Fig. 4: Absence of Mafa inhibitory neurons in sensory and pontine respiratory structures and presence of Mafa inhibitory premotor neurons controlling the geniohyoid muscle.

a Left, transverse sections of P0 wild type pups showing the vagal jugular and nodose ganglia stained for DAPI (white) and by RNAscope using a probe against Mafa (red). Right, top, close-up views from the jugular ganglion probing Mafa (red), VGlut2 (green), Phox2b (blue) and the merge. Right, bottom, close-up views from the nodose ganglion. b Transverse sections of the trigeminal ganglion stained for DAPI (white) and Mafa (red). Right, close up views of the inset in (b) probing Mafa (red), VGlut2 (green) and Gad1&Gad2 (blue). Note the absence of Mafa expression in the nodose ganglion, expression of Mafa in the jugular (sparse) and trigeminal ganglia restricted to VGlut2-expressing excitatory neurons. c Summary of RNAscope probing for co-expression of Mafa/VGlut2- and Mafa/Gad1&2-expression in vagal (5 sections, n = 3 ganglia) and trigeminal (7 sections, n = 3 ganglia) ganglia, nucleus tractus solitarius (nTS, 8 sections, n = 2 pups) and peri12 (8 sections, n = 2 pups). Plots represent mean ± sem. d Left, transverse hemi-section of the brainstem at pontine level of a VGAT^Cre/+; Mafa^FloxLacZ/+ P8 pup, showing absent Mafa⁺ inhibitory neurons (black) in the PB/KF, Pr5 areas but their presence in the SPO and DLL. Right, representative transverse hemi-section of the brainstem at medullary level showing the presence of Mafa⁺ inhibitory neurons in the peri12, peri-nAmb but their virtual absence in the Sp5, Pa5 and nTS (n = 2 pups). e Monosynaptic tracing scheme showing unilateral injection of G-deleted Rabies viruses encoding mCherry and of a helper G- and YFP-encoding HSV virus into the geniohyoid muscle to transynaptically trace the position of premotor neurons. f Representative transverse section (from n = 4 VGAT^cre/+; Mafa^floxLacZ/+pups) immunostained for βgal (white) in the caudal medulla showing mCherry⁺ virally labeled premotor neurons (red) and rare (at this axial level) seeding mCherry⁺/YFP⁺ geniohyoid motoneurons (yellow) in the accessory hypoglossal motor nucleus (arrowhead). g Close-up view of the inset in f showing that a fraction of mCherry⁺ premotor neurons indicated by arrows are Mafa⁺ (mCherry⁺/βgal⁺, white nuclear labeling). h 2D-reconstruction of the position of Mafa⁺ geniohyoid premotor neurons (red dots) and Mafa⁺ cells (gray dots) showing their location in the peri12 area. DLL dorsal nucleus of the lateral lemniscus, KF Kölliker-Fuse nucleus, PB parabrachial nuclei, Pa5 paratrigeminal nucleus, Pr5 principal sensory trigeminal nucleus, scp superior cerebellar peduncle, SPO superior paraolivary nucleus. Scale bars (µm): 100 (a, b left, c), 50 (a, b right), 1000 (d), 500 (f, h), 100 (inset).