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1. Genome-wide analysis of cis-regulatory changes in the metabolic adaptation of cavefish

   https://doi.org/10.1101/2020.08.27.270371  

   Krishnan, Jaya SeidelChris W. Zhang ( August 2020 , bioRxiv)

   null (Ed.)

   Changes in cis-regulatory elements play important roles in adaptation and phenotypic evolution. However, their contribution to metabolic adaptation of organisms is less understood. Here we have utilized a unique vertebrate model, Astyanax mexicanus, different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave water to uncover gene regulatory networks in metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissue of one surface and two independently derived cave populations. We find that many cis-regulatory elements differ in their epigenetic status/chromatin accessibility between surface fish and cavefish, while the two independently derived cave populations have evolved remarkably similar regulatory signatures. These differentially accessible regions are associated with genes of key pathways related to lipid metabolism, circadian rhythm and immune system that are known to be altered in cavefish. Using in vitro and in vivo functional testing of the candidate cis-regulatory elements, we find that genetic changes within them cause quantitative expression differences. We characterized one cis-regulatory element in the hpdb gene and found a genomic deletion in cavefish that abolishes binding of the transcriptional repressor IRF2 in vitro and derepresses enhancer activity in reporter assays. Genetic experiments further validated a cis-mediated role of the enhancer and suggest a role of this deletion in the upregulation of hpdb in wild cavefish populations. Selection of this mutation in multiple independent cave populations supports its importance in the adaptation to the cave environment, providing novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to a food-deprived cave environment. 

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2. Dark‐rearing uncovers novel gene expression patterns in an obligate cave‐dwelling fish

   https://doi.org/10.1002/jez.b.22947  

   Sears, Connor R. ; Boggs, Tyler E. ; Gross, Joshua B. ( May 2020 , Journal of Experimental Zoology Part B: Molecular and Developmental Evolution)

   Extreme environments often result in the evolution of dramatic adaptive features. The Mexican tetra,Astyanax mexicanus, includes 30 different populations of cave‐dwelling forms that live in perpetual darkness. As a consequence, many populations have evolved eye loss, reduced pigmentation, and amplification of nonvisual sensory systems. Closely‐related surface‐dwelling morphs demonstrate typical vision, pigmentation, and sensation. Transcriptomic assessments in this system have revealed important developmental changes associated with the cave morph, however, they have not accounted for photic rearing conditions. Prior studies reared individuals under a 12:12 hr light/dark (LD) cycle. Here, we reared cavefish under constant darkness (DD) for 5+ years. From these experimental individuals, we performed mRNA sequencing and compared gene expression of surface fish reared under LD conditions to cavefish reared under DD conditions to identify photic‐dependent gene expression differences. Gene Ontology enrichment analyses revealed a number of previously underappreciated cave‐associated changes impacting blood physiology and olfaction. We further evaluated the position of differentially expressed genes relative to QTL positions from prior studies and found several candidate genes associated with these ecologically relevant lighting conditions. In sum, this work highlights photic conditions as a key environmental factor impacting gene expression patterns in blind cave‐dwelling fish.

    

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3. Natural selection versus neutral mutation in the evolution of subterranean life: A false dichotomy?

   https://doi.org/10.3389/fevo.2023.1080503  

   Culver, David C. ; Kowalko, Johanna E. ; Pipan, Tanja ( January 2023 , Frontiers in Ecology and Evolution)

   Throughout the evolutionary tree, there are gains and losses of morphological features, physiological processes, and behavioral patterns. Losses are perhaps nowhere so prominent as for subterranean organisms, which typically show reductions or losses of eyes and pigment. These losses seem easy to explain without recourse to natural selection. Its most modern form is the accumulation of selectively neutral, structurally reducing mutations. Selectionist explanations include direct selection, often involving metabolic efficiency in resource poor subterranean environments, and pleiotropy, where genes affecting eyes and pigment have other effects, such as increasing extra-optic sensory structures. This dichotomy echoes the debate in evolutionary biology in general about the sufficiency of natural selection as an explanation of evolution, e.g., Kimura’s neutral mutation theory. Tests of the two hypotheses have largely been one-sided, with data supporting that one or the other processes is occurring. While these tests have utilized a variety of subterranean organisms, the Mexican cavefish,Astyanax mexicanus, which has eyed extant ancestral-like surface fish conspecifics, is easily bred in the lab, and whose whole genome has been sequenced, is the favored experimental organism. However, with few exceptions, tests for selection versus neutral mutations contain limitations or flaws. Notably, these tests are often one sided, testing for the presence of one or the other process. In fact, it is most likely that both processes occur and make a significant contribution to the two most studied traits in cave evolution: eye and pigment reduction. Furthermore, narrow focus on neutral mutation hypothesis versus selection to explain cave-evolved traits often fails, at least in the simplest forms of these hypotheses, to account for aspects that are likely essential for understanding cave evolution: migration or epigenetic effects. Further, epigenetic effects and phenotypic plasticity have been demonstrated to play an important role in cave evolution in recent studies. Phenotypic plasticity does not by itself result in genetic change of course, but plasticity can reveal cryptic genetic variation which then selection can act on. These processes may result in a radical change in our thinking about evolution of subterranean life, especially the speed with which it may occur. Thus, perhaps it is better to ask what role the interaction of genes and environment plays, in addition to natural selection and neutral mutation.

