Fig. 1: Phox2b-expressing RTN neurons preferentially express Kcnq2 but not Kcnq3 channels. | Nature Communications

Fig. 1: Phox2b-expressing RTN neurons preferentially express Kcnq2 but not Kcnq3 channels.

From: Phox2b-expressing neurons contribute to breathing problems in Kcnq2 loss- and gain-of-function encephalopathy models

Fig. 1

Coronal sections from Phox2Cre/+::Ai14 reporter mice were obtained for multiplex in situ hybridization analysis of Kcnq2 and Kcnq3 transcript expression in for Phox2b-expressing brainstem populations. A Photomicrographs of coronal sections from the RTN, 7N, NTS, and LC show tdT-labeled Phox2b+ neurons (yellow) in the RTN express Kcnq2 transcript (cyan) but low Kcnq3 signal (magenta). Insets (right), shows fluorescent in situ hybridization results for the boxed region at a higher magnification where Kcnq2, Kcnq3, and Phox2b-tdT (yellow), the DAPI (blue). Signal was filtered to improve visualization. White arrows identify cells that express Kcnq2 but not Kcnq3, red arrows identify cells that co-express both transcripts, and yellow arrows designate cells that express Kcnq3 but not Kcnq2. Scale bars are 100 µm and 50 µm (insets). B Summary of fluorescent in-situ hybridization results (n = 3 mice, 30–40 days postnatal) show that 90% of tdT-Phox2b labeling in the ventral parafacial region co-localized with Kcnq2 labeling (white) and of these only 24% also showed Kcnq3 labeling (yellow), whereas 10% of tdT-Phox2b neurons in this region lacked both Kcnq2 and Kcnq3 signal (red). In contrast to the RTN, the majority of Kcnq2 signal detected in the 7N, LC and NTS co-localized with Kcnq3 labeling and in some cases only Kcnq3 labeling was observed (cyan). C Summary data plotted as mean ± maximum and minimum show the proportion of Phox2b-expressing cells per slice that express Kcnq2 transcript in the absence of Kcnq3 in the RTN (n = 66 slices, red), 7N (n = 17 slices, white), NTS (n = 23 slices, white), LC (n = 16 slices, white). Significance was determined using the Kruskal-Wallis test; RTN vs NTS (p < 0.0001), RTN vs LC (p < 0.0001), RTN vs. 7N (p < 0.0001). These results show that Phox2b-expressing neurons in the ventral parafacial region have a higher proportion of Phox2b+ neurons that uniquely express Kcnq2 mRNA than other medullary Phox2b-expressing neural populations. 7N, facial motor nucleus; LC, locus coeruleus; nucleus tractus solitarius (NTS).

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