RTN neurons originate from Lbx1- and Egr2-expressing precursors. A–J, Transverse sections through E15.5 brains of Egr2^cre/+; Tau^GFPnLacZ (A–E) and Lbx1^cre/+; Tau^GFPnLacZ (F–J) embryos. A–D, F–I, Anti-β-galactosidase (blue, A and F), combined anti-Islet1,2 (nuclear labeling) and anti-TH (cytoplasmic labeling) (green, B and G), and anti-Phox2b (red, C and H) immunofluorescence. A white line delimits nVII, and a dotted line marks the ventral medullary surface. There are a few β-galactosidase⁺;Phox2b⁺;TH⁺ cells in the RTN from Egr2^cre/+; Tau^GFPnLacZ embryos (white arrows) and one Phox2b⁺; TH⁻;Islet1/2⁻ cell that is β-galactosidase-negative in the RTN from Lbx1^cre/+; Tau^GFPnLacZ embryo (white arrowhead). Asterisks, nonspecific signal from blood vessels. E, J, ISH with an Atoh1 probe (blue) followed by anti-β-galactosidase immunohistochemistry (orange). A black dotted line marks the ventral medullary surface, lateral is to the right.