Sauert K , Kahnert S , Roose M , Gull M , Brändli AW , Ryffel GU , Waldner C .

	Figure 1. Effects of gastrula-stage overexpression of the P328L329del HNF1b mutation on larval development. Representative larvae of crossings between heterozygous HSPCre13 males and heterozygous 328del4 females. The F1 offspring were heat-shocked for two hours at 34uC at early gastrula (stage 11). Embryonic development was monitored until the larvae were free swimming at stage 416. 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 HNF1b 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 t-test). N refers to the number of single pronephri or eyes that were measured. Scale bar = 1 mm.
	Figure 2. Effects of neurula-stage overexpression of P328L329del HNF1b mutation on larval development. Representative larvae of crossings between heterozygous HSPCre13 males and heterozygous 328del4 females. The F1 offspring were heat-shocked for two hours at 34uC at neurula (stage 19). A: Comparison of a larva with overexpressing the P328L329del HNF1b 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 HNF1b 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 t-test). N refers to the number of single pronephri or eyes that were measured. Scale bar = 1 mm.
	Figure 3. Analysis of pronephric marker gene expression in larvae overexpressing P328L329del HNF1b mutation. Whole mount in situ hybridization of non-transgenic controls (A) and mutant animals (A99) heat-shocked at early gastrula (stage 11) were performed for the indicated markers. Abbreviations: OT: otic vesicle: PT, proximal tubules; PD: pronephric duct; G: Quantitation of the marker gene expression domains in mutant and control animals. * = p,0.01 (Student t-test). N refers to the number of measurements performed.
	Figure 4. Analysis of nephron segment marker gene expression in larvae overexpressing P328L329del HNF1b mutation. Whole mount in situ hybridization of non-transgenic controls (A) and mutant animals (A99) heat-shocked at early gastrula stage were performed for the indicated marker genes. A00: Quantitation of the marker gene expression domains in mutant and control animals. * = p,0.01 (Student t-test). N refers to the number of measurements performed.
	Figure 5. Effects of overexpressing the A263insGG HNF1b on larval development. Representative larvae of a crossing between heterozygous HSPCre13 males and heterozygous A263ins6 females. The F1 offspring were heat-shocked for two hours at 34uC at early gastrula (stage 11). A: Comparison of a larva overexpressing the A263insGG HNF1b mutation with a non-transgenic control. Abbreviations: a: thickened anus; g: abnormal gut; e: reduced eye; t: abnormal tail; bracket: edema. Red fluorescence: HSPCre13 transgene. Blue fluorescence: 328del4 transgene. HS: heat-shock treatment. B: Analysis of the pronephric kidneys overexpressing the A263insGG HNF1b mutation Mutant animals with or without edemas developed malformations of the pronephric kidneys 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 t-test). N refers to the number of single pronephri or eyes that were measured. Scale bar = 1 mm.
	Figure 6. Analysis of pronephric marker gene expression in larvae overexpressing A263insGG HNF1b mutation. Whole mount in situ hybridization of non-transgenic controls (A) and mutant animals (A99) 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 t-test). N refers to the number of measurements performed
	Figure S1. Cre activator and effector strains for the overexpression of HNF1β mutants. A: Scheme of the construct present in the HSPCre13 activator strain. Located on the same plasmid, Cre recombinase is under the control of the HSP70 promotor, whereas expression of the tdTomato reporter gene is driven by the CMV promotor. B: Domain structure of HNF1β and its mutant derivatives P328L329del and A263insGG. POUS and POUH refer to the POU specific domain and POU homeodomain, respectively. C: Scheme of the transgenic effector constructs used in the present study. The CMV-driven blue fluorescent protein ECFP is used as a reporter. It is flanked by loxP sites (black triangles) followed by the open reading frame of HNF1β harboring either the P328L329del mutations (328del4 strain) or the A263insGG mutation (A263ins6 strain). Successful recombination of the effector construct upon HSPCre13 activation leads to the expression of either P328L329del or A263insGG. The effector strains contain in addition a FRT site, which is not shown here [20].
	Figure S2. Expression of P328L329del transcripts upon crossing the HSPCre13 with the 328del4 strain. Quantitative RT-PCR analysis of a crossing between a heterozygous HSPCre13 male and a heterozygous P328L329 female. The F1 offspring were heat-shocked for two hours at 34 at early gastrula (stage 11). Double transgenic larvae were identified by the presence of red and blue fluorescence at stage 40 (see Fig. S1). mRNA levels of P328L329del were analyzed by quantitative RT-PCR using specific primers (see Material and Methods) and the results were normalized to odc expression levels. The single bars represent the mean of N larvae +/− standard deviation. n.d. = no detection of P328L329del transcripts. E = edema M = malformations.
	Figure S3. Effects of tailbud-stage overexpression of P328L329del HNF1β mutation on larval development. Representative larvae of crossings between heterozygous HSPCre13 males and heterozygous 328del4 females. The F1 offspring were heat-shocked for two hours at 34 at tailbud stage (st. 25). A: Comparison of a larva with overexpressing the P328L329del HNF1β mutation with a non-transgenic control. Red fluorescence: HSPCre13 transgene. Blue fluorescence: 328del4 transgene. 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.

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