Figure 5. Loss of DL Subnucleus FMNs Positional and Transcriptional Identities in Etv1 Mutant Mice.

(A–D) Facial nucleus cytoarchitecture in E17.5 Etv1^nlz/+ (A and C) and Etv1^nlz/nlz (B and D) embryos as defined by Isl1 ISH (A and B) and ISL1 immunostaining, blue in (C) and (D). An anatomically distinct DL subnucleus is absent from Etv1 mutant embryos, arrows in (B) and (D); and the β-gal reporter marking DL FMNs in Etv1^nlz/+ embryos, green in (C), is absent from Etv1-mutant FMNs (D). The location of FOXP1^ON I FMNs (red in C and D) is unchanged in Etv1 mutants. Green puncta, in (C) and (D), are non-specific background binding of the anti β-gal antibody.

(E and F) Immunofluorescent detection of cleaved caspase-3 apoptosis marker (CASP3, green) in control (E) and Etv1 mutant (F) ISL1^ON (red) FMNs at E14.5.

(G) Ratio of CASP-3^ON, ISL1^ON to CASP-3^OFF, ISL1^ON FMNs detected in Etv1^nlz/nlz and control E14.5 embryos in (E) and (F) represented as means ± SE, n = 3 Etv1 mutant (208 FMNs) and control (238 FMNs) littermate pairs (unpaired t test, p = 0.7639).

(H and I) Retrograde labeling (green cells, white arrows) of DL FMNs in neonatal control (H) and Etv1^nlz/nlz (I) mice with Rh-Dx application to the transected Z/T nerve branch.

(J) Similar numbers of DL FMNs are marked in neonatal control and Etv1^nlz/nlz mice, represented as ISL1^ON FMNs and Rh-Dx-labeled FMNs means ± standard error. n = 3 mice for each genotype: 139 Etv1 mutant and 152 control DL FMNs (unpaired t test, p = 0.4769).

(K) Rh-Dx-labeled OO FMNs assigned to one of four mediolateral positional bins. OO FMNs are more broadly distributed in Etv1 mutant mice than they are in littermate controls, represented as the percentage of OO FMNs assigned to each bin ± SEM. n = 3 Etv1^nlz/nlz and control littermate pairs, 121 and 114 OO FMNs analyzed, respectively, from the two genotypes (unpaired t test, *p < 0.005, **p < 0.0005).

(L–S) Expression of the motor neuron marker Isl1 (L); the class 1 DL markers Etv1 (M), Cntn3 (N), and Sostdc1 (O); class 2 DL markers C1ql1 (P), and Gria3 (Q); and the class 3 DL markers Ldb2 and Alcam in control (upper panels) and Etv1^nlz/nlz (lower panels) facial nuclei at E16.5. Black arrowheads in (M)–(R) indicate DL motor-pool markers present in wild type and their absence from Etv1 mutant, facial motor nuclei; arrows in (P)–(S) indicate FMN marker expression maintained in Etv1 mutants. Open arrowheads in (S) mark Alcam^ON FMNs ectopically occupying the L subnucleus.

Scale bars: 100 μm in (A–D) and (L–S); 50 μm in (E) and (F); and 200 μm in (H) and (I), n ≥ 3 mice for each gene.

See also Figures S3 and S4.