CROCCP2 acts as a human-specific modifier of cilia dynamics and mTOR signaling to promote expansion of cortical progenitors

Abstract

The primary cilium is a central signaling component during embryonic development. Here we focus on CROCCP2, a hominid-specific gene duplicate from ciliary rootlet coiled coil (CROCC), also known as rootletin, that encodes the major component of the ciliary rootlet. We find that CROCCP2 is highly expressed in the human fetal brain and not in other primate species. CROCCP2 gain of function in the mouse embryonic cortex and human cortical cells and organoids results in decreased ciliogenesis and increased cortical progenitor amplification, particularly basal progenitors. CROCCP2 decreases ciliary dynamics by inhibition of the IFT20 ciliary trafficking protein, which then impacts neurogenesis through increased mTOR signaling. Loss of function of CROCCP2 in human cortical cells and organoids leads to increased ciliogenesis, decreased mTOR signaling, and impaired basal progenitor amplification. These data identify CROCCP2 as a human-specific modifier of cortical neurogenesis that acts through modulation of ciliary dynamics and mTOR signaling.

Keywords: CROCC; CROCCP2; cerebral cortex; cilia; evolution; human brain development; mTOR; neurogenesis; rootlet; rootletin.

Graphical abstract

Figure 1

CROCCP2 is an HS duplicate highly expressed in human corticogenesis (A) Schematic representation of CROCC and human-specific paralog CROCCP2 gene structure. Conserved exons are highlighted in light gray. Protein-coding and noncoding exons are depicted in blue and gray, respectively. CROCCP2 corresponds to duplication of exons 13–21 and is composed of three coding exons. (B) Putative protein structure of CROCC and CROCCP2. CROCC is composed of a small head domain and a large coiled-coil repetitive domain. CROCCP2 corresponds to partial duplication of coiled-coil domain. (C) RNA-seq profile of CROCC HS gene family during human corticogenesis. (D) RNA in situ hybridization using specific probes for CROCC and CROCCP2 at nine gestational weeks (GWs). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar, 100 μm. (E) CROCC protein expression on whole human embryo at GW7, scale bar, 5 mm. (F) CROCC expression study during human corticogenesis (GW 12 cortical wall). (F′) Inset at apical surface of ventricular zone, note elongated bundles located underneath basal body stained by gamma-tub. Scale bar, 100–5 μm in inset. (G) CROCC expression during mouse corticogenesis (E14 cortex). (G′) Inset of one primary cilia. Scale bar, 50–3 μm in inset. (H) Schematic representation of interphase apical progenitors. See also Figures S1 and S2.

