Murine model indicates 22q11.2 signaling adaptor CRKL is a dosage-sensitive regulator of genitourinary development

Abstract

The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although ∼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKLCRKL encodes a src-homology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1, a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2.

Keywords: congenital defects; del22q11.2; genitourinary; haploinsufficient; urogenital.

Fig. 1.

Map of GU abnormal patients with CNVs covering 22q11.2 indicates CRKL as candidate gene. CRKL is within the minimal region of overlap and harbors the highest pLI score (pLI = 0.16). Data are gleaned from DECIPHER database, literature (18), as well as original data from D.J.L.’s laboratory (14). Blue indicates duplication; pink indicates deletion. Arrows indicate CNV expands beyond map. Red dotted box indicates minimal region of maximum CNV overlap.

Fig. 2.

Murine Crkl and human CRKL expression patterns. Crkl antisense (A–C) and sense control (D–F) probes were used to stain E12.5 (A, B, D, and E) whole embryos and E16.5 isolated GU tracts (C and F). Blue areas indicate probe hybridization. Crkl is expressed at moderate levels (higher than sense probe background) throughout the developing embryo, including genital tubercle (red dotted outline), kidneys (K), bladder (B), and testes (black arrows). (Scale bars, 2 mm.) CRKL qPCR was performed on cDNAs (G) from spontaneously aborted human fetuses. Expression levels of tissues shown relative to heart expression. Error bars represent SEM.

Fig. 3.

Generation of Crkl exon2Δ/exon2Δ embryos. (A) A multigenerational cross was used to generate Crkl exon2Δ/exon2Δ embryos. (B) Deletion was validated by Sanger sequencing of cDNA indicating splicing of exon 1 to exon 3 (red arrow). (C) Exon 2-deleted protein sequence showing premature stop, and Inset chart comparing predominant phenotypes of Crkl^−/− to Crkl exon2Δ/exon2Δ.

Fig. 4.

Crkl exon 2 deletion results in dose-dependent intrauterine growth restriction and Crkl exon2Δ/exon2Δ embryos exhibit 23% incidence of upper-tract phenotypes. Crkl^+/+, Crkl+/exon2Δ, and Crkl exon2Δ/exon2Δ embryos were collected at E16.5 (A), E17.5, and E18.5. Loss of one copy of wild-type Crkl is sufficient to confer intrauterine growth restriction as early as E16.5 (B). n for each group ≥ 7. Error bars represent SEM. GU tracts were microdissected from E16–E18. Upper-tract phenotypes were observed and penetrance documented (Inset chart). Phenotypes included mild (C) to moderate (D and F) hydronephrosis, unilateral renal agenesis (E), and unilateral renal hypoplasia. Asterisks indicate abnormalities. Asterisk in table indicates P < 0.05 by Fisher exact test. (Scale bars, 2 mm.)

Fig. S1.

Crkl+/exon2Δ age matched males exhibit higher-trending but statistically nonsignificant rates of VUR compared with Crkl^+/+ males. Methylene blue dye is injected into the bladders of anesthetized age-matched males until urethral leakage. When VUR is present, blue dye can be seen flowing retrograde from the bladder through the ureters (black arrows) toward the kidneys (K). VUR was observed in 32.7% (17 of 52) of Crkl+/exon2Δ males versus 21% (12 of 57) of Crkl^+/+ controls (P = 0.19).

Fig. S2.

Crkl+/exon2Δ age matched males exhibit normal bladder capacity to body weight ratios. After aspiration of any present urine, methylene blue dye is injected into the bladders of anesthetized age-matched males until urethral leakage. Bladder capacity is defined as total injected volume before leakage.

Fig. S3.

Cystic kidney found in 1 of 52 Crkl+/exon2Δ adult males. Micro-CT scan was performed to visualize seven independent cysts resulting from apparent UPJ obstruction. Images were taken from 3D reconstructions showing sagittal (A) and transverse (B) sections of the cystic kidney. (Voxel size, 9.5 μm.)

Fig. 5.

Fifty-two genes are differentially expressed in Crkl exon2Δ/exon2Δ E16.5 kidneys compared with Crkl^+/+ E16.5 kidneys at a normalized false-discovery rate (FDR) < 0.05. Total RNA collected from E16.5 kidneys was extracted and next-generation sequenced. 2C indicates two copies of wild-type Crkl; 0C indicates zero copies of wild-type Crkl.

Fig. 6.

A subset of genes differentially expressed by RNA-sequencing were chosen to be validated by qPCR. RNAs from the same samples used for sequencing were reverse-transcribed and used to validate differential expression via the higher sensitivity TaqMan qPCR assay. Most of the validated genes showed more robust fold change values by qPCR than indicated by RNA-seq analysis. Error bars represent SEM.

Fig. S4.

Differentially expressed genes between Crkl+/exon2Δ and Crkl exon2Δ/exon2Δ E16.5 kidneys. Several genes from this analysis overlap to those found in wild-type versus homozygous mutant analysis. More total genes were found to be differentially expressed in this comparison partially because of lower interreplicate variability. 1C indicates one copy of wild-type Crkl; 0C indicates 0 copies of wild-type Crkl.

Fig. S5.

A subset of genes differentially expressed between wild-type and homozygous mutants by RNA-sequencing were assayed by qPCR to compare expression between wild-type and heterozygous mutants. RNAs from the same samples used for sequencing were reverse-transcribed and used in TaqMan qPCR to interrogate differential expression. Although all of the genes shown here were differentially expressed between wild-type and homozygous mutants, no differences were apparent when comparing wild-type and heterozygous mutants. Biological triplicates; error bars represent SEM.

Fig. 7.

Crkl+/exon2Δ males exhibit cryptorchidism, smaller testes, fewer sperm, and subfertility compared with Crkl^+/+ controls. Eighteen- to 20-wk-old males (n = 11 per group) were analyzed for cryptorchidism (A) (testicles circled in red; bladder in green, penis indicated by white star), testicular size (B) (Inset: Left wild-type, Right Crkl+/exon2Δ). H&E staining of testes (C) confirmed abnormal testicular histology in approximately half of the Crkl+/exon2Δ seminiferous tubules with atresia and vacuolization of tubules apparent (*). (Magnification, 10×.) Distribution of cryptorchidism was graphed (D). Testicle weight was graphed as a proportion of body weight (E). Genotype-blinded sperm count was assessed by hemacytometry (F). n = 11 cages per group were mated at 11 wk of age and assessed for fertility across three litters (G). Error bars represent SEM.

Fig. S6.

Litter frequency is unaffected in Crkl+/exon2Δ adult males. Although they exhibit fewer pups per litter compared with wild-types, Crkl+/exon2Δ adult males mate and produce their first three litters with normal frequency. Error bars represent SEM.

Fig. S7.

Sperm motility is unaffected in Crkl+/exon2Δ adult males. Although they exhibit fewer pups per litter and lower sperm count compared with wild-types, Crkl+/exon2Δ adult males do not exhibit significant motility defects. n = 11 per group. Error bars represent SEM.

Fig. S8.

Average pups per litter is decreased in Crkl heterozygote mutants of both sexes. n ≥ 50 litters per group (5 consecutive litters with n ≥ 11 cages per group) were analyzed for pup number. Heterozygote mutants of both sexes exhibit subfertility. Male subfertility was further assessed; female subfertility is the subject of future studies.

Fig. S9.

RNA-sequencing samples and read quality. Chart indicates raw number of reads per sample, raw number of uniquely mapped reads per sample, and percent of uniquely mapped reads per sample.