Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex

Journal name:
Nature Neuroscience
Volume:
19,
Pages:
888–896
Year published:
DOI:
doi:10.1038/nn.4307
Received
Accepted
Published online
Corrected online

Abstract

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

Figures

  1. SmoM2 induces neocortical expansion and folding.
    Figure 1: SmoM2 induces neocortical expansion and folding.

    (a) Control and GFAP::Cre; SmoM2loxP/+ (SmoM2 mutant) brains at P10. (b) Nissl staining of control and SmoM2 brain sections at P7. The arrowhead indicates white matter extended into the induced gyrus. Scale bar, 1 mm. Pictures represent at least 5 repeats. (c) Expression of layer-specific markers: Satb2 (layers II–III), Bcl11b (Ctip2, red, layer V), Tbr1 (green, layer VI), and DAPI (blue). Scale bar, 0.2 mm. Pictures represent at least 3 repeats. (d) Cortical volume calculated from serial MRI scans of P7 control and SmoM2 mutants. Results were normalized to fold change compared to controls. Two-tailed unpaired t-test, P = 0.0380, n = 3 mice per group. We assumed normal distribution although the number of samples was too small for normality tests. F-test for variance, P = 0.3510, F(2,2) = 4.697. (e) Quantification of cell density in the cingulate and medial cortex. Two-tailed unpaired t-test, P = 0.0006 (Satb2), P = 0.3634 (Ctip2), P = 0.0250 (Tbr1); t(10) = 4.877 (Satb2), t(10) = 0.9522 (Ctip2), t(10) = 2.635 (Tbr1); n = 6 brain slices from 3 mice per group. All data passed Kolmogorov–Smirnov (KS) test for normality (P > 0.1) and F-test for variance: P = 4.255 (Satb2), P = 0.8609 (Ctip2), P = 0.2070 (Tbr1); F(5,5) = 0.1380 (Satb2), F(5,5) = 1.179 (Ctip2), F(5,5) = 3.385 (Tbr1). *P < 0.05; ***P < 0.001. Error bars represent s.d.

  2. SmoM2 expands IPCs and bRGs.
    Figure 2: SmoM2 expands IPCs and bRGs.

    (a) E16.5 cortices labeled for a radial glia marker, Pax6 (red), and an IPC marker, Tbr2 (green). The thin dotted line demarcates the medial (M) and dorsal (D) cortices. The thick dotted line marks the boundary between the ventricular zone (VZ) and SVZ. Pax6+Tbr2 cells separated from the VZ by bands of Tbr2+ IPC cells were counted as bRGs (white arrows indicate examples). Pictures represent at least 6 repeats. (b) Quantification of Pax6+Tbr2 radial glia and (c) Tbr2+ IPCs at E16.5. Two-tailed unpaired t-test (aRG, IPC, SVZ IPC, equal variance) or two-tailed unpaired t-test with Welch's correction (bRG, unequal variance), P = 0.7888 (aRG M), P = 0.2648 (aRG D), P = 0.0003 (bRG M), P = 0.0001 (bRG D), P = 0.0017 (IPC M), P = 0.0458 (IPC D), P = 0.0000 (SVZ IPC M), P = 0 (SVZ IPC D); t(14) = 0.2730 (aRG M), t(14) = 1.162 (aRG D), t(7) = 6.503 (bRG M), t(9) = 8.780 (bRG D), t(13) = 3.926 (IPC M), t(13) = 2.208 (IPC D), t(13) = 11.92 (SVZ IPC M), t(13) = 11.52 (SVZ IPC D). All data passed Kolmogorov–Smirnov (KS) testing for normality P > 0.1. bRG did not pass an F-test for equal variance, P = 0.6628 (aRG M), P = 0.1556 (aRG D), P = 0.0003 (bRG M), P = 0.0369 (bRG D), P = 0.2378 (IPC M), P = 0.6396 (IPC D), P = 0.7069 (SVZ IPC M), P = 0.1388 (SVZ IPC D); F(7,7) = 1.408 (aRG M), F(7,7) = 3.127 (aRG D), F(7,7) = 28.04 (bRG M), F(7,7) = 5.603 (bRG D), F(6,7) = 2.596 (IPC M), F(6,7) = 1.441 (IPC D), F(6,7) = 1.336 (SVZ IPC M), F(6,7) = 3.350 (SVZ IPC D), n = 8 brain slices from 3 mice per group. (d,e) E16.5 cortices labeled with Pax6 (green) and Tbr2 (blue) after tamoxifen injection at E13.5. Dotted lines in d indicate intermediate zone (IZ) areas enlarged in e. (f) Quantification of bRGs (tdTomato+Pax6+Tbr2). Two-tailed unpaired t-test with Welch's correction, P = 0.0051, t(6) = 4.300. All data passed Kolmogorov–Smirnov (KS) test for normality, P > 0.1. F-test for equal variance, P = 0.0059, F(6,4) = 3.350, n = 5 (control) and 7 (SmoM2) brain slices from 3 mice per group. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant. Scale bars in a and d, 50 μm; in e, 25 μm. Error bars represent s.e.m.

