Sprouty1 Controls Genitourinary Development via its N-Terminal Tyrosine.

BACKGROUND Studies in mice suggest that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of congenital anomalies of the kidney and urinary tract (CAKUT). Mutations in Sprouty1, an intracellular Ret inhibitor, results in supernumerary kidneys, megaureters, and hydronephrosis in mice. But the underlying molecular mechanisms involved and which structural domains are essential for Sprouty1 function are a matter of controversy, partly because studies have so far relied on ectopic overexpression of the gene in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for the function of Sprouty1 in vitro. METHODS We generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We characterized the development of the genitourinary systems in these mice via different methods, including the use of reporter mice expressing EGFP from the Ret locus, and whole-mount cytokeratin staining. RESULTS Mice lacking this tyrosine grow ectopic ureteric buds that will ultimately form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. CONCLUSIONS Tyrosine 53 is absolutely necessary for Sprouty1 function during genitourinary development in mice.

背景 在小鼠中的研究表明，GDNF - Ret信号通路的紊乱是肾脏和泌尿系统先天性异常（CAKUT）的一个主要遗传原因。作为一种细胞内Ret抑制剂的Sprouty1发生突变，会导致小鼠出现多余的肾脏、巨输尿管和肾积水。但是所涉及的潜在分子机制以及哪些结构域对Sprouty1的功能至关重要仍存在争议，部分原因是到目前为止的研究都依赖于该基因在细胞系中的异位过表达。一个保守的N末端酪氨酸经常（但不总是）被确定为对体外Sprouty1的功能至关重要。 方法 我们培育了Sprouty1基因敲入小鼠，其在第53位的酪氨酸被替换为丙氨酸，这对应于Sprouty1保守的N末端酪氨酸。我们通过不同的方法对这些小鼠泌尿生殖系统的发育进行了表征，包括使用从Ret基因座表达绿色荧光蛋白（EGFP）的报告小鼠以及整装细胞角蛋白染色。 结果 缺乏这种酪氨酸的小鼠会长出异位的输尿管芽，最终会形成多余的肾脏，其表型与Sprouty1基因敲除小鼠无法区分。Sprouty1基因敲入小鼠还会出现巨输尿管和膀胱输尿管反流，这是由于输尿管未能从中肾管分离并迁移到其最终位置所致。 结论 在小鼠泌尿生殖系统发育过程中，酪氨酸53对Sprouty1的功能是绝对必要的。