    

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4. 3D spheroid culturing of Astyanax mexicanus liver‐derived cell lines recapitulates distinct transcriptomic and metabolic states of in vivo tissue environment

   https://doi.org/10.1002/jez.b.23236  

   Biswas, Tathagata ; Rajendran, Naresh ; Hassan, Huzaifa ; Li, Hua ; Zhao, Chongbei ; Rohner, Nicolas ( January 2024 , Journal of Experimental Zoology Part B: Molecular and Developmental Evolution)

   In vitro assays are crucial tools for gaining detailed insights into various biological processes, including metabolism. Cave morphs of the river‐dwelling fish species,Astyanax mexicanus, have adapted their metabolism allowing them to thrive in the biodiversity‐deprived and nutrient‐limited environment of caves. Liver‐derived cells from the cave and river morphs ofA. mexicanushave proven to be excellent in vitro resources to better understand the unique metabolism of these fish. However, the current 2D cultures have not fully captured the complex metabolic profile of theAstyanaxliver. It is known that 3D culturing can modulate the transcriptomic state of cells when compared to its 2D monolayer culture. Therefore, to broaden the possibilities of the in vitro system by modeling a wider gamut of metabolic pathways, we cultured the liver‐derivedAstyanaxcells of both surface and cavefish into 3D spheroids. We successfully established 3D cultures at various cell seeding densities for several weeks and characterized the resultant transcriptomic and metabolic variations. We found that the 3D culturedAstyanaxcells exhibit an altered transcriptomic profile and consequently represent a wider range of metabolic pathways, including cell cycle changes and antioxidant activities, associated with liver functioning as compared to its monolayer culture. Enzymatic assay measuring antioxidants in 2D culture and 3D spheroids also revealed enhanced antioxidative capacity of 3D cultured spheroids, in line with the differential gene expression data. Additionally, the spheroids also exhibited surface and cave‐specific metabolic signatures, making it a suitable system for evolutionary studies associated with cave adaptation. Notably, cavefish derived spheroids enriched for genes responding to xenobiotic stimulus, while the ones from surface enriched for immune response, both of which resonated with known physiologically adaptations associated with each morph. Taken together, the liver‐derived spheroids prove to be a promising in vitro model for widening our understanding of metabolism inA. mexicanusand of vertebrates in general.

    

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5. Unique transcriptional signatures of sleep loss across independently evolved cavefish populations

   https://doi.org/10.1002/jez.b.22949  

   McGaugh, Suzanne E. ; Passow, Courtney N. ; Jaggard, James Brian ; Stahl, Bethany A. ; Keene, Alex C. ( April 2020 , Journal of Experimental Zoology Part B: Molecular and Developmental Evolution)

   Animals respond to sleep loss with compensatory rebound sleep, and this is thought to be critical for the maintenance of physiological homeostasis. Sleep duration varies dramatically across animal species, but it is not known whether evolutionary differences in sleep duration are associated with differences in sleep homeostasis. The Mexican cavefish,Astyanax mexicanus, has emerged as a powerful model for studying the evolution of sleep. While eyed surface populations ofA. mexicanussleep approximately 8 hr each day, multiple blind cavefish populations have converged on sleep patterns that total as little as 2 hr each day, providing the opportunity to examine whether the evolution of sleep loss is accompanied by changes in sleep homeostasis. Here, we examine the behavioral and molecular response to sleep deprivation across four independent populations ofA. mexicanus. Our behavioral analysis indicates that surface fish and all three cavefish populations display robust recovery sleep during the day following nighttime sleep deprivation, suggesting sleep homeostasis remains intact in cavefish. We profiled transcriptome‐wide changes associated with sleep deprivation in surface fish and cavefish. While the total number of differentially expressed genes was not greater for the surface population, the surface population exhibited the highest number of uniquely differentially expressed genes than any other population. Strikingly, a majority of the differentially expressed genes are unique to individual cave populations, suggesting unique expression responses are exhibited across independently evolved cavefish populations. Together, these findings suggest sleep homeostasis is intact in cavefish despite a dramatic reduction in overall sleep duration.

    

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