The genetic basis of novel trait gain in walking fish

Amy L Herbert, Corey AH Allard, Matthew J McCoy, Julia I Wucherpfennig, Stephanie P Krueger, Heidi I Chen, Allex N Gourlay, Kohle D Jackson, Lisa A Abbo, Scott H Bennett, Joshua D Sears, Andrew L Rhyne, Nicholas W Bellono, David M Kingsley

Preprint posted on 14 October 2023 https://www.biorxiv.org/content/10.1101/2023.10.14.562356v1.full

Ever wondered how a species can create new body parts? Walking fish hold the answer!

Selected by Roberto Rodríguez-Morales

Categories: developmental biology, evolutionary biology, genetics

Background

A central question in evolutionary biology is how organisms change across evolutionary timescales. Broadly speaking, evolutionary changes come in one of two ways. Species can “lose traits”, a term referred to as regressive evolution, or “gain new traits”, a term referred to as constructive evolution. Most studies in this field have uncovered the genetic and environmental underpinnings of regressive evolution, including those underlying eye and pigmentation loss in blind cavefish (Gross, 2012), loss of bristles in flies (Suscena, 2000), and loss of legs in snakes (Leal, 2016). These studies identified discrete genetic loci encoding critical developmental regulators, including cis-regulatory element mutations, that underlie trait loss. However, whether constructive evolution uses similar genetic mechanisms to produce new traits, particularly in vertebrates, remains poorly understood.

The authors of this preprint established a new marine fish model for studying the genetics underlying constructive evolution: the sea robin. These fish are particularly suitable for the study of constructive evolution as they have remarkable evolutionary novelties. These include winglike pectoral fins and six prominent detached “legs” that provide sensory information and the ability to walk, dig, and find food on the ocean floor (Fig. 1a). These new “leg-like” structures are accompanied by increases in muscle, sensory and motor systems which include the formation of novel enlarged lobes in the central nervous system.

In this study, the authors use two sea robin species, Prionotus carolinus and Prionotus evolans, that have differences in pectoral fin size, pigmentation pattern and leg size to uncover the genetic underpinnings of evolutionary novelty in vertebrates.

Figure 1. Sea robin leg development. Novel leg-like structures in sea robins (A, black arrowheads) develop from the bottom three ray fins (B, white arrowheads) of the pectoral fin. Scale bar: 1 mm. (B) Schematic representing tissues collected for RNA-seq before and after leg separation. (D) PCA showing clustering of gene expression profiles of legs with pelvic fins before and after separation. (E) Volcano plot showing upregulated gene expression, including tbx3a, in the future legs compared with the top portion of the pectoral fin before leg separation.

Key findings:

Developmental mechanisms of leg development

Sea robin “legs” form from the three ventral-most pectoral fin rays that separate from the pectoral fin during development (Yuschak, 1984 and 1985). In this preprint, the authors molecularly characterized sea robin leg development by sequencing the genome of P. carolinus and performing tissue-specific RNA sequencing (RNA-seq) before and after the leg rays separate from the pectoral fin. They observed expression of several well-known developmental transcription factors in the separating leg rays, including members of the hox gene family. However, the most upregulated gene in leg rays was the T-box transcription factor tbx3a, which is associated to skeletal alterations of posterior forelimb development in humans.

Functional testing of leg developmental genes

Next, the authors established CRISPR/Cas9 genome editing in sea robins to test the functional relevance of candidate genes derived from their genomics experiment. Using the CRISPR/Cas9 injected fish (or “crispants”), they found that mutating hox genes resulted in a reduced number of legs in sea robins, while mutating tbx3a resulted in variable phenotypes, with the most common being smaller legs. The authors point out that humans heterozygous for TBX3 mutations also exhibit variable forelimb associated phenotypes.

Functional and molecular changes in tbx3a targeted fish

To determine if tbx3a is a genetic driver of leg specification, the authors performed RNA-seq on tbx3a crispants and control siblings before and after leg separation. They found a number of pectoral fin marker genes upregulated in tbx3a crispant leg rays before separation, indicating that these become more “fin-like” in the absence of tbx3a expression.

Next, they examined the novel dorsal accessory spinal lobes of sea robins which are organized in a one-to-one relationship with legs. They found significant reductions in the size of these lobes in tbx3a crispants compared to their control siblings, which were potentially related to their reduced ability to sense and find buried mussels. These results highlight the role of tbx3a in sea robin leg development, behavior and neural function.

Cis- and trans-regulatory control of species-specific differences

Leveraging phenotypic differences between P. carolinus and P. evolans, the authors fully assembled a genome for P. evolans to uncover the genetic underpinnings of trait variation between the two species. Next, using in vitro fertilization, the authors created an F1 hybrid generation between the two species to identify allele-specific expression, specifically the contribution of cis– and trans-acting changes to species-specific phenotypes. A large fraction of the genes differentially expressed between both species showed allele-specific expression in the hybrids, suggesting most of the expression differences between the two species are through cis-regulatory mechanisms. However, the authors provide evidence for trans-regulatory control of tbx3a in the formation of novel epithelial papillae that enhance digging and sensory capabilities in P. carolinus, suggesting that trans-regulation can also be a driver of evolutionary change.

What I like about the preprint/Why this work is important:

Unconventional and non-model organisms most often hold the answers to many exciting questions in evolutionary biology. While some of these have helped uncover the mechanisms underlying trait loss, we still know very little about how novel traits emerge. This preprint from the Kingsley and Bellono labs dives into this question by examining the genetic underpinnings of leg development in two species of sea robin, P. carolinus and P. evolans. Through transcriptomic analysis and the establishment of CRISPR/Cas9 for gene editing in both species, the authors provide a new model for uncovering the genetic underpinnings of constructive evolution.

Questions for the Authors:

Do you expect tbx3a phenotypic variability to be reduced if you isolate one allele in a stable mutant line? Is there a limitation to create these types of lines (potentially space limitation)?
Have you thought about creating F2 hybrids to perform QTL analysis of papillae formation and leg development, which could potentially reveal a number of genetic loci and gene candidates involved in this process? Is creating these F2s feasible?
Are there any plans to study evolution of new lobes in the CNS associated to legs? How do those develop? Are there any known or suspected molecular pathways involved?

References:

Gross, J. B., Borowsky, R. & Tabin, C. J. A novel role for Mc1r in the parallel evolution of depigmentation in independent populations of the cavefish Astyanax mexicanus. PLoS Genet. 5, (2009).
Sucena, E. & Stern, D. L. Divergence of larval morphology between Drosophila sechellia and its sibling species caused by cis-regulatory evolution of ovo/shaven-baby. Proc. Natl. Acad. Sci. U.S.A 97, 4530–4534 (2000).
Leal, F. & Cohn, M. J. Loss and re-emergence of legs in snakes by modular evolution of sonic hedgehog and HOXD enhancers. Curr. Biol. 26, 2966–2973 (2016).
Yuschak, P. & Lund, W. A. Eggs, larvae and osteological development of the northern searobin, Prionotus carolinus (Pisces, Triglidae). J. Northwest Atl. Fish. Sci. 5, 1–15 (1984).
Yuschak, P. Fecundity, eggs, larvae and osteological development of the striped searobin, (Prionotus evolans) (Pisces, Triglidae). J. Northwest Atl. Fish. Sci. 6, 65–85 (1985).

Posted on: 14 February 2024

doi: https://doi.org/10.1242/prelights.36519

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