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First published online 12 October 2005
doi: 10.1242/dev.02090

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Sonic hedgehog maintains the identity of cortical interneuron progenitors in the ventral telencephalon

Qing Xu^1,*, Carl P. Wonders^2,* and Stewart A. Anderson^1,

¹ Department of Psychiatry, Weill Medical College of Cornell University, New York, NY 10021, USA
² Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA

View larger version (76K): [in a new window]   Fig. 1. Telencephalic loss of Shh reduces Nkx2.1 expression in progenitors of the MGE. Shown is the S-phase marker BrdU (red) and Nkx2.1 (green) immunolabeling on coronal 12 µm cryosections from E12.5 embryos. The outline in A indicates the bins of MGE used for quantification in G,H. Boxed areas in B and E are shown at higher magnification in C and F. (A-C) In NsCre(–):ShhFl/+ controls, roughly 90% of BrdU-labeled nuclei co-label with Nkx2.1. Over the entire MGE, this proportion falls to roughly 50% in the NsCre:Shhfl/fl mutants, although effect was strongest in the dorsal MGE (t-test: *P<0.05, **P<0.01). Note, the bin 1 result in G and the bin2 result in H are not significant after adjustment for multiple comparisons. Scale bar: 200 µm in A,B,D,E; 50 µm in C,F.

View larger version (94K): [in a new window]   Fig. 2. NsCre:ShhFl/Fl mutants lose the expression of Shh responsive genes at the MGE-LGE sulcus, but maintain other aspects of MGE patterning. (A-C') In situ hybridization of E12.5 forebrain for (A,A') Gli1, (B,B') Nkx6.2 and (C,C') Oct6 in wild-type (A,B,C) and NsCre:ShhFl/Fl mice (A',B',C'). (D-F') Immunofluorescence of E12.5 forebrain for (D,D') Gsh2, (E,E') Dlx2 and (F,F') Olig2 in NsCre(–):ShhFl/wt control (D,E,F) and NsCre:ShhFl/Fl mutant mice (D',E',F'). Despite the loss of expression of Gli1 and Nkx6.2 in the mutant MGE, other markers of MGE patterning in the VZ (Olig2) and SVZ (Oct6) appear to be unaltered. Scale bar: 200 µm.

View larger version (69K): [in a new window]   Fig. 3. Cyclopamine reduces Nkx2.1 expression and alters MGE progenitor fate in vitro. (A-B') Immunofluorescence labeling of re-sectioned (12 µm) E12.5 telencephalic explants cultured for 24 hours. (A,B) Nkx2.1 and BrdU labeling of control sections cultured in 0.05% EtOH. (A') Slices cultured in 5 µM cyclopamine have reduced expression of Nkx2.1 in the ventricular zone, whereas (B') BrdU incorporation appears unchanged. (C-F) Immunofluorescence labeling of somatostatin (Som, red), GFP (green) and BrdU (blue) in cell cultures grown for 14 DIV. The vast majority of transplanted cells from donors of this age have neuronal morphologies. Arrows indicate neurons triple-labeled for Som/GFP/BrdU. (G) Overall transplant cell number is unaffected by cyclopamine. (H) Cyclopamine reduces the percentage of Som- and Pv-expressing interneurons generated from MGE transplants. (I) The percentage of surviving, transplanted neurons (GFP+) that incorporated BrdU prior to transplant is unaffected by cyclopamine after 5 DIV, and slightly reduced after 14 DIV. (J) Cyclopamine dramatically reduces the number of BrdU+ transplanted cells co-labeling for somatostatin. t-test: *P<0.05, **P<0.005. Scale bar in A: 200 µm.

View larger version (67K): [in a new window]   Fig. 4. Cell-autonomous blockade of Shh signaling also reduces Nkx2.1 expression in the MGE of telencephalic slices. Shown are 12 µm sections made from slices cultured from E12.5 + 1 DIV. Slices had been transfected with either pCAGGS-ires-GFP control vector (A-C) or pCAGGS-mPTC1loop2-ires-GFP (D-F) by electroporation. Sections were immunolabeled with GFP (A,E) and Nkx2.1 (B,F). The boxed areas in A and D are enlarged, and the GFP signal intensity equally reduced to show co-labeling of GFP with Nkx2.1 in C and F, respectively. The area of pCAGGS-mPTC1loop2-ires-GFP expression in D has a marked decrease in Nkx2.1-expressing cells (E), and a corresponding loss of GFP-Nkx2.1 co-labeled cells (compare C with F). (G) Quantification of the percentage of GFP-expressing cells that co-labeled with Nkx2.1 in the MGE of these slices found a significant reduction in the mPTC1loop2 transfected slices (n=6; **P<0.004). Scale bar: 50 µm.

