An analysis of lateralized neural crest marker expression across development in the Mexican tetra, Astyanax mexicanus.

The biological basis of lateralized cranial aberrations can be rooted in early asymmetric patterning of developmental tissues. However, precisely how development impacts natural cranial asymmetries remains incompletely understood. Here, we examined embryonic patterning of the cranial neural crest at two phases of embryonic development in a natural animal system with two morphotypes: cave-dwelling and surface-dwelling fish. Surface fish are highly symmetric with respect to cranial form at adulthood, however adult cavefish harbor diverse cranial asymmetries. To examine if lateralized aberrations of the developing neural crest underpin these asymmetries, we used an automated technique to quantify the area and expression level of cranial neural crest markers on the left and right sides of the embryonic head. We examined the expression of marker genes encoding both structural proteins and transcription factors at two key stages of development: 36 hpf (∼mid-migration of the neural crest) and 72 hpf (∼early differentiation of neural crest derivatives). Interestingly, our results revealed asymmetric biases at both phases of development in both morphotypes, however consistent lateral biases were less common in surface fish as development progressed. Additionally, this work provides the information on neural crest development, based on whole-mount expression patterns of 19 genes, between stage-matched cave and surface morphs. Further, this study revealed 'asymmetric' noise as a likely normative component of early neural crest development in natural Astyanax fish. Mature cranial asymmetries in cave morphs may arise from persistence of asymmetric processes during development, or as a function of asymmetric processes occurring later in the life history.

In-Frame Indel Mutations in the Genome of the Blind Mexican Cavefish, Astyanax mexicanus.

Organisms living in the subterranean biome evolve extreme characteristics including vision loss and sensory expansion. Despite prior work linking certain genes to Mendelian traits, the genetic basis for complex cave-associated traits remains unknown. Moreover, it is unclear if certain forms of genetic variation (e.g., indels, copy number variants) are more common in regressive evolution. Progress in this area has been limited by a lack of suitable natural model systems and genomic resources. In recent years, the Mexican tetra, Astyanax mexicanus, has advanced as a model for cave biology and regressive evolution. Here, we present the results of a genome-wide screen for in-frame indels using alignments of RNA-sequencing reads to the draft cavefish genome. Mutations were discovered in three genes associated with blood physiology (mlf1, plg, and wdr1), two genes associated with growth factor signaling (ghrb, rnf126), one gene linked to collagen defects (mia3), and one gene which may have a global epigenetic impact on gene expression (mki67). With one exception, polymorphisms were shared between Pachón and Tinaja cavefish lineages, and different from the surface-dwelling lineage. We confirmed the presence of mutations using direct Sanger sequencing and discovered remarkably similar developmental expression in both morphs despite substantial coding sequence alterations. Further, three mutated genes mapped near previously established quantitative trait loci associated with jaw size, condition factor, lens size, and neuromast variation. This work reveals previously unappreciated traits evolving in this species under environmental pressures (e.g., blood physiology) and provides insight to genetic changes underlying convergence of organisms evolving in complete darkness.

Wholemount In Situ Hybridization for Astyanax Embryos.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.