Beeinflussung der Genregulation durch Rekombinasen in der Entwicklung von Xenopus laevis

Der afrikanische Krallenfrosch Xenopus laevis ist ein attraktives Modell, um die frühe Entwicklung von Vertebraten auf molekularer und zellulärer Ebene zu untersuchen. Aufgrund der Transparenz der Larven kann die Organogenese mit ihren zahlreichen morphologischen Veränderungen gut beobachtet werden. In der vorliegenden Arbeit wurden zwei Aktivator-Stämme generiert, in denen die Cre-Rekombinase durch einen Hitze-Schock aktiviert werden kann, der HSPCre1- und der HSPCre13-Stamm. Durch Kreuzung mit dem Reporter-Stamm wurde gezeigt, dass in dem HSPCre1-Stamm die Expression der Rekombinase zu unterschiedlichen Zeitpunkten in der Entwicklung induziert werden kann. Die vollständige Rekombination der DNA erfolgte innerhalb von vier Stunden ab Beginn des Hitze-Schocks und führte zur Aktivierung des gelb fluoreszierenden Proteins im Reporter-Gen. Des Weiteren wurde gezeigt, dass in den drei verwendeten Effektor-Stämmen eine Cre-vermittelte Rekombination der transgenen DNA stattfindet. Die damit verbundene Überexpression von HNF1beta-P328L329del hatte einen Nieren-Phänotyp zur Folge und interferierte mit einer normalen Entwicklung des Magen-Darm-Traktes. Mit dem HSPCre1-Aktivator-Stamm und den drei Effektor-Stämmen, wt2, 328del-4 und ins263-6, wurde im Xenopus laevis ein binäres System etabliert, in dem die Expression der Effektor-Gene durch Cre-vermittelte Rekombination zu beliebigen Zeitpunkten in der Entwicklung aktiviert werden kann

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

Transcription factor HNF1β and novel partners affect nephrogenesis

Heterozygous mutations of the tissue-specific transcription factor hepatocyte nuclear factor (HNF)1β, cause maturity onset diabetes of the young (MODY5) and kidney anomalies including agenesis, hypoplasia, dysplasia and cysts. Because of these renal anomalies, HNF1β is classified as a CAKUT (congenital anomalies of the kidney and urinary tract) gene. We searched for human fetal kidney proteins interacting with the N-terminal region of HNF1β using a bacterial two-hybrid system and identified five novel proteins along with the known partner DCoH. The interactions were confirmed for four of these proteins by GST pull-down assays. Overexpression of two proteins, E4F1 and ZFP36L1, in Xenopus embryos interfered with pronephros formation. Further, in situ hybridization showed overlapping expression of HNF1β, E4F1 and ZFP36L1 in the developing pronephros. HNF1β is present largely in the nucleus where it colocalized with E4F1. However, ZFP36L1 was located predominantly in the cytoplasm. A nuclear function for ZFP36L1 was shown as it was able to reduce HNF1β transactivation in a luciferase reporter system. Our studies show novel proteins may cooperate with HNF1β in human metanephric development and propose that E4F1 and ZFP36L1 are CAKUT genes. We searched for mutations in the open reading frame of the ZFP36L1 gene in 58 patients with renal anomalies but found none

Measuring root system traits of wheat in 2D images to parameterize 3D root architecture models

Background and aimsThe main difficulty in the use of 3D root architecture models is correct parameterization. We evaluated distributions of the root traits inter-branch distance, branching angle and axial root trajectories from contrasting experimental systems to improve model parameterization.MethodsWe analyzed 2D root images of different wheat varieties (Triticum aestivum) from three different sources using automatic root tracking. Model input parameters and common parameter patterns were identified from extracted root system coordinates. Simulation studies were used to (1) link observed axial root trajectories with model input parameters (2) evaluate errors due to the 2D (versus 3D) nature of image sources and (3) investigate the effect of model parameter distributions on root foraging performance.ResultsDistributions of inter-branch distances were approximated with lognormal functions. Branching angles showed mean values <90°. Gravitropism and tortuosity parameters were quantified in relation to downwards reorientation and segment angles of root axes. Root system projection in 2D increased the variance of branching angles. Root foraging performance was very sensitive to parameter distribution and variance.Conclusions2D image analysis can systematically and efficiently analyze root system architectures and parameterize 3D root architecture models. Effects of root system projection (2D from 3D) and deflection (at rhizotron face) on size and distribution of particular parameters are potentially significant

Effects of gastrula-stage overexpression of the P328L329del HNF1β mutation on larval development.

<p>Representative larvae of crossings between heterozygous HSPCre13 males and heterozygous 328del4 females. The F1 offspring were heat-shocked for two hours at 34°C at early gastrula (stage 11). Embryonic development was monitored until the larvae were free swimming at stage 41–46. A: (a) Representative deformed larva after heat-shock activated overexpression of the P328L329del mutation. (b): Double-transgenic control larva without heat-shock treatment (HS). (c) and (d): Control larvae lacking either the effector (c) or the activator (d) transgene. Black arrows indicate misformed structures and tissues. Abbreviations: g: abnormal gut, a: thickened anus, e: smaller eye, t: abnormal tail, bracket: edema. Red fluorescence: HSPCre13 positive animals. Blue fluorescence: 328del4 positive animals. B: Pronephros defects caused by overexpression of the P328L329del HNF1β mutation. Larvae were immunostained with a mixture of the 3G8 and 4A6 antibodies to visualize the entire pronephric kidney. For each larvae, both pronephric kidneys are shown. Mutant animals with or without edemas developed malformations of the pronephros and the eyes. Abbreviations: e, eye; pt, proximal tubules. White arrowheads indicate more distal parts of the pronephric kidney (pronephric duct). C and D: Quantitation of the average pronephric kidney (C) and eye sizes (D) in mutant and control animals. * = p<0.01 (Student's t-test). N refers to the number of single pronephri or eyes that were measured. Scale bar = 1 mm.</p

Analysis of pronephric marker gene expression in larvae overexpressing A263insGG HNF1β mutation.

<p>Whole mount <i>in situ</i> hybridization of non-transgenic controls (A–F) and mutant animals (A′–F′) were performed for the indicated markers. G: Quantitation of the marker gene expression domains in control and mutant animals. * = p<0.01 ** = p<0.001 (Student's t-test). N refers to the number of measurements performed.</p

Effects of neurula-stage overexpression of P328L329del HNF1β mutation on larval development.

<p>Representative larvae of crossings between heterozygous HSPCre13 males and heterozygous 328del4 females. The F1 offspring were heat-shocked for two hours at 34°C at neurula (stage 19). A: Comparison of a larva with overexpressing the P328L329del HNF1β mutation with a non-transgenic control. Abbreviations: g, abnormal gut; e, reduced eye; t, abnormal tail; bracket: edema. Red fluorescence: HSPCre13 transgene. Blue fluorescence: 328del-4 transgene. HS: heat-shock treatment. The development of control larvae lacking either the effector or the activator transgene was not affected (data not shown). B: Analysis of the pronephric kidneys after overexpression of P328L329del HNF1β mutation at neurula stage. Mutant animals developed malformations of the eyes. Abbreviations: e, eye; pt, proximal tubules. White arrowheads indicate more distal parts of the pronephric kidney (pronephric duct). C and D: Quantitation of the average pronephric kidney (C) and eye sizes (D) in mutant and control animals. * = p<0.01 (Student's t-test). N refers to the number of single pronephri or eyes that were measured. Scale bar = 1 mm.</p