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Files in this Data Supplement:
Fig. S1. LHX6 and NKX2.1 expression pattern in embryonic ventral forebrain. All panels are from the 12 µm cryosection through the medial ganglionic eminence (MGE) of an E12.5 embryo. (A-I) Increasing magnification of the same section co-labeled for NKX2.1 (red) and LHX6 (green). The boxed region in F is shown at higher magnification in G-I. NKX2.1 is highly expressed in proliferation zone (see PCNA labeling in K) of the MGE and is gradually downregulated as cells migrate into the mantle region. LHX6 is mainly expressed in a complementary pattern in the MGE mantle, but some co-expression occurs within the proliferative zone-mantle zone transition (F,I). (J-O) Co-immunolabeling for LHX6 (green) and proliferating cell nuclear antigen (PCNA, red). Most LHX6-expressing cells are PCNA-negative, but co-labeling (arrows in M-O) occurs in the subventricular zone (SVZ), suggesting that some MGE progenitors begin to express LHX6 during the final cell cycle. Scale bars: 560 µm in A; 100 µm in panel in J; 20 µm in M.
Fig. S2. Rescue of interneuron phenotypes by expression of Nkx2.1 in the MGE* of slices from Nkx2.1−/− embryos. (A) A slice from an Nkx2.1−/− embryo was electroporated with pNkx2.1-GFP. The region outlined in white corresponds to the dorsoventral level of the wild-type MGE. This region was dissected, dissociated, and plated onto a high-density feeder layer of neonatal cortical cells as previously described (Xu et al., 2004; Xu et al., 2005). After 14 (for SST and NPY) to 28 (for PV) days in vitro (DIV), the cultures were fixed and stained with the nuclear marker DAPI (B) and immunolabeled for the indicated proteins. (B-D) The same view of a culture stained with DAPI (B) to reveal the high-density nature of these cultures. C (GFP, green) and D (Nkx2.1, red) show that pNkx2.1-GFP drives Nkx2.1 expression in this system. (E-G′) Examples of co-labeling for GFP and parvalbumin (PV; E,E′), somatostatin (SST; F,F′) and neuropeptide Y (NPY; G,G′). In control experiments with pGFP vector, almost no cells expressing any of these markers are detected after transplantation of Nkx2.1−/− MGE-like progenitors (see Table 1). The control results are consistent with previous studies showing failed generation of interneurons that express these markers in Nkx2.1-null mice (Anderson et al., 2001; Xu et al., 2004).
Anderson, S. A., Marin, O., Horn, C., Jennings, K. and Rubenstein, J. L. (2001). Distinct cortical migrations from the medial and lateral ganglionic eminences. Development 128, 353-363.
Fig. S3. Time-course of reduction of LHX6 expression by RNAi and its effect on MGE cell migration to neocortex. Small hairpin RNAi for Lhx6 shLhx6; sequence as in Alifragis et al. (Alifragis et al., 2004) or scramble control sequence (Ambion) were subcloned into the pCAG-GFP vector. The constructs were electroporated into the MGE of E12.5 wild-type slices, maintained in culture for 16 hours, then dissociated and placed into primary culture. Cells were fixed after 1, 3 or 5 days in culture and examined by immunofluorescence for GFP (green) and LHX6 (red). (A-H) Examples of LHX6 and GFP after 5 days in culture. (A-D) LHX6 is expressed in most MGE-derived cells in these cultures, including in control-transfected cells (quantification in I). By contrast, Lhx6 RNAi reduces LHX6 expression (two examples shown in E-H). (I) Quantification of the percentage of transfected cells expressing LHX6. After 1 day (plus the 16 hours in slice culture following electroporation) there is already a significant reduction in the percentage of LHX6/GFP+ cells in the shLhx6 condition relative to control (n=3; P<0.001), but the effect size becomes larger at 3 and 5 days (n=3 at each time point, P<0.001 and P<0.003, respectively).
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