Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?

Background Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene. Changes in gene expression underlie the arteriosclerosis and T-cell immunodeficiency of SIOD; therefore, we hypothesized that SMARCAL1 deficiency causes the focal segmental glomerulosclerosis (FSGS) of SIOD by altering renal gene expression. We tested this hypothesis by gene expression analysis of an SIOD patient kidney and verified these findings through immunofluorescent analysis in additional SIOD patients and a genetic interaction analysis in Drosophila. Results We found increased expression of components and targets of the Wnt and Notch signaling pathways in the SIOD patient kidney, increased levels of unphosphorylated β-catenin and Notch1 intracellular domain in the glomeruli of most SIOD patient kidneys, and genetic interaction between the Drosophila SMARCAL1 homologue Marcal1 and genes of the Wnt and Notch signaling pathways. Conclusions We conclude that increased Wnt and Notch activity result from SMARCAL1 deficiency and, as established causes of FSGS, contribute to the renal disease of most SIOD patients. This further clarifies the pathogenesis of SIOD and will hopefully direct potential therapeutic approaches for SIOD patients. Electronic supplementary material The online version of this article (doi:10.1186/s13023-016-0519-7) contains supplementary material, which is available to authorized users.

optical field of view, the AxioCam MR microscope camera captures images of approximately 448 µm Í 336 µm. The analyzed images are presented as the overview images in Figure 2 and Additional file 1: Figure S3. Using ImageJ, the glomeruli were outlined and the area and integrated density were measured, as well as the mean fluorescence of three adjacent background regions. The corrected total fluorescence (CTF = integrated density -(glomerular area Í mean fluorescence of background readings) was calculated for each glomerulus. Where there was more than one glomerulus present in the image, an average CTF of all glomeruli was calculated. Data were plotted as box and whisker plots and analyzed by the 2-tailed Student's t-test. A p value of less than 0.05 was considered statistically significant. The Bonferroni correction was applied to correct for multiple comparisons.

Drosophila melanogaster genetic screen to determine the effect of Wnt and Notch mutant alleles on the Marcal1 overexpression phenotype
Drosophila wings have five longitudinal veins (L1, L2, L3, L4, and L5) plus anterior and posterior cross veins (ACV and PCV) (Additional file 1: Figure S1B). The overexpression of Marcal1 induces an ectopic vein parallel and anterior to L2, an ectopic vein extending laterally from the PCV, a partially missing or completely absent ACV or PCV, and distal bending or splitting of longitudinal veins L2, L4, and L5 (Additional file 1: Figure S1C) [1]. These wing vein alterations are dependent on enzymatic activity since overexpression of the enzymatically inactive mutant Marcal1 K275R does not alter wing venation [1]. We screened for Wnt and Notch alleles that lead to the suppression or enhancement of the ectopic wing veins induced by the overexpression of Marcal1 [1].
Crosses for analyzing the genetic interaction between the Marcal1 gene with genes encoding for components of the Wnt and Notch signaling pathways were maintained at 25°C for three days. The crosses were then transferred to 28°C. Upon eclosion, the desired F 1 progeny were selected and their wings were mounted, imaged, and assessed as previously described [1].
Ectopic wing veins observed in the F 1 progeny of the crosses were scored according to the following guidelines. For each of the features scored, a phenotype resembling the wild type wing was given a score of 0. Ectopic veins parallel and anterior to the L2 longitudinal vein were given a score of 0 to 2 based on the length of the ectopic vein. The distal portion of the L2 vein was given a score of 0 or 1 based on the absence or presence of bending or splitting. The distal portions of the L4 and L5 veins were given a score of 0 to 4 proportionate to the degree of deviation from the wild type phenotype. Ectopic veins extending perpendicularly from the PCV were given a score of 0 to 3 based on the length of the ectopic vein. A partially or completely absent ACV or PCV were each given a score of 1. Representative images for these phenotypes and their respective scores are present in Additional file 1: Figure S1C. The reference wing vein phenotype was determined by crossing Marcal1 overexpression flies to w 1118 mutants of three genetic backgrounds; the scores from these crosses were averaged to provide a reference score.
To determine whether there was any non-specific interaction between the various mutant alleles and the GAL4-UAS system, all mutant lines were crossed to the C96-GAL4, UAS-Hrs/MKRS transgenic line and the degree of wing margin scalloping in the desired F 1 progeny was scored [2]. Any mutant alleles that interacted with the GAL4-UAS system (i.e., those that enhanced or suppressed the wing margin scalloping phenotype of the C96-GAL4, UAS-Hrs/MKRS transgenic line) were excluded; all mutant alleles presented here had no detectable non-specific interactions with the GAL4-UAS system.
Ten or more wings were analyzed for each cross and scored by two independent readers. Scores for each cross were compared to the reference scores to determine whether the wing vein phenotype was suppressed or enhanced. Where there was a discrepancy between the first two reads, a third read was completed by C. F. B.

