Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate

The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear β-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human β-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when β-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain

S2 Fig -

(A, B) KEGG pathway analysis of Ncp and Hcp enriched genes related to Fig 1B. (C-D) Motif analysis shows known motifs from 70 DARs (Ncp) and 14804 DARs (Hcp) related to Fig 2B. (E) Expression of many of the transcription factors identified among the top 10 motifs is undetectable in the E11.5 Ncp or Hcp (as obtained from; Allen Mouse Brain Atlas, http://mouse.brain-map.org/). Links to images represented in E: Dlx1Dlx2Dlx5Lhx1Lhx3Isl1Nkx6.1En1Rfx2Rfx5Xbp1. (TIF)</p

Worksheets containing raw data of identifying DEGs of mutNcp vs wtNcp Fig 5A, DEGs of mutHcp vs wtHcp in Fig 5B, a list of all genes identified by Venn diagram in Fig 6E.

Worksheets containing raw data of identifying DEGs of mutNcp vs wtNcp Fig 5A, DEGs of mutHcp vs wtHcp in Fig 5B, a list of all genes identified by Venn diagram in Fig 6E.</p

Chromatin accessibility changes upon loss of <i>Lhx2</i> in the Ncp and Hcp.

(A, B) Scatter plots comparing control versus Lhx2 mutant chromatin identify loci for which the global accessibility has changed upon loss of Lhx2 in the Ncp (0) and Hcp (463+1 DARs which map to 405+1 DAGs). (C) Plot profile comparisons of wild type and mutant chromatin in the Hcp showing that the mutant chromatin is less accessible at LHX2 binding sites, TSS, and regions identified in Fig 2B to be differentially accessible (DARs) between wtNcp and wtHcp. (D) Histone modification profiles in the Hcp focusing on the TSS reveal that the loss of Lhx2 appears to be associated with an increase in the repressive mark H3K27Me3, a reduction in the mark H3K4Me3, and no apparent change in H3K27Ac. (E) A Venn diagram illustrates the majority of the down-regulated DARs are associated with an LHX2 binding peak in the Hcp. (F) Examples of genomic loci showing LHX2 binding regions at which chromatin accessibility is decreased upon loss of Lhx2 (also see S6D Fig).</p

S4 Fig -

(A-F) GO: BPs corresponding to both up-and down-regulated genes upon loss of Lhx2 in the Ncp (A-C) and Hcp (D-F). (A, D) show the GSEA analysis and (B, C, E, F) show the overrepresentation test analysis. (TIF)</p

Transcriptomic analysis of the wild type E12.5 Ncp and Hcp.

(A) Schematic representation of the E12.5 mouse brain. (B) A volcano plot comparing Ncp and Hcp mRNA expression identifies preferentially enriched genes in the Ncp (1248) and the Hcp (1364). (C) A heatmap of the top 100 enriched genes in each tissue type; color bar: blue (low expression), red (high expression), clustering method: K means. (D, E) A tree plot depicting the top GO Biological Processes (GO: BPs) from (B). (F) Bar plot of selected differentially expressed genes between Ncp and Hcp. (G, H). In situ hybridization for selected genes identified in (B).</p

Gene Regulatory Networks modulated by LHX2 in the Ncp and Hcp.

(A, C) Venn diagrams depicting the number of genes occupied by LHX2 and dysregulated (blue: downregulated, red: upregulated) upon loss of Lhx2 in the Ncp (A) and Hcp (C) respectively to identify direct targets of LHX2 in the Ncp and Hcp. (B, D) Genes dysregulated upon loss of Lhx2 in the Ncp (B) and Hcp (D) respectively, categorized by “Direct” or (direct + indirect) = “All” targets, mapped to the cell-type specific gene enrichment profiles in [28]) to identify progenitor-enriched (grey) and neuron-enriched genes (black). (E) Venn diagram comparing the direct targets of LHX2 that are dysregulated upon loss of Lhx2 in the Ncp (112 downregulated; 118 upregulated) and Hcp (70 downregulated; 153 upregulated), and in both tissues (43 downregulated; 35 upregulated). (F) Comparison of LHX2 occupancy in the E10.5 dorsal telencephalon (dtel; blue circle) with that in the E12.5 Ncp (red) and Hcp (green) results in genes occupied in all these three tissues (716, yellow), in the E10.5 dtel and the E12.5 Ncp (286, red) or the E12.5 Hcp (1252, green). (G) Venn diagram comparing the genes in E (LHX2 direct targets) that are also occupied by LHX2 at E10.5. In the Ncp, there are 62 downregulated 59 upregulated genes. In the Hcp there are 33 downregulated upregulated 94 upregulated. 37 downregulated and 25 upregulated are common to both tissues. (H) Heatmaps displaying genes occupied by LHX2. Cluster 1: Occupancy at both E10.5 (dtel) and E12.5 (Ncp and Hcp). Cluster 2: Occupancy at only E10.5. (I-L) KEGG pathway analysis (GO: BP) of genes identified in (E, G) reveals 4 pathways dysregulated upon loss of Lhx2 in the Ncp (red bars) and Hcp (green bars). Individual fold changes are plotted from the RNA-seq data (black: genes occupied by LHX2 at E12.5 and E10.5; blue: occupied only at E12.5).</p

LHX2 occupancy in the E12.5 mouse neocortical and hippocampal primordia (Ncp and Hcp, respectively).

(A) Schematic representation of the E12.5 mouse brain. (B) Progenitor markers PAX6, LHX2, and TBR2 immunostaining/in situ hybridization (Lhx2) in the Ncp and Hcp. (C-G) LHX2 ChIP-seq data in the Ncp and Hcp. Plots of PePr peak-called regions in the Ncp and Hcp show TF LHX2 occupancy in each tissue. Only statistically significant peaks were used for further analysis (p-value 0.0001 and fold change over input: cut off >10 fold) (C); The number of LHX2 occupancy peaks and associated number of genes (D); common genes occupied by LHX2 between the Ncp and Hcp (E); Percentage of LHX2 occupancy peaks categorized by type of genomic region (F); LHX2-occupied genes enriched in different cortical cell types identified by gene enrichment profiles in [28](G). The scale bars in B are 100 μm.</p

S5 Fig -

(A, B) KEGG pathway analysis of direct targets of LHX2 from Fig 5 (F) reveal 4 dysregulated pathways common to the E12.5 Ncp and Hcp. (C-F) KEGG pathway analysis for these pathways includes both direct (*) and indirect targets of LHX2. (E, F). (TIF)</p

Worksheet containing raw data of DARs identified in Fig 2B, a list of annotated DARs identified in Fig 2B, a comparative analysis of genes associated with DARs and gene expression in wtHcp vs wtNcp related to S1B Fig.

Worksheet containing raw data of DARs identified in Fig 2B, a list of annotated DARs identified in Fig 2B, a comparative analysis of genes associated with DARs and gene expression in wtHcp vs wtNcp related to S1B Fig.</p