Unique transcriptional signatures of sleep loss across independently evolved cavefish populations.
Journal of experimental zoology. Part B, Molecular and developmental evolution
Sleep Regulates Glial Plasticity and Expression of the Engulfment Receptor Draper Following Neural Injury.
Current biology : CB
2020; 30 (6): 1092?1101.e3
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 of A. mexicanus sleep 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 of A. 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.
View details for DOI 10.1002/jez.b.22949
View details for PubMedID 32351033
An Adult Brain Atlas Reveals Broad Neuroanatomical Changes in Independently Evolved Populations of Mexican Cavefish.
Frontiers in neuroanatomy
2019; 13: 88
Chronic sleep disturbance is associated with numerous health consequences, including neurodegenerative disease and cognitive decline . Neurite damage due to apoptosis, trauma, or genetic factors is a common feature of aging, and clearance of damaged neurons is essential for maintenance of brain function. In the central nervous system, damaged neurites are cleared by Wallerian degeneration, in which activated microglia and macrophages engulf damaged neurons . The fruit fly Drosophila melanogaster provides a powerful model for investigating the relationship between sleep and Wallerian degeneration . Several lines of evidence suggest that glia influence sleep duration, sleep-mediated neuronal homeostasis, and clearance of toxic substances during sleep, raising the possibility that glial engulfment of damaged axons is regulated by sleep . To explore this possibility, we axotomized olfactory receptor neurons and measured the effects of sleep loss or gain on the clearance of damaged neurites. Mechanical and genetic sleep deprivation impaired the clearance of damaged neurites. Conversely, treatment with the sleep-promoting drug gaboxadol accelerated clearance, while genetic induction of sleep promotes Draper expression. In sleep-deprived animals, multiple markers of glial activation were delayed, including activation of the JAK-STAT pathway, upregulation of the cell corpse engulfment receptor Draper, and innervation of the antennal lobe by glial membranes. These markers were all enhanced following genetic and pharmacological sleep induction. Taken together, these findings reveal a critical association between sleep and glial activation following neural injury, providing a platform for further investigations of the molecular mechanisms underlying sleep-dependent modulation of glial function and neurite clearance.
View details for DOI 10.1016/j.cub.2020.02.057
View details for PubMedID 32142708
Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods.
Molecular ecology resources
2019; 19 (2): 456?64
A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus, is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant, ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus, and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate brain atlases of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions that have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an enlargement of the hypothalamus in all cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and prethalamus. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior.
View details for DOI 10.3389/fnana.2019.00088
View details for PubMedID 31636546
View details for PubMedCentralID PMC6788135
Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.
Developmental dynamics : an official publication of the American Association of Anatomists
2019; 248 (8): 679?87
RNA sequencing is a popular next-generation sequencing technique for assaying genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field-based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome-wide expression patterns in 30-day-old fry collected from the same brood at the same time point that were flash-frozen in liquid nitrogen and stored at -80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash-freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field-based studies are substantial and should not be ignored.
View details for DOI 10.1111/1755-0998.12965
View details for PubMedID 30447171
View details for PubMedCentralID PMC6393184
Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish.
Journal of visualized experiments : JoVE
Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems.Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish.The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap.
View details for DOI 10.1002/dvdy.32
View details for PubMedID 30938001
View details for PubMedCentralID PMC6675643
Manipulation of Gene Function in Mexican Cavefish.
Journal of visualized experiments : JoVE
Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.
View details for DOI 10.3791/59198
View details for PubMedID 30958465
Evolutionary shift towards lateral line dependent prey capture behavior in the blind Mexican cavefish.
2018; 441 (2): 328?37
Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.
View details for DOI 10.3791/59093
View details for PubMedID 31058898
Convergence on reduced stress behavior in the Mexican blind cavefish.
