Lifelong single-cell profiling of cranial neural crest diversification in zebrafish

Abstract

The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.

Gill developmental program in the teleost mandibular arch

“…At 14 dpf, the ucmaa-p1 enhancer, active in gill filament but not hyaline cartilage in the face, drives GFP expression in both pseudobranch and gill filament cartilage (Figure 2b), as seen for endogenous expression of ucmaa (Figure 3-figure supplement 1). In our single-cell chromatin accessibility analysis of neural crest-derived cells (Fabian et al, 2022), we also identified an irx5a proximal enhancer selectively accessible in pillar cells, a specialized type of endothelial cell in the gill secondary filaments (Figure 3-figure supplement 2). At 13, 20, 60 dpf and one-year-old adult fish, the irx5a-p1 enhancer drives GFP expression in pillar cells of the pseudobranch and gills (Figure 2c; Figure 3-figure supplement 2).…”

“…Zebrafish mutant for gata3 fails to form gill buds (Sheehan-Rooney et al, 2013), and single-cell chromatin accessibility analysis of neural crest-derived cells had implicated Gata3 and Gata2a in development of gill filament cell type differentiation (Fabian et al, 2022). We find that gata3 and gata2a are prominently expressed in both the developing pseudobranch and gill buds at 3 and 5 dpf (Figure 3a; Figure 3-figure supplement 1).…”

“…In skate, the pseudobranch and gills share expression of foxl2, shh, gata3 , and gcm2 (Hirschberger and Gillis, 2021). To test whether this reflects conserved gene regulatory mechanisms indicative of serial homology, we examined activity of several gill-specific enhancers (Fabian et al, 2022). At 5 dpf, the gata3-p1 enhancer drives GFP expression in the growing tips of both the pseudobranch and gill buds (Figure 2a).…”

“…In teleost gills, two rows of filaments are anchored to a prominent gill bar skeleton. Both the filaments and supportive gill bars develop from the embryonic pharyngeal arches that consist of mesenchyme of neural crest and mesoderm origin and epithelia of endodermal and ectodermal origin (Fabian et al, 2022; Mongera et al, 2013). The third through sixth arches generate gills in most fishes, and in cartilaginous fishes the second (hyoid) arch forms a hemibranch (one row of gill filaments).…”

Integrative analysis of transcriptomics in human craniofacial development reveals novel candidate disease genes

“…Thus far gene expression during craniofacial development has been primarily studied in model organisms such as mouse, chicken, and zebrafish (Askary et al, 2017; Brugmann et al, 2010; Chang et al, 2014; ENCODE Project Consortium, 2012; Fabian et al, 2022; Hooper et al, 2017; Li et al, 2019; Potter and Potter, 2015). Due to ethical and logistical reasons, gene expression data from primary craniofacial developing human tissue are rare (Cai et al, 2005; Samuels et al, 2020) compared to the thousands of gene expression data sets produced in adult tissues by large-scale consortia such as Encode and GTEx.…”