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Fig. 4 (A) Whole mount confocal (20X z = 14, 5 μm voxel depth) imaging of Tg(mpeg1:GFP; nbt:dsRed) at 3 dpf highlighting rare gut-associated mpeg1GFP+ mφ in proximity to maturing nbtDsRed+ gut neural tracts. Dashed line indicates margins of developing intestinal tube. (B) Outside of the gut, at 3 dpf mφ are often associated with neural processes of the peripheral nervous system (PNS) (20 X with Nyquist sampling z = 25, 2 μm voxel depth). (B′) Inset shows mφ-neural process bridging (20 X with Nyquist sampling z = 10, 2 μm voxel depth). (C) Whole mount in vivo imaging of 5-6 dpf Tg(mpeg1:GFP;nbt:dsRed) fish reveals mφ are more frequently found throughout the body with increased gut-associated mφ frequency (20 X tiled image z = 32, 3 μm voxel depth). (C′ and C″) Mφ at this stage are well developed and often observed along the lateral line nerve where mφ bodies lie along the lateral line itself and mφ processes contact and bridge adjacent nbtDsRed+ neural processes. (C‴) At 5-6 dpf gut mpeg1GFP+ mφ are nbtDsRed+- associated in the enteric nervous system (ENS). (D–G) (D) Ex vivo intact whole gut explant confocal imaging (20 X tiled image, z = 30, 3 μm voxel depth) at 6 dpf showing distribution and location of macrophages (mpeg1GFP+) and enteric neurons (nbtDsRed+) along the intestine (A, anterior; and P, posterior), and (E) elaborately extended ‘mature’ and differentiated morphologies of gut-associated macrophages (20 X, z = 8, 3 μm voxel depth). Both in vivo whole mount imaging (20 X, z = 18, 5 μm voxel depth) (F) and ex vivo whole gut explant imaging (G) at 9 dpf shows increased density of gut macrophages relative to the distribution of neurons in the gut and along the body (20 X tiled image, z = 20, 5 μm voxel depth). (H) Higher magnification in vivo imaging shows contact between intestinal macrophages with enteric neuronal cell bodies and processes (20 ×, z = 16, 5 μm voxel depth). (I) 3D volumetric rendering of 12-14 dpf ex vivo whole gut explant demonstrates increased density of mpeg1GFP+ mφ in the surrounding gut wall, suggesting a possible ‘outside-in’ development of gut macrophages (20 X, z = 37, 1 μm voxel depth). (J) Whole mount in vivo imaging of 12-14 dpf Tg(mpeg1:GFP;nbt:dsRed) fish shows increased density of whole-body and gut-associated mpeg1:GFP+ macrophages (20 X tiled image, z = 50, 5 μm voxel depth). Dotted lines demarcate the intestinal tube. (K) Ex vivo intact whole gut explant confocal imaging at 12-14 dpf shows gut-specific residency of dense mpeg1:GFP+ cells (20 X tiled image, z = 16, 5 μm voxel depth). (L) mpeg1GFP+ macrophages, arrow, can be seen abutting neuron-containing musculature and intercalating processes around intestinal epithelial cells (20 X with Nyquist sampling, z = 15, 1 μm voxel depth). (M) Transillumination imaging demonstrates stereotypical ‘adult-like’ S-shape of the gut becomes apparent around 30 dpf (20 X tiled image z = 13, 5 μm voxel depth). (N) The developed gut is densely innervated and populated with mpeg1GFP+ mφ and appears mature in phenotype (20 X tiled image z = 17, 5 μm voxel depth). (O) Standard length (mm) of zebrafish increases linearly across larval development. Pooled data are represented as average +/− S.E.M., n = 3–20 per group. (P) Gut length increases as a function of development nonlinearly, with two ‘spurts’ of growth including between 6 and 9 days (just after complete yolk resorption and onset of exogenous feeding) and at the onset of the juvenile development stage (~3-4 weeks postfertilization). (Q) The number of total mpeg1GFP+ gut mφ at key stages of larval development increases exponentially as shown by a linear increase on a base ten logarithmic scale. Representative data (n = 3/group) are shown as mean ± S.E.M.; numbers show the average total macrophage number per gut. (R) Increase in total mpeg1GFP+ gut mφ as a function of gut length shows a similar developmental pattern as in (P) Representative data (n = 3/group) are shown as mean ± S.D. (S) Individual mφ area (μm2) remains constant across larval development. Graph shows area calculations of n = 20–40 individual macrophages pooled from n = 3 individuals at the indicated developmental checkpoints. Mean ± S.E.M. is depicted, significance determined by one-way ANOVA. Q-S, log-linear plots shown on a base ten logarithmic scale on the y axis. A = anterior, P = posterior. Scale bars shown as indicated.