A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10—another important ENS developmental regulator gene—GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood

Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota

<div><p>The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this “microbial organ” for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the <i>ret</i> locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host–microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.</p></div

The enteric nervous system promotes intestinal health by constraining microbiota composition

<div><p>Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, <i>sox10</i>, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health.</p></div

Intestinal microbiota are necessary and sufficient to induce increased intestinal neutrophil accumulation in <i>sox10</i> mutants.

<p><b>(A)</b> Quantification of intestinal neutrophil number per 140 μm of distal intestine. Neutrophil accumulation was inhibited when <i>sox10</i> mutants were raised GF compared to CV controls. <i>n</i> > 21 per condition. <b>(B)</b> Schematic of fish used as donors in the transmission experiment. Intensity of red indicates level of intestinal inflammation. <b>(C)</b> Schematic of the experimental protocol. Intestines of GF, CV WT, <i>sox10</i> mutants, or <i>iap</i> MO were dissected for use as inoculum for 4 dpf GF WT recipients. Recipient fish were colonized for 2 d before examination of intestinal neutrophil number. <b>(D)</b> Transfer of intestinal microbes from inflamed intestines of <i>sox10</i> mutants causes increased intestinal neutrophil number in WTs. <i>n</i> ≥ 10, *<i>p</i> < 0.05, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, ANOVA with Tukey’s range test. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000689#pbio.2000689.s002" target="_blank">S2 Fig</a>.</p

Populations of <i>Vibrio</i> and <i>Aeromonas</i> exhibit different dynamics within the zebrafish intestine.

<p>(A) An optical section of the intestinal bulb from a larval zebrafish mono-associated with <i>Vibrio</i> (the cyan box in the diagram below outlines the region imaged). The population consists of discrete, highly motile individuals (inset: single <i>Vibrio</i> cells). (B) A montage of images taken from the time-series in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s009" target="_blank">S3 Movie</a> shows that the highly motile and planktonic <i>Vibrio</i> cells maintain their overall distribution despite repeated intestinal contractions. Time between frames: 1 second. (C) An optical section of the intestinal midgut from a larval zebrafish mono-associated with <i>Aeromonas</i> (the magenta box in the diagram below outlines the region imaged). Cells are largely non-motile and densely aggregated. (D) A montage of images taken from the time-series in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s013" target="_blank">S7 Movie</a> shows an aggregate of <i>Aeromonas</i> in the midgut that is spatially dynamic, entering and exiting the field of view multiple times. Time between frames: 1 second. (A–D) Scale bars: 50 μm.</p

Intestinal motility and bacterial competition are altered in <i>ret</i> mutant zebrafish hosts.

<p>(A) Amplitudes of periodic contraction along the intestine for wild-type (wt) and <i>ret</i> mutant zebrafish at 5 and 6 dpf. Shown are histograms with the percent of individual larvae within different amplitude ranges. Vertical dashed lines indicate median amplitudes for respective curves. <i>n =</i> 28 (5 dpf, wt); <i>n =</i> 22 (5 dpf, <i>ret</i>); <i>n =</i> 29 (6 dpf, wt); <i>n =</i> 21 (6 dpf, <i>ret</i>). (B) GF wild-type and <i>ret</i> heterozygous hosts (wt) were raised together with <i>ret</i> homozygous mutant hosts (<i>ret</i>) and colonized at 4 dpf with <i>Aeromonas</i>. At 5 (left) or 6 (right) dpf <i>Vibrio</i> was added to the water column for 24 hr prior to whole gut dissection and serial plating to enumerate bacterial abundances. Additionally plotted are respective <i>Aeromonas</i> mono-association reference (ref.) populations from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.g001" target="_blank">Fig 1B</a> (left, 4–6; right, 4–7). The difference between <i>Aeromonas</i> abundance during challenge and mono-association was determined by an unpaired <i>t</i> test. CFU = colony-forming units; *** = <i>p</i> < 0.0001; ns = not significant; <i>n</i> > 18/condition. Gray and black dashed lines denote limits of quantification and detection, respectively. Underlying data for A and B are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s001" target="_blank">S1 Data</a>.</p

Inflamed intestines are rescued by anti-inflammatory bacterial isolates or transplantation of WT ENS into <i>sox10</i> mutants.

