DEV031781 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Birth-dating of Isl1⁺ OM/Pou4f1⁺ RN neurons in WT and En1^+/Cre; Otx2^flox/flox embryos, and of serotonergic (5-HT⁺) neurons in En1^+/Cre; Otx2^flox/flox embryos. (A-X) Representative coronal midbrain sections of WT (A-P) and En1^+/Cre; Otx2^flox/flox (Q-X) embryos pulse-labeled with BrdU at E9.2 (A,E), E10.5 (B,F), E11.5 (C,G) or E12.5 (D,H) and sacrificed ∼2 days later at E12.5 (A,B,E,F), E13.5 (C,G) and E15.5 (D,H); or cumulative-labeled with BrdU (4 times every 3 hours) between E9.2-E9.7 (I,M,Q,U), E10.2-E10.7 (J,N,R,V), E11.2-E11.7 (K,O,S,W) or E12.2.-E12.7 (L,P,T,X) and sacrificed at E15.5 (I-X). In WT embryos, full colocalization of Isl1⁺ and BrdU⁺ cells was only seen after BrdU administration at E9.2, and of Pou4f1⁺ and BrdU⁺ cells only after BrdU administration at E10.5, indicating that Isl1⁺ OM neurons are born around E9.5, whereas Pou4f1⁺ RN neurons are born around E10.5. Compared with WT embryos, no difference was observed in the generation of Isl1⁺ OM neurons in the En1^+/Cre; Otx2^flox/flox mutants. Ectopic 5-HT⁺ neurons were generated between E10.2 and E11.7 in the conditional mutants.

• Supplemental Table S1 -

• Supplemental Figure S2 -

  Fig. S2. Isl1⁺ MNs are not induced ectopically in the caudal rhombomere 1 (r1) of En1^+/Wnt1 embryos at E12.5. (A-P) Representative sagittal (A-D) and coronal sections from the rostral (E,F,I,J,M,N) or caudal (G,H,K,L,O,P) r1 of WT (A,C,E,G,I,K,M,O) and En1^+/Wnt1 (B,D,F,H,J,L,N,P) embryos at E12.5, hybridized with a probe for Wnt1 (A-D) or immunostained for Otx2 (E,F) Otx2/Pitx3 (G,H), Shh (I-L) and Isl1 (M-P). At rostral r1 levels (red bar in B), ectopic Isl1⁺ MNs are found lateral to the ectopic Otx2⁺ medial FP in En1^+/Wnt1 mutants. At more caudal r1 levels (red bar in D), ectopic Isl1⁺ MNs are not detected in En1^+/Wnt1 embryos, although ectopic Pitx3⁺ mDA neurons (and Pou4f1⁺ RN neurons, see Fig. 3) are generated at this caudal level of the mutant rostral hindbrain.

• Supplemental Figure S3 -

  Fig. S3. Proliferation and apoptotic cell death are not affected in the VM of Nkx6-1^−/− embryos. (A-H) Immunodetection of phosphorylated Histone H3 (pH3; A-D) and of cleaved caspase 3 (cCasp3; E-H) on representative coronal midbrain sections of E9.0-9.5 (A,C), E10.0-10.5 (E,G) and E11.0 (B,D,F,H) WT (A,B,E,F) and Nkx6^−/− (C,D,G,H) embryos reveals no decrease of mitotic cells or increase of apoptotic cells in the mutants. (I-L) Detection of Neurog1 and Neurog2 on representative coronal midbrain sections does not show obvious differences between WT (I,K) and Nkx6-1^−/− (J,L) embryos at E12.5. (M) pH3⁺ cell numbers in the VM of E11.0 Nkx6-1^−/− embryos (mean±s.d: 6.911×10⁻⁵±1.17×10⁻⁴ cells/µm²; n=4) are similar to the WT (mean±s.d: 7.091×10⁻⁵±6.55×10⁻⁵ cells/µm²; n=4) (paired Student’s t-test, P=0.78). Broken lines in B and D indicate the approximate dorsal limit of the VM area used for pH3⁺ cell counting.

• Supplemental Figure S4 -

  Fig. S4. Ectopic Dbx1 expression starts at E10.5 in the Nkx6-1^−/− VM. (A-L) Representative coronal midbrain sections of WT (A,C,E,G,I,K) and Nkx6-1^−/− (B,D,F,H,J,L) embryos at E9.5 (A,B,E,F,I,J) and E10.5 (C,D,G,H,K,L). (A-D) Ectopic Dbx1 expression (arrows in D) in the Nkx6-1^−/− VM is first detected at E10.5. (E-H) Phox2a expression is unaffected in the mutants. (I-L) Dbx2 is not expressed in the WT midbrain and is not induced ectopically in the mutants.