Notch mutant phenotypes
Several Notch mutant alleles cause phenotypes that manifest in the wing, eye, and bristle.
We screened for the suppression or enhancement of these phenotypes in the context of Marcal1 loss or gain. For the loss-of-function screen, selected Notch pathway mutant alleles from the Bloomington Drosophila Stock Center (Bloomington, IN, USA) were crossed into the Marcal1 loss-of-function background. These crosses were carried out and maintained at 20°C. For the overexpression screen, selected Notch pathway mutant alleles were crossed into the Marcal1 overexpression background as detailed above. Upon eclosion, the desired F 1 progeny were selected, and the relevant phenotype analyzed. Since several of the alleles are temperaturedependent, the Notch mutant phenotypes were also assessed at 28°C.
A minimum of 40 wings was scored for each genotype where the suppression or enhancement of a wing phenotype was being assessed. Notch alleles N nd-1 and N nd-3 exhibit wing notching in homozygous females and hemizygous males; all Delta alleles in this study exhibit deltas, wing vein thickening, and ectopic veins in heterozygous flies; the Serrate allele Ser 1 exhibits serrated wings in heterozygous flies; the fringe allele fng 13 occasionally exhibits wing notching in heterozygous flies; and all Hairless alleles in this study exhibit shortened longitudinal veins in heterozygous flies as previously described [3][4][5]. The presence or absence of the phenotype of interest was scored for each wing.
Eighty bristles of the relevant type were scored for each genotype where the suppression or enhancement of a bristle phenotype was being assessed. Notch allele N spl-1 exhibits missing, double, or ectopic anterior and posterior scutellar bristles in homozygous females and hemizygous males, while all Hairless alleles in this study exhibit missing bristles on the head and notum in heterozygous flies as previously described [5,6]. A total of 80 anterior and posterior scutellar bristles were scored for the N spl-1 allele, while 80 bristles of each type on the notum were scored for the Hairless alleles. The presence or absence of the phenotype of interest was scored for each bristle type.
Eighty eyes were scored for each genotype where the suppression or enhancement of an eye phenotype was being assessed. The Notch allele N spl-1 exhibits rough and reduced eyes in homozygous females and hemizygous males as previously described [7].
Wings exhibiting a blistered phenotype and eyes exhibiting a rough and reduced eye phenotype were imaged using an MZ16 Stereomicroscope (Leica Microsystems Inc., Concord, ON, Canada).   Table S3. Primer sequences used in this study.