<p><b>(A)</b> Addition of a representative <i>Escherichia</i> isolate, <i>E</i>. <i>coli</i> HS, to CV <i>sox10</i> mutants reduces intestinal neutrophil accumulation. Monoassociation of <i>sox10</i> mutants with <i>E</i>. <i>coli</i> HS does not increase neutrophil level over that observed in GF zebrafish. <i>n</i> > 20, from at least three independent experiments. <b>(B)</b> Correlation between absolute abundance of <i>E</i>. <i>coli</i> HS and log₁₀(intestinal neutrophil number + 1) in experiments with added <i>E</i>. <i>coli</i> HS. Linear regression analysis with 95% confidence intervals. For A, B: <i>n</i> > 35, from three to six independent experiments. <b>(C)</b> Representative images of distal intestine from WT, <i>sox10</i>^<i>-</i>, and <i>sox10</i>^<i>-</i> rescued by WT ENS precursor transplantation. Anti-ElavI1–labeled enteric neurons are white (white arrow); neutrophils are black (black arrow). Scale bar = 100 μm. <b>(D)</b> Quantification of intestinal neutrophil number per 140 μm of distal intestine. <i>n</i> > 6 for all conditions, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ****<i>p</i> < 0.0001, ANOVA with Tukey’s range test. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000689#pbio.2000689.s004" target="_blank">S4 Fig</a>.</p

<i>Aeromonas</i> experiences sharp drops in population size that are intensified during <i>Vibrio</i> challenge.

<p>(A) MIPs of <i>Aeromonas</i> (magenta) and <i>Vibrio</i> (cyan) in a larval zebrafish intestine. Scale bar: 200 μm. The fish was initially colonized at 4 dpf with <i>Aeromonas</i>, challenged 24 hr later by inoculation with <i>Vibrio</i>, and then imaged every 20 min for 14 hr. The times indicated denote hours post-challenge. In all images, the region shown spans about 80% of the intestine, with the anterior on the left. Image contrast in both color channels is enhanced for clarity. Yellow dotted line roughly indicates the lumenal boundary. As time progresses, the anterior growth of <i>Vibrio</i> as well as abrupt changes in the <i>Aeromonas</i> distribution (arrows) are evident. (B,C) Total bacterial abundance, derived from image data, for <i>Aeromonas</i> and <i>Vibrio</i> in two representative fish inoculated and challenged as in panel A, as a function of time following the <i>Vibrio</i> inoculation. Sharp drops of over an order of magnitude in the <i>Aeromonas</i> population, but not the <i>Vibrio</i> population, are evident. (D,E) Total abundance for <i>Aeromonas</i> in mono-associations as a function of time post-inoculation, in two representative fish. Sudden declines are also observed, though in general the populations recover to approximately pre-collapse levels. (F) The ratio, <i>f</i>, of the abundance immediately after to that before population drops, for <i>Aeromonas</i> challenged by <i>Vibrio</i>; this ratio spans many orders of magnitude (horizontal axis). At the same time points, the <i>Vibrio</i> populations are essentially unchanged, with ratios of populations afterward to before being close to one (vertical axis). (G) Characteristics of <i>Aeromonas</i> population collapses. Circles and bars indicate the mean and standard deviation, respectively, of <i>f</i> and <i>p</i>_c, the magnitude and rate of collapse occurrence, for both mono-associations and <i>Aeromonas</i> challenged by <i>Vibrio</i>. The dashed line at <i>f</i> = 0.1 indicates the threshold for identification of collapses. Underlying data for B–G are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002517#pbio.1002517.s001" target="_blank">S1 Data</a>.</p