Figure 2

CROCCP2 gain of function leads to basal cortical progenitor amplification IUE of pCIG (CONTROL) or Pcig-MYC-CROCCP2 (CROCCP2) at E13.5. (A and B) Immunofluorescence analysis of coronal sections of E14.5 brains stained for DAPI and GFP analyzing the distribution of transfected cells in CONTROL (A) and CROCCP2 (B). (C) Histograms showing the percentage of GFP⁺ cells in ventricular zone (VZ), subventricular zone and intermediate zone (SVZ/IZ) and cortical plate (CP). n = 19 CONTROL embryos, 16 CROCCP2 embryos, p = 0.1977 (VZ), p = 0.1631 (SVZ). (D, E, G, H, J, and K) Immunofluorescence analysis of coronal sections of E14.5 brains stained for GFP and Pax6 (D and E) or GFP and Tbr2 (G and H) or GFP and Neurod2 (J and K) for CONTROL (D, G, and J) and CROCCP2 (E, H, and K). Arrows point to double-positive cells. (F, I, and L) Scatter plots showing the percentage of GFP⁺ cells co-expressing either Pax6 (F), Tbr2 (I), or Neurod2 (L). n = 12 CONTROL embryos, 26 CROCCP2 embryos, p = 0.6356 (PAX6); n = 27 CONTROL embryos, 26 CROCCP2 embryos, ^∗∗p = 0.0082 (TBR2), n = 14 CONTROL embryos, 11 CROCCP2 embryos, p = 0.787 (Neurod2). (M and N) Immunofluorescence analysis of coronal sections of E14.5 brains stained for GFP and phosphohistone H3 (PH3) in (M) CONTROL and (N) CROCCP2. (N′, N″, and N‴). Inset showing GFP, PH3, and Tbr2 immunofluorescence of basally dividing cells. Vast majority of cells are Tbr2 positive. (O) Scatter plot showing the percentage and distribution of PH3⁺/GFP⁺ cells. n = 19 CONTROL embryos, 16 CROCCP2 embryos, ^∗∗∗p = 0.0003 (ALL mitosis), ^∗∗p = 0.0073 (APICAL), ^∗∗∗p = 0.0002 (BASAL). (P and Q) Immunofluorescence analysis of coronal sections of E14.5 brains stained for GFP, EDU, and Tbr2 in CONTROL (P) and CROCCP2 (Q). (P′ and Q′) Inset focusing on triple-positive cells pointed by arrows. (R) Scatter plot showing the proportion and distribution of EDU⁺/GFP⁺ cells. n = 23 CONTROL embryos, 19 CROCCP2 embryos, ^∗p = 0.0367 (ALL cells), ^∗p = 0.0183 (VZ), ^∗∗∗p < 0.001 (SVZ/IZ). (S) Scatter plot showing proportion of triple-positive cells EDU⁺, Tbr2⁺, GFP⁺ n = 6 CONTROL embryos, 7 CROCCP2 embryos, ^∗∗p = 0.007. (A, B, D, E, G, H, J, K, M, N, P, and Q) Scale bar, 25 μm. (T–W) Immunofluorescence analysis of coronal sections of E15.5 brains stained for GFP and DAPI (T and V) or GFP and Neurod2 (U and W) in CONTROL (T and U) or CROCCP2 (V and W). Inset showing double positive (U′ and W′) and GFP-positive only (U″ and W″) cells. Scale bar, 50 μm. (X) Histograms showing the percentage of GFP⁺ cells in ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP). n = 16 CONTROL embryos, 13 CROCCP2 embryos, p = 0.3422 (VZ), p = 0.6590 (SVZ), ^∗p = 0.050 (IZ), p = 0.3511 (CP). (Y) Scatter plot showing proportion of GFP⁺ Neurod2⁺ cells. (n = 12 CONTROL embryos, 7 CROCCP2 embryos, ^∗∗∗∗p < 0.0001). (C, F, I, L, O, R, S, X, and Y) Data are represented as mean ± SEM. Each dot represents an embryo, p values by Student’s t test. See also Figure S3.

Figure 3

CROCCP2 gain of function leads to reduced ciliary length in apical cortical progenitors (A and B) Mouse in utero co-electroporation of ARL13B-RFP fused vector with pCIG (CONTROL) (A) or pCIG-MYC-CROCCP2 (CROCCP2) (B) at E13.5, followed by analysis at E14.5 through immunofluorescence staining of RFP and GFP. Scale bar, 10 μm. (A′ and B′) Inset illustrates primary cilia at the VZ apical surface. Scale bar, 5 μm. (C) Scatter plot showing primary cilia size at the VZ apical surface. n = 210 cilia, 5 CONTROL embryos, 221 cilia, 4 CROCCP2 embryos, ^∗∗∗p < 0.001. (D, E, H, and I) Immunofluorescence of primary culture of mouse embryonic cortex infected with either pLenti-CIG-CONTROL (CONTROL) (D and H) or pLenti-CIG-MYC-CROCCP2 (CROCCP2) (E and I) expressing lentivirus at E13 followed by analysis 72 h later. Scale bar, 10 μm. (D and E) Immunostaining of DAPI, GFP, Sox2, and Arl13b. (H and I) Immunostaining of DAPI, GFP, acetylated tubulin (AC TUB), and Sox2. (F and G) (F) Scatter plots showing ciliary length in Sox2⁺ cells using Arl13b (n = 4 experiments, 322 cilia CONTROL, 326 cilia CROCCP2, ^∗∗∗∗p < 0.0001) and (G) the percentage of nonciliated Sox2⁺ cells (n = 4; ^∗p = 0.286). (J) Scatter plot showing ciliary length in Sox2⁺ cells using acetylated tubulin (n = 2, 22 CONTROL cilia, 24 CROCCP2 cilia, ^∗∗p = 0.0029). (C, F, and J) Data are presented as mean ± SEM, one dot represents one cilium, p values by Student’s t test. (G) Data are presented as median ± IQ, each dot represents mean percentage of nonciliated cells per experiment, p values by Mann-Whitney test. See also Figure S4.

