Generation of MITF-A transgenic mice.

<p><b>A)</b> Schematic representation of the Ksp-cadherin-FLAG-MITF-A transgene. <b>B)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic mice (line 42) 2 months after birth. Data are means ± SEM; n = 4–6 per each genotype. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: ** P < 0.01, *** P < 0.001. <b>C)</b> MITF-A protein expression evaluated by western blot on kidney nuclear protein extracts from WT, HE and HO MITF-A transgenic mice 2 months after birth. This is a representative image of three experiments. Nuclear protein extracts from <i>Mitfa</i>^-/- kidneys were used as a negative control; crude extracts from renal cells transfected with either FLAG-MITF-A plasmid (lane 1) or MITF-A plasmid (lane 2) were used as a positive control. Lamin A/C was used as control of nuclear protein amount. IB = immunoblot.</p

Characterization of MITF-A transgenic mice.

<p>Characterization of MITF-A transgenic mice.</p

Expression pattern of MITF-A during kidney development.

<p><b>A-B)</b><i>In situ</i> hybridization of <i>Mitf-A</i> of E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos using an antisense RNA probe directed against a sequence encompassing exon 1A, specific for <i>Mitf-A</i>, and exon 1B common to <i>Mitf-A</i>, <i>Mitf-H</i>, <i>Mitf-C</i>, <i>Mitf-J</i> and <i>Mitf-Mc</i> isoforms. The inset shows the staining of E13.5 kidneys using the sense RNA probe. Magnifications are X100 (left panels), X200 (middle panels) and X400 (right panels). In WT kidneys <b>(A)</b> a weak staining is observed in branches of UB (black arrow), in S-shaped body (blue arrow) and in metanephric mesenchyme (asterisk). Consistent with the use of the Ksp-cadherin promoter, the signal in MITF transgenic kidneys <b>(B)</b> was strongly increased in UB and tips (black arrow), in ureteric tip (black arrow) and to a lesser extent in S-shaped body (blue arrow). <b>C)</b> <i>In situ</i> hybridization of <i>Mitf-A</i> in transgenic HO kidneys after laminin immunohistochemistry (red). Note <i>Mitf</i> expression in ureteric bud and tip (black arrow), in and S-shaped body (blue arrow). Magnification X400. Sections are representative images of 4 kidneys per genotype. <b>D</b>) Immunostaining of MITF-A in WT and HO MITF-A transgenic metanephroi at E13.5. Note the increase of MITF-A expression in UB stalks, tips and S-bodies. Magnification X400.</p

<i>Mitfa</i> inactivation results in reduced glomeruli number.

<p><b>A)</b> Schematic representation of the targeting strategy used to inactivate <i>Mitfa</i>. <b>B-C)</b> <i>Mitf-A</i> <b>(B)</b> and total <i>Mitf</i> mRNA <b>(C)</b> expression evaluated by quantitative RT-PCR in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. <b>D)</b> Glomerular number in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. Data are means ± SEM, n = 8–10 per each genotype. Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> <i>versus Mitfa</i>^<i>+/+</i>: *** P < 0.001.</p

Impact of MITF-A overexpression on cell survival.

<p><b>A-B)</b> Cell proliferation in E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos. Proliferating cells were identified using an anti-phospho-histone H3 (pH3) <b>(A)</b> and an anti-PCNA antibody <b>(B)</b>. Magnifications are X400 and X600, respectively. Left panels: representative images of 5 kidneys; right panels: quantification of the number of pH3-positive and PCNA-positive cells per UB structure. <b>C)</b> Apoptosis was evaluated by TUNEL assay in E13.5 kidneys from WT and HO MITF-A transgenic embryos. Left panels: representative images of 5 kidneys (magnification X400); right panels: quantification of the number of TUNEL-positive cells per microscopic field. Data are means ± SEM. Quantifications were performed on three sections for each kidney (n = 5 mice per genotype). Mann-Whitney test; transgenic <i>versus</i> wild-type mice: *** <i>P</i> < 0.001.</p

Expression of candidate MITF-A targets in E13.5 kidneys.

<p><b>A)</b><i>In situ</i> hybridization of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> in wild-type (WT) and homozygous (HO) MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). <b>B)</b> Quantitative RT-PCR analysis of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> mRNA expression in E13.5 kidneys of WT, heterozygous (HE) and HO MITF-A transgenic embryos (n = 6–9 per genotype). <b>C)</b> <i>In situ</i> hybridization of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> in WT and HO MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). Note the increased staining of <i>Re</i>t mRNA in transgenic kidneys at E13.5. <b>D)</b> Quantitative RT-PCR analysis of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> mRNA expression in E13.5 kidneys of WT, HE and HO MITF-A transgenic embryos (n = 6–9 per genotype). Data are means ± SEM. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: * P < 0.05, ** P < 0.01.</p

MITF-A modulates kidney branching morphogenesis.

<p><b>A)</b> Whole mount E13.5 metanephroi in wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic embryos (line 42) after staining with anti-Calbindin antibody. These are representative images of at least 6 embryos for each genotype. Bar = 100 μm. <b>B)</b> Morphology of kidneys in WT, HE and HO MITF-A transgenic embryos at E13.5. These are representative images of at least 6 embryos for each genotype. <b>C-D)</b> Ureteric bud (UB) branching, as assayed by counting the number of UB tips in (<b>C</b>) WT (n = 17), HE (n = 14) and HO (n = 25) MITF-A transgenic embryos and (<b>D</b>) <i>Mitfa</i>^<i>+/+</i> (n = 15) and <i>Mitfa</i>^<i>-/-</i> (n = 20) embryos at E13.5. <b>E-F)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from (<b>E</b>) WT, HE and HO MITF-A transgenic embryos (n = 7–8 per each genotype) and (<b>F</b>) <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> embryos (n = 7 and 3 per genotype, respectively) at E 13.5. Data are means ± SEM. For transgenic MITF-A mice: ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: *** P < 0.001, HE v<i>ersus</i> HO MITF-A transgenic mice: ## P < 0.01, ### P < 0.01. For <i>Mitfa</i> knockout mice: Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> versus: <i>Mitfa</i>^<i>+/+</i>: * P < 0.05, *** P < 0.001.</p

RET heterozygosis reverts MITF-A-induced phenotype.

<p><b>A-B)</b> Glomeruli number per kidney <b>(A</b>) and kidney weight/body weight ratio (KW/BW) (<b>B)</b> in double transgenic mice generated by crossing mice overexpression MITF-A with heterozygous <i>Ret</i> knockout mice. Four groups of mice were studied: double wild-type mice (WT), heterozygous (HE) mice bearing an allele of <i>Ret</i>, HE mice overexpressing MITF-A and double HE MITF-A and <i>Ret</i> transgenic mice. Data are means ± SEM, n = 3–8 per each genotype. ANOVA followed by Tukey-Kramer test; transgenic versus wild-type mice: *** P < 0.001; MITF-A^wt/tgMITF-A mice <i>versus</i> MITF-A^wt/tgMITF-A;<i>Ret</i>^<i>+/-</i> mice: § P < 0.05, §§ P < 0.01.</p