Enteric nervous system development in avian and zebrafish models

AbstractOur current understanding of the developmental biology of the enteric nervous system (ENS) and the genesis of ENS diseases is founded almost entirely on studies using model systems. Although genetic studies in the mouse have been at the forefront of this field over the last 20 years or so, historically it was the easy accessibility of the chick embryo for experimental manipulations that allowed the first descriptions of the neural crest origins of the ENS in the 1950s. More recently, studies in the chick and other non-mammalian model systems, notably zebrafish, have continued to advance our understanding of the basic biology of ENS development, with each animal model providing unique experimental advantages. Here we review the basic biology of ENS development in chick and zebrafish, highlighting conserved and unique features, and emphasising novel contributions to our general understanding of ENS development due to technical or biological features

Ret signalling integrates a craniofacial muscle module during development

An appropriate organisation of muscles is crucial for their function, yet it is not known how functionally related muscles are coordinated with each other during development. In this study, we show that the development of a subset of functionally related head muscles in the zebrafish is regulated by Ret tyrosine kinase signalling. Three genes in the Ret pathway (gfra3, artemin2 and ret) are required specifically for the development of muscles attaching to the opercular bone (gill cover), but not other adjacent muscles. In animals lacking Ret or Gfra3 function, myogenic gene expression is reduced in forming opercular muscles, but not in non-opercular muscles derived from the same muscle anlagen. These animals have a normal skeleton with small or missing opercular muscles and tightly closed mouths. Myogenic defects correlate with a highly restricted expression of artn2, gfra3 and ret in mesenchymal cells in and around the forming opercular muscles. ret+ cells become restricted to the forming opercular muscles and a loss of Ret signalling results in reductions of only these, but not adjacent, muscles, revealing a specific role of Ret in a subset of head muscles. We propose that Ret signalling regulates myogenesis in head muscles in a modular manner and that this is achieved by restricting Ret function to a subset of muscle precursors.</jats:p

A Novel Zebrafish <i>ret</i> Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for <i>mapk10</i> as a Modifier of Enteric Nervous System Phenotype Severity

<div><p>Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and <i>in vivo</i> physiological studies. We show that <i>ret</i> mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in <i>ret</i> heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in <i>ret</i> mutants, and partially impaired in <i>ret</i> heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant <i>ret</i> heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated <i>mapk10</i> loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of <i>mapk10</i> mutations into <i>ret</i> heterozygotes enhanced the ENS deficit, supporting <i>MAPK10</i> as a HSCR susceptibility locus. Our studies demonstrate that <i>ret</i> heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.</p></div

<i>mapk10</i> as candidate to account for varying expressivity in HSCR.

<p> (A) Enteric neuron number in the distal gut quantified in 4dpf larvae resulting from crosses between <i>mapk10</i>^Δ10/+ and <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+ zebrafish. No difference was detected between WT and heterozygous <i>mapk10</i>^Δ10 larvae, but <i>mapk10</i>^Δ10/Δ10 larvae have a small, but statistically significant reduction in ENS cell number in the gut relative to WT (one-way ANOVA, p = 0.0442, Tukey post-hoc), indicating a role for <i>mapk10</i> in normal ENS development. No significant difference was detected between <i>ret</i>^{<i>hu2846</i>/+} and <i>mapk10</i>^Δ10/+;<i>ret</i>^{<i>hu2846</i>/+} larvae, however, loss of <i>mapk10</i> in the <i>ret</i>^{<i>hu2846</i>/+} background leads to statistically significant reduction in neuron number (Welch’s one-way ANOVA, <i>p</i> = 0.048). (B) To examine phenotype distribution, individual zebrafish were binned according to neuron number in the distal gut. WT, <i>mapk10</i>^Δ10/+ and <i>mapk10</i>^Δ10/Δ10 larvae show normal distribution of phenotypes (blue, red, and green bars, respectively, Shapiro-Wilk normality test, for WT: p = 0.776, for <i>mapk10</i>^Δ10/+: p = 0.910, and for <i>mapk10</i>^Δ10/Δ10: p = 0.1149). Although <i>ret</i>^{<i>hu2846</i>/+} larvae guts are phenotypic, they display a range of neuron numbers in the distal gut region, reflecting normal distribution of mild and progressively more severe colonization phenotypes (Shapiro-Wilk normality test, p = 0.5720, blue bars), counts of neuron number in <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+ (red bars) and <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 larval guts (green bars) exhibit a non-normal distribution pattern (Shapiro-Wilk normality test, for <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+: p = 0.0054, and for <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 p = 0.0014), and counts of enteric neuron number in <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 larvae, showed a statistically significant increase in standard deviation compared with <i>ret</i>^{<i>hu2846</i>/+} larvae (Brown-Forsythe, 0.0445).</p

Intestinal motility is impaired in <i>ret</i>^{<i>hu2846</i>/+} and <i>ret</i>^{<i>hu2846/2846</i>} larvae.

