Fig 1.
Overexpressing Otx2 in the hindbrain leads ventrally to a specific expansion of the mdDA neuronal population.
Representative coronal (A-P) and parasagittal (Q-T) sections of midbrain and hindbrain regions of WT (A-H, Q-R) and En1+/Otx2 embryos (I-P, S-T). (A-C, I-K) In the ventral midbrain of WT and of En1+/Otx2 mutants, NKX6.1 and the RN as visualized by POU4F1 are laterally adjacent to the mdDA neurons. (F-H) In the ventral hindbrain of WT mdDA markers are not observed and the NKX6.1 shows an expression pattern typical for the rostal hindbrain. (N-P) In the ventral hindbrain of En1+/Otx2mutants the mdDA markers NURR1 and TH are present, but laterally not flanked by the RN or by the ventricular NKX6.1 expression domain (arrow). (D-E, L-M) In the dorsal midbrain of En1+/Otx2embryos, POU4F1 and the proliferation marker PHH3 are increased. (Q, R) In WT, Otx2 and Lmx1b expression extend until the isthmic constriction (indicated by arrow) marking the caudal border of the midbrain. In En1+/Otx2 mutants the caudally extended Otx2 expression domain leads to a concomitant caudal extension of the Lmx1b expression domain. (Scale bar, 100 μm).
Fig 2.
Otx2 requires Lmx1b for the development of mdDA neurons.
Representative coronal midbrain section of E12.5 WT (A-B, K-L), En1+/Otx2(C-D, M-N), Lmx1b-/- (E-F, O-P) and En1+/Otx2; Lmx1b-/- (G-H, Q-R) embryos. (I-J) Quantification of TH+ neurons and ratio of TH+-NURR1+neurons in different gentypes. (C-D) In En1+/Otx2 embryos the number of TH+ and NURR1+ neurons is increased. (E, F) In Lmx1b-/- embryos, Otx2 is downregulated and TH+ neurons are reduced in the medial region of the mdDA precursor domain and missing in the lateral domain (dotted line indicate border between medial and lateral region). (G, H) In En1+/Otx2;Lmx1b-/- mutants Otx2 is not sufficient to induce the terminal differentiation of NURR+TH- cells to fully differentiated NURR+TH+ mdDA neurons and to rescue the lateral TH+ mdDA neurons. (I) Compared to the number of TH+ neurons in WT, the number of TH neurons in En1+/Otx2 mutants are significantly increased and decreased in Lmx1b-/- and En1+/Otx2; Lmx1b-/- embryos. (K, L) In WT there is a clear border between the mdDA neurons and the POU4F1 and LIM1/2 expression domain. (M, N) In En1+/Otx2 mutants the border is maintained, but the mdDA neuronal population is expanded laterally, without affecting the size of the RN. (O, P) In Lmx1b-/-, POU4F1+ and LIM1/2+ cells are mixed with NURR1+ neurons. (Q, R) In En1+/Otx2;Lmx1b-/- mutants Otx2 did not re-establish the lateral border of the mdDA precursor field. (Scale bar, 100 μm).
Fig 3.
Otx2 does not require Lmx1b for the induction of OMNs.
Representative coronal midbrain section of E12.5 WT (A, E, I, M, Q, U), En1+/Otx2 (B, F, J, N, R, V), Lmx1b-/- (C, G, K, O, S, W) and En1+/Otx2;Lmx1b-/- (D, H, L, P, T, X) embryos. In WT (A) and En1+/Otx2 (B) mutants OMNs are present as visualized by ISLET1. In Lmx1b-/- (C) ISLET1 expression is missing. In En1+/Otx2;Lmx1b-/- (D) the restored Otx2 expression leads to a significant rescue of OMNs, as indicated by ISLET1 expression. (E-H, I-L) In all genotypes the patterning of the ventral midbrain is unperturbed as indicated by normal expression of NKX2.2 and NKX6.1. (M-P) En1+/Otx2 mutants show an increase in proliferation in the mdDA precursor domain as marked by an increase in PHH3. In contrast Lmx1b-/- and En1+/Otx2;Lmx1b-/- mutants do not show any abnormalities in the expression of PHH3. (Q-T) Apoptosis as visualized by CASPASE 3 expression was normal in all genotypes. (U-X) Trigeminal ganglia showing strong CASPASE 3 expression were used a positive control. (Scale bar, 100 μm).