View larger version (59K): [in a new window]   Fig. 5. Exogenous Shh rescues Nkx2.1 expression and somatostatin fate of NsCre:ShhFl/Fl MGE progenitors in vitro. (A,B) Sections (12 µm) from slices cultured from E13.5 NsCre:ShhFl/Fl + 1 DIV. BrdU was added to the culture medium 6 hours before fixation. Control sections (A) show a marked reduction of Nkx2.1 in the MGE, primarily in the proliferative region. When cultured with 10 nM Shh (B), Nkx2.1 expression is rescued in the proliferative region of the MGE. (C) Quantification of similarly treated sections co-labeled for Nkx2.1 and BrdU. Although the addition of Shh does not affect the overall percentage of BrdU+ cells (left), the percentage of these cells that co-express Nkx2.1 is increased nearly 3-fold (right). To determine the fate potential of the BrdU-labeled cells, the proliferative region of the MGE was transplanted onto cortical feeders and immunolabeled for BrdU and somatostatin (D). (E) Quantification of the percentage of BrdU+ cells that co-labeled with Som, showing a marked reduction in NsCre:ShhFl/Fl compared with wild type, and a partial restoration of Som-BrdU co-labeling following the addition of Shh (n=3). Scale bar in D: 200 µm.

View larger version (119K): [in a new window]   Fig. 6. Reductions in somatostatin and parvalbumin-expressing interneuron subgroups in NsCre:ShhFl/Fl cortex. Shown are examples of immunofluorescence labeling with antibodies to the neuronal marker NeuN (A,A'), a marker of projection neurons, Tbr1 (B,B'), the interneuron marker GABA (C,C'), and three markers of MGE-derived, Nkx2.1-dependent interneuron subgroups, parvalbumin (Pv; D,D'), somatostatin (Som; E,E') and neuropeptide Y (Npy; F,F'). (A-F) Wild type; (A'-F') mutant. Data in G-I was collected from cryosections through the somatosensory cortex of three mutants and three controls at P12. Despite the trend towards a slightly increased density in both NeuN-labeled cells and in all cells in the mutants (G,H), there is a significant reduction in the percentage of neurons that express GABA (H). In the superficial cortex, this reduction is offset by an increased percentage of neurons that express Tbr1. Consistent with the reduction in GABA, reductions in cells expressing Pv, Som and Npy were observed, although after adjustment for multiple comparisons this effect was significant for only Pv and Som in layers 2-4. *P=0.05; **P<0.01. Scale bar in F: 100 µm for A-F.

View larger version (92K): [in a new window]   Fig. 7. Interneuron losses in NsCre:ShhFl/Fl mutants do not appear to result from defects in postmitotic development. (A) RT-PCR experiments on samples from P0 cortex collected from genotypes NsCre:ShhFl/Fl (lanes 1, 2 and 3), NsCre:ShhFl/Wt (lanes 4 and 5), NsCre(–):ShhFl/Wt (lanes 6 and 7), and NsCre(–):ShhFl/Fl (lanes 8 and 9). (B,C) Anti-calbindin immunolabeling of 12 µm cryosections from E16.5 embryos shows a reduction of calbindin-expressing cells in the cortical subventricular zone (SVZ; arrows) of the NsCre:ShhFl/Fl section (C). (D) Dlx5/6Cre:floxed ß-gal reporter expression in a 12 µm section at E16.5. Little reporter recombination is seen at E14.5 (not shown), but by E16.5 many stained cells are evident in the striatum (St) and cortex. (E,F) Immunofluorescence co-labeling of Dlx5/6Cre:floxed-GFP reporter and Pv (E) or Som (F) in layers III-V of somatosensory cortex at P25. Over 90% of cortical interneurons expressing either of these interneuron subgroup markers co-label with GFP. (G) Counts of interneuron subgroups in the postnatal (P25) somatosensory cortex of Dlx5/6Cre:SmoFl/FL mutants. CP, cortical plate; Ctx, neocortex; IZ, intermediate zone; MZ, marginal zone; VZ, ventricular zone. Scale bar: 50 µM.

View larger version (46K): [in a new window]   Fig. 8. Three actions of sonic hedgehog on embryonic telencephalon development. (A) Early in development (E9.0-E12.5), Shh forms a reciprocal gradient with the repressor form of Gli3. The relative expression levels of these two signals then work to establish dorsoventral patterning of the telencephalon. (B) Through prenatal development and possibly throughout life, Shh supports the expansion of radial glial stem cells (blue), promoting overall growth of the developing brain. (C) During neurogenesis in the MGE, Shh maintains the expression of Nkx2.1 (green nuclei) in progenitor cells (blue) that asymmetrically divide into an additional radial glial cell and a transient-amplifying progenitor (red). In the presence of Shh signaling (right pathway), these progenitors migrate into the SVZ where they continue to divide to generate striatal and cortical interneurons. In the absence of Shh (left pathway), the progenitors continue to divide (albeit with reduction of progenitor self-renewal), but Nkx2.1 expression is lost, as is their fate to become Pv- or Som-expressing interneurons.