<p>(A) Spatiotemporal maps (STMs) generated to quantify 400 second recordings of gut motility in live 7dpf zebrafish larvae. WT (<i>ret</i>^+/+) larvae exhibit cyclical retrograde (anal to oral) motility patterns in the intestinal bulb (brackets), and anterograde (oral to anal) peristaltic waves in the intestine starting at the intestinal bulb/intestine junction (white arrows); black arrows denote motility end points. STMs of <i>ret</i>^{<i>hu2846</i>/+} larvae with ENS phenotypes and <i>ret</i>^{<i>hu2846/2846</i>} larvae show alterations in cyclical retrograde motility patterns (brackets) and frequency and distance of anterograde motility waves (white to black arrows). (B) Anterograde contraction characterization. Phenotypic (<i>ret</i>^{<i>hu2846</i>/+}P) and non-phenotypic (<i>ret</i>^{<i>hu2846</i>/+}NP) larvae identified immunohistochemically. Reduced peristaltic frequency in <i>ret</i>^{<i>hu2846/2846</i>} larvae and <i>ret</i>^{<i>hu2846</i>/+} P relative to WT (one-way ANOVA, p = 0.0004; Bonferroni’s post-hoc, p = 0.0001 and p = 0.0367), and also reduced in <i>ret</i>^{<i>hu2846/2846</i>} larvae as compared to <i>ret</i>^{<i>hu2846</i>/+} NP (p = 0.0002) and <i>ret</i>^{<i>hu2846</i>/+} P (p = 0.0035). Reduced contraction travel distance in <i>ret</i>^{<i>hu2846/2846</i>} larvae relative to WT (one-way ANOVA, p = 0.0005; Bonferroni’s post-hoc, p = 0.0002), and relative to both <i>ret</i>^{<i>hu2846</i>/+} P (p = 0.0232) and <i>ret</i>^{<i>hu2846</i>/+} NP (p = 0.0237). Reduced contraction velocity in <i>ret</i>^{<i>hu2846/2846</i>} relative to WT (one-way ANOVA, p = 0.0212; Bonferroni’s post-hoc, p = 0.0138). Increased contractions interval in <i>ret</i>^{<i>hu2846/2846</i>} larvae relative to WT and <i>ret</i>^{<i>hu2846</i>/+} NP (one-way ANOVA, p = 0.0078; Bonferroni’s post-hoc, p = 0.0053 and p = 0.0097). (C) Loss of anterograde contractions in <i>ret</i>^{<i>hu2846/hu2846</i>} larvae compared to WT and <i>ret</i>^{<i>hu2846</i>/+} NP (#, Fisher’s exact, p = 0.0198 and p = 0.028). <i>ret</i>^{<i>hu2846/2846</i>} larvae show retrograde motility changes relative to all other genotypes, with contractions losing cyclic patterns (Fisher’s exact, vs. WT: p = 0.0001, vs. <i>ret</i>^{<i>hu2846</i>/+} NP: p = 0.001, and vs. <i>ret</i>^{<i>hu2846</i>/+} P: p = 0.0082). (D) Equivalent analysis in the SAGFF234A;UAS:GFP background, expressing GFP in ENS cells and their processes, enables mapping of contraction endpoints relative to cell body position (GFP⁺HuC/D⁺ cells, r_s = 0.8857, p = 0.0333) and cell process (GFP⁺HuC/D⁺ process, r_s = 0.942, p = 0.0167). Black lines indicate linear fits.</p

Using <i>ret</i>^{<i>hu2846</i>/+} as sensitized background to test role of <i>mapk10</i> as ENS development modifier gene.

<p>(A) RNA <i>in situ</i> hybridization shows <i>mapk10</i> expression correlates with location of the ENS in WT (<i>ret</i>^+/+), <i>ret</i>^{<i>hu2846</i>/+}, and <i>ret</i>^{<i>hu2846</i>/hu2846} 3dpf larvae (asterisks indicate end of gut, filled arrows indicate <i>mapk10</i>⁺ cells, and open arrows denote gut areas lacking <i>mapk10</i>⁺ cells). (B) MO gene knock-down of <i>mapk10</i> (using a splice blocking MO, <i>mapk10</i>MO) performed on embryos from WT x <i>ret</i>^{<i>hu2846</i>/+} cross to allow knock-down in both WT and <i>ret</i>^{<i>hu2846</i>/+} embryos, and compared to a control MO. Immunostaining with HuC/D at 4dpf shows that most severe phenotypes are observed when <i>mapk10</i>MO is injected into <i>ret</i>^{<i>hu2846</i>/+} embryos. Asterisks indicate end of gut tube (anal pore), and arrows denote position of last HuC/D⁺ neuron. (C) Quantification of neuron number in the last 5 somite lengths shows that injection of <i>mapk10</i>MO into WT embryos causes a modest, statistically significant reduction of enteric neurons in the gut relative to WT larvae injected with control MO (one-way ANOVA, p<0.001; Bonferroni’s post-hoc test, p = 0.0033), revealing a role for <i>mapk10</i> in normal ENS development. Injection of <i>mapk10</i>MO into <i>ret</i>^{<i>hu2846</i>/+} embryos causes severe reduction of enteric neurons, which is statistically different from either <i>ret</i>^{<i>hu2846</i>/+} injected with control MO (one-way ANOVA, p<0.001, Bonferroni’s post-hoc test, <i>P</i><0.0001) or <i>mapk10</i>MO injected into WT embryos (one-way ANOVA p<0.001, Bonferroni’s post-hoc test p = 0.006).</p