Prdm16 mutant ChP cells fail to exit cell cycle and are still neural progenitors

(A) RNA in situ hybridization with a Prdm16 probe, showing expression of Prdm16 in the E10.5 and E12.5 brains. The line and bracket indicate the developing ChP. (B) Staining of PRDM16 using a PRDM16 antibody on the E13.5 control and null mutant brains. Red: PRDM16; blue: the nucleus dye DAPI. White arrows indicate the ChP in the control. (C) RNA in situ hybridization using a Ttr probe on E12.5 control and Prdm16 null mutant forebrains. (D) Two-hour EdU labeling in the E12.5 control and homozygous brains. The brain sections are co-stained with an antibody against B-gal. Note: the lacZ gene trap is present in the heterozygous and homozygous mutants but not in wild-type animals, and the highest expression of B-Gal is seen at the ChP indicated by brackets. Red: B-Gal; blue: the nucleus dye DAPI; green: EdU. (E) DCX antibody staining on the E12.5 control and homozygous brain slices. White arrows indicate some of the DCX positive cells. Quantification of EdU+ (D) or DCX+ (E) cells is shown on the right. Biological replicates: N=3. Error bars represent standard deviation (SD). Statistical significance is calculated using unpaired t-test. ***, p<0.001. (F) Double S-phase labeling as illustrated on the top. Immunostaining of the control and Prdm16 KO brain slices using a PRDM16 and a BrdU antibodies and the EdU detection kit. An enlarged view highlight the ChP area is presented below. (G) Quantification of EdU+ and BrdU+ cells from the double labeling experiment. Biological replicates: N=3. Error bars represent standard deviation (SD). Statistical significance is calculated using unpaired t-test. ***, p<0.001.

Prdm16 is required for BMP4-induced NSC quiescence

(A) Illustration of the NSC culture assay. (B) Immunostaining of mKI67 in red and SOX2 in green and EdU labeling in blue on indicated NSC genotypes and treatment. (C-D) Quantification of the fraction of mKI67+ (C) and EdU+ (D) cells among the total cell number marked by SOX2. (E) RT-qPCR measurement of Ttr levels relative to 18S RNAs. Biological replicates: N=3. Error bars represent standard deviation (SD). Statistical significance is calculated using unpaired t-test. ***, p<0.001; **, p< 001; *, p<0.05; n.s., non-significant

BMP signaling represses cell proliferation genes to induce cell quiescence

(A) Heatmaps of ChIP-seq coverage (normalized by library depths and input) centered at peak summits for SMAD4, pSMAD1/5/8 and SMAD3 in Prdm16_E cells with or without BMP4. (B) A volcano plot of differential H3K4me3 read counts at TSS in Prdm16_E cells with BMP4 versus those with no BMP4. Significant changes (P < 0.01) are colored. (C) A bubble plot showing GO terms significantly enriched in SMAD-repressed genes (labelled in dark green in B). (D) A volcano plot of differential H3K4me3 read counts at TSS in Prdm16_KO cells with BMP4 versus Prdm16_E cells with BMP4. Significant changes (P < 0.01) are colored. (E) A heatmap of H3K4me3 normalized read counts at TSS of the 31 genes co-repressed by PRDM16 and SMADs (overlapping genes from those in dark green in B and those in dark pink in D). (F) RT-qPCR measurement of Wnt7b and Cdkn1a mRNA levels in indicated genotypes and conditions from three biological replicates, each with two technical replicates. Statistical significance was calculated using unpaired t-test (***, p<0.001; **, p< 0.01; *, p<0.05; n.s., non-significant). (G) Genome browser snapshots showing the H3K4me3 CUT&TAG tracks in the Wnt7b and Cdkn1a loci.

PRDM16 anchors the SMAD proteins at specific genomic regions to mediate repression

(A) A Venn diagram showing the overlap of CUT&TAG peak in BMP4-treated and non-treated Prdm16_E cells. (B) The mean coverages (normalized by library depths) centered at peak summits are shown for the PRDM16 CUT&TAG signal in BMP4-treated and non-treated Prdm16_E cells and Prdm16_KO mutant samples (4 replicates combined). (C) Metaplots of SMAD4 and pSMAD1/5/8 ChIP-seq coverage centered at SMAD or pSMAD peak summits that have PRDM16 co-binding (top) and those without PRDM16 co-binding (bottom) in BMP4 treated Prdm16_E and Prdm16_KO cells. The average coverage is plotted in the bar plots on the right. Only the peaks with PRDM16 co-binding show significant reduction in the KO cells (*** P<0.001; ** P<0.01; * P<0.05; n.a. P>0.05). (D-E) Genome browser view of the Wnt7b and Id1 loci, showing PRDM16 CUT&TAG and SMAD ChIP-seq tracks. The co-bound regions are highlighted. (F-G) ChIP-qPCR validation of SMAD4 binding to the highlighted regions in D-E from Prdm16_E and Prdm16_KO cells with two replicates. (H) The occurrence frequency of four types of SMAD motifs is shown in five binned SMAD ChP-seq peaks scored from low to high. The palindromic motif GTCTAGAC shows the highest occurrence frequency in peaks with PRDM16 co-binding (solid red) and is the only one that has reduced occurrence frequency in peaks without PRDM16 co-binding (dashed red).

