Consistent with the observation

that Atoh1 is essential for the formation of RL descendants Erastin ( Machold and Fishell, 2005; Wang et al., 2005), RL-derived Atoh1 populations in the ventral medulla, including the lateral reticular nucleus (LRt) and spinal trigeminal neurons (Sp5I), were virtually abolished in the Atoh1 null brainstem at E18.5 ( Figure 1, compare C to B). In contrast, Atoh1 null mice still retain the RL-independent RTN neurons, but the somas cluster at the dorsal surface of nVII, likely as a result of a migration defect (white arrowheads) ( Figures 1B and 1C). Moreover, the closely localized nVII neurons, which do not express Atoh1, show normal marker expression and localization ( Figures S1A and S1B available online), suggesting their development is Atoh1 independent. During embryonic development, the RTN neurons migrate radially to assume their final location around the nVII, with the majority of them lining the ventral medullar surface (Dubreuil et al., 2009; Rose et al., 2009b). In Atoh1 null mice, the mislocalized RTN neurons retain expression of lineage markers such as Phox2b and ladybird homeobox homolog 1 (Lbx1), similar to WT mice ( Figures 1D and 1E), indicating that their lineage identities are unchanged. This defect is different from the CCHS mouse model, in which these neurons do not form ( Dubreuil et al., 2008). selleck chemicals llc We then stained for myristoylated

GFP to ask whether loss of Atoh1 affects neuronal connectivity of lower brainstem circuitry. In unless the preBötC region (orange dotted circled neurons marked by somatostatin, Sst) of the E18.5 WT brainstem ( Figure 1F), we detected neuronal processes extending from both rostral (white open arrowheads) and caudal (white arrowheads) Atoh1 populations. The rostral neuronal bundles correspond to the pontine Atoh1 respiratory populations and the RTN neurons, while the caudal processes belong predominantly to the LRt neurons ( Abbott et al., 2009; Rose et al., 2009a, 2009b). This early connectivity is consistent with connectivity in adult

rodents and functional connectivity occurring prior to the onset of inspiratory behaviors in utero ( Feldman and Del Negro, 2006). In the Atoh1 null brain, the preBötC received little to no Atoh1-dependent rostral and caudal inputs ( Figure 1G). Notably, neurites of the mislocalized RTN neurons accumulate at the dorsal side of nVII and do not extend to the preBötC. This suggests that Atoh1 null RTN neurons not only mislocalize but also lack direct targeting to the primary breathing center. In an effort to identify the Atoh1 subpopulations critical for neonatal survival, we applied conditional knockout strategies. We have previously shown that removal of Atoh1 using a HoxB1Cre allele that covers all tissues caudal to the rhombomere 3/4 boundary results in 50% neonatal lethality ( Maricich et al., 2009).