DEV010082 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Prospective prethalamus is still present at the 6- to 8-somite stage in the Six3-null brain. Otx1 is expressed in the mesencephalon and prospective diencephalon (A) of the wild-type embryo at the 6- to 8-somite stage. Otx1 expression is expanded in the anterior neural fold of the Six3-null brain at this stage (A′). (B) Six3^+/− and (B′) Six3^−/− embryos stained with S-Gal to detect the presence of lacZ in the Six3⁻ allele (red) (Lagutin et al., 2003) and Irx1 (blue). Irx1 expression is observed in the mesencephalon and prospective caudal diencephalon of the wild-type brain (B) and is expanded in the anterior neural fold of the Six3-null brain (B′). Arrows in A-B′ point to the anterior limit of expression. Lhx5, Arx1, Fezf1 and Fezf2 are expressed in the prospective prethalamus of the wild-type brain (arrows, C-F), but dorsal expression of these markers is lost in the Six3-null brain (arrows, C′-F′) at these stages. (G) Nkx2.1 is expressed in the prospective prethalamus and hypothalamus. (H) Six6 is expressed in the anterior hypothalamus of the wild-type brain. Nkx2.1 is reduced (G′) and Six6 expression is absent (H′) in the Six3-null brain.

• Supplemental Figure S2 -

  Fig. S2. Irx3 and Tcf4 expression expands into the most-anterior part of the Six3-null brain at the 22- to 24-somite stage. Sagittal sections of whole-mount in situ hybridizations shown in Fig. 1I′,K′. (A) Irx3 is expressed in the caudal diencephalon and midbrain of the wild-type brain. (B) Tcf4 is expressed in the caudal diencephalon and ventral prethalamus. Irx3 (A′) and Tcf4 (B′) are expressed in the most-anterior region of the Six3-null brain (arrows) at this stage. PT, prethalamus; T, thalamus.

• Supplemental Figure S3 -

  Fig. S3. Lhx2 expression is not detected in the anterior part of the Six3-null brain. Sagittal sections of E12.5 wild-type (A,E) and Six3-null (B,F) brains and E14.5 Wnt1-null (C,G) and Six3;Wnt1 double-mutant brains (D,H). (A,D) DAB immunohistochemistry with a goat anti-Tcf4 antibody (Santa Cruz, Santa Cruz, CA) (brown). (E-H) Double DAB immunohistochemistry with Tcf4 (brown) and Lhx2 in situ hybridization (blue). Lhx2 is expressed only in the border between the tegmentum and the pretectum of the Six3-null mutant brain (F). PTC, pretectum; T, thalamus.

• Supplemental Figure S4 -

  Fig. S4. The thalamus is absent in the anterior region of the Six3-null brain at late embryonic stages. Sagittal sections of E14.5 wild-type (A,C,E) and Six3-null (B,D,F) brains. Gbx2 (A), Ngn2 (C) and Prox1 (E) are expressed in the wild-type thalamus (T). No thalamic expression of these markers was detected in the anterior region of the Six3-null brain at this stage. PT, Prethalamus.

• Supplemental Figure S5 -

  Fig. S5. The subcommisural organ (SCO) is expanded dorsally in the Six3-null brain. Sagittal sections of wild-type (A-G) and Six3-null (A′-G′) brains The SCO is a secretory gland of ependymal cells located in the anterior part of the pretectum, between the pineal gland and the sylvian aqueduct (asterisk, A). At E14.5, the SCO is composed of a lower layer of ependymal cells (B) that are Otx2 (asterisk, C) and vimentin (asterisk, D) positive, with the fibers of the posterior commisure that are Gap43-positive on the top (asterisk, E). The SCO secretes the glycoprotein SCO-spondin (detected by in situ in F,G). In the Six3-null, the SCO was located in the most-anterior part of the brain (A′-G′). (H) The SCO length was nearly twice that of the wild-type littermate controls. Length estimated as number of cells, and length in arbitrary units; number of microns=value×20; blue, wild-type; red, Six3^−/−. n=9, **P<0.01. Rabbit Otx2 antibody (1:2000). Guinea pig vimentin antibody (1:500; RDI, Concord, MA). Anterior is to the left.

• Supplemental Figure S6 -

  Fig. S6. Pretectum identity is not established in the anterior region of the Six3-null brain until E11.0. Lim1 in situ hybridization of sagittal sections of 25- to 30-somite stage (E10.0), 35- to 40-somite stage (E11.0) and E12.5 wild-type and Six3-null embryos. Lim1 is expressed in the pretectum of the wild-type brain (A) but not in the anterior region of the Six3-null brain (B) at the 25- to 30-somite stage. Lim1 is expressed in both the pretectum and the anterior region of the Six3-null brain at the 35- to 40-somite stage (E11.0) (C,D). A similar result was observed at E12.5 (E,F). HY, hypothalamus; PTC, pretectum.