Figure 4

CROCCP2 co-localizes with IFT20 at the Golgi level (A and B) Immunofluorescence staining of HEK cells co-transfected with pCIG-HA-CROCCP2 (HA (CROCCP2) (B) or pCIG (CONTROL) (A) & p3xFLAG-IFT20 (FLAG(IFT20)). Note co-localization of CROCCP2 and IFT20 tagged proteins in CROCCP2 expressing cells. (C and D) Immunofluorescence staining with GM130 on HEK cells transfected either with p3xFLAG-IFT20 (C) or pCIG-HA-CROCCP2 (D) expressing vector. Note partial co-localization of IFT20 with GM130 (C) and CROCCP2 with GM130 (D). (E and F) Human 2D in vitro corticogenesis (D32), infected with pLenti-CIG-CONTROL (CONTROL) (E) or pLenti-CIG-MYC-CROCCP2 (MYC(CROCCP2) (F) at D25 and analyzed 6 days after infection. Note Myc signal co-localization with Ift20 endogenous signal (F). (G–J) Immunofluorescence staining of DAPI, GFP, and Arl13b on mouse cortex primary culture after ex utero at E13.5 using scramble shRNA (G), Ift20 shRNA (shIFT20) (H), pCIG (CONTROL) (I), PCIG-MYC-CROCCP2 (CROCCP2) (J) vectors. Data analyzed 72 h after transfection. Arrows indicate short primary cilia. (K) Scatter plot showing ciliary length measured through Arl13b immunostainings. Data are represented as mean ± SEM. Each dot represents one cilium. n = 2, 45 cilia CONTROL, 90 cilia scramble shRNA, 49 cilia CROCCP2, 72 cilia shIFT20, p = 0.8053 (CONTROL versus scramble shRNA), ^∗∗∗p = 0.0005 (CONTROL versus CROCCP2), ^∗∗∗p = 0.0008 (CONTROL versus shIFT20), ^∗∗p = 0.0020 (scramble shRNA versus shIFT20), ^∗∗p = 0.0015 (scramble shRNA versus CROCCP2), and p = 0.7801 (CROCCP2 versus shIFT20). p values by one-way ANOVA followed by post hoc Tukey test. (A–J) Scale bar, 10 μm. See also Figure S4.

Figure 5

CROCCP2 affects cilia and neurogenesis through IFT20 downregulation (A–D, G–J, and L–O) IUE of scramble shRNA, shRNA against Ift20 (shIFT20), pCIG (CONTROL), PCIG+p3XFLAG-IFT20 (CONTROL+IFT20), pCIG-MYC-CROCCP2 (CROCCP2), PCIG-MYC-CROCCP2+p3XFLAG-IFT20 (CROCCP2+IFT20) at E13.5. (A, B, and G–J) Immunofluorescence of coronal sections of E14.5 brains stained with GFP and phosphohistone H3 (PH3) or (C, D, and L–O) stained with GPF and Tbr2. Scale bar, 25 μm. (E) Scatter plot showing the percentage of PH3+/GFP+ cells and their localization. n = 9 scramble shRNA embryos, 7 shIFT20 embryos, ^∗p = 0.0399 (ALL mitosis), p = 0.05482 (APICAL), ^∗∗∗∗p < 0.0001 (BASAL). (F) Quantification of Tbr2⁺/GFP⁺ progenitors (n = 10 scramble shRNA embryos, 7 shIFT20 embryos, ^∗∗∗∗p < 0.0001). (K) Scatter plot showing the percentage of PH3+/GFP+ positive cell and their distribution. n = 30 CONTROL embryos, 5 CONTROL+IFT20 embryos, 34 CROCCP2 embryos, 13 CROCCP2+IFT20 embryos; ALL mitosis: p = 0.8110 (CONTROL versus CONTROL+IFT20), ^∗∗∗∗p < 0.0001 (CONTROL versus CROCCP2), p = 0.7257 (CONTROL versus CROCCP2+IFT20), p = 0.4684 (CROCCP2 versus CONTROL+IFT20), p = 0.4280 (CONTROL+IFT20 versus CROCCP2+IFT20), ^∗∗∗p = 0.0002 (CROCCP2 versus CROCCP2+IFT20). APICAL mitosis: p = 0.5865 (CONTROL versus CONTROL+IFT20), ^∗∗p = 0.0059 (CONTROL versus CROCCP2), p = 0.9964 (CONTROL versus CROCCP2+IFT20), p = 0.9763 (CONTROL+IFT20 versus CROCCP2), p = 0.5271 (CONTROL+IFT20 versus CROCCP2+IFT20), ^∗p = 0.0434 (CROCCP2 versus CROCCP2+IFT20). BASAL mitosis: p = 0.9929 (CONTROL versus CONTROL+IFT20), ^∗∗∗∗p < 0.0001 (CONTROL versus CROCCP2), p = 0.9990 (CONTROL versus CROCCP2+IFT20), p = 0.0531 (CONTROL+IFT20 versus CROCCP2), p = 0.9982 (CONTROL+IFT20 versus CROCCP2+IFT20), ^∗∗p = 0.0033 (CROCCP2 versus CROCCP2+IFT20). (P) Scatter plot showing the percentage of TBR2⁺/GFP⁺. n = 7 CONTROL embryos, 3 CONTROL+IFT20 embryos, 7 CROCCP2 embryos, 8 CROCCP2+IFT20 embryos: p = 0.9835 (CONTROL versus CONTROL+IFT20), ^∗∗∗p = 0.0002 (CONTROL versus CROCCP2), p = 0.9409 (CONTROL versus CROCCP2+IFT20), ^∗∗p = 0.0013 (CROCCP2 versus CONTROL+IFT20), p = 0.9999 (CONTROL+IFT20 versus CROCCP2+IFT20), ^∗∗∗∗p < 0.0001 (CROCCP2 versus CROCCP2+IFT20). (E, F, K, and P) Data are presented as mean ± SEM; each dot represents an embryo. (E and F) p values by Student’s t test. (K and P) p values by one-way ANOVA followed by Tukey post hoc test. See also Figure S5.

