Using the zebrafish to model central conducting lymphatic anomaly driven by pathogenic variants in KRAS

Authors

• BA Sempowski
• SM Paulissen
• DP Shukla
• J Iben
• F Rueda-Faucz
• G Margolin
• RK Dale
• CM Marshall
• SE Sheppard

Abstract

Central conducting lymphatic anomaly (CCLA) is a devastating disease characterized by the inability of the lymphatic system to facilitate circulation of lymphatic fluid due to malformations in the thoracic duct or other central lymphatics. Currently only 41% of affected individuals receive a genetic diagnosis leaving the majority without answers or proper treatment. Prior studies in zebrafish and mice have characterized the effect of KRAS p.G12D on the developing vasculature showing that lymphatic vessel dilations and malformations respond to MEK inhibition. The purpose of this study was to generate zebrafish models of two additional KRAS variants to continue to elucidate the underlying molecular mechanisms of KRAS-driven CCLA and to determine if different variants respond similarly to the same therapies. We mosaically expressed activating KRAS variants (KRAS p.G12C, p.G12D, and A146T) in zebrafish to characterize their effect on the developing lymphatic system. Mosaic expression of p.G12C, p.G12D, and p.A146T increased pericardial edema (53%(n=140), 69%(n=64), and 55%(n=20) respectively) and cystic malformations (38%, 69%, and 75% respectively). Following initial characterization of the variants, we used confocal imaging to examine vessel-specific differences in the trunk lymphatics. All three variants caused an increase in the diameter of the intersegmental lymphatic vessels and posterior cardinal vein. Additionally, all three variants caused dysmorphology of the thoracic duct (75%, 81%, and 60%, respectively). Initial RNA-sequencing analysis for p.G12D demonstrates an up-regulation of lymphangiogenesis-related genes and pathways. These models closely resemble CCLA in humans and show promise for continued use in studying additional pharmacological candidates for patients with KRAS-driven CCLA.

Scientific Focus Area: Developmental Biology

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