Restricted proliferation during neurogenesis contributes to regionalization of the amphioxus nervous system

Abstract

The central nervous system of the cephalochordate amphioxus consists of a dorsal neural tube with an anterior brain. Two decades of gene expression analyses in developing amphioxus embryos have shown that despite the lack of overt segmentation the amphioxus neural tube is highly regionalized at the molecular level. However, little is known about the mechanisms that generate such precise regionalization. Proliferation is a key driver of pattern formation and cell type diversification, but in amphioxus it has never been studied in detail nor in the specific context of neurogenesis. Here, we describe the dynamics of cell division during the formation of the central nervous system in amphioxus embryos and its contributions to the regionalization of the neural axis. We show that after gastrulation, proliferation pauses to become spatially restricted to the anterior and posterior ends of the neural tube at neurula stages. Only at the onset of larval life, proliferation resumes in the central part of the nervous system. By marking specific populations and inhibiting cell division during neurulation, we demonstrate that proliferation in the anterior cerebral vesicle is required to establish the full cell type repertoire of the frontal eye complex and the putative hypothalamic region of the amphioxus brain, while posterior proliferating progenitors, which were found here to derive from the dorsal lip of the blastopore, contribute to elongate the caudal floor plate. Between these proliferative domains, we find trunk nervous system differentiation is independent from cell division, which decreases during neurulation and resumes at the early larval stage. Taken together, our results highlight multiple roles for proliferation in shaping the amphioxus nervous system.

An ancient gene regulatory network sets the position of the forebrain in chordates

“…In line with this being part of the amphioxus hypothalamus, we find the middle Bsx domain absent from embryos in which cell division has been arrested from the 7ss (Supplementary Figure 7B). These results recapitulate and expand what we already reported for Otp 77 , corroborating that the amphioxus hypothalamus develops by intercalated growth in neurulating embryos and demonstrating that Bsx positive cells in the Six3/6-/FoxD+/Otp+ domain are part of this developmental program (Figure 6C).…”

“…Unlike FoxQ2 however, Six3/6 and Frz5/8 remain in the nervous system. At 7ss, Frz5/8 marks the anterior most part of the neural tube, while Six3/6 expression is separated into an anterior and posterior population of cells separated by an intercalated Six3/6 -negative region, as previously described 77 . During the phase of Six3/6 and Frz5/8 restriction, sFRP1/2/5a remains expressed at the boundary between the anterior neural tube and non-neural ectoderm (Figure 2B).…”

“…In normal development, the posterior limit of Fezf would meet the anterior limit of Nkx2 . 1 expression at the intercalated Six3/6 -negative region, which we recently defined as hypothalamic for co-expressing the vertebrate hypothalamic markers FoxD and Otp 77 . Given this, we reasoned that the loss of Six3/6 expression in AZA treated embryos might imply a loss of the hypothalamic brain.…”

“…Second, we found that Nk2.1 and Fezf, two early markers of hypothalamic fate in vertebrates (see supplementary Figure 1F) 86 , are lost from the amphioxus anterior CNS upon aGRN silencing by Wnt overactivation (see Figure 5). In normal development, the posterior limit of Fezf would meet the anterior limit of Nkx2.1 expression at the intercalated Six3/6-negative region, which we recently defined as hypothalamic for co-expressing the vertebrate hypothalamic markers FoxD and Otp 77 . Given this, we reasoned that the loss of Six3/6 expression in AZA treated embryos might imply a loss of the hypothalamic brain.…”

“…Fixed embryos were processed for immunofluorescence as described in 77 . Briefly, embryos were rehydrated and bleached as described above for in situ HCR and permeabilized overnight in NPBS + 1% DMSO + 1% Triton.…”