  3. SmoM2 expands bRGs by increasing their self-renewal and changing the aRG division angle toward bRG production.
    Figure 3: SmoM2 expands bRGs by increasing their self-renewal and changing the aRG division angle toward bRG production.

    (a) The experimental scheme and E16.5 cortices labeled for Pax6 (blue), Tbr2 (green), BrdU (gray) and EdU (red). The boxed areas are enlarged in the right. Pictures represent at least 3 repeats. (b) Proliferation (EdU+, left) and self-renewal (EdU+BrdU+, right) of radial glia and IPCs. All data collected from 8 brain slices from 3 mice per group passed Kolmogorov–Smirnov (KS) test for normality, P > 0.1. Two-tailed unpaired t-test for proliferation: aRG, P = 0.2680, t(14) = 1.154; bRG, P = 0.0103, t(14) = 2.960; IPC, P = 0.0001, t(14) = 15.75. F-test for equal variance: aRG, P = 0.5533, F(7,7) = 1.594; bRG, P = 0.3659, F(7,7) = 2.045; IPC, P = 0.1751, F(7,7) = 2.963. Two-tailed unpaired t-test with Welch's correction for self-renewal of bRG: P = 0.0056, t(9) = 3.618; F-test for variance: P = 0.0274, F(7,7) = 6.245. Two-tailed unpaired t-test for self-renewal of IPC: P = 0.0001, t(14) = 5.321; F-test for variance: P = 0.0869, F(7,7) = 4.013. (c) Distribution of the distances of the EdU+ bRGs from the ventricular surface. Mann-Whitney test, P = 0.0001, sum of ranks = 1,392, 10,540, U = 571.5, n = 40 bRGs for control and 114 bRGs for SmoM2. N = 8 brain slices from 3 mice per group. (d) E15.5 cortices labeled for TuJ1 (red), Pax6 (blue), and Tbr2 (green). (e) Numbers of neurons in the ventricular zone (VZ; top) and cell fate (IPC vs. radial glia (RG)) in the VZ (bottom) for the same cortices used in d. Pictures represent at least 3 repeats. For relative density of TuJ1 in the VZ, Mann Whitney test, P = 0.0006, sum of ranks = 134, 37, U = 1.000, n = 10 and 8 for control and SmoM2, respectively, brain slices from 3 pairs of mice. For ratio of radial glia and IPC in the VZ, Mann Whitney test, P = 0.0021, sum of ranks = 28, 77, U = 0, n = 7 brain slices from 3 mice per group. (f,g) Mitotic aRGs at E14.5 labeled for phospho-vimentin (P-Vim, green) and DAPI (blue) (f) and quantification of the division angle (g). We blindly analyzed 101 cells (SmoM2 mutants) and 71 cells (controls) from 3 pairs of mice. In f, α indicates the angle between the apical surface (dotted line) and the plane of division (solid line). The divisions are termed 'horizontal' if 60° < α ≤ 90°, 'oblique' if 30° < α ≤ 60°, and 'vertical' if 0° < α ≤ 30°. Mann Whitney test, P = 0.0092, sum of ranks = 6,937, 7,769, U = 2,719. P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.001; ns, nonsignificant. Error bars represent s.e.m. Scale bars in a and d, 50 μm; f, 5 μm.

