirm that the two genes are expressed in the same cells in (G) and (H), combined and split channels of a single Apotome section are provided (the region of the embryo shown in the right insets is highlighted by white box). Scale bars: 50 um. Nuclei (blue) in fluorescent images stained with Hoechst.

Figure 5. Perturbations show Lv-neurogenin is proneural for ciliary band neurons

(A–H) Perturbations using two different Ngn MOs show Lv-ngn is upstream of delta (MO1: n=83, 86% downregulated; MO2: n=60, 82% downregulated) and Lv-chat expression in the ciliary band at 48 hrs (MO1: n=67, 75% downregulated; MO2: n=107, 87% downregulated), which suggests that Lv-ngn is proneural for neurons in the ciliary band. See Fig. S2 for explanation of focal planes shown in (C–D, G–H). ‘n’ represents the total number of embryos scored, and the percentage indicates the percent of embryos scored with the shown effect. Arrowheads in (D) and (H) show the Lv-chat-expressing postoral neurons are still specified in Ngn knockdown embryos. (I–J) Fluorescent whole mount double in situ hybridization shows that Lv-ngn co-expresses with delta. (I) A single confocal section shows that Lv-ngn expressing cells in the ectoderm also express delta at a time before the Lv-ngn cells are in the ciliary band. (J) A maximum intensity projection on the left panel shows that later in development, once Lv-ngn cells are in the ciliary band, they continue to express Lv-delta. (K) Shows Lv-Ngn cells in the ciliary band express Lv-chat at 40 hpf. To confirm that the two genes are expressed in the same cells, combined and split channels of a single confocal section are provided (the region of the embryo shown in the right insets is highlighted by white box). Scale bars: 50 um. Nuclei (blue) in fluorescent images stained with Hoechst.

Figure 6. Perturbations show Lv-orthopedia is necessary for differentiation of postoral neurons

(A–B, G–H) Perturbations using two different Otp MOs show Lv-otp is not upstream of delta expression in the postoral neuroblasts (MO1: n=131, 96% unchanged; MO2: n=101, 91% unchanged) or in other neural cells of the embryo. Arrowheads show examples of postoral neural cells that express delta. (C–F, I–L) Lv-otp is upstream of Lv-chat (MO1: n=196, 66% downregulated; MO2: n=201, 75% downregulated) and Lv-th (MO1: n=104, 76% downregulated; MO2: n=31, 97% downregulated) in the oral ectoderm. ‘n’ represents the total number of embryos scored, and the percentage indicates the percent of embryos scored with the shown effect. These data suggest that Orthopedia is required for the differentiation of the dopaminergic/cholinergic postoral neurons in L. variegatus, acting downstream of delta. (M) A single confocal section shows that Lv-otp expressing cells in the oral ectoderm also express delta. (N) A maximum intensity projection on the left panel shows that later in development, the Lv-otp expressing cells in the oral ectoderm will become the postoral neurons, which express Lv-chat. To confirm that the two genes are expressed in the same cells, combined and split channels of a single confocal section are provided (the region of the embryo shown in the right insets is highlighted by white box). Scale bars: 50 um. Nuclei (blue) in fluorescent images stained with Hoechst.

Figure 7. Gene regulatory network subcircuits operating in each neural subtype

(A–C) Schematics show the effect of transcription factor perturbations on the L. variegatus nervous system. Biotapestry models show the gene regulatory network subcircuit each gene is acting in (Longabaugh et al., 2005). (A) Knockdown and overexpression of Lv-ac/sc results in less or more serotonergic neurons in the L. variegatus apical organ, respectively. (B) Knockdown of Lv-ngn results in a loss of ciliary band cholinergic neurons (C) Knockdown of Lv-otp results in loss of cholinergic/catecholaminergic postoral neurons. (D) Phylogenetic tree showing relative positions of vertebrates, echinoderms, and protostome groups. Shapes represent the different transcription factors; colors represent function found in each clade. Data from Platynereis, vertebrates, and sea urchins suggest shared functions of Achaete-Scute and Neurogenin. We propose the GRN subcircuit containing Orthopedia is conserved with vertebrates and is likely ancient to the deuterostome lineage, however further analyses need to be carried out with Orthopedia orthologues in protosome groups to determine if the subcircuit evolved earlier (blue triangle). Data taken from (Simionato et al., 2008) (Lu, et al., 2012) (Stolfi et al., 2015)(Vervoort and Ledent, 2001)(Huang et al., 2014)(Bertrand et al., 2002)(Ghysen and Dambly-Chaudière, 1988)(Skeath and Carroll, 1994)(Ma et al., 1998)(Ma et al., 1999)(Ma et al., 1996)(Sommer et al., 1996)(Roybon et al., 2010)(Bush et al., 1996)(Yuan et al., 2016)(Ryu et al., 2007)(Fernandes et al., 2013)(Mummery-widmer et al., 2009).