Background: The myocardin (MYOCD) gene, highly expressed in detrusor smooth muscle cells (SMC), can cause a Prune Belly Syndrome-like disorder when mutated. MYOCD is a nuclear co-transcription factor that activates expression of SMC contractile genes, converting embryonic mesenchyme to SMC. Myocd knockout mice die in early embryonic life from malformed vascular SMC. However, megabladder-/- (Mgb) mice, having a chromosome rearrangement disrupting bladder-specific Myocd regulatory regions, manifest normal vascular and visceral SMC development except in the detrusor. These mgb mice, lacking detrusor SMC development at embryonic day 15.5(E15.5), survive fetal life and survive postnatally only with vesicostomy. Here, we use cutting-edge technology to spatially resolve differences in gene expression between wild type (WT) and Mgb E15.5 embryos to better understand SMC differentiation and bladder organogenesis.Methods: Duplicate WT and Mgb E15.5 embryos were formalin-fixed, paraffin-embedded, and cross-sectioned. Slices were placed on 10X Genomics Visium mRNA spatial transcriptomics slides(55μm diameter spatially barcoded spots arrayed 100μm apart). After tissue permeabilization and mRNA reverse-transcription, cDNA was amplified and Illumina NovaSeq6000 sequenced. Spatial transcriptomics data were processed and statistically analyzed with SPATA2 (selection of organ/tissue regions), 10X Genomics Space Ranger-2.1.1, and Seurat-5.0.3(dimensional reduction/clustering/cell-deconvolution), referencing mouse GRCm38-2020. Gene Ontogeny(GO) analysis identified functional attributes of top up- and down-regulated gene transcripts, with validation by RNAscope or immunofluorescence.Results: Histologically, E15.5 WT and Mgb bladders are the same size but Mgb bladders have no detrusor SMC visible (Figure-1A). Histologically, WT bladders were spatially divided into WT-urothelium, WT-intermediate, and WT-outer (SMC) layers, while Mgb bladders could only be divided into Mgb-urothelium and Mgb-outer (Figure-1B). Transcripts from WT SMC spots were compared to Mgb-outer by gene expression analysis, identifying 21 upregulated and 48 downregulated genes (Figure-1C), including downregulation of 18 known SMC genes (Figure-1D-F). For secondary validation of SMC downregulated genes, we confirmed significant reduction of Myocd expression using RNAscope and of α-SMA/Acta2 and Tagln/Sm22 by immunofluorescence (Figure-1E) in WT versus mgb bladders. Additionally, intestine's SMC layer showed no SMC gene differences between WT and Mgb, reinforcing the bladder-specific phenotype (Figure-1B and 1G). Of the 21 upregulated mgb genes, 10 relate to extracellular matrix by GO(Figure-1H) and 6 have unique attributes not previously associated with Myocd deficiency, including Creb3l1 (collagen gene transcriptional factor), Itm2b, S100a6, Islr, and Bcl2l1 (stem cell maintenance/ differentiation inhibition and regulation of apoptosis/proliferation) and Mkrn1 (E3-ubiquitin ligase targeting PPARγ, a crucial transcription factor for urothelium differentiation) (Figure1I). Mgb-outer maintains a mesenchymal-like molecular signature by cell deconvolution (Figure-1J).Conclusions: Spatial transcriptomics of Myocd-deficient Mgb E15.5 bladders has identified 1) novel genes dysregulated, 2) bladder-specific SMC gene dysregulation, since gut SMC differentiation is not altered, and 3) a pattern of gene dysregulation revealing that in the absence of Myocd, the upregulated genes prevent the outer layer from differentiating, maintaining the mesenchymal phenotype. These findings deepen our comprehension of Myocd’s role in bladder organogenesis and open new avenues for targeted therapeutic interventions. Grants:NIH-R01DK100483,DK127589,AUA/UCF,OSU-NCH
