, 2011). The paratrigeminal (pTRI) neurons surrounding the trigeminal motor nucleus (nV) also express Atoh1, Phox2b, and Lbx1 ( Figure S2A). They are also targeted by the Phox2bCre allele ( Figure S2B) and showed Atoh1-independent lineage specification ( Figure S2C). However, unlike the RTN neurons, the pTRI neurons do not require Atoh1 for proper localization, as shown by marker analyses ( Figures S2D and S2E) and
cell number quantification ( Figure S2F) from serial sections. Phox2bCre-mediated conditional knockout do not affect RL-derived Atoh1 neurons, as mRNA in situ hybridization ( Figure 4A) and fate mapping analyses ( Figures 4B–4E) showed that Atoh1 expression and the development of RL populations are normal in the Selleck Cilengitide Atoh1Phox2bCKO mice. We conclude that Atoh1Phox2bCKO mice show a selective RTN mislocalization phenotype while the rest of the RL-derived Atoh1 populations remained unaffected. We monitored the Selleckchem BGB324 birth of conditional mutants and discovered that although the birth rate of all genotypes conformed to Mendelian ratios, 43% (20/46) of Atoh1Phox2bCKO mice died within the first hour after birth; none of the other genotypes showed postnatal lethality. We were surprised to find erroneous RTN migration in surviving Atoh1Phox2bCKO mice, similar to the mice that died
at P0 ( Figures S3A–S3D), suggesting that loss of Atoh1 increases respiratory vulnerability specifically during the newborn period. To determine whether the RTN and caudal HoxA4-derived Atoh1 neurons affect newborn viability synergistically, we generated Phox2bCre; HoxA4Cre-mediated Atoh1 mutant animals, which showed neonatal lethality (52%, 9/17) not significantly different from that of Phox2bCre alone (two-tailed p value = 0.4477, Fisher’s exact test). Taken together, we conclude that Atoh1-mediated development of the RTN neurons is critical for neonatal respiratory fitness. To ascertain whether loss of Atoh1 in the RTN has a direct effect on the respiratory rhythm-generating networks right before birth, we recorded the inspiratory activity from razoxane the C4 root of E18.5 brainstem-spinal cord
preparations. Interestingly, the baseline fictive respiratory frequency of the Atoh1Phox2bCKO mice was significantly slower than that of their WT littermates (Atoh1Phox2bCKO: 37.44% ± 2.48%, n = 5, versus WT: 100% ± 22.24%, n = 9, p < 0.05) ( Figure 5A). To test the response of respiratory circuit to excitatory neuropeptides, we recorded the inspiratory activity of WT and Atoh1Phox2bCKO brainstems 5 min before and after 1 μM Substance P (SP) treatment ( Figure 5B). The Atoh1Phox2bCKO mice show consistently depressed baseline motor activity when compared with WT (Atoh1Phox2bCKO: 23.45% ± 5.60%, n = 5, versus WT: 100% ± 34.61%, n = 6, ∗p < 0.05, paired t test). Interestingly, SP application significantly increased the motor activity of WT (174.