Figure 6

CROCCP2 affects cilia and neurogenesis through mTOR signaling upregulation (A and B) Flat-mount analysis of IUE using pCIG (CONTROL) (A) or pCIG-MYC-CROCCP2 (CROCCP2) (B) performed at E13.5 and fixed 24 h later (A, B, D, and E). Whole-mount staining of en-face view of the ventricular zone using DAPI, GFP and ZO1 to stain apical endfoot. (A and B) Arrows point to interphase progenitor apical endfoot—stars mark mitotic progenitors. Scale bar, 10 μm. (C) Scatter plot of of apical endfoot surface measurement. Data are represented as mean ± SEM. One dot represents one apical endfoot. n = 136 apical endfeet CONTROL—3 embryos,116 apical endfeet CROCCP2—4 embryos, 152 apical endfeet of non-electroporated (NOT ELEC) cortex sides—2 embryos, ^∗∗∗∗p < 0.0001 (CONTROL versus CROCCP2), ^∗∗∗∗p < 0.0001 (NOT ELEC versus CROCCP2), p = 0.8261 (NOT ELEC versus CONTROL). p values by one-way ANOVA followed by Tukey post hoc test. (D and E) Immunofluorescence analysis of mouse embryonic cortex primary culture infected with either plenti-CIG (CONTROL) (D) or plenty-CIG-MYC-CROCCP2 (CROCCP2) (E) at E13.5 and analyzed 72 h later. DAPI, GFP, and pS6 immunostainings as a readout of the MTOR pathway activity. Scale bar, 25 μm. (F) Scatter plot showing the percentage of pS6⁺/GFP⁺ cells. Data are represented as mean ± SEM, one dot represents one microscopic field (n = 4, 22 microscopic fields CONTROL, 20 microscopic fields CROCCP2, 22,631 cells CONTROL, 20,382 cells CROCCP2) ^∗∗∗p = 0.004 using Student’s t test. (G–J and N–Q) IUE of pCIG + scramble shRNA (CO+SHSCR), pCIG-MYC-CROCCP2 + shRNA (CRP2+SHSCR), pCIG-MYC-CROCCP2 + mix of shRNAs against mTOR (CRP2+SHMTOR), pCIG + mix of shRNAs against mTOR (CO+SHMTOR) at E13.5. (G–J) Immunofluorescence of coronal sections of E14.5 brains stained with GFP and phosphohistone H3 (PH3) or (N–Q) stained with GPF and TBR2. Scale bar, 25 μm. (K–M) Scatter plots showing the percentage and distribution of PH3+/GFP+ cells. BASAL: ^∗∗p = 0.0091 (CRP2+SHSCR versus CRP2+Shmtor), ^∗∗p = 0.0035 (CRP2+SHSCR versus CO+Shmtor), ^∗∗p = 0.0017 (CRP2+SHSCR versus CO+SHSCR), p = 0.7267 (CRP2+Shmtor versus CO+Shmtor), p = 0.8762 (CRP2+Shmtor versus CO+SHSCR), p = 0.9779 (CO+Shmtor versus CO+SHSCR) using one-way ANOVA followed by Tukey post hoc test. APICAL: nonsignificant differences among means, F = 1.503, p = 0.2372 using one-way ANOVA. ALL: nonsignificant differences among means, F = 2.955, p = 0.0511 using one-way ANOVA. (n = 7 CO+SHSCR embryos, n = 11 CRP2+SHSCR embryos, n = 8 CRP2+Shmtor embryos, n = 4 CO+Shmtor embryos). Data are presented as mean ± SEM; each dot represents an embryo. (R) Scatter plot showing the percentage of TBR2+/GFP+ cells. ^∗∗∗∗p < 0.0001 (CRP2+SHSCR versus CRP2+SHmtor), ^∗∗∗∗p < 0.0001 (CRP2+SHSCR versus CO+SHMTOR), ^∗∗∗∗p < 0.0001 (CRP2+SHSCR versus CO+SHSCR), p = 0.4249 (CRP2+Shmtor versus CO+Shmtor), p = 0.8844 (CRP2+Shmtor versus CO+SHSCR), p = 0.1556 (CO+Shmtor versus CO+SHSCR) using one-way ANOVA followed by Tukey post hoc test (n = 8 CO+SHSCR embryos, n = 7 CRP2+SHSCR embryos, n = 6 CRP2+Shmtor embryos, n = 3 CO+Shmtor embryos). Data are presented as mean ± SEM; each dot represents an embryo. See also Figure S6.

