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Abstract: FR-OR073

Tight Regulation of Crkl Isoforms Account for the Etiology and Pleiotropic Effect of the 22q11.2 Microdeletion Syndrome (DiGeorge) on Kidney and Urinary Tract Development

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Martino, Jeremiah, Columbia University Medical Center, New York, New York, United States
  • Liu, Qingxue, Columbia University Medical Center, New York, New York, United States
  • Lim, Tze Yin, Columbia University Medical Center, New York, New York, United States
  • Steers, Nicholas J., Columbia University Medical Center, New York, New York, United States
  • D'Agati, Vivette D., Columbia University Medical Center, New York, New York, United States
  • Sanna-Cherchi, Simone, Columbia University Medical Center, New York, New York, United States
Background

We recently showed that haploinsufficiency and point mutations in CRKL drive kidney and urinary tract malformations in the DiGeorge, or 22q11.2, syndrome and in sporadic CAKUT, respectively. We examined the developmental necessity of two Crkl transcript isoforms using conditional mutant mouse models.

Methods

Bulk RNAseq data from E15.5 mouse kidneys were analyzed for expression of Crkl T1 and T2 expression. Phenotypic analyses were carried out on conditional mouse mutants generated by breeding Crkl(Ex1)f/f or Crkl(Ex2)f/f with either Six2-, Hoxb7- or Pax2-Cre mice to delete either both splice variants by removing the first of three exons (Ex1), or by removing T1 only via Ex2 excision.

Results

Analysis of RNAseq data show T1 and T2 expression at a mean TPM of 39.5 and 9.3, respectively, (n=4) in E15.5 mouse kidneys. This is comparable to TPM data for housekeeping (Gapdh=55.5) and kidney & urinary tract genes (Pax2=10.4, Hoxb7=54.9, Six2=24.1). The two isoforms exist also in human embryonic kidneys. At P0, phenotypic analysis of Six2-Cre;Crkl(Ex1)f/f and Six2-Cre;Crkl(Ex2)f/f mice shows severe renal hypoplasia. When both Crkl transcripts were removed in ureteric-bud derived structures via Hoxb7-Cre, both unilateral and bilateral ureteropelvic dilation and early hydronephrosis developed by P0. However, in Hoxb7-Cre;Crkl(Ex2)f/f mice, where only T1 is absent, there was a decreased frequency and severity of obstructive phenotypes. Lastly, when bred with Pax2-Cre mice, both crosses show mild renal hypoplasia at P0 and E18.5.

Conclusion

These data suggest a tight regulation of both Crkl isoforms during mouse embryonic development with possible tissue-specific synergistic and modulatory effects. On the one hand, the presence of both Crkl isoforms in the metanephric mesenchyme is necessary for normal kidney development, but when inactivated in both compartments, hypoplasia was less severe. When T1 was removed from UB-derived structures, the presence of only T2 was sufficient to prevent abnormal development and obstructive uropathy, which were only observed with absence of both Crkl isoforms. These findings provide further insight into the pleiotropic effect of human CRKL mutations on kidney and urinary tract development.

Funding

  • NIDDK Support