# A genetic modifier links integrin α5 to the phenotypic variation in fibronectin 1a mutant zebrafish

## TL;DR

This study finds that a genetic modifier, integrin alpha 5, influences the severity of heart defects in zebrafish with a fibronectin mutation.

## Contribution

The study identifies integrin alpha 5 as a genetic modifier of phenotypic variation in fibronectin 1a mutant zebrafish.

## Key findings

- natter/fn1a mutants show variable cardiac phenotypes similar to those with a large fn1a deletion.
- Upregulation of fn1b is observed in mutants with severe phenotypes, but it does not modulate the mutant phenotype.
- A genetic modifier at the integrin alpha 5 (itgα5) locus was identified through selective breeding and whole-genome sequencing.

## Abstract

Phenotypic variation is often observed in individuals with the same mutation. However, the mechanisms that contribute to this variation remain largely unknown. Fibronectin mutants in both mouse and zebrafish fail to form a functional cardiovascular system, although the penetrance and expressivity of this phenotype vary depending on the genetic background. Here we investigate the variation of the zebrafish natter phenotype, which is caused by a nonsense mutation in fibronectin 1a (fn1a). natter/fn1a mutants exhibit incompletely penetrant cardia bifida, a phenotype caused by the failure of cardiac progenitors to migrate to the midline. To examine whether this variation is related to the nonsense mutation, we first generated a large deletion in fn1a that removes the proximal promoter and first 17 exons. Characterisation of this allele found that mutants display variable cardiac phenotypes indistinguishable from those observed in natter/fn1a mutants. As phenotypic variation is often associated with changes in paralogous gene expression, we next examined the expression of the fn1a paralogue, fn1b, and observed its upregulation specifically in the natter/fn1a mutants that exhibit a severe phenotype. However, overexpression and double mutant analyses suggest that fn1b expression levels do not modulate the natter/fn1a mutant phenotype. During these studies, we observed a small proportion of natter/fn1a mutants with a wild-type (WT)-like phenotype. Selectively raising WT looking mutant larvae increased the proportion of natter/fn1a mutants displaying the WT-like phenotype from 1.7% to 38.6% in just three generations, indicating the selection of a genetic modifier of the mutant phenotype. We mapped this modifier to the integrin alpha 5 (itgα5) locus through whole-genome sequencing. Furthermore, we found that manipulating itgα5 expression influenced the severity of the fn1a mutant phenotype, and that the variance in itgα5 expression was increased in fn1a mutants exhibiting a severe phenotype. Taken together, these results indicate that itgα5 modifies the fn1a mutant phenotype.

Recent studies have identified apparently healthy individuals with mutations that would be expected to lead to disease. Why these individuals are disease resistant is not understood. Studies in zebrafish and other organisms have revealed that environmental factors, stochastic fluctuations in gene expression, and genetic modifiers can contribute to such phenotypic variation. However, the precise mechanisms underlying phenotypic variation are likely unique to each mutation. Here, we present an analysis of zebrafish natter/fibronectin 1a (fn1a) mutants. These mutants display variable cardiac, brain, and somite defects. Fibronectin mutants in mouse display similar phenotypes, and compelling evidence that they are affected by a genetic modifier has been reported. We find that the zebrafish natter/fn1a mutant phenotype is dependent on developmental temperature, a genetic modifier, and an age-dependent parental factor. Using a simple breeding strategy, we selected for mutants that display milder phenotypes. Coupling selection with whole-genome sequencing, we mapped a genetic modifier to the integrin alpha 5 (itgα5) gene, the primary receptor for Fibronectin. Further analyses of itgα5 expression in fn1a mutants and manipulating itgα5 expression in fn1a mutants indicate that itgα5 modifies the natter/fn1a mutant phenotype. The results presented here provide new insights into the mechanisms regulating phenotypic variation in natter/fn1a mutants.