The epidermal differentiation-associated Grainyhead gene Get1/Grhl3 also regulates urothelial differentiation

Abstract

Skin and bladder epithelia form effective permeability barriers through the activation of distinct differentiation gene programs. Using a genome-wide gene-expression study, we identified transcriptional regulators whose expression correlates highly with that of differentiation markers in both the bladder and skin, including the Grainyhead factor Get1/Grhl3, which is already known to be important for epidermal barrier formation. In the bladder, Get1 is most highly expressed in the differentiated umbrella cells and its mutation in mice leads to a defective bladder epithelial barrier formation due to the failure of apical membrane specialization. Genes encoding components of the specialized urothelial membrane, the uroplakins, were downregulated in Get1(-/-) mice. At least one of these genes, uroplakin II, is a direct target of Get1. The urothelial-specific activation of the uroplakin II gene is due to selective binding of Get1 to the uroplakin II promoter in urothelial cells, which is most likely regulated by histone modifications. These results show a crucial role for Get1 in urothelial differentiation and barrier formation.

Figure 1

Shared transcriptional regulatory programs of bladder and skin differentiation. (A) Global analysis of common and unique transcriptional regulators regulated during bladder and skin differentiation at E14.5, E16.5, and E18.5. TR, transcription regulator. (B) The expression patterns of terminal differentiation markers of bladder epithelium and dorsal skin at the indicated embryonic age. (C) Heat map of transcriptional regulators with expression highly correlated to terminal differentiation markers of the bladder and dorsal skin. Note the Grhl3 (Get1) in bold. (D) Heat map of transcriptional regulators that are expressed in the bladder but not in dorsal skin, and that are highly correlated to bladder terminal differentiation markers. Note that Foxq1 has multiple probe sets. For panels C and D, expression levels are normalized across time points and indicated by the colorimetric ratio scale.

Figure 2

Get1 is required for bladder epithelium umbrella-cell development. (A–C) WT mouse bladder epithelial sections from E16.5 (A) and E18.5 (B) were immunostained with Get1 antibody, whereas Get1 immunostaining in the E18.5 Get1^−/− bladder was used as negative control (C). Arrowheads point to umbrella cells. Get1 is specifically expressed in the developing and mature umbrella cells of the urothelium. (D, E) Urinary bladder (Bl) and kidneys (K) at E18.5 from WT (D) and Get1^−/− mice (E). The Get1^−/− urinary bladder is enlarged, whereas the kidneys appear normal. (F–I) Hematoxylin–eosin staining of the bladder epithelium from WT (F, H) and Get1^−/− (G, I) mice at E16.5 (F, G) and E18.5 (H, I). Arrowheads point to the umbrella cells in WT mice at E16.5 and E18.5. Arrows point to the surface-layer cells in Get1^−/− urothelium at E18.5. The dashed lines indicate the border between the epithelium and mesenchyme. Umbrella cells are absent from the Get1^−/− bladder epithelium. Scale bar: (A–C) and (F–I), 12.5 μM.

Figure 3

Get1 is required for apical cell-membrane specialization in the bladder epithelium. (A–D) Scanning electron microscopy of the bladder epithelial surface of WT (A, B) and Get1^−/− (C, D) mice at E18.5. Red arrows mark the outline of superficial cells. Yellow arrowheads point to hinge regions on the plicated surface of WT mice. Yellow arrows point to microvilli in the Get1^−/− urothelium. The superficial cells of the Get1^−/− urothelium are much smaller than in the WT, and their surface contains microvilli rather than ridges. (E, F) Transmission electron microscopy of WT (E) and Get1^−/− (F) bladder epithelium. Arrowheads in (E) point to fusiform vesicles. Arrowheads in (F) point to discoidal vesicles. Arrows in (F) point to microvilli. The Get1^−/− urothelium lacks urothelial apical plaques, and uroplakin-containing fusiform vesicles are much less prominent than in the WT. (G) Quantification of vesicle number in bladder superficial cells. The Y-axis shows the vesicle number per μm². The results represent the mean and s.e.m. for three WT and three Get1^−/− embryos. (H) Model for the role of Get1 in urothelial cell differentiation.

Figure 4

Get1 is required for effective permeability-barrier formation and terminal differentiation in mouse bladder epithelium. (A, B) Gross pictures of methylene-blue penetration assay of WT (A) and Get1^−/− (B) urinary bladders. (C, D) Post-sections of WT (C) and Get1^−/− (D) urinary bladders after methylene-blue assay. Arrows in (C) and (D) point to the bladder epithelium. The methylene-blue dye infiltrates the bladder wall of Get1^−/− mice, indicating impaired permeability barrier. (E–N) Immunostaining showing expression of K18 (E, F), K20 (G, H), K6 (I–L) and phospho-histone 3 (M, N) in WT (E, G, I, K, M) and Get1^−/− (F, H, J, L, N) bladder epithelium at the indicated stages. Arrows in (I) and (K) point to normal umbrella cells. Expression of the umbrella-cell markers K18 and K20 is decreased, indicating impaired differentiation. The expression of K6 is markedly upregulated and extends to the surface in Get1^−/− bladder epithelium, where it is normally excluded. (O) Quantification of phospho-histone-3-positive cells in WT and Get1^−/− bladder epithelium. The number of phospho-histone-3-positive cells is normal, indicating that the cell proliferation is unchanged in the Get1^−/− bladder epithelium. Scale bar: (C, D) 50 μM; (E–N) 12.5 μM.