  4. Retrovirus expressing SMOM2 promotes bRG production at the clonal level.
    Figure 4: Retrovirus expressing SMOM2 promotes bRG production at the clonal level.

    (a) Micrographs showing examples of different types of clones. Cortices were labeled with GFP (green), Pax6 (red), and Tbr2 (blue) to determine cell fates 48 h (E15) or 72 h (E16) after in utero intraventricular injection of GFP or SMOM2 GFP retroviruses at E13. IPC/N clones contained IPCs and/or neurons but no radial glia; aRG + IPC/N clones had one aRG and IPC/N; 2aRG + IPC/N clones had two aRGs with or without IPC/N; and bRG clones had at least one bRG and other cell types. Gray panels include single-channel images for GFP; colored insets show Tbr2 and Pax6 staining for the GFP+ cells. Dotted lines outline the cell bodies of GFP+ cells. Scale bar, 20 μm. (b) Composition of individual clones at E16: GFP clones (top) and SMOM2 GFP clones (bottom). Each vertical column of cells above a mark on the x axis represents a single clone. (c) Distribution of clone types. At E15 (left two charts), we found 23 IPC/N, 23 aRG + IPC/N, and 4 2aRG + IPC/N clones for GFP; and 20 IPC/N, 34 aRG + IPC/N, and 7 2aRG + IPC/N clones for SMOM2 GFP. At E16 (right two charts), we found 35 IPC/N, 26 aRG + IPC/N, 2 2aRG + IPC/N, and 3 bRG clones for GFP; and 31 IPC/N, 36 aRG + IPC/N, 11 2aRG + IPC/N, and 23 bRG clones for SMOM2. Two-sided Fisher's exact test, P = 0.0823 at E15 (50 GFP clones, 61 SMOM2 GFP clones), or chi-square test, P = 0.1659, χ2(2) = 3.593 at E15; chi-square test, P = 0.0002, χ2(2) = 17.61 at E16 (66 GFP clones, 101 SMOM2 GFP clones).

  5. Smo is required to expand IPCs, bRGs and upper-layer neurons.
    Figure 5: Smo is required to expand IPCs, bRGs and upper-layer neurons.

    (a) E16.5 cortices labeled for Pax6 (red) and Tbr2 (green). The circles indicate examples of Pax6+Tbr2 bRGs. Scale bar, 50 μm. We analyzed 9 sections from 3 mice per group. (b) Quantification of radial glia. Mann Whitney test; medial aRG, P = 0.5076, sum of ranks = 93.50, 77.50, U = 32.50; dorsal aRG, P = 0.3401, sum of ranks = 97, 74, U = 29.00; medial bRG, P = 0.0005, sum of ranks = 122, 49, U = 4.000; dorsal bRG P = 0.0002, sum of ranks = 124, 47, U = 2.000. (c) Quantification of IPCs. Mann Whitney test; medial IPC, P = 0.0188, sum of ranks = 112, 59, U = 14.00; dorsal IPC, P = 0.0078, sum of ranks = 115, 56, U = 11.00. (d) Quantification of aRGs dividing non-horizontally (0° ≤ α ≤ 60°). We blindly analyzed 144 cells (Smo mutants) and 190 cells (controls) in 7 sections from 3 mice per group. Two-tailed unpaired t-test with Welch's correction, P = 0.0124, t(7) = 3.343; F test for variance, P = 0.0098, F(6,6) = 11.18. (e) Expression and quantification of layer-specific markers: Satb2 (red), Ctip2 (blue) and Tbr1 (green). Scale bar, 0.2 mm. Two-tailed unpaired t-test: for Tbr1, P = 0, t(16) = 5.244; for Ctip2, P = 0.0271, t(16) = 2.432; for Satb2, P = 0, t(11) = 7.947. n = 9 sections from 3 mice per group. All data passed Kolmogorov–Smirnov (KS) test for normality, P > 0.1 and F-test for equal variance: P = 0.4990, F(8,8) = 1.642 (Tbr1), P = 0.0701, F(8,8) = 3.928 (Ctip2), P = 0.0418, F(8,8) = 4.719 (Satb2). ns, P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.001. Error bars represent s.e.m. in bd and s.d. in e.

