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CNE_22755_sm_SuppFigure1.tif3317KSupplementary Figure 1. DiI labeling of YFP+ P7 Ednrb+/-sacral fibers. (A) DiI crystals were placed on large sacral fascicles near the pelvic ganglia (PG). The fibers were labeled (magenta) and visualized as they entered the colon. (B) Higher magnification of box in A showing the sacral fibers in proximity to myenteric ganglia (green). The identity of the DiI labeled structures along the fibers (arrowheads) is uncertain. Arrow inside scale bar indicates oral direction. Scale bar: (A) 500 μm, (B) 50 μm.
CNE_22755_sm_SuppFigure2.tif2299KSupplementary Figure 2. (A) P23 Ednrb-/-whole-mount showing Hu/YFP just oral to the pelvic ganglion. Hu cells (green) accompany sacral nerve fascicles (magenta). (B) The same P23 preparation as A at the transition site between ganglionic and aganglionic regions. Ganglia and single Hu+ cells (green, arrows) are found along a YFP+ fascicle (magenta) superficial to the longitudinal muscle. The most aboral myenteric ganglion (green, arrowhead) is found in a deeper location in the same area. Arrow inside scale bar indicates oral direction. Scale bar: (A, B) 50 μm.
CNE_22755_sm_SuppFigure3.tif5337KSupplementary Figure 3. (A magenta-green copy of Fig. 6 for the assistance of color blind readers.) Cells on nerve fibers in Ednrb-/-preparations. (A) Surface view. E14.5 YFP+ SNCCs (red) appear along βIII-tubulin-immunoreactive nerve fibers (green) within the distal colon. (B) Surface view. E15.5 YFP+ cells are found along nerve fibers in the distal colon heading in an oral direction (arrow inside scale bar indicates oral direction). (C) Section through another E15.5 distal colon just oral to the pelvic ganglion. A βlll-tubulin-immunoreactive fiber (green) and YFP+ cells (red) together enter the wall of the colon where many YFP+ cells are already present. (D) Section through the E15.5 distal colon in a position more oral than C. A YFP+ cell (red) and Blll-tubulin-immunoreactive fibers (green) are colocalized (arrow); numerous YFP+ cells are found between the muscle layers. (E) Whole mount of P7 distal colon. Hu+ (green) cells are found on YFP+ (red) nerve fibers near the pelvic ganglia. (F-H) Cross section of P31 distal colon. (F) Both a faintly–stained YFP+ cell and neurofilament-immunoreactive nerve fibers (green, arrowhead) are located between the muscle layers while nerve fibers are also found at the edge of the muscle-submucosa interface. The submucosa and the mucosa were removed. (G) A Hu+(red) neuron is found between the muscle layers usually occupied by the myenteric ganglia. (H) Overlay of images with bright field image and the circular (CM) and longitudinal muscles (LM) labeled. Scale bars: (A,C-H) 50 μm, (B) 100 μm.
CNE_22755_sm_SuppFigure4.tif3103KSupplementary Figure 4. (A magenta-green copy of Fig. 7 for the assistance of color blind readers.) (A) E16.5 Ednrb-/-. TH+ cells (arrows) and fibers accompany sacral fibers projecting orally from the PG into the colon. Note the absence of ganglia. Inset, upper right: Two TH+ cells within box are shown at higher magnification. (B) P27 Ednrb-/-. Changes in position of extrinsic fibers when encountering intrinsic ganglia. Neural crest-derived cells are labeled with β-galactosidase. Cells and fibers on the serosal surface are sharp and dark while cells and fibers within the muscle layers are lighter and blurry. (C) Overlay of fibers shown in B. At the site where the sacral fibers extend into the region of vagal ganglia in the midcolon, large sacral fibers (green) are located between the muscle layers, the same level as vagal ganglia (red). There are also ganglia in the serosal region (blue), above the vagal ganglia, whose origin could be either cells that traveled from sacral axons and/or from the vagally-derived intrinsic neurons in the hypoganglionic region. These different elements appear to intersect (arrows). (D) Diagram summarizing the findings for Ednrb-/-aganglionic colon. Sacral extrinsic fibers and cells (green) projecting from the pelvic ganglia (PG) intersect with the vagal myenteric plexus (red) in the mid-colon. A population of cells and smaller fascicles (blue) appear at the vagal wavefront. They exit the myenteric locale and reside in the serosal region superficial to the longitudinal muscle (LM) where they appear to have connections with vagal ganglia. The larger sacral fascicles continue orally between the muscle layers where they appear to intermingle with the vagal ganglia. (CM: circular muscle) (E) Ednrb+/-. YFP+ sacral fibers (green) extending from the pelvic ganglia (PG) into the distal colon contain TH+ cells (red, arrows). A single TH+ cell (arrowhead) is incorporated into ganglia formed by VNCCs. (F) E16.5 Ednrb+/-colon. A confocal image showing TH+ (red, arrows) YFP+ SNCCs among the YFP+ VNCCs (green) forming nascent ganglia. Note a single TH+ cell that isn't YFP+ (arrowhead). (G) Diagram summarizing the findings for Ednrb+/-ganglionated colon. Fibers leaving the pelvic ganglion travel the longest distance superficial to the longitudinal muscle before penetrating the muscle and terminating between the muscle layers. SNCC (green) cells move along extrinsic fibers to reach VNCC myenteric ganglia (red) where they become incorporated into the ganglia. Arrows inside scale bar indicate oral direction. Scale bar: (A, E) 50 μm, (B, C) 250 μm, (F) 25 μm, (D, G) not to scale.

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