CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell–cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

Background: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. Methods: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy) that abrogates its kinase ability. Vegfr3Chy/+ mice were examined for developmental abnormalities and kidney specific outcomes. Control and Vegfr3Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue clearing, light-sheet microscopy, and computational analyses. Results: In the kidney, VEGFR3 is expressed not only in lymphatic vessels, but also in various blood capillaries. Vegfr3Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in blood pressure, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low dose cisplatin (5 mg/kg), although Vegfr3Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length, but showed increased lymphatic density due to decreased cortical volume. Conclusions: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histological changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in chronic kidney disease requires assessment of changes to cortical volume.

41CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, 42 including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition 43 and transcriptional signaling. Due to advances in next generation sequencing, CTNND1 has been 44 implicated in human diseases including cleft palate and blepharocheilodontic syndrome (BCD) albeit 45 only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 46 thirteen participants presenting with craniofacial dysmorphisms including cleft palate and 47 variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader 53 velocardiofacial-like syndrome. 54 55 of p120-catenin, and, given the range of phenotypes seen in our cohort, should be considered more 105 broadly to cause a VCF-like syndrome. 107 Subjects and Methods 108

BackgroundLymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease.MethodsWe utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy) that abrogates its kinase ability. Vegfr3Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using a combination of tissue clearing, light-sheet microscopy and computational analyses.ResultsIn the kidney, we found Vegfr3 is expressed not only in lymphatic vessels, but also various blood vessels. Vegfr3Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in blood pressure, renal function and histology. Similarly, there was no difference in the degree of renal injury after cisplatin, although Vegfr3Chy/+ mice developed more perivascular inflammation by histology. Control mice treated with cisplatin had a measurable increase in cortical lymphatic density despite no change in cortical lymphatic volume and length.ConclusionsWe demonstrate that Vegfr3 is required for development of renal lymphatics, but a reduction in lymphatic density does not alter renal function and induces only modest histological changes after injury. Our data suggests that an increase in lymphatic density after cisplatin injury may reflect the loss of cortical volume associated with chronic kidney disease rather than growth of lymphatic vessels.SIGNIFICANCE STATEMENTDefects in renal lymphatics occur in various kidney diseases, but their role in maintaining kidney structure and function is unknown. We combine tissue clearing, light-sheet microscopy and computational analysis to characterize lymphatics and find that mice with a heterozygous mutation in Vegfr3 (Vegfr3Chy/+) have severely reduced renal lymphatics. Strikingly, these mice have indistinguishable renal function and histology compared with controls. Even after cisplatin injury, there are no differences in renal function, although Vegfr3Chy/+ mice developed more perivascular inflammation. Our data present a novel method of lymphatic quantification and suggest that a normal complement of renal lymphatics is dispensable for renal structure and function.