The unique mental abilities of humans are rooted in the immensely expanded and folded neocortex, which reflects the expansion of neural progenitors, especially basal progenitors including basal radial glia (bRGs) and intermediate progenitor cells (IPCs). We found that constitutively active Sonic hedgehog (Shh) signaling expanded bRGs and IPCs and induced folding in the otherwise smooth mouse neocortex, whereas the loss of Shh signaling decreased the number of bRGs and IPCs and the size of the neocortex. SHH signaling was strongly active in the human fetal neocortex but Shh signaling was not strongly active in the mouse embryonic neocortex, and blocking SHH signaling in human cerebral organoids decreased the number of bRGs. Mechanistically, Shh signaling increased the initial generation and self-renewal of bRGs and IPC proliferation in mice and the initial generation of bRGs in human cerebral organoids. Thus, robust SHH signaling in the human fetal neocortex may contribute to bRG and IPC expansion and neocortical growth and folding.
At a glance
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- Supplementary Figure 1: Boundaries used to quantify cells in E16.5 brains and SmoM2 expression patterns induced by multiple Cre lines. (699 KB)
a. Tangential boundaries were determined based on the trajectories of radial processes from the VZ to the pial surface. The processes of RGs were visualized by RC2 staining. Note that radial processes originating from the medial roof of the lateral ventricle curve to reach the medial surface of the hemisphere instead of extending straight to the dorsal surface of the brain. We used that boundary point (arrow) as our landmark to define the medial (M) and dorsal (D) parts of the cortex. The arrowhead indicates the dorsal medial corner of the lateral ventricle. A small part of the medial roof of the lateral ventricle was omitted in the RC2 tilting (*). b. Separate channels for images shown Fig. 2a. The thin dotted lines indicate the boundary between the medial (M) and dorsal (D) cortex. The thick dotted lines indicate a boundary between the SVZ and VZ. c. Immunofluorescence showing SmoM2 expression induced by different Cre lines. Anti-GFP antibody was used to detect SmoM2-YFP fusion protein expressed by SmoM2 mutants. The GFAP::Cre; SmoM2loxP/+ cortex displays a high-medial to low-lateral gradient, whereas the cortices of Nestin::CreER; SmoM2loxP/+ (injected with tamoxifen at E12.5) or Nestin::Cre; SmoM2loxP/+ brains showed no clear gradient of SmoM2 expression. All the micrographs have been repeated for more than 3 times.
- Supplementary Figure 2: Diverse morphology of bRGs (oRGs) in SmoM2 mutants. (684 KB)
a. E16.5 SmoM2 cortex labeled for RC2 (green), Glast (blue), and Sox2 (red). Inset A shows an example of a Sox2+ cell attached to the pial surface by a single basal process that resembles the classic morphology of bRGs. Inset B shows a Sox2+ cell that has just divided and bears a basal process with a growth cone–like structure at the end (arrowhead). Inset C shows a Sox2+ cell with bipolar processes positioned tangentially. Scale bar = 20 μm. b. Diverse morphology of bRGs in GFAP::CreER; SmoM2loxP/+; tdTomatoloxP/+ cortex at E16.5 after tamoxifen injection at E13.5, as shown by labeling for tdTomato reporter (red), Pax6+ (green), and Tbr2− (blue): bRGs bearing apical (A), bipolar (B), basal (C), or multipolar (D) processes. The multipolar cells may correspond to transient bRGs observed in monkeys, which alternate between stages showing unipolar or bipolar radial processes and stages without a radial process22; however, we cannot rule out the possibility that these cells may be mis-differentiated bRGs or IPCs. The arrows point to the processes. Note that the bRGs are Pax6+ (green) Tbr2− (blue). The pie chart quantifies Pax6+ Tbr2− tdTomato+ cells in each morphologic category. All the micrographs have been repeated for more than 3 times.
