XB-ART-11233Proc Natl Acad Sci U S A. April 25, 2000; 97 (9): 4695-700.
The mutated human gene encoding hepatocyte nuclear factor 1beta inhibits kidney formation in developing Xenopus embryos.
The transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1beta 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 HNF1beta, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1beta 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 HNF1beta can be functionally characterized in Xenopus. Our findings imply that HNF1beta 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.
PubMed ID: 10758154
PMC ID: PMC18295
Article link: Proc Natl Acad Sci U S A.
Genes referenced: hnf1a hnf1b lhx1 odc1
Antibodies referenced: Kidney Ab1 Kidney Ab2
Article Images: [+] show captions
|Figure 1 DNA binding and dimerization of the P328L329 mutant. (a) Scheme of the related human transcription factor HNF1α and HNF1β with the frameshift mutations P328L329fsdelCCTCT (P328L329) and R137-K161del. The various domains are indicated, and the numbers refer to the amino acid positions. The homology between HNF1α and HNF1β is given (2). (b) Gel-retardation assay using the 32P-labeled HNF1-binding site as a probe. The additions of the various in vitro translation products and of the antibodies are given. The heterodimers are marked with an asterisk. The methods used are described (21).|
|Figure 2 Transactivation potential of the P328L329 mutant. (a) Increasing amounts of expression vector encoding P328L329 or HNF1β were cotransfected into HeLa cells with a luciferase reporter containing four HNF1-binding sites (23). The fold activation compared with transfections lacking any transfected factor is given. The SDs from at least six determinations are presented, and the methods used have been described (23). (b) RNA encoding HNF1α, HNF1β, or P328L329 was injected into fertilized Xenopus eggs, and the RNA was extracted at the late gastrulae stage (stage 12.5). Endogenous HNF1α and ODC (ornithine desoxycarboxylase) transcripts were determined by reverse transcription–PCR as described (24). The control is RNA of a stage 20 embryo. Stages are as given in ref. 25.|
|Figure 3 Phenotypic changes in pronephros formation observed in living Xenopus larvae (stages 45–48). Two-cell-stage embryos were injected with 0.25 ng of GFP RNA together with 1 ng of RNA encoding P328L329 (a and b), HNF1β (c and d), or HNF1α (e and f). Larvae expressing green fluorescence on the left or right side are given in a, c, and e. The same animals were examined for pronephros formation at higher magnification (b, d, and f). The injected side and the pronephros are marked by black arrowheads and by red arrows, respectively. The techniques have been described (24).|
|Figure 4 Quantification of the phenotypic changes in pronephros formation observed in living Xenopus larvae. Injected normal larvae that showed green fluorescence on either the right or left side were scored at stage 45–48 for pronephros development on the injected side. The percent distribution of pronephros of normal or reduced size as well as the absence is given for the various mutants analyzed. Reduction in size of at least one-fourth was used as criteria to classify as reduced whereas in cases defined as absent, no tubular structures were visible. The number of analyzed larvae is given (N). Larvae that had unusual pigmentation, differences in the size of the eyes, or a narrower head structure were included. These minor phenotypes approximated to about 20% and were not correlated to the type of transcription factor injected.|
|Figure 5 Whole-mount staining of the pronephros of larvae derived from injected embryos. (a– i) Whole-mount immunostainings with a mixture of the mAbs 3G8 and 4A6, which recognize terminal differentiation markers of the pronephric tubules and duct, respectively (30). A Cy-3-conjugated secondary anti-mouse antibody was used to get red fluorescence (31). (a– c) Stage 37–39 larvae obtained from P328L329-, HNF1β-, and HNF1α-injected embryos. Each image shows an individual animal with its lateral and dorsal views. The injected side is marked by a white arrowhead. (d– i) Individual stage 45–48 larvae that had received the indicated constructs. The injected sides are marked by white arrowheads in the dorsal views, and the lateral views represent the injected sides. Pronephric tubules (pt), ducts (d), coiled ducts (cd), and cysts (c) are indicated in the lateral views.|
|Figure 6 Whole-mount staining of the pronephros anlage by Xlim-1. (a and b) Whole-mount in situ hybridizations using an Xlim-1 antisense probe on HNF1β- and HNF1α-injected embryos. The left and right side of the same animal is shown with the cement gland (cg) marked to indicate the anterior part of the stage 20 tail-bud embryos. The arrow points to the pronephros anlage stained by Xlim-1 hybridization. The staining involved 8, 9, or 4 embryos injected with HNF1α, HNF1β, or P329L329 (not shown) and was indistinguishable from 10 uninjected embryos.|