Fig 2: 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 VGATCre/+; MafaFloxLacZ/+ 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 VGATcre/+; MafafloxLacZ/+pups) immunostained for ßgal (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+/ßgal+, 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).

Fig 3: Differences in PDX1 and MAFA levels contribute to islet signaling.a–c Ca2+ fluxes (a) in response to glucose (b) or glucose + KCl (c) are impaired in B-MAT islets, also shown by representative images (d) (n = 34 islets/4 animals; unpaired t-test) (scale bar = 40 µm). Inset in (a) shows an inverse correlation between glucose-stimulated Ca2+ amplitude and BFP expression in individual ß-cells (Pdx1-BFP; n = 6 islets/3 animals; R2 = 0.21, P < 0.0001) (G11, 11 mM glucose; KCl, 10 mM). e No diffeences in the % glucose-responsive ß-cells are detected in B-MAT islets (n = 34 islets/4 animals; unpaired t-test). f–h As for (a–c), but using Fura2 (n = 33 islets/4 animals; unpaired t-test). i Expression of genes encoding CACNA1D and CACNB2 Ca2+ channel subunits is reduced in B-MAT islets (n = 8 animals; paired t-test). j, k Ca2+ pulse duration is reduced in B-MAT islets, as shown by summary bar graph (j) and traces (k) (n = 8 islets/4 animals; unpaired t-test). l, m ATP/ADP ratios are reduced in B-MAT islets, as shown by mean traces (l) and summary bar graph (m) (n = 40 islets/4 animals; unpaired t-test). n, o GCK expression (n) tends to be reduced in B-MAT islets (n = 10 islets/2 animals; paired t-test), although Gck levels are normal (o) (n = 7 animals; paired t-test) (scale bar = 15 µm). p, q Ca2+ (p) and ATP/ADP (q) responses to increasing glucose concentration are decreased in B-MAT islets (Ca2+: n = 11 islets/5 animals; two-way ANOVA F = 20.36, DF = 4) (ATP/ADP: n = 37 islets/5 animals, two-way ANOVA; F = 6.10, DF = 4) (Bonferroni’s multiple comparison). r, s Mean traces (r) and bar graph (s) showing reduced cAMP levels in response to glucose and forskolin (FSK, 100 µM) in B-MAT islets (n = 13 islets; unpaired t-test). t Adcy8 expression remains unchanged in B-MAT islets (n = 6 animals; paired t-test). u G6pc2 and Ascl1 are up- and down-regulated, respectively, in B-MAT islets (n = 6 animals; paired t-test). Color scale shows Ca2+ as min (0%) to max (100%) value. Bar graphs and traces show the mean ± SEM. All tests are two-sided where relevant. CTCF-corrected total cell fluorescence.

Fig 4: MafA expression is strongly decreased in mice lacking p43. (A) Representative pictures of MafA expression (green) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth. (B) Quantification of beta-cells expressing MafA by islet (%) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth (n = 5 animals for each age and each genotype). (C) Quantification of relative MafA intensity by islet (%) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth (n = 5 animals for each age and each genotype). (D) Representative pictures of PDX1 expression (green) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth. (E) Quantification of beta-cells expressing PDX1 by islet (%) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth (n = 5 animals for each age and each genotype). (F) Quantification of relative PDX1 intensity by islet (%) in pancreatic sections from WT and p43-/- mice at 2 days, 7 days, 14 days and 3 months after birth (n = 5 animals for each age and each genotype). Statistical significance: ** p < 0.01; *** p < 0.001, Student’s t-test. Results are expressed as mean ± SEM.

Fig 5: Chemogenic activation of Mafa inhibitory neurons triggers apneas.a Genetic scheme to introduce the hM3Dq DREADD in Mafa+ inhibitory neurons. b Plethysmographic traces of the ventilation of a P1 MafaFlpo/+; VGATCre/+; RC::hM3Dq/+ pups before (top) and 30 min after a subcutaneous injection of CNO (5 mg/kg). Note the presence of a click-like upward pressure shift terminating the breath holding apnea. c Injection of CNO in P1-2 mutants caused a five-fold increase of the apneic time fraction (n = 12 pups, Two-way ANOVA comparing the CNO effect on mutants and on Cre+/+ siblings P = 0.001, df = 1, F = 14.7). This effect was usually reversible (n = 10/12 pups) but caused irreversible (n = 2/12 pups) apneic breathing (black traces filled and empty symbols) followed by death briefly after. d, e Experimental controls showing no effects of CNO on Cre+/+ pups (d, n = 10 and no effect of saline (PBS) on mutant pups (e, n = 4). f Quantification of the apneic time fraction of apnea types before and 60 min after injection of CNO showing significant increases for both types (n = 12 pups; breath holding: two-sided Wilcoxon test P = 0.0005 and central-like: two-sided Wilcoxon test P = 0.0005). Plots represent mean ± sem.