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

Abstract

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.

HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2

“…In contrast to these observations, the overexpression of two human HNF1B mutants in Xenopus embryos has recently been shown to result in modest effects on pronephros development, although proximal tubules were the most affected (Sauert et al, 2012). Since the mutants employed in that study either retained transcriptional activity (P328L329del) or did not bind DNA (A263insGG), it is likely that they were less efficient than our construct in decreasing Hnf1b activity.…”

HNF1β Is Essential for Nephron Segmentation during Nephrogenesis

“…40,43 Experiments in frogs have further implicated Hnf1b factors in nephron formation but determining their precise function has been elusive. 36,44,45 Here, using the zebrafish model, we demonstrate that Hnf1b factors act downstream of pax2a and pax8 to restrict podocyte fate and to establish the proximo-distal segmentation pattern of the nephron ( Figure 7). In addition, evidence for transcriptional regulatory loops was identified between hnf1ba/b, pax2a, pax8, and irx3b.…”

Metabolic alterations caused by HNF1β expression in ovarian clear cell carcinoma contribute to cell survival

“…Heterodimer of rBAT and BAT1 (b 0,+ AT), which is coded by SLC7A9 , transports cystine and dibasic amino acids, such as lysine, arginine, and ornithine, and thus is critical for reabsorption of these amino acids in the proximal tubule. With regard to the relation of HNF1β and rBAT, Sauert K. et al reported that HNF1β mutation decreased rBAT expression [ 46 ]. And the results of our metabolome analysis that intracellular lysine, arginine, and ornithine were significantly higher in HNF1β-high cells ( Fig.…”