DEV027003 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Activation of the tOtx2^bov allele by En1-driven Cre recombinase in progenitors expressing En1. (A-F′′) Immunohistochemistry and in situ hybridization at the anatomical level corresponding to the posterior pretectum/anterior mesencephalon (A-C′′) and to the intermediate mesencephalon (D-F′′) of E10.5 En1^Cre/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov embryos to detect En1-driven Cre recombinase (A-A′′,D-D′′), GFP transcripts (B-B′′,E-E′′) and En1 mRNA (C-C′′,F-F′′) show that, at E10.5, GFP and consequently the transgenic Otx2 allele(s) have been activated in the progenitor domains of the posterior pretectal area and in the whole mesencephalon, even though at this stage En1-driven Cre recombinase and En1 mRNA are restricted to an area including the MHB. (G-I′′) Whole-mount in situ hybridization performed at E9.2 on En1^Cre/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov embryos with Cre (G-G′′), GFP (H-H′′) and En1 (I-I′′) probes shows that GFP and therefore the Otx2 transgene(s) are activated in the En1 domain, which at this stage includes also the presumptive ventral pretectal area (arrow). However, compared with En1^Cre/+; tOtx2^ov/ov mutants (H′′) the GFP expression is less pronounced in En1^Cre/+; tOtx2^ov/+ embryos (H′).

• Supplemental Figure S2 -

  Fig. S2. Otx2 overexpression generates mild abnormality at the MHB. (A-O) In situ hybridization performed on adjacent sagittal sections of E10.5 and E12.5 tOtx2^bov/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov embryos with Otx1 (A,F,K,D,I,N), Fgf8 (B,G,L,E,J,O) and Gbx2 (C,H,M) probes shows that at E10.5 in En1^Cre/+; tOtx2^ov/+ the expression of Otx1 and Fgf8 retains a fairly normal positioning, even though a few scattered spots of Fgf8 expression can be detected in the rostralmost hindbrain where the Otx2 overexpressing allele is ectopically activated; in En1^Cre/+; tOtx2^ov/ov a higher level of Otx2 overexpression induces a more pronounced posterior expansion of Otx1 and Fgf8 expression, whereas the rostral border of Gbx2 is apparently unaffected; at E12.5, the expression of Otx1 and Fgf8 in En1^Cre/+; tOtx2^ov/+ embryos is very similar to that of the tOtx2^ov/+ control, whereas the En1^Cre/+; tOtx2^ov/ov mutants exhibit only a slight enlargement of the Fgf8 domain. The arrow indicates the posterior border of Otx1 expression. Abbreviations: Mes, mesencephalon; Hb, hindbrain.

• Supplemental Figure S3 -

  Fig. S3. Otx2 overexpression induces selective expansion of the mdDA domain with increasing severity along the A-P axis. (A-L′) Immunohistochemistry performed at six sequential anatomical levels corresponding to the area that includes the pretectum and anterior mesencephalon, the intermediate mesencephalon and the posterior mesencephalon of E10.75 wild-type and En1^Cre/+; tOtx2^ov/ov embryos with Foxa2 and Nkx6.1 (A-F′) and Otx2 and Nkx2.2 (G-L′) shows that in the Otx2 overexpressing mutant, the Foxa2⁺-Nkx6.1^- mdDA domain (ventral to the arrow) gradually increases its extent, moving towards the posterior mesencephalon. The Foxa2⁺-Nkx6.1⁺-Nkx2.2⁻ (between arrow and arrowhead) and the Foxa2⁺-Nkx6.1⁺-Nkx2.2⁺ (dorsal to the arrowhead) domains, by contrast, are very similar in wild-type and mutant embryos, and are apparently not influenced by the Otx2 overexpression. Abbreviation: Ant., anterior.

