Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells

Fluorescence confocal images of embryonic ureter explants.

<p>E14 ureters were explanted and maintained for six days in organ culture, without (<i>Ctrl</i>) or with addition of FGF7 (<i>FGF7</i>). In these longidutinal sections, all nuclei were stained blue with DAPI and the red color indicates positive signal after immunostaining for α-smooth muscle actin, thus marking the muscle layer (small arrows). In each frame the asterisk indicates the (collapsed) lumen and the white brackets span the epithelial layer. Note that the heterozygous <i>p63</i> explant displays the expected normal epithelium with 2-4 cell layers (<b>A</b>) and that the epithelium is also multilayerd upon exposure to FGF7 (<b>B</b>). In contrast, the urothelium in homozygous mutant p63 (-/-) explants generally have only one or two layers, both without (<b>C</b>) or with (<b>D</b>) FGF7 treatment. Bars are 10 μm.</p

Double immunostaining for CK15/P63 and CK15/CK19.

<p>Fluorescence images of cross sections of wild type E17 mouse ureters. In each frame, the asterisk indicates the ureteric lumen and the dotted lines indicate the border between the urothelium and differentiating smooth muscle. <b>A</b>-<b>C</b>. Double immunostaining for CK15 (green in <b>A</b>) and P63 (red in <b>B</b>), with the merged images shown in <b>C</b> where nuclei are stained blue with DAPI. The same two CK15+ cells are arrowed in A and C; note the presence of P63 in their nuclei. <b>D</b>-<b>F</b>. Double immunostaining for CK15 (red in <b>D</b>) and CK19 (green in <b>E</b>), with the merged images shown in <b>F</b> where nuclei have been stained blue with DAPI. The same two CK15+ cells are arrowed in <b>D</b> and <b>F</b>. Note that CK19 has an overlapping but more extensive distribution than CK15. Bars are 100 μm.</p

Fetal kidneys and ureters of wild type and <i>p63</i> mutant mice.

<p>All frames are from E14 organs. <b>A</b>, <b>C</b> and <b>E</b> are from wild type mice (-/-) and B, D and <b>F</b> are from <i>p63</i> homozygous mutant mice (-/-). <b>A</b> and <b>B</b>. Whole mount images showing similar gross appearance of renal tracts in the two genotypes. <b>C</b>-<b>F</b>. Histology sections of the cortex (<b>C</b> and <b>D</b>) and medulla (<b>E</b> and <b>F</b>) of the metanephric kidney, with nuclei stained with haematoxylin. Both show several layers of forming nephrons in the cortex and a normal-looking nascent kidney pelvis (asterisks) in the medulla. Bars are 100 μm.</p

TUNEL labeling of ureters.

<p>Transverse sections through ureters with all nuclei stained blue with DAPI; apoptotic nulei are green. The asterisk in each image indicates the ureteric lumen. <b>A</b>. Wild type E16 ureter shows no apoptosis in the double-layered epithelium or in adjacent differentiating smooth muscle. There is a single apoptotic nucleus (arrow) in the nearby peritoneal mesothelium. <b>B</b>. Homozygous <i>p63</i> mutant ureter with two apoptotic nuclei; one (indicated by the arrow on the left) is in the stromal layer immediately outside the epithelial monolayer, while the other (indicated by the arrow on the right) is within loose cells just outside the muscle layer. Bars are 50 μm.</p

CK15 in human urinary bladder cancer.

<p><b>A</b>-<b>F</b>. Low power bright field images of histology from adult urinary bladders, from normal healthy (<b>A</b>) and pathological (<b>B</b>-<b>F</b>) tissues, as indicated in the frames (Inflam = inflamed tissue). <b>G</b>. Quantification (in arbitrary units) of CK15 immunohistochemical signals across urothelia and tumour sections of individual samples. On the horizontal axis, the first number below each group designation is the average age in years of the set of patients; the number of patient samples (n) is shown in brackets. SCC = squamous cell carcinoma of the bladder. Bar is 200 μm. </p

Growth of explanted embryonic ureters.

<p>E14 wild type mouse ureters were explanted into organ culture and maintained for up to six days. <b>A</b> and <b>B</b>. Phase contrast images of whole explants on day six of culture. Ureters cultured in the presence of FG7 (<b>B</b>) appeared bulkier than controls (Ctrl in <b>A</b>). <b>C</b>. Groups of explants cultured with FGF7 were significantly (#; p<0.05, as assessed by t-tests) longer than controls at three and six days of culture (each dot represents one ureter). <b>D</b>. Whole mount CK15 immunostaining of ureteric explant on day six of culture showed positive cells in the central, urothelial, core of the organ; the dotted line indicates the perimeter of the ureter and the top/proximal end is on the left of the frame. <b>E</b>-<b>J</b>. Cross sectional images of explanted ureters fed for six days with control media (<b>E</b>-<b>G</b>) or media supplemented with FGF7 (<b>H</b>-<b>J</b>). In each frame, the dotted line marks the border between the urothelium and the surrounding differentiating smooth muscle, and an asterisk has been placed in the lumen. Images E and H demonstrate that CK15+ (green) urothelial cells were present in both experimental groups. Images F and I show BrdU+ cell nuclei in each experimental group. Most of them were in the developing muscle layer but some were present in the urothelium. In the merged images (G and J, where DAPI/blue nuclear staining is also shown), it is apparent the the BrdU+ cells in the urothelium usually do not correspond to the CK15+ cells. In each of the frames <b>E-G</b>, the locations of the same two CK15+ cells are arrowed; the upper cell is BrdU- while the lower one is BrdU+. In <b>H</b>-<b>J</b>, there is a cluster of BrdU+ epithelial nuclei at ‘6-9 o’clock’ and they are separate from CK15+ cells. Bars are 50 μm. </p