# Combined mutations of hoxa13a, hoxa13b, and hoxd13a lead to structural shifts in zebrafish soft fin rays providing insight into spiny ray evolution

## TL;DR

Deleting three hox genes in zebrafish causes their soft fin rays to resemble spiny rays, offering clues about how spines evolved in fish.

## Contribution

The study shows how mutations in hoxa13a, hoxa13b, and hoxd13a genes can lead to structural changes resembling spiny rays, providing insight into fin ray evolution.

## Key findings

- Triple hox13 mutant zebrafish develop fin rays with shorter, thicker bones and fewer joints, similar to spiny rays.
- Mutant zebrafish show altered gene expression patterns resembling those seen in acanthomorph spiny rays.
- Hoxa13a and hoxa13b are more critical for soft ray formation than hoxd13a.

## Abstract

The zebrafish hoxa13a, hoxa13b, and hoxd13a genes have been shown to be essential for proper pectoral fin patterning. Combined mutations in these genes cause an elaboration of the pectoral fin endoskeleton, and a size reduction of the rays. In this study, we further examine the impact of partial deletions in these genes on the fin exoskeleton. Using morphological and micro-CT scan analyses, we found that rays of all fins of triple hox13 mutants are strongly affected, except for the caudal fin that is much less perturbed. Rays are shorter and thicker than wildtype rays, and present a loss of joints, bifurcations, and actinotrichia. Altogether, they lose many of the typical soft ray features and resemble more to the spiny rays of acanthomorphs. In these species, actinotrichia are present in spiny rays but organized differently than in soft rays, and spiny rays develop in the absence of hoxa13a/b expression. Gene expression analysis of triple hox13 mutant zebrafish larvae shows an expansion of the alx4a expression domain relative to the size of the dorsal and anal fin primordia and a reduction in grem1b expression that are reminiscent of the differences observed in acanthomorph spiny versus soft rays. Using various combinations of genotypes, hoxa13a and hoxa13b were found to be more important for normal soft ray formation than hoxd13a. In conclusion, our results demonstrate the importance of the hox13 paralogs for proper soft ray development and suggest a lack of hox13 expression could be important for their transformation into spiny rays.

Fins of bony fish present incredible diversity in the shape, structure, and number of the fin rays. Among teleosts, acanthomorph fish have sharp spiny rays in their fins that are thought to serve as a defense mechanism from predators. These spines evolved in teleost fish around 133–150 million years ago, but how exactly they evolved, and the genetic mechanisms involved in the appearance of this structure are not totally clear. Zebrafish (Danio rerio), a common fish model amenable for genetic studies, do not normally contain spines in their fins and only have soft rays formed by the stacking of short bone segments. In our study, we show that genetically modified zebrafish develop fin rays that show close similarities to spines, including shorter length, lack of segments and bifurcations, and thicker bone that are all seen in spines but not in soft rays. Our mutant zebrafish have deletions in the hoxa13a, hoxa13b, and hoxd13a paralogous genes, that code for transcription factors with the ability to control the expression of downstream target genes. Our findings show that modifications to the expression of these hox genes could have influenced the evolutionary appearance of spines in fish fins.