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. 2024 Feb 1;17(2):dmm050323.
doi: 10.1242/dmm.050323. Epub 2024 Feb 27.

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis

Max J van Essen  1   2 , Elizabeth J Apsley  1   2 , Joey Riepsaame  3 , Ruijie Xu  4 , Paul A Northcott  4 , Sally A Cowley  5 , John Jacob  1 , Esther B E Becker  1   2
Affiliations

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis

Max J van Essen et al. Dis Model Mech. .

Abstract

Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

Keywords: CRISPR; Cerebellum; Development; Medulloblastoma; Patched 1; Sonic hedgehog; iPSCs.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Generation of PTCH1 loss-of-function mutations in human iPSCs using CRISPR. (A) CRISPR gene editing strategy to target exon 3 of the PTCH1 gene. Guide RNAs (green) were designed to result in the excision of a 243-bp region spanning the splice donor (SD) site of exon 3. (B) PCR genotyping confirmed the generation of heterozygous (Het) PTCH1 iPSC clones producing both control (CTRL) (676 bp) and mutant (433 bp) PCR amplicons as well as homozygous (Hom) clones producing only the mutant amplicon. (C) Sanger sequencing of PTCH1 mRNA revealed the exclusion of exon 3 in the mutant mRNA, resulting in a frameshift mutation in exon 4. (D) Schematic depiction of the effect of exon 3 exclusion, resulting in a frameshift mutation and multiple premature translation termination codons in the new exon 4. See also Figs S1-S6.
Fig. 2.
Fig. 2.
Cerebellar differentiation of PTCH1-mutant iPSCs reveals changes in morphology and cerebellar lineage markers. (A) Schematic overview of the cerebellar differentiation protocol. Ri, Rho kinase inhibitor Y-27632; SB, SB431542. (B) Representative images of organoids generated from control (CTRL), PTCH1+/− (Het) and PTCH1−/− (Hom) iPSCs during differentiation. Arrowheads indicate regions of enhanced polarisation within the homozygous organoids. (C) Relative size change of organoids at day 35 compared to day 7. The data are from 46 (CTRL), 48 (Het) and 35 (Hom) individual organoids across three differentiations. Error bars indicate s.d. Statistical significance was computed by one-way ANOVA [F(2,126)=28.31, P<0.001] with Tukey's post hoc test (CTRL versus Het, P<0.001, 95% c.i. [0.3011, 0.9412]; CTRL versus Hom, P<0.001, 95% c.i. [0.7406, 1.4364]; Het versus Hom, P=0.005, 95% c.i. [0.1226, 0.8121]). (D) Expression of cerebellar and SHH pathway genes, measured by RT-qPCR. Each data point represents an experimental replicate. Data are from at least three biological replicates from separate differentiations. Error bars indicate s.d. Statistical significance was computed by one-way ANOVA: ATOH1 [F(2,46)=227.272, P<0.001], Dunnett's post hoc test (CTRL versus Hom, P<0.001, 95% c.i. [5.9792, 7.4879]); PAX6 [F(2,45)=581.687, P<0.001], Dunnett's post hoc test (CTRL versus Het, P<0.001, 95% c.i. [−1.5630, −0.6926]; CTRL versus Hom, P<0.001, 95% c.i. [5.0842, 5.9291]); KIRREL2 [F(2,19)=6.141, P=0.009], Dunnett's post hoc test (CTRL versus Hom, P=0.009, 95% c.i. [0.13853, 0.95927]); GLI1 [F(2,20)=25.272, P<0.001], Dunnett's post hoc test (CTRL versus Het, P<0.001, 95% c.i. [−2.8026, −0.9513]; CTRL versus Hom, P<0.001, 95% c.i. [−3.3523, −1.5857]). **P<0.01; ***P<0.001; ****P<0.0001. (E) Immunofluorescence staining of day 35 organoids with antibodies specific to the rhombic lip lineage marker PAX6 (green) and the ventricular zone marker KIRREL2 (green). KIRREL2 is primarily expressed in the small neuroepithelial lumina present in cerebellar organoids (arrows). Nuclei are visualised in blue by Hoechst staining. Images are representative of six independent experiments. Scale bars: 100 μm (top); 50 μm (bottom). See also Fig. S2.
Fig. 3.
Fig. 3.
Homozygous PTCH1 loss-of-function prevents cerebellar organoid differentiation. (A) Principal component analysis using the 1000 most variable genes showing the separation of samples in principal component (PC) 1 and PC2. Control (CTRL) samples were generated with the AH017 line, PTCH1+/− (Het) samples with lines A3 and C6, and PTCH1−/− (Hom) samples with lines A2, B3 and H3. (B) Volcano plot of the ten genes with the most significant change in either direction. Genes upregulated in homozygous organoids are depicted in red, downregulated genes are blue. (C) Expression of genes associated with the ventral neural tube, dorsal neural tube, hindbrain, forebrain, WNT inhibition and WNT signalling. Values are scaled within rows. The heatmap shows CTRL samples generated with the AH017 line, and Hom (PTCH1−/−) samples generated with clones A2, B3 and H3. (D) Immunofluorescence of day 35 organoids with antibodies specific to the pan-neuronal marker NeuN (green) and ventral neural tube marker NKX2-2 (red). Nuclei are visualised in blue by Hoechst staining. Images are representative of four independent experiments. Scale bars: 250 μm.
Fig. 4.
Fig. 4.
SHH pathway inhibition rescues cerebellar marker expression. (A) Simplified schematic of the SHH pathway with the effect of cyclopamine (red line, inhibitory) and SAG (green arrow, activating) on SMO. (B) Schematic of the experimental design to test the effect of SHH pathway inhibition on PTCH1−/− cerebellar organoid differentiation. Ri, Rho kinase inhibitor Y-27632; SB, SB431542. (C) Changes in gene expression of day 35 organoids in different treatment conditions as measured by RT-qPCR and plotted as log2(fold change or FC). Expression is relative to that of the DMSO control condition of each line and normalised using ACTB and GAPDH. n=4 per condition from two differentiations. Error bars indicate s.d. Statistical significance was computed by one-way ANOVA: CTRL organoids {PAX6 [F(3,12)=8.123, P=0.003], Dunnett's post hoc test (DMSO versus 500 nM SAG, P=0.004, 95% c.i. [0.9618, 4.5269]); ATOH1 [F(3,12)=13.631, P<0.001], Dunnett's post hoc test (DMSO versus 500 nM SAG, P=0.001, 95% c.i. [2.4912, 8.5367]); SHH [F(3,12)=201.872, P<0.001], Dunnett's post hoc test (DMSO versus 1 µM cyclopamine, P=0.026, 95% c.i. [0.2447, 3.8296]; DMSO versus 500 nM SAG, P<0.001, 95% c.i. [−13.9309, −10.3460]); NKX2-1 [F(3,12)=168.315, P<0.001], Dunnett's post hoc test (DMSO versus 500 nM SAG, P=0.001, 95% c.i. [−10.4973, −7.8783])}; Hom organoids {ATOH1 [F(2,9)=19.421, P=0.001], Dunnett's