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. Author manuscript; available in PMC: 2017 Sep 30.
Published in final edited form as: Science. 2017 Mar 30;355(6332):1411–1415. doi: 10.1126/science.aai7984

Fig. 1. Identification and genetic ablation of Cdh9/Dbx1 double positive neurons in preBötC.

Fig. 1

A, Cdh9 mRNA expression (blue) in section of E14.5 mouse embryo (11). Insets, ventrolateral medulla, ventral cerebellum. B, (Top) Cdh9 locus on chromosome 15 (numbers, distance from centromere). (Middle) BAC RP23-318N6. Vertical lines, Cdh9 exons. (Bottom) Cdh9-LOSL-DTR BAC transgene: insertion at Cdh9 start codon of mOrange sequence and polyadenylation (pA) signals, flanked by loxP sites (triangles), followed by DTR sequence. C,D, Ventrolateral medulla sections of P0 Cdh9-LOSL-DTR mouse immunostained for mOrange to show Cdh9 expression (red) and P0 wild type mouse immunostained for Somatostatin (SST, green), shown aligned (registered by compact nucleus ambiguus (NAC), cranial nerve 7 (nVII), and ventral brainstem surface) in sagittal plane (upper) and transverse projection (lower panels). d, dorsal; c, caudal; m, medial. Bar, 200 μm. E–I, preBötC of P0 Cdh9 LOSL-DTR (E,F) or Cdh9-LOSL-DTR;Dbx1-lacZ (G–I) mouse immunostained for Cdh9-mOrange (E–G,I, red), SST (E, green), NeuN (F, green) or beta-galactosidase (Dbx1-LacZ, H,I, blue). Among Cdh9-neurons, none co-expressed SST (n=43 cells), all co-expressed NeuN (n=57), and 56% co-expressed Dbx1 reporter (n=292, arrowheads). Bar (for E–I), 50 μm. J,K, Whole cell voltage clamp recordings (top, pA, picoAmp) of Cdh9/Dbx1 preBötC neurons in slice preparations (top) and simultaneous integrated cnXII activity (bottom). Neuron in J (neuron 1, Table S1) shows bursts in all inspiratory events (“inspiratory pattern”). Neuron in K (neuron 3) shows more widespread activity but bursts only during some events (“inspiratory-associated”). Bars, 500 msec. L, Schematic of ventrolateral medulla. Cdh9/Dbx1 neurons (blue border) intermingle with SST (green) and other Cdh9 neurons (red) in preBötC. M,N, Intersectional genetic labeling of Cdh9/Dbx1 preBötC neurons with DTR (immunostain, green) in ~P35 Cdh9-LOSL-DTR;Dbx1-cre mice before (M) and after (N) intraperitoneal DT injection to ablate them. Bar, 50 μm.