Additional file 4 of Detect tissue heterogeneity in gene expression data with BioQC

Supplementary Document 3. This document can also be assessed on the BioQC website under [29] respectively. (ZIP 325 kb

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

Characterization of MITF-A transgenic mice.

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

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

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

Novel <i>NEK8</i> Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

<div><p>Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. <i>NEK8/NPHP9</i> mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, <i>situs inversus</i>, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of <i>NEK8</i> mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to <i>NEK8</i> loss of function, highlighting their likely gain-of-function effect. We also showed that <i>NEK8</i> missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of <i>Nek8</i>-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of <i>Jck</i> mice. Moreover, co-injection of <i>nek8</i> MO with WT or mutated <i>NEK8-GFP</i> RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human <i>YAP</i> RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of <i>Nek8</i>-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that <i>NEK8</i> human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.</p></div