The mutated human gene encoding hepatocyte nuclear factor 1β inhibits kidney formation in developing Xenopus embryos

Abstract

The transcription factor hepatocyte nuclear factor 1␤ (HNF1␤) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1␤ gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the Xenopus embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in Xenopus the wild-type HNF1␤, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1␤ mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in Xenopus. Thus, the overexpression of both human mutants has a different effect on renal development in Xenopus, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1␤ can be functionally characterized in Xenopus. Our findings imply that HNF1␤ not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.

Distinct Molecular and Morphogenetic Properties of Mutations in the Human HNF1β Gene That Lead to Defective Kidney Development

“…However, transfecting low amounts of Y352 expression vector, the transactivation potential of the mutant is lower compared with the wild type, whereas at higher amounts, the mutant reaches the transactivation potential of the wild type ( Figure 3B). We previously found a similar behavior for the mutant P328L329, although in this case, at high input, the mutant was even more active than the wild type, suggesting a gain of function mutation (15). To compare directly the transactivation potential between the two mutants, we transfected HeLa cells with increasing concentrations of either of these expression vectors.…”

“…The expression vector encoding human HNF1␤ wild-type, P328L329del CCTCT, and R137-K161del have been described (15). The additional HNF1␤ mutations were made by site-directed mutagenesis (Quick Change site-directed mutagenesis kit; Stratagene, La Jolla, CA) on the myc-Rc/CMVHNF1␤ construct using the following primer pairs: S151P, 5'-CGCACCTCCCCCAGCATC-3', 5'-GAT-GCTGGGGGAGGTGCG-3'; E101X, 5'-CTCAACACCTAGGAGGC-GG-3', 5'-CCGCCTCCTAGGTGTTGAG-3'; R177X, 5'-CAGAAAGC-AATGAGAGATC-3', 5'-GATCTCTCATTGCTTTCTG-3'; P159delT, 5'-CAAGGGCACCCCATGAAGACCCAG-3', 5'-CTGGGTCTTCAT-GGGG-TGCCCTTG-3'; Q243delC, 5'-CCC-GCGTCCAGCAAATC-3', 5'-GATTTGCTGGACGC-GGG-3'; A263insGG, 5'-GAGAGAGAGG-GGCCTTAGTGGAGG-3', 5'-CCTCCACTAAGGC-CCCTCTCTCTC-3'; Y352insA, 5'-GAGTGCGCTAACAGCCAGCAG-3', 5'-CTGCTG-GCTGTTAGCGCACTC-3'.…”

“…In amphibians, the pronephros is the functional kidney throughout larval development, and the frog Xenopus laevis is most attractive to analyze the molecular and cellular events in pronephros formation (13,14). Using the Xenopus system, we have shown that the introduction of a mutated human HNF1␤ transcription factor into the developing frog embryo leads to partial or even complete agenesis of the pronephric tubules and duct (15).…”

Regulation of Pancreatic .BETA.-cell Function by the HNF Transcription Network: Lessons from Maturity-Onset Diabetes of the Young (MODY)

“…We found the S36F mutation in a Japanese MODY family and functional analysis showed that it was a gain-of-function mutation [43]. It has been reported that overexpression of wild-type HNF-1b in Xenopus embryos leads to impaired development of the pronephros [44].…”

Identification of a gain-of-function mutation in the HNF-1β gene in a Japanese family with MODY

“…Gain-of-function could be a third mechanism, although its pathogenesis of impaired insulin secretion is not known. Recently, it has been reported that a frameshift mutation (P328L329fsdelCCTCT), identified in a European MODY5 family with renal abnormalities, acts as a gain-of-function mutation (~twofold) [8]. Thus, S36F mutation in the HNF-1b gene might affect the clinical phenotypes in this MODY family.…”