• Supplemental Figure S5 -

  Fig. S5. Expression of Shh/Shh signaling-related genes, Lmx1a, Lmx1b, Wnt1 and Wnt5a, is not affected in the Nkx6-1^−/− VM. (A-P) Representative coronal midbrain sections of E12.5 WT (A,C,E,G,I,K,M,O) and Nkx6-1^−/− (B,D,F,H,J,L,N,P) embryos, showing no differences in Shh (C,D), Foxa2 (E,F), Nkx2-2 (G,H), Lmx1a (I,J), Lmx1b (K,L), Wnt1 (M,N), or Wnt5a (O,P) expression between WT and mutant embryos, although Pou4f1 (A,B) expression is strongly reduced in Nkx6-1^−/− embryos.

• Supplemental Figure S6 -

  Fig. S6. VM neurons are not re-specified into other neurotransmitter phenotypes in the absence of Nkx6-1. (A-H′) Representative coronal sections from the caudal midbrain of E12.5 WT (A,C,C′,E,E′,G,G′) and Nkx6-1^−/− (B,D,D′,F,F′,H,H′) embryos. (A,B) Ectopic induction of Dbx1 (arrows in B) in the mutant VM. (C-D′) The Vglut2⁺ domain does not appear to be altered in the Nkx6-1^−/− VM. (E-H′) Expression of Helt/Mgn (E-F′) and Gad1 (G-H′) does not overlap with the ectopically induced Dbx1 in the mutant VM. (C′-H′) Pseudo-colored overlays of consecutive sections hybridized with probes for Pou4f1 (light blue in C′,D′) or Dbx1 (light blue in E′-H′) and Vglut2 (C′,D′), Helt (E′,F′) or Gad1 (G′,H′) (red). Overlapping expression domains appear in dark blue.

• Supplemental Figure S7 -

  Fig. S7. Nkx6-1 controls the proper migration and neurotransmitter expression of Isl1⁺ OM/TN neurons at E12.5. (A-X) Representative coronal sections at consecutive anterior-posterior (ArP, left to right) levels of the midbrain/rostral hindbrain of E12.5 WT (A-D,I-L,Q-T) and Nkx6-1^−/− (E-H,M-P,U-X) embryos. (A-H) Some Isl1⁺ OM/TN neurons are ectopically positioned (arrows in E,F,H) in the Nkx6-1^−/− VM/rostral hindbrain, but do not intermingle with Th⁺ mDA neurons. (I-P) Ectopically positioned Isl1⁺ OM neurons are located within the strongly reduced Pou4f1⁺ RN domain. (Q-X) Normal expression of Phox2a (Q,R,U,V) but strong reduction of the cholinergic marker Vacht (S,T,W,X) in the E12.5 mutant VM. DA, dopaminergic neurons; OM, oculomotor nucleus; RN, red nucleus; TN, trochlear nucleus.

• Supplemental Figure S8 -

  Fig. S8. Nkx6-2 is not required for RN and OM neuron generation. (A-P) Representative coronal midbrain sections of WT (A,E,I,M), Nkx6-1^−/− (B,F,J,N), Nkx6-2^−/− (C,G,K,O) and Nkx6-1^−/−; Nkx6-2^−/− (D,H,L,P) embryos at E12.5 (A-H) and E18.5 (I-P). Pou4f1⁺ RN neurons are generated normally in Nkx6-2^−/− embryos (C,K) and still develop in Nkx6-1^−/−; Nkx6-2^−/− double mutants (D,L), albeit in similar reduced numbers (not shown) as in Nkx6-1^−/− embryos (B,J). Isl1⁺ OM neurons are not affected in Nkx6-2^−/− mice (G,O) but ectopically positioned (arrows in F,H) in the Nkx6-1^−/− (F,N) and Nkx6-1^−/−; Nkx6-2^−/− (H,P) VM.

• Supplemental Figure S9 -

  Fig. S9. Dorsal Dbx1 expression is shifted ventrally in the Nkx6-2^−/− midbrain. (A-H) Representative coronal midbrain sections of E12.5 WT (A,E), Nkx6-1^−/− (B,F), Nkx6-2^−/− (C,G) and Nkx6-1^−/−; Nkx6-2^−/− (D,H) embryos. (A-D) Ectopic expression of Otx2 (arrows in B,D) in the MZ of the Nkx6-1^−/− and Nkx6-1^−/−; Nkx6-2^−/− double mutant VM. Note that some Nkx6-2⁺ cells coexpress Otx2 (arrow in B). (E-H) Ectopic induction of Dbx1 (black arrows in F,H) in the Nkx6-1^−/− and Nkx6-1^−/−; Nkx6-2^−/− VM. The dorsal Dbx1⁺ domain in the lateral midbrain is shifted ventrally (red brackets in F,G) in Nkx6-2^−/− embryos.