ISL1 (blue), NR2F1 (red) and GATA3 (green) immunofluorescent staining of hindbrain cross-sections at r4 (top row) and r6 (middle row) axial levels in E14.5 Gata3 flox/+ ;Phox2b-Cre − WT ( a , b ), Gata3 tlz/flox ;Phox2b-Cre + conditional Gata3 knockout ( c , d ), cRE1 dup/+ ; Gata3 flox/+ duplication ( e , f ) and cRE1 dup/+ ; Gata3 tlz/flox ;Phox2b-Cre + rescue ( g , h ) embryos. i , j , A rescue embryo with ISL1 (blue), GATA2 (red) and GATA3 (green) immunofluorescence (for WT and cRE1 dup/+ comparators, see Fig. 7a,c,e,g ). Dotted blue squares surround IEE OCNs in a , c , e , g and i and are magnified (bottom row). Dotted white squares marking the right facial nucleus are boxed in b , d , f , h and j and magnified (bottom row). Rescue embryos lack OCNs ( g , i ) and form an FBMN nucleus ( h , j ) intermediate in cross-sectional area between WT ( b ) and cRE1 dup/+ ( f ) embryos. White arrows in magnification of g highlight r4 ISL1 ON ;NR2F1 ON FBMNs. White open arrowheads show trigeminal motor neurons and asterisks the abducens nucleus. Dorsal and ventral borders of the hindbrain are outlined in gray. Scale bar, 200 μm in a applies to a – j ( n = 3 ( a , b ), 3 ( c , d ), 4 ( e , f ), 3 ( g , h ) and 5 ( i , j ) embryos).
