Identification of distinct epithelial factors in the developing bladder and urethra

The lower urinary is derived from the endodermal precursor, the cloaca. Despite common embryological origins, the epithelial linings and surrounding mesenchyme of the bladder and urethra show divergence. The bladder forms from the anterior part of the urogenital sinus, a cloacal derivative formed after septation of the cloaca to give rise to the anorectal and urogenital tracts. The urethra elongates posteriorly and shows sex-specific differences as a result of hormonal action. The bladder is a target for regenerative therapies and tissue reconstruction strategies. A significant shortcoming of these strategies has been the inability to recapitulate all layers of bladder epithelial cells, most importantly the apical uroplakin positive layer that forms a tight, impermeable barrier against components in the urine. The urethral epithelium is contiguous with the bladder epithelium but has distinct cellular architecture and lacks a uroplakin expressing apical layer. We hypothesized that comparison of the developing urethra and bladder epithelium will identify early genes that are responsible for urethra and bladder-specific differentiation. In this study, we compared the epithelial compartments of the developing mouse bladder and urethra to identify genes that are differentially expressed in these two compartments. Our study identified transcription factor genes and other tissue-specific markers that are confined to either the urethra or bladder epithelium. Additionally, we describe sex-specific differences in the lower urinary tract epithelium which are more pronounced between the male and female urethral epithelium and less evident in the bladder epithelium between sexes. The objective of this study was to identify compartment specific gene expression in the developing bladder and urethra. We obtained timed-pregnant CD-1 female mice and collected embryos at embryonic day 16.5. The lower urinary tract, including bladder and pelvic urethra, were dissected from the embryos. The epithelial layer of the bladder and urethra were mechanically separated from the mesenchymal layer after enzymatic digest with trypsin. Male and female samples from a litter were separately pooled. Each litter yielded 4 samples: Female bladder epithelium, Female urethral epithelium, Male bladder epithelium, Male urethral epithelium. A total of 4 biological replicates for each sample were obtained from 4 independent litters.下尿路源自内胚层前体——泄殖腔。尽管二者具有共同的胚胎学起源，但膀胱与尿道的上皮衬里及其周围间充质却呈现出分化差异。膀胱由尿生殖窦的前部发育而来；尿生殖窦是泄殖腔分隔后形成的结构，可分化为肛直肠道与尿生殖道。尿道向后延伸，并因激素作用而表现出性别特异性差异。膀胱是再生治疗与组织重建策略的重要靶标。此类策略的重大缺陷在于无法重现膀胱上皮细胞的全层结构，其中最为关键的是形成紧密不透尿成分屏障的顶端尿斑蛋白(uroplakin)阳性层。尿道上皮与膀胱上皮连续分布，但二者具有截然不同的细胞结构，且尿道上皮缺乏表达尿斑蛋白的顶端层。本研究假设，对发育中的尿道上皮与膀胱上皮进行比较分析，可识别出介导尿道与膀胱特异性分化的早期基因。本研究中，我们对发育中小鼠的膀胱与尿道上皮区域进行比较，以鉴定在这两个区域中差异表达的基因。本研究鉴定出了仅局限于尿道上皮或膀胱上皮的转录因子基因与其他组织特异性标志物。此外，本研究还描述了下尿路上皮的性别特异性差异：雄性与雌性尿道上皮间的差异更为显著，而膀胱上皮的性别差异则相对不明显。本研究的研究目标为鉴定发育中膀胱与尿道的区域特异性基因表达谱。我们选取了定时受孕的CD-1品系雌性小鼠，于胚胎发育第16.5天收集胚胎。从胚胎中分离取下尿路组织，包括膀胱与骨盆段尿道。经胰蛋白酶(trypsin)酶解后，通过机械分离法将膀胱与尿道的上皮层与间充质层分离。将同窝胚胎的雄性与雌性样本分别合并。每窝胚胎可获得4份样本：雌性膀胱上皮、雌性尿道上皮、雄性膀胱上皮、雄性尿道上皮。每类样本均通过4个独立窝的胚胎获得4份生物学重复样本。