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. Author manuscript; available in PMC: 2021 Jun 7.
Published in final edited form as: Nat Neurosci. 2020 Dec 7;24(1):34–46. doi: 10.1038/s41593-020-00736-x

Figure 4. Assessment of IPAN characteristics in ENC6, 7 and 12.

Figure 4

a-c) Immunohistochemical detection of IPAN markers in ENC6, 7 and 12. d) Graph showing the proportions of cells among ENC6, 7 and 12 expressing IPAN markers. Each dot indicates one animal (n= 3-6). NF-M was detected in 63,6±5,43% of ENC6 (1326 cells), 82,5±7,42% of ENC7 (903 cells) and 100% of ENC12 neurons. CALB was found in 2,3±2,96% of ENC6 (1999 cells) and 14,9±4,76% of ENC7 (1588 cells) neurons and considered a defining marker of ENC12. Bar graphs show mean SD. We did not attempt to quanitify CGRP as faithful detection required colchicine treatment, compromising other marker expression. e-g) Representative examples of ENC6, 7 and 12 cell morphologies in Nmu-Cre;R26RTomato, Cck-IRES-Cre;AAV-DIO-Eyfp/Ruby3 mice and wildtype mice labelled with NTNG1/CALB, respectively. See Extended Data Fig. 6 for more examples. h) Quantification of neuron sizes in morphological groups of ENC6, 7 and 12. Each circle represents one cell. Box-and-whisker plots indicate max-min (whiskers), 25-75 percentile (boxes) with median as a centre line. Two-sided Student’s t-test was used for statistical analysis. ****p <0.0001. i) Quantification of proportions of different morphological types among ENC6, 7, and 12. Number of cells analysed (each from 2 animals): 689 ENC6 neurons; 440 ENC7 neurons; 321 ENC12 neurons. j-l) Cell bodies and projections originating from ENC6,7 and 12 are visible in the myenteric plexus plane. m-o) Immunohistochemical analysis in the circular muscle layer showing clear labelling of axons (PGP9.5+) and ICCs (ANO1+) but absence of ENC6, 7 and 12 axons. p) Abundance of motor neuron projections (NOS1+, ENK+ or CALR+) in the circular muscle layer. q-s) Transverse sections showing the direction of projections originating from NMU+, CCK+ and NTNG1/CALB neurons. Many NMU+ axons (but not CCK+ or NTNG1/CALB) were found to cross the circular muscle layer and project down into submucosa and villi. Stars indicate positive axons and dotted stripes demarcates the submucosal layer. Color channels were individually adjusted. Pictures show either myenteric peel preparations or transverse sections at P21-P90. Scale bars indicate 20μm in a-g, and 50μm in j-s. ANO1: Anoctamin 1; CALB: Calbindin; CALR: Calretinin; CCK: Cholecystokinin; CGRP: Calcitonin Gene Related Peptide; EYFP: Enhanced Yellow Fluorescent Protein; ENK: Enkephalin; HUC/D: ELAV-like Protein 3/4; ICC: Interstitial Cell of Cajal; NF-M: Neurofilament M; NMU: Neuromedin U; NOS1: Nitric Oxide Synthase 1; NTNG1: Netrin G1; PGP9.5: Neuron Cytoplasmic Protein 9.5; TOM: tdTomato, DAPI: 4′,6-diamidino-2-phenylindole