Changes to immune cell composition in the gut is Myd88 dependent. a Schematic representation of the developmental features of early zebrafish larvae which form the foundation of this experimental approach. b Representative images of distal intestines of germ-free (GF) and conventionalized (CONVD) wildtype (WT) larvae following L-plastin staining. Composite images of bright-field and L-plastin signal from confocal maximum intensity Z-projection in red. c WT larvae under germ-free conditions exhibit a significantly elevated leukocyte presence in the distal intestine, compared to conventionalized. This elevated leukocyte presence was no longer detectable under the same conditions in myd88 deficient larvae. Figure is representative of three independent replications. d Representative live microscopy images of distal intestine of GF and CONVD larvae of the Mpx:GFP reporter zebrafish line. Bright-field overlaid with confocal maximum intensity Z-projection in green. e Germ-free larvae exhibit significantly reduced neutrophil infiltration in the distal intestine compared to conventionalized larvae. No significant difference was observed in myd88 deficient backgrounds. Figure is made from pooled data from three biological replicates. b, d an area of intestine extending 4 somites proximal to the cloaca, representing the area of counting, is outlined in white dashed lines. scale bars represent 100 µm. c, e each data point represent cell counts from one larvae based on confocal z-stacks acquired at 20 times magnification. Error bars represent standard error of the mean. *P ≤ 0.05 by two-way ANOVA with Bonferroni correction for multiple comparisons
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