The SMAD complex switches genome localization in the absence of Prdm16

(A-B) Genome browser snapshots of the Id3 (top) and Wnt7b (bottom) gene loci, showing ectopic SMAD peaks, indicated by the arrows. (C) The volcano plots showing differential SMAD4 and pSMAD1/5/8 ChIP-seq read counts in BMP4-treated Prdm16_KO cells compared to BMP4-treated Prdm16_E cells. The sites in blue are co-bound by PRDM16, while the sites in red are not bound by PRDM16 but within the 200kb of a PRDM16-cobound site. The average log2 fold-change is plotted on the right. (D-E) De novo motif discovery from SMAD4 and pSMAD1/5/8 ChIP-seq peaks in Prdm16_E cells (D) and Prdm16_KO cells (E). (F-G) ChIP-qPCR validation of increased SMAD4 and c-FOS occupancy at the highlighted ectopic sites in Prdm16_KO mutant cells versus Prdm16_E cells. The locations of PCR amplicons are shown in A and B. (H) An enhancer switch model demonstrating that SMADs change co-factors and become into a transcriptional activator in the absence of PRDM16.

PRDM16 and SMADs co-regulated genes are differentially expressed in the ChP and neural cells

(A) An illustration of the CH and ChP regions with opposite Wnt and BMP gradients and a violin plot of scRNA-seq gene expression at E12.5 for the indicated genes in several selected cell type clusters reanalyzed from 37. Definition of the clusters is based on marker gene expression from E9.5-E18.5 brain cells. These include the choroid plexus clusters 1-5 (Chpl), with cluster Chpl5 identified by this study based on Ttr expression, Radial glia clusters (RglCH 1-3, RglDF 6-10, and RglF7). CH: cortical hem; DF: dorsal forebrain; F: forebrain. Per cell expression was normalized to give a log2 normalized counts. (B) Metaplots and heatmaps of PRDM16 and IgG CUT&TAG coverage (normalized by library depths) centered at peak summits from E12.5 dorsal midline tissues. (C) A genome browser snapshot of the Wnt7b locus, showing PRDM16 CUT&TAG and SMADs ChIP-seq tracks.

PRDM16 suppresses Wnt signaling and cell proliferation in ChP epithelial cells

(A) SCRINSHOT images of E11.5 wild-type and Prdm16 mutant brains, with BMP4, Ttr, Wnt7b and Wnt2b probes. Note: The red arrows indicate a strong unspecific signal in all channels due to autofluorescence of blood cells. The cells selected for quantification are highlighted in yellow in the DAPI channel. (B) Enlarged views of SCRINSHOT images at the area indicated in A, showing an increase of Wnt and neural gene expression levels and decrease of Ttr and Prdm16. (C) A violin plot with the dot counts measuring expression levels of the 24 genes examined. Each gene is measured in 330 CH and 330 ChP cells from three wild-type and three Prdm16 mutant samples. (D) SCRINSHOT images for E12.5 wild-type and Prdm16 mutant brains, with Axin2, Ttr, Wnt 7b, Wnt2b and mKi67 probes. (E) Enlarged views of the areas indicated in the dash-lined rectangles in D. The ChP parts are highlighted by dashed lines.(F) Distribution of dot counts for indicated genes in the wild-type (wt) and Prdm16 mutant (mut) CH and ChP areas. The X-axis shows the 110 cells in each replicate, and the Y-axis shows the mean value of the number of counts in three biological replicates. P value was calculated using area under the ROC curve in Prism Graphpad. (G) Multi-variant linear regression of mKi67, Axin2 and Ttr using the dot counts for each gene in three pairs of wild-type and Prdm16 mutant samples. All 660 cells including both CH and ChP tissues in each genotype are included. Pearson correlation and statistical difference were calculated in Prism Graphpad. (H) Illustration of the regulatory circuit, which is required for transitioning proliferative NSCs to quiescent NSCs, a prerequisite for the specification of ChP epithelial cells.