Fig 4.
In Lmx1b-/- embryos NURR1+ neurons express LMX1A and FOXA2 but not EN1.
Representative coronal midbrain section of E12.5 WT (A, E, I, M), En1+/Otx2(B, F, J, N), Lmx1b-/- (C, G, K, O) and En1+/Otx2; Lmx1b-/-(D, H, L, P) embryos. LMX1A (A-H) and FOXA2 (I-L), which are important for the formation of the mdDA precursor domain do not show any changes in expression in Lmx1b-/- or in En1+/Otx2;Lmx1b-/- embryos. In contrast EN1 (O) is lost in NURR1+ neurons in Lmx1b-/- mutants. In En1+/Otx2;Lmx1b-/- embryos (P), Otx2 is not sufficient to rescue the EN1 expression lost in Lmx1b-/-. (Scale bar, A-D, I-P, 100 μm, E-H 6 μm).
Fig 5.
Wnt1 and Fgf8 expression is induced but not maintained in Lmx1b-/- embryos.
Representative sagittal sections of the mesencephalic flexure (A-C, A'-C', J-L, J'-L') and the lateral (D-F, D'-F', M-O, M'-O') and dorsal (G-I, G'-I', P-R, P'-R') aspect of the MHO of E9.5 (A-I, A'-I') and E10.5 (J-R, J'-R') WT (A-R) and Lmx1b-/- (A'-R') embryos. Genes were visualized by radioactive mRNA in situ hybridization. (B, B') At E9.5 Wnt1 expression is normally induced in the MF of Lmx1b-/-. (D'-E', G'-H') At E9.5, Fgf8 and Wnt1 expression are reduced but present in Lmx1b-/- embryos at the lateral and dorsal aspect of the MHO. (C, C', F, F', I, I') Overlay of adjacent sections indicate that Fgf8 is expressed in Lmx1b-/- as in WT directly posterior to the Otx2 expression domain. (K, K') At E10.5 Wnt1 expression is maintained at the MF of Lmx1b-/-. (M'-N', P'-Q') At E10.5, expression of Fgf8 and Wnt1 are lost in the lateral MHO domain, but still present in the dorsal region of the MHO. (L-R, L'-R') Overlay of adjacent sections indicate that Fgf8 is expressed in Lmx1b-/- as in WT directly posterior to the Wnt1 expression domain. (Scale bar, 250 μm).
Fig 6.
Lmx1b-/- embryos show a specific spatio-temporal loss of En1 and Wnt1 expression.
Representative sagittal sections of the mesencephalic flexure of WT (A-R) and Lmx1b-/- (A'-R') embryos at E11.5 (A-I, A'-I') and E12.5 (J-R, J'-R'). Genes were visualized by radioactive mRNA in situ hybridization. (A-F, A'-F') At E11.5, En1 and Wnt1 expressions are lost in Lmx1b-/- embryos at the rostrolateral aspect of the mesencephalic flexure (arrow) and only the caudomedial expression domain is detectable (arrow head). (G-I, G'-I') The expression of Lmx1a is similar between the WT and the Lmx1b-/-, including the rostrolateral domain (arrow). (J-L, J'-L') At E12.5, the caudomedial En1 expression domain is lost in Lmx1b-/-, while the hindbrain En1 expression is still present. (P-R, P'-R') Th expression is significantly reduced. (M-O, M'-O'). No significant difference was found in the expression of Nurr1. Red dotted line represent boundary between midbrain and hindbrain. (Scale bar, 250 μm).