  6. Strong SHH signaling in human aRGs.
    Figure 6: Strong SHH signaling in human aRGs.

    (a) GLI1 expression during human fetal development (left) and a comparison of GLI1 and Gli1 expression levels in developing human and mouse cortices (right). The log GLI1/SOX2 ratio represents log2(mean FPKM GLI1) – log2(mean FPKM SOX2) for humans and log2(mean FPKM Gli1) – log2(mean FPKM Sox2) for mice, where FPKM is fragments per kilobase of transcript per million base pairs mapped. (b) Top left: hematoxylin and eosin (H&E) staining and in situ hybridization for GLI1 (purple dots) in the human fetal brain at 14 pcw. The top right panel is an enlargement of the boxed area in the top left panels. Boxed areas in the top right panel are enlarged in the bottom two panels. Scale bars, 1 mm in top left two panels and 50 μm in the enlarged images on the right and bottom. Images represent results from 3 independent tissue samples. (c) Left three panels: human fetal brain at 14 pcw stained with anti-SHH antibody (green) and DAPI (purple). Co-incubation with SHH peptide blocks the strong SHH staining at the ventricular surface. This anti-SHH antibody specifically stained Purkinje cells in the mouse cerebellum (right panel), which express Shh. Scale bars, 20 μm in human brain panels and 200 μm in mouse panel. Each figure represents at least 3 repeats on two different tissue samples.

  7. SHH signaling promotes bRG production in human cerebral organoids.
    Figure 7: SHH signaling promotes bRG production in human cerebral organoids.

    (a) Examples of horizontal (60° < α ≤ 90°), oblique (30° < α ≤ 60°) and vertical (0° ≤ α ≤ 30°) divisions of aRGs in cerebral organoids stained with phospho-vimentin (P-VIM, red) and DAPI (blue) and quantification of the division angles. We blindly analyzed 123 (DMSO), 133 (SAG) and 135 (SANT-1) cells from 15 organoids per group from 3 independent experiments. Mann Whitney test, DMSO vs. SANT-1, P = 0.0007, sum of ranks = 14,080, 19,590, U = 6,330; DMSO vs. SAG, P = 0.2681, sum of ranks = 15,336, 17,817, U = 7,586. Scale bar, 5 μm (b) Human cerebral organoids labeled for PAX6 (green) and TBR2 (purple) and quantification of bRGs (PAX6+TBR2 cells separated from dense PAX6+ cells by stretches of TBR2+ cells that are indicated by dotted lines). We analyzed 21 (DMSO), 20 (SAG) and 21 (SANT-1) VZ and SVZ structures from 11 (DMSO), 13 (SAG) and 15 (SANT-1) organoids from 3 independent experiments. Mann Whitney test, DMSO vs. SANT-1, P = 0, sum of ranks = 627.5, 275.5, U = 44.50; DMSO vs. SAG, P = 0.2565, sum of ranks = 485, 376, U = 166.0. Scale bar, 50 μm. (c) Apical surfaces of aRGs in organoids labeled for cilia markers (ARL13B, red), SMO (green) and DAPI (blue) and quantification of cilia containing SMO. Scale bar, 2 μm. We examined 294 (DMSO), 292 (SANT-1) and 400 (SAG) cilia from 4 (DMSO), 3 (SANT-1) 3 (SAG) organoids from two independent experiments, of which 145 (DMSO), 53 (SANT-1) and 190 (SAG) cilia contained SMO. Two sided Fisher's exact test, P = 0.0001 (DMSO vs. SANT-1), P = 0.6454 (DMSO vs. SAG). ns, P > 0.05; *P < 0.01; ***P < 0.001. Error bars represent s.e.m in b and s.d. in c.

