Developmental origin of peripheral ciliary band neurons in the sea urchin embryo

Abstract

In the sea urchin larva, most neurons lie within an ectodermal region called the ciliary band. Our understanding of the mechanisms of specification and patterning of these peripheral ciliary band neurons is incomplete. Here, we first examine the gene regulatory landscape from which this population of neural progenitors arise in the neuroectoderm. We show that ciliary band neural progenitors first appear in a bilaterally symmetric pattern on the lateral edges of chordin expression in the neuroectoderm. Later in development, these progenitors appear in a salt-and-pepper pattern in the ciliary band where they express soxC, and prox, which are markers of neural specification, and begin to express synaptotagminB, a marker of differentiated neurons. We show that the ciliary band expresses the acid sensing ion channel gene asicl, which suggests that ciliary band neurons control the larva's ability to discern touch sensitivity. Using a chemical inhibitor of MAPK signaling, we show that this signaling pathway is required for proper specification and patterning of ciliary band neurons. Using live imaging, we show that these neural progenitors undergo small distance migrations in the embryo. We then show that the normal swimming behavior of the larvae is compromised if the neurogenesis pathway is perturbed. The developmental sequence of ciliary band neurons is very similar to that of neural crest-derived sensory neurons in vertebrates and may provide insights into the evolution of sensory neurons in deuterostomes.

Keywords: Ciliary band; Neurogenin; Neuronal progenitor; Peripheral neurons; Sea urchin.

Figure 1:. Regulatory landscape of ciliary band neurons

(A-D) Maximum intensity projections of whole mount in situ hybridizations (WMISH) show ngn expression. At 20 hpf (late gastrula stage) and 22hpf (prism stage) ngn is expressed in bilateral patches in the neuroectoderm (A,B). Ngn expressing cells enter the ciliary band by 24 hpf (early pluteus stage), and by 32 hpf (pluteus stage) ngn is found in a salt-and-pepper pattern throughout the ciliary band. (E-E’) Maximum intensity projection of double WMISH at 20 hpf shows ngn-expressing neural cells are bilaterally symmetric in the neuroectoderm on the borders of chordin expression. (F-F’) Maximum intensity projection WMISH at prism stage shows ngn expression is in cells in the neuroectoderm outside the ciliary band, marked here by hnf6 expression. (G) By 32 hpf ngn neural cells are inside the ciliary band, within the expression domain of hnf6. Inset image shows an aboral view of a different embryo. (H-I) Maximum intensity projection WMISH shows co-expression of some ngn-expressing cells with soxC (H), synb (I), and prox (J) in the ciliary band. Panels in (H-J) show combined and split single confocal slices of the region highlighted by the white boxes. ((K) Schematic showing lateral and oral views of a pluteus larva. Green marks the ciliary band, magenta marks ngnexpressing neural cells.

Figure 2:. The ciliary band expresses asicl and ciliary band neuronal progenitors require MAPK signaling

A) Maximum intensity projection WMISH shows asicl expression. (B) Maximum intensity projection WMISH shows asicl expression is in the ciliary band, marked here by hnf6. Inset image shows an aboral view of a different embryo. (C) Maximum intensity projection WMISH shows ngn-expressing neural cells are within the ciliary band at the time that asicl is expressed there. Arrowhead show ngn-expressing cells. Panels in (C) show combined and split single apotome slices of the region highlighted by the white box. (D-F) Maximum intensity projections of whole mount in situ hybridizations (WMISH) of ngn. Out of 1468 control embryos, 99.7% show ngn-expressing cells scattered throughout the ciliary band. Ngn expression is lost in 75% of embryos treated with U0126 at hatched blastula stage. In 25% of embryos treated with U0126 at hatched blastula, ngn expression is reduced to 1–2 cells in the ciliary band.

Figure 3:. Ciliary band neural progenitors project neurites and migrate small distances

(A) Whole mount in situ hybridization show that neural progenitors co-express ngn with brn1/2/4 in neural progenitors in the ciliary band. A single confocal section is shown. Side panels show multichannel and single channel images of highlighted regions. (B) Quantifications of cell counts done on double whole mount in situ hybridizations for ngn and brn1/2/4. A total of 68 embryos were assessed (embryos were either 26, 28, 30, or 32 hpf) and cells that expressed both genes or only ngn or brn1/2/4 were counted and scored as such. “n” represents the number of cells counted with the given condition. (C-E) Shows still images of embryos injected with Brn1/2/4 GFP BAC displaying neurites. Inset images show combined or split channel of the GFP expressing cell. Arrowhead indicates neurite projections. (F-F’) Shows still images from Supplementary Video 1 of an embryo injected with Brn1/2/4 GFP BAC. (F) Shows stills of combined DIC and GFP channels. (F’) Only the GFP fluorescent channel is shown. Time given in min:sec. Arrowhead indicates cell undergoing migration.