Identification of distinct epithelial factors in the developing mouse bladder and urethra (E14.5 and E18.5). Identification of distinct epithelial factors in the developing mouse bladder and urethra (E14.5 and E18.5)

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. Overall design: 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 14.5 and E18.5. 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. Tissues from female embryos from a litter were separately pooled. Each litter yielded 2 sample types: Female bladder epithelium and female urethral epithelium. A total of n=4 biological replicates for each sample type were obtained from 4 independent litters at E14.5. A total of n=3 biological replicates for each sample type were obtained from 3 independent litters at E18.5.

下尿路（lower urinary）起源于内胚层前体——泄殖腔（cloaca）。尽管二者拥有共同的胚胎学起源，但膀胱与尿道的上皮衬里及周围间充质却呈现出分化差异。膀胱由尿生殖窦（urogenital sinus）的前部发育而来，而尿生殖窦是泄殖腔经分隔后形成的结构，可分化为肛肠道与尿生殖道。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靶器官。这类策略的一个显著缺陷在于，无法重现膀胱上皮细胞的所有层结构，其中最为关键的是顶端尿蛋白（uroplakin）阳性层——该层可形成紧密不透水的屏障，阻挡尿液中的成分。尿道上皮与膀胱上皮连续分布，但具有独特的细胞结构，且不表达尿蛋白的顶端层。我们提出假设：对发育中的尿道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。本研究对发育中小鼠的膀胱与尿道上皮隔室进行了比较，以鉴定在这两个隔室中差异表达的基因。本研究鉴定出了仅局限于尿道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，我们还描述了下尿路上皮的性别特异性差异：这种差异在雌雄尿道上皮之间更为显著，而在不同性别间的膀胱上皮中则不甚明显。实验设计：本研究旨在鉴定发育中膀胱与尿道的隔室特异性基因表达谱。我们获取了定时受孕的CD-1品系雌性小鼠，分别在胚胎第14.5天（E14.5）与胚胎第18.5天（E18.5）收集胚胎。从胚胎中解剖分离下尿路组织，包括膀胱与骨盆尿道。经胰蛋白酶酶解后，将膀胱与尿道的上皮层与间充质层机械分离。同窝雌性胚胎的组织分别合并为样本。每一同窝可获得2种样本类型：雌性膀胱上皮与雌性尿道上皮。在E14.5天，我们从4个独立同窝中获取了每种样本类型的n=4个生物学重复；在E18.5天，我们从3个独立同窝中获取了每种样本类型的n=3个生物学重复。