DEV02090 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Reduced cortical size in NsCre:Shh^Fl/Fl mice. (A) Cortical surface area is reduced by 26.6±1.3% in conditional mutants relative to controls. Measurements were made of freshly dissected brains at P12, after perfusion and prior to post-fixation. Controls included both the NsCre(–):Shh^Fl/Wt and the NsCre(–):Shh^Fl/Fl genotypes. Scale bar: 2 mm. (B) Reduced cortical thickness in NsCre:Shh^Fl/Fl mutants. Cortical thickness measurements were obtained by averaging the length of the medial, midpoint and lateral edge of the counting boxes (see Materials and methods) of four cresyl violet-stained sections spaced 120 µm apart. The neocortical thickness of NsCre:Shh^Fl/Fl mice is significantly reduced through layers 2-6, with most of this difference occurring in the superficial layers (layers 2-4). The thickness of layer 1 was not affected (not shown). n=3 mutants and 3 controls, *P<0.05.

• Supplemental Figure 2 -

  Fig. S2. Normal appearance of PH3 staining in NsCre:Shh^Fl/Fl mice. Shown are 12μm thick cryosections from E12.5 embryos stained for the Mphase marker PH3. There is no apparent difference in the number or distribution of labeled cells in the mutant section. Combined with data on the S-phase marker BrdU after a 1 hour pulse (Fig. 1), this finding suggests that in the NsCre:Shh^Fl/Fl mutants most progenitors are progressing through the cell cycle despite the loss of SHH.

• Supplemental Figure 3 -

  Fig. S3. Expression of Lhx6, Lhx7 and islet 1 in the subcortical mantle region of NsCre:Shh^Fl/Fl mutants at E14.5. The fates of all cells that ever express these genes is unknown, but the expression domain of Lhx6 includes cortical and striatal interneurons (Lavdas et al., 1999; Cobos et al., 2005), whereas the fates of islet 1⁺ cells include projection neurons and cholinergic interneurons of the striatum (Wang and Liu, 2001; Stenman et al., 2003), and the fates of Lhx7⁺ cells includes cholinergic neurons (Zhou et al., 2003; Fragkouli et al., 2005). NsCre:Shh^Fl/Fl mutants express reduced levels of Lhx6 (A,A¢) but the expression of Lhx7 (B,B¢) and islet 1 (C,C¢) is grossly normal. These results are consistent with data shown in Fig. 6 and in Fig. S5, showing that, in the NsCre:Shh^Fl/Fl mutants, GABAergic interneurons of the striatum and cortex are affected more than cholinergic interneurons of the striatum. One reason for this difference could be that cholinergic interneurons are born earlier than most GABAergic interneurons, and thus they may have escaped the effect of Shh inactivation on mitotic progenitors. Cx, cerebral cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MGE*, MGE-like region of the NsCre:Shh^Fl/Fl mutant. At E14.5, MGE-LGE sulcus is absent, but an MGE-like region that expresses Lhx6 is present (A¢).

• Supplemental Figure 4 -

  Fig. S4. Reduced detection of interneuron markers in NsCre:Smoothened^Fl/Fl mutants. Shown are 12 µm coronal cryosections through the somatosensory cortex of conditional smoothened-null mutants and controls at age P12. Reductions in detectable cell profiles of GABA (A,A¢), parvalbumin (Pv; B,B¢), somatostatin (Som; C,C¢) and Npy (D,D¢) are evident, with the greatest effect being on Som and Npy. These results are consistent with the effect of NsCre-mediated inactivation of Shh expression (Fig. 6), although the NsCre:Smo^Fl/Fl phenotype may be less severe. This notion is supported by the finding that these mutants generally survive into their fourth postnatal week, about one week longer than the NsCre:Shh^Fl/Fl mutants. The reason for this difference is unclear, but possibilities include a reduced efficiency of recombination at the Smo locus, or an increased stability or functional potency of the Smo protein relative to Shh. Of note, our findings of reduced interneuron markers in these mutants differ from data presented in Machold et al. (Machold et al., 2003), which found the areal density of GABA+ profiles in cortex to be grossly normal in NsCre:Smo^Fl/- mutants. Other interneuron markers were not tested in that paper. Scale bar in A: 100 µm

• Supplemental Figure 5 -

  Fig. S5. Reductions of striatal interneurons in NsCreShh^Fl/Fl mutants at P12. Shown are 12 µm coronal cryosections through the striatum of conditional Shh null mutants and controls at age P12. Large reductions in detectable cell profiles expressing Somatostatin (Som; C,D) and Npy (G; bar graph) are evident (n=3; P<0.01). No significant reduction of either parvalbumin (Pv; A,B) or choline-actyl-transferase (ChAT; E,F) cells are detected. Despite the trend towards reduced numbers of ChAT+ cells, the lack of a statistically significant reduction (n=3; P<0.17) is consistent with the grossly normal expression of islet 1 and Lhx7 in Fig. S3. The lack of reduction in Pv-expressing cells is more surprising, but may relate to the immaturity of Pv expression at P12, resulting in a bias towards the detection of the earliest born cells that could have escaped the effects of Shh inactivation.

• Supplemental Figure 6 -

  Fig. S6. Dlx5/6Cre-mediated recombination occurs very rarely in cells expressing the S-phase marker BrdU. (A) GFP and (B) BrdU (1 hour pulse of 100 mg/kg) labeling of a 12 µm section from a Dlx5/6Cre:Z/EGFP embryo at E16.5. Boxed areas are enlarged in C,D,E. The lack of BrdU/GFP co-labeling suggests that Dlx5/6Cre-driven recombination occurs primarily in postmitotic cells.

• Supplemental Figure 7 -

  Fig. S7. Grossly normal Nkx2.1 expression in the striatum of Dlx5/6-Cre:Smo^Fl/Fl mutants. (A,B) GFP (A) and GFP/Nkx2.1 (B) immunofluorescence signals from the same section of striatum from a P25 Dlx5/6-Cre:Z/EGFP reporter mouse. Arrows in A indicate GFP expression in each of the cells that are immunopositive for Nkx2.1 in B. Examination of multiple sections from this reporter cross revealed that about 85% of Nkx2.1+ cells in the postnatal striatum have recombined the GFP reporter under the control of the Dlx5/6-Cre. (C,D) Immunofluorescence for Nkx2.1 and the blue nuclear stain DAPI in the P25 striatum of a Dlx5/6Cre+:Smo^Fl/Fl mutant (D) and a Dlx5/6Cre–:Smo^Fl/Wt control (C). There was no gross difference in labeling for Nkx2.1 expression between striatal sections from mutant and control brains (n=3). Although it is not known whether smoothened receptors are present in Nkx2.1+ cells outside of the embryonic proliferative region of the ventral telencephalon, taken together the results shown above suggest that Shh is not required to maintain Nkx2.1 expression in these differentiated cells.