Heat-Shock Mediated Overexpression of HNF1β Mutations Has Differential Effects on Gene Expression in the Xenopus Pronephric Kidney

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B.由TCF2基因编码的转录因子HNF1B（Hepatocyte Nuclear Factor 1B）在脊椎动物的器官发生过程中发挥关键作用。在人类中，HNF1B的杂合突变与多种疾病相关，包括引发青少年起病的成人型糖尿病（MODY5）的胰岛β细胞功能障碍、肾脏发育缺陷、肝功能紊乱、胰腺萎缩以及生殖道畸形。非洲爪蟾（Xenopus laevis）是研究胚胎发生与器官发生过程的优秀模型生物，因其可通过一系列实验方法进行便捷操作。本研究中，我们在发育中的非洲爪蟾胚胎内过表达HNF1β突变体，以探究其在器官发生过程中的作用，尤其聚焦于发育中的前肾（pronephric kidney）。为此，我们构建了一套包含激活菌株与效应菌株的热激诱导型双元Cre/loxP系统。对突变体HNF1B变体P328L329del与A263insGG进行热激激活后，可引发多种器官畸形，受影响的幼虫会出现严重水肿。前肾的缺陷主要局限于畸形的近端小管。此外，两种均参与氨基酸转运的近端小管标记基因tmem27与slc3a1的表达均受到影响。P328L329del与A263insGG均能下调slc3a1的表达。除此之外，P328L329del会降低tmem27的表达，而A263insGG过表达则会下调氯离子通道clcnk与转录因子pax2的表达。两种HNF1B突变体的过表达会引发不同的表型，表现为前肾体积缩小或增大。过表达HNF1B突变体时，所选的前肾标记基因的表达会受到差异性影响。基于本研究结果，我们推测HNF1B突变体在过表达时会以特定且差异化的方式影响基因调控。此外，本研究证实，我们新构建的针对非洲爪蟾胚胎的Cre/loxP系统，是探究转录因子在发育前肾中基因调控潜能的理想替代方案，本研究以HNF1B为例验证了这一点。