- Supplementary Figure 3: Increase of RGs at the expense of IPCs and neurons in the VZ and increase of RGs dividing non-apically. (1,114 KB)
a. Sections rostral and caudal to the images shown in Fig. 3d labeled for TuJ1 (white or red), Pax6 (blue), and Tbr2 (green). Both rostral and caudal sections showed patterns of cell composition in the VZ similar to that in the medial section shown in Fig. 3d. Arrows point to examples of bRGs. Scale bar = 50 μm. b. Non-apically dividing RGs at E15.5 indicated by the M-phase marker phospho-histone 3 (PH3, grey or red), Sox2 (green), and Tbr2 (blue). The arrows indicate examples of non-apically dividing RGs (PH3+ Sox2+ Tbr2−). Scale bar = 20 μm. c. Higher magnification of cells A, B, and C in panel (b). d. Quantification of dividing IPCs (PH3+ Tbr2+) and RGs (PH3+ Sox2+ Tbr2−). Mann Whitney test, for IPC (PH3+ Tbr2+), P = 0.0004, Sum of ranks = 45, 126, Mann-Whitney U = 0.0000, for AP RGs (PH3+ Sox2+ Tbr2−), P = 0.2878, Sum of ranks = 98, 73, Mann-Whitney U = 28.00, for nonAP RGs (PH3+ Sox2+ Tbr2−), P = 0.0016, Sum of ranks = 50, 121, Mann-Whitney U = 5.000. 9 sections from 4 pairs of control and mutant mice were analyzed. AP, apical; ns, P > 0.05; ***P < 0.001. Error bars = standard error of the mean.
- Supplementary Figure 4: SmoM2 induces folding outside the cingulate cortex. (973 KB)
a. Nissl staining of brain sections of Nestin::Cre; SmoM2loxP/+ mice at P7. Only a few Nestin::Cre; SmoM2loxP/+ mutant embryos survived to birth. The boxed regions showing folds (A–D) are enlarged below. The arrows in (B) and (D) point to folds in the lateral cortex. Scale bar = 1 mm. These were only observed in two rare survivors as Nestin::Cre; SmoM2loxP/+ mice die prenatally. b. Cortex corresponding to the boxed area in panel (B) labeled for layer-specific markers, Satb2 (white or red, layers II–IV), Ctip2 (blue, layer V), and Tbr1 (green, layer VI). The cortices of the rare surviving Nestin::Cre; SmoM2loxP/+ mice maintained normal layering. c. Nissl staining of coronal sections of control (SmoM2loxP/+) and Nestin::CreER; SmoM2loxP/+ brains at P3. A relatively low dose of tamoxifen (1.5 mg/40 g of body weight, IP injection at E12.5) was used to avoid embryonic lethality. The arrows point to folds in the lateral cortices that are enlarged in the images on the right. Scale bar = 0.5 mm. Cortical folding was observed in approximately 30% of the Nestin::CreER; SmoM2loxP/+ brains examined.
- Supplementary Figure 5: Expression of Ascl1 and Dlx2 in the cortices of GFAP::Cre; SmoM2loxP/+ mice. (629 KB)
a. qPCR quantification of Ascl1 and Dlx2 mRNA in microdissected medial E14.5 cortices of control and SmoM2 mutants. b. E16.5 brains labeled for Ascl1 (green), Pax6 (red), and Tbr2 (blue). c. E16.5 cortices labeled for Dlx2 (green) and Tbr2 (purple) or Sox2 (purple). All the micrographs have been repeated for more than 3 times. GE: ganglionic eminence
- Supplementary Figure 6: Primary cilia and Smo are required for neocortical growth. (362 KB)
a. The upper pair of images show the whole brains of a SmoM2 mutant (GFAP::Cre; SmoM2loxP/+) and a SmoM2 mutant lacking cilia (GFAP::Cre; SmoM2loxP/+; Kif3aloxP/loxP) at P2. The lower row pair shows the cingulate cortex stained with hematoxylin. Note the absence of folding in the SmoM2 mutants without cilia. b. Whole brains of a mutant lacking Smo (GFAP::Cre; SmoloxP/loxP) and a control mouse at P21. Images represent results from more than 3 pairs of mice. Scale bar = 2 mm.
