Transcriptional regulation of bladder-ureter development

描述（由申请人提供）：转录因子Hoxa 13作为泌尿生殖系统（GU）生长发育的调节剂起着重要作用。在人类中，HOXA 13突变导致手足生殖器综合征（HFGS）和Guttmacher综合征（GS），这两种常染色体显性遗传病深刻影响肢体、膀胱、输尿管、子宫和外生殖器的发育。在小鼠中，Hoxa 13的GU功能是保守的，因为Hoxa 13的突变也会导致膀胱、输尿管、子宫和外生殖器的畸形。与Hoxa 13功能丧失相关的GU畸形的有趣之处在于，许多这些缺陷反映了泌尿生殖窦模式的变化，泌尿生殖窦是外生殖器、子宫和大部分膀胱的发育区域。更重要的是，虽然膀胱、子宫和输尿管的发育起源已经被很好地理解，但令人惊讶的是，我们对这些结构正常个体发育所需的细胞和分子信号知之甚少。最近，我们证明Hoxa 13缺陷小鼠可以用来阐明外生殖器正常发育所需的分子和细胞机制。本研究确定尿道下裂与Hoxa 13功能丧失相关是由Fgf-8和Bmp7在泌尿生殖窦和尿道板上皮中的表达缺失引起的。在细胞水平上，这种生长因子信号的缺失直接影响细胞的增殖和凋亡，导致前尿道闭合缺陷和排泄道畸形。认识到Hoxa 13的突变也会影响膀胱和输尿管的形成，我假设形成这些结构所需的许多细胞和分子机制可以通过检查Hoxa 13功能的丧失如何影响细胞信号、基因表达和这些受影响结构的发育模式来阐明。为了验证这一假设，将从膀胱和输尿管中纯化表达突变Hoxa13-GFP等位基因的细胞，以确定基因表达的变化。接下来，将鉴定由Hoxa 13结合的顺式作用DNA调控元件，该元件指导受影响靶基因的组织特异性表达。将对这些顺式作用元件进行序列比较，以确定哪些DNA序列作为膀胱或输尿管特异性基因表达的调节因子。这些指导Cre重组酶表达的候选顺式作用元件将在转基因胚胎中进行测试，以评估其以膀胱或输尿管特异性方式突变基因的能力，为选择性调节GU区域基因的组织特异性功能提供新的资源。

DESCRIPTION (provided by applicant): The transcription factor Hoxa 13 plays an essential role as a regulator of genitourinary (GU) growth and development. In humans, mutations in HOXA 13 cause Hand Foot Genital (HFGS) and Guttmacher (GS) Syndromes, two autosomal dominant disorders that profoundly affect the development of the limb, bladder, ureter, uterus, and external genitalia. In mice, the GU function(s) of Hoxa 13 are conserved, as mutations in Hoxa 13 also cause malformations of the bladder, ureter, uterus, and external genitalia. What is intriguing about the GU malformations associated with the loss of Hoxa 13 function is that many of these defects reflect changes in the patterning of urogenital sinus, a developmental region from which the external genitalia, uterus, and most of the bladder are derived. More importantly, while the developmental origins of the bladder, uterus, and ureters are well understood, surprisingly little is known about the cellular and molecular signals required for the normal ontogeny of these structures. Recently, we demonstrated that Hoxa 13 deficient mice could be used to elucidate the molecular and cellular mechanisms required for normal development of the external genitalia. Here hypospadia associated with loss of Hoxa 13 function was determined to be caused by the loss of Fgf-8 and Bmp7 expression in the urogenital sinus and urethral plate epithelium. At the cellular level this loss in growth factor signaling directly affected cell proliferation and apoptosis, causing defects in the closure of ventral urethra as well as malformation of the excretory meatus. Recognizing that mutations in Hoxa 13 also affect the formation of the bladder and ureter, I hypothesize that many of the cellular and molecular mechanisms required for the formation of these structures can be elucidated by examining how loss of Hoxa 13 function impacts cell signaling, gene expression, and developmental patterning of these affected structures. To test this hypothesis, cells expressing a mutant Hoxa13-GFP allele will be purified from the bladder and ureter to identify changes in gene expression. Next the cis-acting DNA regulatory elements bound by Hoxa 13 that direct the tissue-specific expression of affected target genes will be identified. Sequence comparisons of these cis-acting elements will be performed to identify which DNA sequences function as bladder or ureter-specific regulators of gene expression. These candidate cis-acting elements directing Cre recombinase expression will be tested in transgenic embryos to evaluate their capacity to mutate genes in a bladder or ureter-specific manner, providing new resources to selectively regulate the tissue-specific function of genes in the GU region.