• Supplemental Figure S7 -

  Fig. S7. Proliferation and apoptosis are normal in the anterior region of the Six3-null brain at the 25- to 30-somite stage. Coronal sections of the wild-type thalamus (C,F) and pretectum (D,G) and the anterior region of the Six3-null brain (E,H) illustrating the number of TUNEL⁺ cells (C-E) and BrdU⁺ cells (F-H). Sections were counterstained with DAPI. Red lines on A and B show the level of section. Asterisks show the position of blood cells stained with FITC. Arrows point to apoptotic cells. TUNEL⁺ or BrdU⁺ cells were counted, and their number per the number of DAPI-stained cells was calculated. Results from counting TUNEL⁺ cells (I), and from counting BrdU⁺ cells (J). The numbers of TUNEL⁺ cells (mean, 63.53/1000 DAPI⁺ cells; s.d., 8.57) and BrdU⁺ cells (mean, 41.64/100 DAPI⁺ cells; s.d., 4.96) in the Six3-null brain were comparable to those of TUNEL⁺ cells (mean, 59.40/1000; s.d., 16.66; P=0.2995) and BrdU⁺ cells (mean, 42.95/100 DAPI⁺ cells; s.d., 2.87; P=0.3135) in the wild-type pretectum (n=6 samples from 3 embryos). Both the anterior region of the Six3-null brain and the pretectum of the wild-type brain have a significantly higher number of TUNEL⁺ cells than the thalamus of the wild-type brain (mean, 1.70/1000 DAPI⁺ cells; s.d., 0.57; P=6.3035×10⁻⁵ and P=0.0011, respectively) (n=6 samples from 3 embryos). By contrast, we observed that the thalamus has more BrdU⁺ cells (mean, 57.27/100 DAPI⁺ cells; s.d., 3.0636) than the pretectum (P=0.003) of the wild-type brain and the anterior region of the Six3-null brain (P=0.0026). T, thalamus; PTC, pretectum.

• Supplemental Figure S8 -

  Fig. S8. Proliferation and apoptosis are normal in the anterior region of the Six3-null brain at the 35- to 40-somite stage (E11.0). Coronal sections of the thalamus (C,F) and pretectum (D,G) of the wild-type brain and the anterior region of the Six3-null brain (E,H) illustrating the number of TUNEL⁺ cells (C-E) and BrdU⁺ cells (F-H). Sections were counterstained with DAPI. Red lines on A and B show the level of section. Asterisks point to blood cells stained with FITC. Arrows point to apoptotic cells. The numbers of TUNEL⁺ or BrdU⁺ cells per DAPI-stained cell were counted. Results from counting TUNEL⁺ cells (I), and from counting BrdU⁺ cells (J). The number of TUNEL⁺ cells in the anterior region of the Six3-null brain (mean, 4.19/1000 DAPI⁺ cells; s.d., 1.30) is significantly lower than that in the pretectum (mean, 6.52/1000 DAPI⁺ cells; s.d., 2.48; P=0.0188) (n=8 samples from 3 embryos). Both structures have fewer TUNEL⁺ cells than the thalamus of the wild-type brain (mean, 9.29/1000 DAPI⁺ cells; s.d., 2.42; P=0.0012 for the anterior region of the Six3-mutant brain, and P=0.0406 for the pretectum of the wild-type brain) (n=8 samples from 3 embryos). However, the reduced number of TUNEL⁺ cells in all of these structures is too small to suggest any important role of cell death in their development at this stage. The proliferation level, as indicated by the number of BrdU⁺ cells, is similar between the pretectum of the wild-type brain (mean, 34.62/100 DAPI⁺ cells; s.d., 3.5643) and the anterior region of the Six3-null brain (mean, 33.30/100 DAPI⁺ cells; s.d., 2.23; P=0.2099) (n=8 samples from 3 embryos). Again, the number of BrdU⁺ cells is lower in these later two structures when compared with the thalamus of the wild-type brain (mean, 64.41/100 DAPI⁺ cells; s.d., 7.8670; P=8.2892×10^-5 and P=0.0002, respectively) (n=8 samples from 3 embryos). T, thalamus; PTC, pretectum; TG, tegmentum.

• Supplemental Figure S9 -

  Fig. S9. Shh expression is detected in the putative ZLI region of the Six3-null brain at the 30- to 35-somite stage. Shh is expressed in the putative ZLI in the wild-type (A, arrow) and Six3-null (B, arrow) brains at the 30- to 35-somite stage.

• Supplemental Figure S10 -

  Fig. S10. Phenotypic rescue of the diencephalon on the Six3-null brain is Wnt1-dose-dependent. Absence of one copy of Wnt1 is enough to partially recover the diencephalic phenotype of Six3-null brains, as indicated by the expression of Lhx2 and Ngn2 in the thalamus (A,B), and Dlx2 in the prethalamus (C). Limited hypothalamic Nkx2.1 staining is also observed (D). Sagittal sections of E13.5 (A,B) and E14.5 (C,D) Six3^−/−;Wnt1^−/− embryos. Anterior is to the left. T, thalamus; PT, prethalamus; HY, hypothalamus.