post hoc test (DMSO versus 5 µM cyclopamine, P<0.001, 95% c.i. [−9.7165, −3.9305]); PAX6 [F(2,9)=111.502, P<0.001], Dunnett's post hoc test (DMSO versus 1 µM cyclopamine, P<0.001, 95% c.i. [−5.2075, −2.8681]; DMSO versus 5 µM cyclopamine, P<0.001, 95% c.i. [−7.7988, −5.4595]); SHH [F(2,9)=9.029, P=0.007], Dunnett's post hoc test (DMSO versus 1 µM cyclopamine, P=0.005, 95% c.i. [0.4002, 1.8379]; DMSO versus 5 µM cyclopamine, P=0.02, 95% c.i. 0.1318, 1.5695]); NKX2-1 [F(2,9)=35.711, P<0.001], Dunnett's post hoc test (DMSO versus 5 µM cyclopamine, P<0.001, 95% c.i. [2.9451, 5.79]); SIX3 [F(2,9)=23.791, P<0.001], Dunnett's post hoc test (DMSO versus 5 µM cyclopamine: P<0.001, 95% c.i. [1.7809, 4.2339])}. *P<0.05; **P<0.01; ***P<0.001. (D) Immunofluorescence of day 35 organoids from different conditions with antibodies specific to PAX6 (green), KIRREL2 (red) or NKX2-2 (green). White arrowheads indicate the apical lumen of the cerebellar plate within the organoids, where typical staining of KIRREL2 can be seen. Nuclei are visualised in blue by Hoechst staining. Images are representative of four independent experiments. Scale bars: 100 μm. (E) Experimental design to test the effect of cyclopamine or SAG treatment cessation on cerebellar marker expression is depicted on the left. Heatmap showing the ΔCT values normalised to ACTB and GAPDH in each condition is shown on the right. n=3. The asterisk indicates statistical significance computed by two-tailed unpaired Student's t-test comparing treatment cessation with continued treatment [t(2)=−5.2331, P=0.03]. *P<0.05. Values are scaled within columns. (F) Principal component analysis showing different treatment and genotype conditions. The contribution of each gene to the principal components PC1 and PC2 is indicated by an arrow. The length of the arrow relates to the size of the contribution, indicating that SHH expression contributes the most to changes in PC1 and PAX6 expression has the greatest effect on PC2. See also Fig. S8.
Fig. 5.
Fig. 5.
PTCH1-heterozygous cerebellar organoids display tissue-specific effects of increased SHH signalling. (A) Principal component analysis using the 1000 most variable genes showing the separation of samples in principal components PC1 and PC2. Control (CTRL) samples were generated with the AH017 line, Het (PTCH1+/−) samples with clones A3, B2, B4 and C6. (B) Expression of genes associated with different stages of rhombic lip (RL) development and RL derivatives. Only differentially expressed genes are displayed. Values are scaled within rows. The heatmap shows control (CTRL) samples generated with the AH017 line, and Het (PTCH1+/−) samples generated with clones A3, B2, B4 and C6. (C) Bar graph of mean fluorescence intensity (MFI) values, measured by flow cytometry of PAX6-stained control (CTRL) and PTCH1+/− (Het) organoids. A total of 50,000 cells per pool were measured. n=4. Error bars indicate s.d. Asterisks indicate statistical significance computed by two-tailed unpaired Student's t-test [t(5)=−5.1567, P=0.005]. **P<0.01. (D) Venn diagram displaying the overlap in differentially upregulated genes. (E) Heatmap showing the Euclidean distance measurements of organoid samples with laser capture microdissected (LCM) regional samples (Aldinger et al., 2021). Samples are organised by region and age. Measurements are scaled in rows. A shorter distance relates to a higher similarity in gene expression profile. The black outline indicates the difference in similarity to RL samples seen between heterozygous and wild-type organoids. In bulk samples, the time points with the highest similarity are marked. (F) Gene sets uniquely enriched in heterozygous organoids. The colour of the dots corresponds to the Benjamini–Hochberg adjusted P-value, and the size of the dots corresponds to the number of genes allocated to each gene set. (G) Volcano plot of selected representative genes associated with the top uniquely differentially expressed pathways in heterozygous organoids. The size of each data point corresponds to the base mean expression of the gene. EGL, external granule cell layer; GCP, granule cell progenitors; PCL, Purkinje cell layer; PCW, post conception weeks; RL, rhombic lip.
Fig. 6.
Fig. 6.
PTCH1-heterozygous organoids display relevant features for medulloblastoma biology. (A) Immunofluorescence of day 35 control (CTRL) and PTCH1+/− (Het) organoids with antibodies specific to the proliferation marker CCNB1 (green). Nuclei are visualised with Hoechst. Scale bars: 100 μm. (B) Relative expression of CCNB1 normalised to Hoechst signals to correct for the number of nuclei in each organoid. Data points represent five different organoids from three differentiations. Error bars indicate s.d. Asterisks indicate statistical significance computed by two-tailed unpaired Student's t-test [t(21)=3.10, P=0.005]. **P<0.01. (C) Schematic of priming sites of primer pairs A and B. CTRL PTCH1 mRNA allows amplification from both primer pairs, whereas the mutant mRNA only allows the amplification from pair B. (D) Dot plot of ΔCT values generated by normalising the expression of pair A to pair B. Linear regression line shows the relation of ΔCT with time. Lower values represent the lower relative expression of pair A, correlating with the respective expression of wild-type PTCH1 mRNA. Data are from more than three biological replicates across separate differentiations. R2 values indicate the fit of the linear regression model. (E) Enrichment of genes upregulated in heterozygous PTCH1+/− organoids (PTCH1-orgs) in subtypes of human medulloblastoma (MB). Genes are significantly enriched in SHH-MB samples. Box plots represent the interquartile range (IQR), the whiskers extend to values within 1.5 times the IQR from the quartiles, and the median is marked with a line. Statistical significance was computed using a two-tailed unpaired t-test with P-values adjusted by the Holm–Bonferroni method. See also Fig. S9. (F) Example genes associated with druggable targets that were found to be upregulated in heterozygous organoids. Values are scaled within rows. The heatmap shows CTRL samples generated with the AH017 line, and Het (PTCH1+/−) samples generated with clones A3, B2, B4 and C6.
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References