Primer Sequence Drosophila Marcal1 overexpression
Gapdh2 Abbreviations: AXIN2, axin 2; CCND1, cyclin D1; CCND2, cyclin D2; F, forward; GAPDH, glyceraldehyde 3phosphate dehydrogenase; HES1, hes family bHLH transcription factor 1; HES2, hes family bHLH transcription factor 2; HEY1, hes related family bHLH transcription factor with YRPW motif 1; HEY2, hes related family bHLH transcription factor with YRPW motif 2; JUN, Jun proto-oncogene; R, reverse.  1 The genes that are involved in the individual KEGG pathway term. 2 The number of genes involved in a given term divided by the total number of input genes (i.e., 2,241 genes). 3 The magnitude of enrichment of a given term represented by the ratio of the proportion of genes involved in the term within the input genes and of the proportion of genes involved in the term within the human genome. 4 Modified Fisher's Exact p values, corrected for multiple comparisons by the Bonferroni method, representing the significance of enrichment of a given term. 5 These 59 genes correspond to 60 RefSeq IDs and are represented by 36 unique DAVID gene IDs. 6 These 31 genes correspond to 32 RefSeq IDs and are represented by 30 unique DAVID gene IDs.      Figure S3. Quantification of β-catenin immunofluorescent staining in SIOD and isolated FSGS kidneys. Expression data are presented as box and whisker plots of relative β-catenin immunofluorescence in unaffected control (n = 3), SIOD patient (n = 7), and isolated FSGS patient (n = 9) kidneys. A single transplanted kidney in an SIOD patient was also available for analysis (SIOD Tx). Boxes represent the interquartile range (25th -75th percentile), horizontal lines within boxes represent the median, and whiskers represent the range. Adjacent to the box and whisker plots are the individual data points from which the box and whisker plots are derived. β-catenin immunofluorescence values were normalized to the median unaffected control value set at 1 (dotted line). Abbreviations: **, p < 0.01; FSGS, focal segmental glomerulosclerosis; SIOD, Schimke immuno-osseous dysplasia.     were measured by qRT-PCR in 15-week-gestation unaffected control kidneys (n = 4) and in a 15-week-gestation SMARCAL1-deficient kidney (n = 1, SD133b). For each sample, the mRNA levels of 3 technical replicates were normalized to the mRNA levels of the housekeeping gene GAPDH and plotted relative to SD133b set at 1 (dotted line). Boxes represent the interquartile range (25th -75th percentile), horizontal lines within boxes represent the median, whiskers represent the range, and individual points represent outliers. Adjacent to the box and whisker plots are the individual data points from which the box and whisker plots are derived. Figure S9. A schematic of the Drosophila Wnt and Notch signaling pathways. (A) Wnt pathway schematic: In the absence of the wingless (wg) ligand, armadillo (arm) is phosphorylated by shaggy (sgg), ubiquinated, and targeted for degradation via the proteasome. In the presence of wg, the complex associates with phosphorylated arrow (arr). Although arm is still phosphorylated by sgg, ubiquitination of arm is inhibited. The complex becomes saturated with phosphorylated arm, and newly synthesized arm accumulates and translocates to the nucleus to activate target gene expression. (B) Notch pathway schematic: In the absence of ligand, the Notch pathway is inactive. Suppressor of hairless (Su(H)) binds Hairless (H) to recruit co-repressors such as C-terminal binding protein (CtBP), groucho (gro), and histone deacetylase (HDAC1) to repress transcription. In the presence of the ligand Delta (Dl) or Serrate (Ser), the ligand binds Notch (N) to trigger consecutive S2 and S3 proteolytic cleavages of N; Presenilin (Psn) is part of the γ-secretase complex which is involved in the S3 cleavage. The Notch intracellular domain (NICD) is then released into the cytoplasm and translocates to the nucleus where it binds Su(H) and recruits the co-activators such as mastermind (mam) and nejire (nej) to activate transcription.   Since several features of the Delta mutant phenotype and the Marcal1 overexpression phenotype overlap, it was not possible to assess these wings (middle right column), however a proportion of flies presented with the new phenotype of blistered wings (right column), indicative of a genetic interaction between Marcal1 gain and Delta. Percentages of blistered wings observed are shown. All wings shown are from female flies, excepting the N nd-1 allele since the N phenotype of interest is only present in homozygous females and hemizygous males and the Marcal1 overexpression cross did not give rise to homozygous female progeny.