  8. Boundaries used to quantify cells in E16.5 brains and SmoM2 expression patterns induced by multiple Cre lines.
    Supplementary Fig. 1: Boundaries used to quantify cells in E16.5 brains and SmoM2 expression patterns induced by multiple Cre lines.

    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.

  9. Diverse morphology of bRGs (oRGs) in SmoM2 mutants.
    Supplementary Fig. 2: Diverse morphology of bRGs (oRGs) in SmoM2 mutants.

    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.

  10. Increase of RGs at the expense of IPCs and neurons in the VZ and increase of RGs dividing non-apically.
    Supplementary Fig. 3: Increase of RGs at the expense of IPCs and neurons in the VZ and increase of RGs dividing non-apically.

    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.

  11. SmoM2 induces folding outside the cingulate cortex.
    Supplementary Fig. 4: SmoM2 induces folding outside the cingulate cortex.

    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 (AD) 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.

  12. Expression of Ascl1 and Dlx2 in the cortices of GFAP::Cre; SmoM2loxP/+ mice.
    Supplementary Fig. 5: Expression of Ascl1 and Dlx2 in the cortices of GFAP::Cre; SmoM2loxP/+ mice.

    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

  13. Primary cilia and Smo are required for neocortical growth.
    Supplementary Fig. 6: Primary cilia and Smo are required for neocortical growth.

    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.

  14. Gli1 expression in the mouse embryonic forebrain and GLI1 expression in the human fetal forebrain.
    Supplementary Fig. 7: Gli1 expression in the mouse embryonic forebrain and GLI1 expression in the human fetal forebrain.

    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&quest;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.

  15. Relative levels of GLI1 in the human fetal neocortex are higher than are those of Gli1 in the mouse embryonic cortex.
    Supplementary Fig. 8: Relative levels of GLI1 in the human fetal neocortex are higher than are those of Gli1 in the mouse embryonic cortex.

    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.

  16. SHH mRNA and SHH protein are expressed in the human hypothalamic VZ.
    Supplementary Fig. 9: SHH mRNA and SHH protein are expressed in the human hypothalamic VZ.

    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.

  17. Blocking SHH signaling decreases SATB2+ neurons in human cerebral organoids.
    Supplementary Fig. 10: Blocking SHH signaling decreases SATB2+ neurons in human cerebral organoids.

    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.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

Corrected online 28 June 2016
In the version of this article initially published, the units on the x axis in Figure 3c were given as mm; the correct units are μm. At the end of the legend to Figure 7, the error bars were described as s.d.; they are actually s.e.m. in b and s.d. in c. In the third sentence of the Online Methods section on human cerebral organoids, 10% knockout serum replacement, 1% GlutaMAX and 1% MEM-NEAA should have been 20%, 1× and 1×, respectively. In the sixth sentence, 1% N2 supplement, 1% GlutaMAX and 1% MEM-NEAA should each have been 1×. In the eighth sentence, 6-mm dishes should have been 6-cm dishes, 0.5% N2 supplement and 0.5% MEM-NEAA should each have been 0.5×, and 1% B27 without vitamin A, 1% GlutaMAX and 1% penicillin/streptomycin should each have been 1×. In Supplementary Figure 10b, the graph lacked error bars. The errors have been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Lei Wang,
    • Shirui Hou &
    • Young-Goo Han
  2. Division of Brain Tumor Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Lei Wang,
    • Shirui Hou &
    • Young-Goo Han

Contributions

L.W. and Y.-G.H. designed and performed the experiments and wrote the manuscript. S.H. performed the in utero retroviral injections. Y.-G.H. conceived and supervised the study.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Supplementary Figures

  1. 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.

  2. 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.

  3. 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.

  4. 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 (AD) 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.

  5. 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

  6. 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.

  7. 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&quest;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.

  8. 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.

  9. 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.

  10. 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.

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