PgmNr M310: A genetic epistasis analysis of an ENU-induced Reln mutant reveals that the C-terminal domain of RELN is required for binding to the receptor VLDLR but not to APOER2.

Authors:
D. R. Beier ^1,2 ; S. Ha ¹

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Institutes
1) Seattle Children's Research Institute, Seattle, WA; 2) University of Washington, Seattle, WA.

Abstract:

We have previously described a forward genetic screen for brain lamination defects using ENU-mutagenesis of a reporter line that marked specific layers of the cerebral cortex (Ha et al., Cereb Cortex, 2015).  In this study we identified a novel hypomorphic allele of reelin, named Reln^CTRdel, which carries a splice-site mutation that results in a truncation of the C-terminal region (CTR) domain of the RELN protein product. The mutant secretes RELN, distinguishing Reln^CTRdel from Orleans reeler, which has a larger C-terminal truncation that completely prevents RELN secretion and phenocopies reeler. In contrast, Reln^CTRdel displays remarkably distinct phenotypes from reeler. First, the size and foliation of the cerebellum is normal, and this mutant is not ataxic. Second, the mutant does not have the inversion of cortical layers; however, both superficial and deep layer neurons migrate beyond normal boundaries. Third, the dentate gyrus morphology of the hippocampus is severely disturbed; most of the infrapyramidal blade is absent, while the suprapyramidal blade is present and better laminated when compared with reeler. Genetic epistasis analysis showed that Reln^CTRdel/Lpr8^tm1Her (AKA Apoer2^null) double homozygotes have phenotypes akin to reeler, while Reln^CTRdel/Vldlr^tm1Her mice do not. Given that Apoer2^null/Vldlr^tm1Her double-knockout mice resemble reeler and single-knockout mice are hypomorphic, we infer that Reln^CTRdel/Apoer2^null double homozygotes have both receptor pathways disrupted. Therefore, the epistasis results suggest that CTR-truncation disrupts an interaction with VLDLR, while the APOER2 signaling pathway remains active, which accounts for the hypomorphic phenotype in Reln^CTRdel mice. A receptor binding assay confirms that CTR-truncation of RELN significantly decreases binding to VLDLR, but not to APOER2. Taken together, the in vitro and in vivo results demonstrate that the absence of the CTR domain can limit receptor-binding specificity of RELN. Our discovery, using a focused neurodevelopmental analysis, of an allele of reeler that would likely not otherwise be identified (as it is not ataxic) illustrates the utility of unbiased forward genetic approaches in the mouse.