Figure 5

Identification of significantly differentially expressed genes in E18.5 Get1^−/− bladder. (A) Downregulated and (B) upregulated bladder differentiation-related genes. Expression levels from microarrays are normalized across time points and indicated by the colorimetric ratio scale. The fold and P-value of significant differential expression, as well as the cluster membership of the WT time course, for each gene are indicated. Note that UpkII (in bold) is significantly downregulated, and that several genes have multiple probe sets. (C) Quantitative RT–PCR validation of selective genes that are differentially regulated in Get1^−/− E18.5 bladders. Expression for each gene is normalized to 1 for WT. Results represent the mean and s.e.m. for three replicates.

Figure 6

Adhesion abnormalities in superficial urothelial cells in the Get1^−/− bladders. (A, B) Transmission electron microscopy images of WT (A) and Get1 mutant (B) bladders at E18.5. The white arrows in panels A and B point to tight junctions. The red arrowheads in panel A point to normal cell–cell adhesions. The red arrowheads in panel B point to separated cell–cell adhesions. There are abundant fusiform vesicles in panel A, whereas only a few discoid vesicles are observed in panel B. Vesicles are indicated by the stars. Scale bar: 0.5 μM. The results are based on three WT and three Get1^−/− E18.5 embryos. (C) The statistically significant differentially expressed cell-adhesion genes in Get1^−/− bladder and skin. NC indicates no significant change; NS indicates not ‘present' in the microarray data set.

Figure 7

Get1 is required for normal uroplakin expression in the bladder epithelium. (A, B) Semi-quantitative qRT–PCR analysis of UpkIa, UpkIb, UpkII, UpkIIIa, and UpkIIIb in WT and Get1^−/− bladder epithelium at E16.5 (A) and E18.5 (B). Expression of all uroplakin transcripts is decreased in the Get1^−/− bladders. (C–N) Immunostaining showing expression of UpkIa (C–F), UpkII (G–J), and UpkIIIa (K–N) in WT (C, E, G, I, K, M) and Get1^−/− (D, F, H, J, L, N) bladder epithelium at the indicated stages. The bladder epithelial expression of UpkIa, UpkII, and UpkIIIa proteins is decreased in Get1^−/− mice, based on four WT and four Get1^−/− mice. Scale bar: (C–N) 12.5 μM.

Figure 8

Get1 directly regulates Uroplakin II expression and is dependent on histone modifications. (A) Identification of an evolutionarily conserved Get1 DNA-binding site (DBS) in the UpkII promoter. Location of the binding site upstream of the transcriptional start site is indicated for both human and mouse UpkII genes. (B) WT and mutated −2800 UpkII luciferase-reporter constructs were transfected into RT4 bladder cells. Mutation of the Get1-binding site resulted in decreased UpkII promoter activity. (C) Chromatin immunoprecipitation (ChIP) assays carried out on RT4 cells using a Get1 antibody or IgG as a control, followed by PCR amplification of a region of the human UpkII promoter containing the Get1 site indicated in (A, upper panel) and a non-specific downstream (DS) region of the UpkII gene as a negative control (lower panel). Input DNA is unprecipitated chromatin from the IgG sample. Get1 binds specifically to the promoter region of the UpkII gene. (D, E) Quantitative real-time PCR of Get1 (D) and UpkII (E) transcripts in undifferentiated (NHEK-U) and differentiated (NHEK-D) normal human epidermal keratinocytes, and RT4 cells. Standard error of the mean and mean fold differences in expression were calculated using three replicates normalized to RPLPO. (F) Get1 and UpkII protein expression in RT4 cells and NHEK-D detected by immunofluorescence staining. Although Get1 protein is expressed in both cell types, UpkII is selectively expressed in RT4 cells. Nuclear DAPI staining is red. (G) ChIP assays carried out on NHEK-D and RT4 cells using antibodies against RNA polymerase II, Get1, H3K9Ac, and H3K27me3, followed by PCR amplification of a 193-bp region of the human UpkII promoter containing the Get1 site in (A). Get1 and RNA polymerase-II binding, and histone modifications were quantified using quantitative PCR (upper) and also analyzed by direct inspection on agarose gels (lower). Black bars indicate binding in RT4 cells and gray bars indicate binding in NHEK-D cells. Get1 binding to the UpkII promoter is associated with increased RNA polymerase II binding and histone modifications associated with active gene expression. TSS indicates transcription start site. (H) Quantitative real-time PCR of UpkII in NHEK-D cells with and without TSA treatment. TSA treatment increased the expression of UpkII in human keratinocytes.

Figure 9

Model of UpkII regulation in the urothelium and epidermis. In the urothelium, the H3K9Ac mark is present and Get1 binds to the promoter and activates transcription of the UpkII gene. In contrast, in the epidermis, where the H3K9-acetylation mark is absent and an H3K27me3-repressive mark is enriched, Get1 does not bind as efficiently to the UpkII gene. We speculate that for some epidermal-specific genes (gene A) the reverse may true; Get1 activates selectively in epidermal cells. For yet other genes that are expressed in both the epidermis and urothelium (gene B), Get1 may bind and activate in both cell types.