Liver-derived cell lines from cavefish Astyanax mexicanus as an in vitro model for studying metabolic adaptation

Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (1). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with a comprehensive biochemical and molecular characterization of the cell lines, which reflects key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.

Cavefish cope with environmental hypoxia by developing more erythrocytes and overexpression of hypoxia inducible genes

Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax, potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 (hif1) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.

The Preservation of PPARγ Genome Duplicates in Some Teleost Lineages: Insights into Lipid Metabolism and Xenobiotic Exploitation

Three peroxisome proliferator-activated receptor paralogues (PPARα, -β and -γ) are currently recognized in vertebrate genomes. PPARγ is known to modulate nutrition, adipogenesis and immunity in vertebrates. Natural ligands of PPARγ have been proposed; however, the receptor also binds synthetic ligands such as endocrine disruptors. Two paralogues of PPARα and PPARβ have been documented in teleost species, a consequence of the 3R WGD. Recently, two PPARγ paralogue genes were also identified in Astyanax mexicanus. We aimed to determine whether the presence of two PPARγ paralogues is prevalent in other teleost genomes, through genomic and phylogenetic analysis. Our results showed that besides Characiformes, two PPARγ paralogous genes were also identified in other teleost taxa, coinciding with the teleost-specific, whole-genome duplication and with the retention of both genes prior to the separation of the Clupeocephala. To functionally characterize these genes, we used the European sardine (Sardina pilchardus) as a model. PPARγA and PPARγB display a different tissue distribution, despite the similarity of their functional profiles: they are unresponsive to tested fatty acids and other human PPARγ ligands yet yield a transcriptional response in the presence of tributyltin (TBT). This observation puts forward the relevance of comparative analysis to decipher alternative binding architectures and broadens the disruptive potential of man-made chemicals for aquatic species.

CaveCrawler: An interactive analysis suite for cavefish bioinformatics

The growing use of genomics data in diverse animal models provides the basis for identifying genomic and transcriptional differences across species and contexts. Databases containing genomic and functional data have played critical roles in the development of numerous genetic models but are lacking for most emerging models of evolution. There is a rapidly expanding use of genomic, transcriptional, and functional genetic approaches to study diverse traits of the Mexican tetra, Astyanax mexicanus. This species exists as two morphs, eyed surface populations and at least 30 blind cave populations, providing a system to study convergent evolution. We have generated a web-based analysis suite that integrates datasets from different studies to identify how gene transcription and genetic markers of selection differ between populations and across experimental contexts. Results can be processed with other analysis platforms including Gene Ontology (GO) to enable biological inference from cross-study patterns and identify future avenues of research. Furthermore, the framework that we have built A. mexicanus can readily applied to other emerging model systems.

Fish can use hydrostatic pressure to determine their absolute depth

Hydrostatic pressure is a global cue that varies linearly with depth which could provide crucial spatial information for fish navigating vertically; however, whether fish can determine their depth using hydrostatic pressure has remained unknown. Here we show that Mexican tetras (Astyanax mexicanus) can learn the depth of a food site and consistently return to it with high fidelity using only hydrostatic pressure as a cue. Further, fish shifted their search location vertically as predicted if using pressure alone to signal depth. This study uncovers new sensory information available to fish which allows them to resolve their absolute depth on a fine scale.

Eye morphogenesis in the blind Mexican cavefish

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviors during evagination which participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations, and multiple independent cave populations which have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Further, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye-loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behavior

Social behavior is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behavior, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behavior. Overall, these results suggest that the antagonistic association between repetitive and social-like behaviors is deeply shared from teleosts through mammals.