Extended Data Fig. 9: Transcriptional and positional r4MN identity is disrupted in the cRE1^dup/+ embryonic hindbrain.

(a,b) Immunofluorescence of WT (left) and cRE1^dup/+ (right) E10.5 r4 axial hindbrain cryosections stained in (a) for ISL1 (blue), GATA2 (red), GATA3 (green), and in (b) for IS1 (blue), NR2F1 (red), GATA3 (green). In WT, a medial population of r4MNs excludes GATA2 and GATA3 expression (a, left, blue cells); in cRE1^dup/+, GATA2 and GATA3 expression overlaps extensively with ISL1 (a, right). A subset of medial r4MNs express NR2F1 in WT (b, left, arrowhead) but not in cRE1^dup/+ hindbrains (b, right). (c-n) E12.5 r4 axial hindbrain cryosections stained in (c-h) as in (a), and in (i-n) as in (b) on WT (left) and cRE1^dup/+ (right) sections. Compound (c,i) and single (d-f,j-l) channels. Arrows mark migrating IEEs (c-f). ISL1, GATA2, GATA3 midline r4MNs are expanded in number in cRE1^dup/+ hindbrains. ISL1^OFF;GATA2^ON;GATA3^ON interneurons form diffuse, bilateral columns overlapping r4MNs in WT and cRE1^dup/+ hindbrains. Midline r4MN clusters in (c) are enlarged in (g,h), and (i) in (m,n). Ventral IEEs are delineated from dorsal FBMNs by GATA2 and GATA3 expression in WT (g,h, left), but not in cRE1^dup/+ hindbrains (g,h right). GATA2^ON; ISL1^OFF cells in the FBMN compartment are likely interneurons (h, left, dorsal red cells). NR2F1 expression detected in WT FBMNs (m,n, left, purple cells) is decreased in cRE1^dup/+ midline r4 MNs (m,n, right). (o-r) E16.5 r4 (o,p) and r6 (q,r) axial hindbrain cryosections with staining as per (a,b) on WT (o-r, left) and cRE1^dup/+ (o-r, right) sections. GATA2 is downregulated in OCNs at this stage in both genotypes (o,q), and NR2F1 is detected in FBMNs but not IEEs (p,r). cRE1^dup/+ but not WT embryos have scattered ectopic ISL1^ON;GATA3^ON r4 neurons (o,p right vs. left). The r6 VEN population is enlarged and FBMN nuclei smaller in cRE1^dup/+ compared to WT (q, r right vs. left). Solid lines = anatomic borders of the hindbrain, dotted ovals encircle IEEs, dashed ovals encircle FBMNs. Open arrowheads = trigeminal motor neurons, asterisks in (q) = abducens nucleus. Scale bars = 200μm. Scale bar in (a) applies to (a,b); (c) applies to (c-f, i-l); (o) and (q) apply to (o,p) and (q,r), respectively. n = 3 (a-n) and 2 (o-r) embryos.