- Supplementary Figure 7: Gli1 expression in the mouse embryonic forebrain and GLI1 expression in the human fetal forebrain. (1,107 KB)
a. In situ hybridization for Gli1 mRNA (dark brown dots) on mouse brain (E15.5) (Images obtained from the Allen Institute for Brain Science website at http://developingmouse.brain-map.org/experiment/siv?id=100051605&imageId=101024922&initImage=ish). The boxed areas are enlarged on the right. Note that Gli1 was only detectable in the ventral forebrain, including the ganglionic eminence. b. Levels of GLI1 mRNA expression (purple dots) in the ganglionic eminence were similar to those in the cortex (Fig. 6b) in the human fetal brain. The boxed area in the upper image is enlarged in the lower image. CP, cortical plate; CTX, cortex; GE, ganglionic eminence; VZ, ventricular zone. Images represent results from 3 independent tissue samples.
- Supplementary Figure 8: Relative levels of GLI1 in the human fetal neocortex are higher than are those of Gli1 in the mouse embryonic cortex. (204 KB)
a. Relative levels of GLI1 mRNA in the human fetal neocortex over time from 8 pcw to 38 pcw. GLI1 expression was normalized to that of SOX2, NES, or PAX6. We constructed these graphs by using RNAseq data from the BrainSpan Developmental Transcriptome database (http://www.brainspan.org). b. Comparisons of Gli1 and GLI1 expression in different cortical areas of the mouse and human brain. Mouse mRNA expression levels were obtained from RNAseq analyses of E14.5 medial and lateral cortices. The human fetal brain results were obtained from the BrainSpan database (12–19 pcw). c. GLI1 and Gli1 expression in sorted human and mouse RGs. Calculations were based on RNAseq data from Florio et al.28.
- Supplementary Figure 9: SHH mRNA and SHH protein are expressed in the human hypothalamic VZ. (1,073 KB)
a. In situ hybridization images for SHH mRNA (purple dots) on human fetal brain at 14 pcw. SHH mRNA was detected in the hypothalamic VZ (*). Each boxed area is enlarged in the adjacent image to the right. Images represent results from 2 independent tissue samples. b. Human fetal hypothalamus at 14 pcw stained with anti-SHH antibody (green) and DAPI (purple). CP, cortical plate; VZ, ventricular zone. Scale bar = 20 μm. Pictures represent at least 3 repeats.
- Supplementary Figure 10: Blocking SHH signaling decreases SATB2+ neurons in human cerebral organoids. (454 KB)
a. Organoids are labeled for SOX2 (green), TBR2 (blue), and phospho-vimentin (red). SOX2+ RGs formed a VZ-like structure surrounding a lumen. TBR2+ IPCs formed an SVZ-like layer basal to the VZ-like structure. The phospho- vimentin labeled RGs in mitosis (arrowheads). Similar to what is observed in vivo, most RGs divided at the apical surface lining lumen; however, some RGs divided outside the VZ, resembling bRGs. The arrows indicate radial fibers of RGs expressing phospho-vimentin. b. The experimental scheme and organoids labeled for SATB2 (green) and CldU (purple) and quantification of SATB2+ CldU+ cells normalized to the total number of SATB2+ cells. Organoids were treated with SANT1 (400 nM) or DMSO for 10 days from 29 days to 39 days after differentiation. To label a cohort of neurons produced during treatment, we treated organoids with CldU (3 μg/mL) for 48 h from 35 days to 37 days after differentiation. The organoids were fixed at 64 days after differentiation. Scale bar = 50 μm. Two tailed unpaired t test, P = 0.0000, t(20) = 5.126; 10 (DMSO) and 12 (SANT-1) 'cortical' regions of 4 organoids each from 2 independent experiments were analyzed; KS normality test, P > 0.1; F test for variance, P = 0.5469, F(9, 11) = 1.458. Error bars = standard error of the mean.