• Supplemental Figure S4 -

  Fig. S4. Selective expansion of the mesDA domain is maintained at E12.5 in the intermediate and posterior mesencephalon of Otx2 overexpressing mutants, while lack of Otx2 remarkably affects the generation of mesDA neurons in the intermediate and posterior mesencephalon. (A-C′′) Immunohistochemistry performed on adjacent frontal sections through the intermediate mesencephalon of E12.5 tOtx2^bov/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov with Lmx1b and Nkx6.1 (A-A′′), Shh and Nkx6.1 (B-B′′), and Foxa2 and Nkx2.2 (C-C′′) shows that, although the expansion of the mesDA domain correlates with the level of Otx2 overexpression, the boundary relationships and the extent of the Nkx6.1⁺-Shh⁺-Foxa2⁺ (between the arrow and arrowhead) and the Nkx6.1⁺-Nkx2.2⁺-Foxa2⁺ domains (dorsal to the arrowhead) are very similar in mutant and control embryos. (D-G′) Immunohistochemistry performed on adjacent frontal sections through the anterior and intermediate mesencephalon of E12.5 wild-type and En1^Cre/+; Otx2^flox/flox embryos with TH (D,D′,F,F′) and 5-HT (E,E′,G,G′) shows that, in contrast to the intermediate mesencephalon, in the anterior mesencephalon the generation of mesDA neurons is moderately affected and 5-HT⁺ neurons are not generated. (H-L′) Immunohistochemistry performed on frontal sections through the pretectum/anterior mesencephalon, the intermediate mesencephalon and the posterior mesencephalon of E12.5 wild-type and En1^Cre/+; Otx2^flox/flox embryos with Pitx3 and Nkx6.1 shows that mesDA neuron generation is gradually impaired, moving towards the intermediate and posterior mesencephalon (J-L′) and that, as previously reported, Nkx6.1 expression is lost at this stage in the subventricular zone of the VM.

• Supplemental Figure S5 -

  Fig. S5. Reduced Qf in the mesDA domain of Otx2 over-expressing mutants. (A-F) Representative adjacent sections through the intermediate mesencephalon of E11.5 tOtx2^bov/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov administered with BrdU at E10.5, are immunostained with Ki67 and BrdU (A,C,E) and Nkx6.1 and AADC (B,D,F) to determine the Qf in the mesDA domain (AADC⁺-Nkx6.1⁻) and in the Nkx6.1⁺ territory. (G,H) Graphic representation of the Qfs shows that, compared with control embryos, in the mesDA domain but not in the Nkx6.1⁺ domain, the percentage of BrdU⁺ progenitors exiting the cell cycle is significantly reduced in response to the level of Otx2 overexpression. The arrow and the broken line (A-F) indicate the Nkx6.1⁺ territory analyzed.

• Supplemental Figure S6 -

  Fig. S6. Abnormalities in Wnt1 expression correlate with the mesencephalic territory showing impaired proliferation of mesDA progenitors by over-expression or lack of Otx2. (A-D′′) Wnt1 in situ hybridization performed on E10.5 tOtx2^bov/+, En1^Cre/+; Otx2^flox/flox and En1^Cre/+; tOtx2^ov/ov embryos at four sequential anatomical levels corresponding to the anterior (A-A′′), intermediate (B-C′′) and posterior (D-D′′) mesencephalon shows that in the rostral region of the mesencephalon the expression of Wnt1 is not remarkably affected by the overexpression or lack of Otx2, while moving towards the caudal mesencephalon, Wnt1 expression is gradually expanded or lost respectively in response to the overexpression or lack of Otx2.

• Supplemental Figure S7 -

  Fig. S7. Otx2 expression in the mesDA domain of wild-type embryos. (A-F′) Adjacent sections through the intermediate mesencephalon of E11.5 and E12.5 wild-type embryos immunostained with Otx2 (A-F′) and Nkx6.1 (A,A′), Sox2 (B,B′), Mash1 (C,C′), Ngn2 (D,D′), Nurr1 (E,E′) and Pitx3 (F,F′) show that Otx2 is fully co-expressed with Sox2, Mash1, Ngn2 and Nurr1 in mesDA progenitors and immature precursors, whereas not all of the Pitx3⁺ neurons exhibit Otx2 expression.