E14.5E16.5E18.5 Normalized mRNA level Get1 Nfix Smarcd3 A Supplementary Figure 1 (A) The microarray expression levels of bladder terminal differentiation markers in skin, and skin terminal differentiation markers in bladder at the indicated embryonic stages. Upk1b and Upk3b are expressed in developing skin at E14.5 and E16.5, other uroplakin genes are not expressed in the developing skin, and no uroplakins are expressed at E18.5. Loricrin and Filaggrin are not expressed in bladder. (B) Validation by quantitive RT-PCR of uroplakin gene expression during bladder development. Uroplakin mRNA expression increases during bladder development. (C) Validation by quantitive RT-PCR of selective transcription factor during skin and bladder development E14.5E16.5E18.5 UpkIb UpkIIIb B E14.5E16.5E18.5 UpkIb UpkIIIa UpkII Bladder E14.5E16.5E18.5 Ivl Ppl Evpl Skin C Bladder Skin Normalized mRNA level Normalized Gene Expression Level

Supplementary Figure S2 – Principle component analysis (PCA) of bladder and dorsal skin samples at E14.5, E16.5, and E18.5. (A) PCA performed based on GC-RMA values for all probe sets. Each array is represented by a single point, and there are multiple biological replicates (2-3) for each tissue/stage. The first three principle components (PC #1, 2, 3) takes into account of 53.4%, 11.3%, and 7.3% (72% combined) of total variability within the data set, respectively. Note that E14.5 dorsal skin has a more similar global gene expression profiles to bladder samples than to E16.5 and E18.5 dorsal skin (B) Global similarity in gene expression between dorsal skin and bladder at E14.5, E16.5, and E18.5 as indicated by average of Pearson product moment correlation coefficients across the all probe sets. (C) Representative genes with more similar expression level between E14.5 dorsal skin and all bladder time points in comparison to E16.5 and E18.5 dorsal skin. Plotted are the average gene expression levels (GC-RMA values) of the replicates for each tissue type at the indicated embryonic age Lce1A1DspCdsnKrt8Krt18Krt19 Bladder E14.5 Bladder E16.5 Bladder E18.5 Skin E14.5 Skin E16.5 Skin E18.5 Avg. expression (GC - RMA) BC A

Normalized gene expression level Embryonic days E14.5 E16.5 E18.5 A B Hdac E14.5E16.5E18.5 Normalized mRNA level Yeats E14.5E16.5E18.5 Normalized mRNA level Fmo E14.5E16.5E18.5 Normalized mRNA level Agr E14.5E16.5E18.5 Normalized mRNA level Snx E14.5E16.5E18.5 Normalized mRNA level

Supplementary Figure S3. Time-course gene expression analysis defines specific functional categories orchestrating bladder development. (A) The average profile for each cluster (#1-6) is indicated by red lines connecting the box plots at each time point. Listed are the key significantly enriched functional categories (Gene Ontology Biological Process), with the associated number of genes and enrichment P-value, found within each cluster. For each cluster, the transcription regulators or chromatin modifiers are listed as gene symbols. Note Grhl3 (Get1) in bold is within cluster 2. The first three clusters have increasing expression during differentiation with different dynamics over the time course. Cluster 1 has high expression already at E16.5 and is enriched for genes involved in metabolic processes and muscle development, suggesting that key steps in bladder muscle development are completed relatively early. This cluster contains PPARγ which through in vitro experiments has been implicated in the control of urothelial differentiation (Varley et al, 2006; Varley et al, 2004). Cluster 2 shows time course dynamics that are intermediate between those of clusters 1 and 3; it has lower increment in expression between E14.5 and E16.5 than cluster 1. This cluster, which contains Get1, has significantly enriched functional categories that are characteristic for cytoskeletal and membrane organization, including the uroplakin genes, as well as lipid metabolism. This is consistent with the specialized lipid composition and the importance of the AUM in differentiated cells of the epithelium and suggests a possible role for Get1 in these processes. Cluster 3 shows later activation with expression increasing at E18.5. There is a striking enrichment of genes involved in cell communication, suggesting that these genes are involved in late umbrella cell differentiation. Clusters 4 to 6 show decreasing expression during bladder development with each cluster showing somewhat different time dynamics. Genes encoding cell cycle regulators and cellular metabolic processes are highly enriched in these clusters, consistent with decreasing epithelial proliferation as differentiation progresses. (B) Quantitative RT-PCR validation of selective genes.

Get1 E18.5 bladder (3 CTC vs. 3KO) 622DEGsbetween CTC and KO One-way ANOVA across genotype (P< , FDR within 10%) 226 highlyDEGs(66 down, 160 up) Removed scan date batch effect Filtered based on fold change ( ? -1.5 or ? 1.5 fold change) 66 bladder differentiation-regulated DEGs(20 down, 46 up) Filtered based on bladder differentiation-regulated genes Supplementary Figure S4 Overview of data processing and statistical analysis to identify significantly differentially expressed genes in E18.5 Get1-/- bladder that are also regulated during normal bladder development. FDR – false discovery rate. DEG – differentially expressed gene.

Supplementary Figure S5. Quantitative RT-PCR validation of Cldn2 and Cldn5

BC A

Supplementary Figure S6. (A) Location of potential Get1 binding sites in the five mouse uroplakin genes. Potential Get1 binding sites with the consensus sequence (AACCGGTT) were identified with BEARR (Batch Extraction and Analysis of cis-Regulatory Regions) analysis using the PMW (position weight matrices) score  5.7 by extracting the sequences (-50kb and +25kb up- and downstream of transcription start site, respectively, or to the nearest gene) of the mouse uroplakin genes (Vega et al., 2004). The sequence and annotation database selected for BEARR analysis was the Mouse Genome Assembly NCBI Build 33. The VISTA Genome Browser (Frazer et al., 2004) was used to generate a graphical representation of the conserved and non-conserved sequences surrounding uroplakin genes. Dark blue boxes represent exons, light blue boxes UTRs, pink boxes conserved non-coding sequence (50% conservation between mouse and human). Triangles indicate the positions of the potential Get1 binding. Conserved Get1 binding sites between mouse and human are marked in green. The number on top of the triangle marks PMW score of the potential Get1/Grhl3 binding site by BEARR. The Get1 sites used in gel shift or chip assay are marked with star. (B) Eletrophoretic mobility shift assays with a 32P-labeled consensus DNA-binding site for the Get-1 protein. Binding was competed with the 1, 4, and 16 ng/uL of unlabeled binding sites from the indicated uroplakin genes, as shown in A. Human UpkIb binding site is located in a conserved region from to bp and human UpkIIIa binding site is located in a conserved region from to -7436bp. (C) Chromatin immunoprecipitation (ChIP) assays performed in RT4 cells using a Get1 antibody or IgG as a control followed by PCR amplification of the regions of the human UpkIB and UpkIIIa promoter containing the Get1 site indicated in B, and human DHFR gene as a negative control

CD AB Supplementary Figure S7 (A,B) Histology of WT and Get1-/- forestomach at E18.5 (C,D) histology of WT and Get1-/- distal intestine at E18.5. Scale bar: A,B, 12.5  M; C,D, 100  M. +/+-/- +/+-/-