Molecular fingerprinting delineates progenitor populations in the developing zebrafish enteric nervous system

Abstract

Background To understand the basis of nervous system development, we must learn how multipotent progenitors generate diverse neuronal and glial lineages. We addressed this issue in the zebrafish enteric nervous system (ENS), a complex neuronal and glial network that regulates essential intestinal functions. Little is currently known about how ENS progenitor subpopulations generate enteric neuronal and glial diversity. Results We identified temporally and spatially dependent progenitor subpopulations based on coexpression of three genes essential for normal ENS development: phox2bb, sox10, and ret. Our data suggest that combinatorial expression of these genes delineates three major ENS progenitor subpopulations, (1) phox2bb+/ret−/sox10−, (2) phox2bb+/ret+/sox10−, and (3) phox2bb+/ret+/sox10+, that reflect temporal progression of progenitor maturation during migration. We also found that differentiating zebrafish neurons maintain phox2bb and ret expression, and lose sox10 expression. Conclusion Our data show that zebrafish enteric progenitors constitute a heterogeneous population at both early and late stages of ENS development and suggest that marker gene expression is indicative of a progenitor’s fate. We propose that a progenitor’s expression profile reveals its developmental state: “younger” wave front progenitors express all three genes, whereas more mature progenitors behind the wave front selectively lose sox10 and/or ret expression, which may indicate developmental restriction.

Gut feelings: Studying enteric nervous system development, function, and disease in the zebrafish model system

“…Ptf1 has been shown to regulate neuronal subtype specification in different parts of the nervous system (Hoshino et al, ; Fujitani et al, ; Dullin et al, ; Kaslin et al, ), putting ptf1 forward as a potential new regulator of neuronal subtype differentiation in the ENS (Uribe et al, ). Additionally, a recent study by Taylor and colleagues () found continued expression of ret and phox2bb in enteric neurons during different stages of ENS development. Interestingly, two populations of enteric neurons were identified, one phox2bb + / ret + and another phox2bb + / ret ‐ , which may represent different neuronal subtypes (Taylor et al, ).…”

“…E,F: Adapted from Ganz et al, . Asterisk (*) indicates that co‐expression patterns have been investigated between genes with an asterisk (Taylor et al, ); # indicates that the gene is expressed in a subgroup of EPCs. For the other genes listed, it has not been tested if they are expressed in all or in a subgroup of EPCs; $ shh is expressed in the distal end of GE, but it is unclear if shh is also found in the anterior part of GE.…”

“…In Zebrafish, phox2bb is expressed in EPCs and later in enteric neurons (Fig. A–C) (Elworthy et al, ; Taylor et al, ). The expression of its co‐ortholog phox2ba in the ENS in Zebrafish remains unexplored.…”

“…In the anterior and mid‐regions of the gut, the number of triple‐positive EPCs decreased and different EPC subpopulations were present, comprising presumptive neuronally and glial‐fated EPCs (Fig. B) (Taylor et al, ). Other genes have also been shown to be expressed in a subset of EPCs, even though their co‐expression patterns with other EPC marker genes have not been investigated in detail.…”

Epigenetic factors coordinate intestinal development

“…EPCs, endoderm, and developing mesoderm are in such close proximity during intestinal development (Wallace et al, 2005a) that previous studies did not differentiate the expression pattern of uhrf1 among these cell types. The transgene Tg(phox2b:EGFP) is expressed in migrating EPCs beginning around 30-32 hours post fertilization [hpf, (Shepherd et al, 2004;Taylor et al, 2016)] allowing identification of EPCs among surrounding intestinal cells. We evaluated uhrf1 RNA expression in embryos homozygous for the phox2b:EGFP transgene at 48 hpf and performed immunohistochemistry to detect GFP expression in EPCs (Fig.…”