    1. Ackerman, S. L., Kozak, L. P., Przyborski, S. A., Rund, L. A., Boyer, B. B. and Knowles, B. B. (1997). The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein. Nature 386, 838-842. 10.1038/386838a0 - DOI - PubMed
    1. Aguilar, A., Meunier, A., Strehl, L., Martinovic, J., Bonniere, M., Attie-Bitach, T., Encha-Razavi, F. and Spassky, N. (2012). Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome. Proc. Natl. Acad. Sci. USA 109, 16951-16956. 10.1073/pnas.1201408109 - DOI - PMC - PubMed
    1. Aldinger, K. A., Thomson, Z., Phelps, I. G., Haldipur, P., Deng, M., Timms, A. E., Hirano, M., Santpere, G., Roco, C., Rosenberg, A. B.et al. (2021). Spatial and cell type transcriptional landscape of human cerebellar development. Nat. Neurosci. 24, 1163-1175. 10.1038/s41593-021-00872-y - DOI - PMC - PubMed
    1. Apte, R. S., Chen, D. S. and Ferrara, N. (2019). VEGF in signaling and disease: beyond discovery and development. Cell 176, 1248-1264. 10.1016/j.cell.2019.01.021 - DOI - PMC - PubMed
    1. Aruga, J. (2004). The role of Zic genes in neural development. Mol. Cell. Neurosci. 26, 205-221. 10.1016/j.mcn.2004.01.004 - DOI - PubMed