Fig. 4: Effects of Wnt signaling knockdown in THY1⁺RXFP2⁺ APMCs.

A Dot plot showing the expression of WNT receptors and co-receptors in THY1⁺ clusters of each tissue. B UMAP plot visualizing the expression of RXFP2 and NEGR1. Note that the expression patterns of these two genes are very similar. C Flow cytometry assay showing the proportion of THY1⁺ APMCs (76%) in the male AP tissue. D Flow cytometry assay showing the proportion of THY1⁺RXFP2⁺ APMCs (16%) and THY1⁺RXFP2^- APMCs (84%) in the THY1⁺ APMC population. E Immunofluorescence staining assay of NEGR1⁺ APMCs, indicating that the sorting of RXFP2⁺ cells was successful. Scale bar: 100 μm. Note that NEGR1 is exclusively expressed in male AP RXFP2⁺ APMCs. F Schematic showing the design of sgRNA to knock down part of exon 1 of LRP6 using CRISPR technology. G Immunoblots of LRP6 protein of RXFP2⁺LRP6^KO and RXFP2⁺ APMCs and internal reference protein (GAPDH) in the AP-m tissue. H Comparison of cell viability of three types of THY1⁺ APMCs. Note that the proliferation of RXFP2⁺ APMCs increased significantly compared to RXFP2^- APMCs and RXFP2⁺LRP6^KO APMCs. ****p < 0.0001. Error bars indicate the SEM. I Cell apoptosis rate of RXFP2⁺LRP6^KO APMCs through TUNEL assay analysis. Note that there is almost no cell apoptosis for RXFP2^- APMCs and THY1⁺RXFP2⁺ APMCs. Yellow arrowhead: apoptotic cells. J Osteogenic ability of three types of THY1⁺ APMCs through Alizarin red staining. Note that the osteogenic ability of RXFP2⁺ APMCs increased significantly compared to RXFP2^- APMCs and RXFP2⁺ LRP6^KO APMCs. K Heat map showing correlations between RXFP2⁺ APMCs, RXFP2^- APMCs and RXFP2⁺ LRP6^KO APMCs based on bulk RNA-seq data. Pearson correlation coefficients are displayed. L Volcano plot showing the DEGs in RXFP2⁺ APMCs vs. RXFP2⁺LRP6^KO APMCs. DEGs (dot) with an absolute log₂FoldChange > 2 and an adjusted p value < 0.001 are highlighted in red. Note that 188 genes are up-regulated and 351 are down-regulated. M Dot plot showing top DAVID-enriched Gene Ontology terms with 188 up-regulated genes in RXFP2⁺ APMCs vs. RXFP2⁺LRP6^KO APMCs. The terms highly relevant to the context of the study are highlighted in red. N Dot plot showing top DAVID-enriched Gene Ontology terms using 351 down-regulated genes in RXFP2⁺ APMCs vs. RXFP2⁺LRP6^KO APMCs. The terms highly relevant to the context of the study are highlighted in red.