In situ hybridization and immunohistochemistry was performed as previously described (Philippidou et al., 2012 (link); Vagnozzi et al., 2020 (link)), on tissue fixed for 2 h in 4% paraformaldehyde (PFA) and cryosectioned at 16 μm. In situ probes were generated from e12.5 cervical spinal cord cDNA libraries using PCR primers with a T7 RNA polymerase promoter sequence at the 5’ end of the reverse primer. All probes generated were 750–1000 bp in length. Wholemounts of diaphragm muscles were stained as described (Philippidou et al., 2012 (link)). The following antibodies were used: rabbit anti-cleaved Caspase 3 (1:1000; Cell Signaling, RRID:AB_2341188 ), mouse anti-olig2-A488-conjugated (1:500; Millipore, RRID:AB_11205039 ), rabbit anti-β-Catenin (1:250; Cell Signaling Technology, RRID:AB_11127203 ), goat anti-Scip (1:5000; Santa Cruz Biotechnology, RRID:AB_2268536 ), mouse anti-Islet1/2 (1:1000, DSHB, RRID:AB_2314683 ) (Tsuchida et al., 1994 (link)), rabbit anti-neurofilament (1:1000; Synaptic Systems, RRID:AB_887743 ), rabbit anti-synaptophysin (1:250, Thermo Fisher, RRID:AB_10983675 ), and α-bungarotoxin Alexa Fluor 555 conjugate (1:1000; Invitrogen, RRID:AB_2617152 ). Images were obtained with a Zeiss LSM 800 confocal microscope and analyzed with Zen Blue, ImageJ (Fiji), and Imaris (Bitplane). Phrenic MN number was quantified using the Imaris “spots” function to detect cell bodies that coexpressed high levels of Scip and Isl1/2 in a region of interest limited to the left and right sides of the ventral spinal cord.
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