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Abstract: SA-PO326

Deletion of Crkl Splice Isoforms in the Mouse Kidney Disrupts Intercompartmental Signaling Required for Progenitor Renewal and Branched Morphogenesis

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine

Authors

  • Martino, Jeremiah, Columbia University Irving Medical Center, New York, New York, United States
  • Lim, Tze Yin, Columbia University Irving Medical Center, New York, New York, United States
  • Liu, Qingxue, Columbia University Irving Medical Center, New York, New York, United States
  • Ke, Juntao, Columbia University Irving Medical Center, New York, New York, United States
  • Whittemore, Gregory Baker, Columbia University Irving Medical Center, New York, New York, United States
  • Gupta, Yask, Columbia University Irving Medical Center, New York, New York, United States
  • Jin, Gina, Columbia University Irving Medical Center, New York, New York, United States
  • Mendelsohn, Cathy L., Columbia University Department of Urology, New York, New York, United States
  • Gharavi, Ali G., Columbia University Irving Medical Center, New York, New York, United States
  • D'Agati, Vivette D., Columbia University Irving Medical Center, New York, New York, United States
  • Sampogna, Rosemary V., Columbia University Irving Medical Center, New York, New York, United States
  • Sanna-Cherchi, Simone, Columbia University Irving Medical Center, New York, New York, United States
Background

We previously showed that haploinsufficiency and point mutations in CRKL drive kidney and urinary tract malformations in the DiGeorge, or 22q11.2, Syndrome (DGS) and in sporadic CAKUT, respectively. Here, we examined the developmental requirement of the two Crkl splice variants (T1 & T2) when conditionally and differentially deleted in the metanephric mesenchyme (MM).

Methods

We characterized phenotypic abnormalities in developing kidneys from mice with conditional deletion of both T1 and T2 ("No Crkl"), or only the T1 isoform ("T2 only"), specifically within the MM. Bulk and scRNAseq data were acquired and analyzed from embryonic kidneys isolated from 13.5 and 15.5dpc wild-type and mutant mice. IHC and ISH analyses were used to validate and identify key signaling pathways.

Results

At P0, both “No Crkl,” and “T2 only” kidney phenotypes were characterized by severe hypoplasia and near-absence of nephrogenic progenitors (NP). Morphometric analysis of the renal collecting system at 13.5 and 15.5dpc shows a hyperbranched phenotype in “No Crkl” mutants, while expression of only T2 led to a hypobranched phenotype. Using our single-cell data to “deconvolve” bulk RNAseq data, we found an overrepresentation of early proximal tubule (ePT) markers in “No Crkl” mice, and an underrepresentation in “T2-only” mice, suggesting a cell type-based contribution to the divergent branching phenotypes. Both groups of mutants also showed an underrepresentation of NP markers, where scRNAseq and ISH analyses confirmed decreased expression of Fgf8 and Fgf9 in NPs and renal vesicle. Lastly, in-depth time-based analyses of bulk RNAseq datasets revealed 3 differentially regulated gene sets that may explain the divergent branching phenotype between the two mutants.

Conclusion

Genetic manipulation of Crkl confirms a crucial role in kidney development, where disrupted Fgf8 expression suggests a cellular mechanism underlying renal hypodysplasia. Furthermore, disruption of splice isoform balance reveals a dichotomous pattern in early branching, suggesting a differential role for each isoform in regulating STOP and GO signaling associated with growth and branching morphogenesis.

Funding

  • NIDDK Support