Figure 7

CROCCP2 is required for amplification of basal progenitors in human cortical organoids (A–H) Cytoarchitecture and cellular identity (FOXG1, SOX2, TBR2, TBR1) of human cortical organoids at day 30 following infection with (A–D) pLenti-CIG-GFP (CONTROL) or (E–H) pLenti-CIG-MYC-CROCCP2 (CROCCP2). Scale bar, 100 μm in (A and E) and 20 μm in (B)–(D) and (F)–(H). (I and J) Immunofluorescence of Hoechst, GFP, and PH3 performed on cryosections of hES-cell derived organoids at day 30. Arrows point to GFP+ PH3+ double-positive cells. Scale bar, 25 μm. (K) Scatter plot with mean ± SEM showing the percentage of phospho-histone H3 population among GFP-positive cells (CO versus CRP2 total PH3: ^∗∗∗∗p < 0.0001, CO versus CRP2 apical PH3: ^∗∗∗∗p < 0.0001, CO versus CRP2 basal PH3: ^∗∗∗p = 0.0003, n = 4, pCIG-control [CO]: 4,179 cells from 56 VZs of 12 organoids, pCIG-CROCCP2 [CRP2]: 2,094 cells from 29 VZs of 9 organoids). (L and M) Immunofluorescence of GFP and TBR2 performed on cryosections of cortical organoids at day 30. Arrows point to GFP+ TBR2+ double-positive cells. Scale bar, 20 μm. (N) Scatter plot with mean ± SEM showing the percentage of TBR2 (^∗p = 0.0149) populations among GFP-positive cells (L and N: n = 3, pCIG-control [CO]: 581 cells from 6 VZs of 6 organoids, pCIG-CROCCP2 [CRP2]: 681 cells from 6 VZs of 6 organoids; M: pCIG-control [CO]: n = 3, 2,011 cells from 10 VZs of 4 organoids, pCIG-CROCCP2 [CRP2]: n = 4, 1,983 cells from 12 VZs of 5 organoids). (O and P) Immunofluorescence of Hoechst, GFP, TBR2, and TUJ1 performed on cryosections of cortical organoids at day 60. Red and yellow arrowheads point to GFP+ TBR2+ and GFP+ TUJ1+ double-positive cells, respectively. Scale bar, 20 μm. (Q–S) Scatter plots with mean ± SEM showing the percentage of (Q) SOX2 (^∗∗p = 0.0036), (R) TBR2 (^∗∗∗p = 0.0006) and (S) TUJ1 (^∗∗p = 0.0013) populations among GFP-positive cells (R and S: n = 3, scramble shRNA [SCR]: 1,567 cells from nine VZs of six organoids, CROCCP2 shRNA [SHCRP2]: 1,301 cells from nine VZs of six organoids; Q: n = 3, scramble shRNA [SCR]: 2,163 cells from 13 VZs of 6 organoids, CROCCP2 shRNA [SHCRP2]: 2,620 cells from 18 VZs of 7 organoids). (T and U) Immunofluorescence of Hoechst, GFP, and SOX2 performed on cryosections of cortical organoids at day 60. Arrows point to GFP+ SOX2+ double-positive cells located outside of VZ structures delimited by dotted lines. Scale bar, 20 μm. (V) Scatter plot with mean ± SEM showing the percentage of basal SOX2 cells (^∗p = 0.0198) among total SOX2-positive GFP-positive cells (n = 3, scramble shRNA [SCR]: 668 cells from 12 VZs of 6 organoids, CROCCP2 shRNA [SHCRP2]: 794 cells from 12 VZs of 6 organoids). See also Figure S7.