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.

Conclusions: 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. Developmental Dynamics 245:1081-1096, 2016. © 2016 Wiley Periodicals, Inc.

Keywords: development; enteric glia; enteric neuron; gene expression; neural crest.

Figure 1. Experimental setup

(A) At 36 hpf, we imaged migrating progenitors in four alternate sections at the wave front (w). (B) At 54, (C) 60 hpf, and (D) 84 hpf we analyzed three regions, an anterior region (a) defined by the most anterior appearance of phox2bb:EGFP+ progenitor cells around the intestinal epithelium; a posterior region (p) defined to be the last section with phox2bb:EGFP+ enteric progenitors, and a mid region (m) defined as the middle three sections between a and p. Schematic side-views of zebrafish embryos at the ages indicated.

Figure 2. phox2bb:EGFP exhibits a high degree of colocalization with phox2bb mRNA

(A) and (C) show confocal images of cross-sections through the trunk. (A) At 54 and (C) 60 hpf phox2bb mRNA (red) largely colocalizes with phox2bb:EGFP (green). Insets show enlargements of outlined cells, overlay, phox2bb:EGFP, phox2bb mRNA (from left to right). (B) At 54 and (D) 60 hpf quantification in percent of phox2bb:EGFP and phox2bb mRNA colocalization in the three regions analyzed. Note that the dotted line between the measured values in (B) and (D) does not show the continuous percentage of coexpression between the anterior, mid and posterior values, but is drawn to facilitate visualization of coexpression trends. ie – intestinal epithelium Scale bar = 10µm.

Figure 3. sox10 and ret are differentially expressed in two different progenitor subpopulations

(A), (C), (E) and (G) show confocal images of cross-sections through the trunk. (A) At 54 and (C) 60 hpf ret mRNA (red) colocalizes with phox2bb (green). Insets show enlargements of outlined cells, overlay, phox2bb, ret mRNA (from left to right). Note that phox2bb refers to phox2bb:EGFP expression as described in the text. (B) At 54 and (D) 60 hpf quantification in percent of phox2bb and ret colocalization in the three regions analyzed. (E) At 54 and (G) 60 hpf sox10 mRNA (red) partially colocalizes with phox2bb (green). Insets show enlargement of outlined cell, overlay, phox2bb, sox10 mRNA (from left to right). (F) At 54 and (H) 60 hpf quantification in percent of phox2bb and sox10 mRNA colocalization in the three regions analyzed. The dotted line between the measured values does not show the continuous percentage of coexpression between the anterior, mid and posterior values, but is drawn to facilitate visualization of coexpression trends. ie – intestinal epithelium Scale bar = 10µm.

Figure 4. Colocalization of ret and sox10 with phox2bb reveals a total of three enteric progenitor subpopulations

(A) and (C) show confocal images of cross-sections through the trunk. (A) At 54 and (C) 60 hpf phox2bb (green), ret mRNA (red) and sox10 mRNA (blue) colocalize in subpopulations. Insets show close-up of outlined cell(s), overlay, phox2bb, ret mRNA, sox10 mRNA (from left to right). Note that phox2bb refers to phox2bb:EGFP expression as described in the text. (B) At 54 and (D) 60 hpf quantification in percent of phox2bb, ret and sox10 colocalization in the three regions analyzed. The dotted line between the measured values does not show the continuous percentage of coexpression between the anterior, mid and posterior values, but is drawn to facilitate visualization of coexpression trends. ie – intestinal epithelium Scale bar = 10µm.

Figure 5. A small population of differentiating neurons express phox2bb and ret

(A), (C), (E) and (G) show confocal images of cross-sections through the trunk. (A) At 54 and (C) 60 hpf ret mRNA (red) colocalizes with Elavl (blue) and phox2bb (green). Insets show close-ups of outlined cell, overlay, phox2bb, ret mRNA, Elavl (from left to right). Note that phox2bb refers to phox2bb:EGFP expression as described in the text. (B) At 54 and (D) 60 hpf quantification in percent of phox2bb, ret and Elavl colocalization in the three regions analyzed. (E) At 54 and (G) 60 hpf sox10 mRNA (red) does not colocalize with Elavl positive (blue), phox2bb positive cells. Insets show close-up of outlined cell, overlay, phox2bb, sox10 mRNA, Elavl (from left to right). (F) At 54 and (H) 60 hpf quantification in percent of phox2bb, sox10 and Elavl colocalization in the three regions analyzed. The dotted line between the measured values does not show the continuous percentage of coexpression between the anterior, mid and posterior values, but is drawn to facilitate visualization of coexpression trends. ie – intestinal epithelium Scale bar = 10µm.

Figure 6. A small population of differentiating neurons express phox2bb and ret at later stages of neuronal differentiation

(A) and (C) show confocal images of cross-sections through the trunk. (A) At 84 hpf ret mRNA (red) colocalizes with Elavl (blue) and phox2bb (green). Insets show close-ups of outlined cell, overlay, phox2bb, ret mRNA, Elavl (from left to right). Note that phox2bb refers to phox2bb:EGFP expression as described in the text. (B) At 84 hpf quantification in percent of phox2bb, ret and Elavl colocalization in the three regions analyzed. (C) At 84 hpf sox10 mRNA (red) does not colocalize with Elavl positive (blue), phox2bb positive (green) cells. Insets show close-up of outlined cell, overlay, phox2bb, sox10 mRNA, Elavl (from left to right). (D) At 84 hpf quantification in percent of phox2bb, sox10 and Elavl colocalization in the three regions analyzed. The dotted line between the measured values does not show the continuous percentage of coexpression between the anterior, mid and posterior values, but is drawn to facilitate visualization of coexpression trends. ie – intestinal epithelium Scale bar = 10µm.

Figure 7. Enteric progenitor subpopulations are present at 36 hpf

(A) and (C) show confocal images of cross-sections through the trunk. At 36 hpf (A) ret mRNA (red) partially colocalizes with phox2bb (green). Insets show close-up of outlined cell, overlay, phox2bb, ret mRNA (from left to right). Note that phox2bb refers to phox2bb:EGFP expression as described in the text. (B) Quantification in percent of phox2bb and ret colocalization at the wave front. (C) sox10 mRNA (red) partially colocalizes with phox2bb (green). Insets show close-up of outlined cell, overlay, phox2bb, sox10 mRNA (from left to right). (D) Quantification in percent of phox2bb and sox10 colocalization at the wave front. ie – intestinal epithelium Scale bar = 10µm.

Figure 8. Summary of proposed enteric subpopulations during zebrafish ENS development

(A) At 54 and (B) 60 hpf enteric progenitors constitute a heterogeneous population of cells based on spatio-temporally dynamic expression of phox2bb, ret, and sox10. Elavl+ differentiating neurons express ret and phox2bb, but do not express sox10. (C) Proposed model for types of enteric progenitor subpopulations. Expression profiles may be indicative of a progenitor’s developmental state and its fate. Note that the phox2bb and the phox2bb/ret positive progenitor populations are likely to give rise to two different suites of neuronal subtypes.