2018; 441 (2): 319?27
Feeding strategies are dependent on multi-modal sensory processing, that integrates visual, chemosensory, and mechanoreceptive cues. In many fish species, local environments and food availability dramatically influence the evolution of sensory and morphological traits that underlie feeding. The Mexican cavefish, Astyanax mexicanus, have developed robust changes in sensory-dependent behaviors, but the impact on prey detection and feeding behavior is not known. In the absence of eyes, cavefish have evolved enhanced sensitivity of the lateral line, comprised of mechanosensory organs that sense water flow and detect prey. Here, we identify evolved differences in prey capture behavior of larval cavefish that are dependent on lateral line sensitivity. Under lighted conditions, cavefish strike Artemia prey at a wider angle than surface fish; however, this difference is diminished under dark conditions. In addition, the strike distance is greater in cavefish than surface fish, revealing an ability to capture, and likely detect, prey at greater distances. Experimental ablation of the lateral line disrupts prey capture in cavefish under both light and dark conditions, while it only impacts surface fish under dark conditions. Together, these findings identify an evolutionary shift towards a dependence on the lateral line for prey capture in cavefish, providing a model for investigating how loss of visual cues impacts multi-modal sensory behaviors.
View details for DOI 10.1016/j.ydbio.2018.04.027
View details for PubMedID 29772227
View details for PubMedCentralID PMC6450390
Hypocretin underlies the evolution of sleep loss in the Mexican cavefish.
Responding appropriately to stress is essential for survival, yet in pathological states, these responses can develop into debilitating conditions such as post-traumatic stress disorder and generalized anxiety. While genetic models have provided insight into the neurochemical and neuroanatomical pathways that underlie stress, little is known about how evolutionary processes and naturally occurring variation contribute to the diverse responses to stressful stimuli observed in the animal kingdom. The Mexican cavefish is a powerful system to address how altered genetic and neuronal systems can give rise to altered behaviors. When introduced into a novel tank, surface fish and cavefish display a stereotypic stress response, characterized by reduced exploratory behavior and increased immobility, akin to "freezing". The stress response in cave and surface forms is reduced by pharmacological treatment with the anxiolytic drug, buspirone, fortifying the notion that behavior in the assay represents a conserved stress state. We find that cave populations display reduced behavioral measures of stress compared to surface conspecifics, including increased time in the top half of the tank and fewer periods of immobility. Further, reduced stress responses are observed in multiple independently derived cavefish populations, suggesting convergence on loss of behavioral stress responses in the novel tank assay. These findings provide evidence of a naturally occurring species with two drastically different forms in which a shift in predator-rich ecology to one with few predators corresponds to a reduction in stress behavior.
View details for DOI 10.1016/j.ydbio.2018.05.009
View details for PubMedID 29803645
View details for PubMedCentralID PMC6444932
The lateral line confers evolutionarily derived sleep loss in the Mexican cavefish.
The Journal of experimental biology
2017; 220 (Pt 2): 284?93
The duration of sleep varies dramatically between species, yet little is known about the genetic basis or evolutionary factors driving this variation in behavior. The Mexican cavefish, Astyanax mexicanus, exists as surface populations that inhabit rivers, and multiple cave populations with convergent evolution on sleep loss. The number of Hypocretin/Orexin (HCRT)-positive hypothalamic neurons is increased significantly in cavefish, and HCRT is upregulated at both the transcript and protein levels. Pharmacological or genetic inhibition of HCRT signaling increases sleep in cavefish, suggesting enhanced HCRT signaling underlies the evolution of sleep loss. Ablation of the lateral line or starvation, manipulations that selectively promote sleep in cavefish, inhibit hcrt expression in cavefish while having little effect on surface fish. These findings provide the first evidence of genetic and neuronal changes that contribute to the evolution of sleep loss, and support a conserved role for HCRT in sleep regulation.
View details for DOI 10.7554/eLife.32637
View details for PubMedID 29405117
View details for PubMedCentralID PMC5800846
Distinct genetic architecture underlies the emergence of sleep loss and prey-seeking behavior in the Mexican cavefish.
2015; 13: 15
Sleep is an essential behavior exhibited by nearly all animals, and disruption of this process is associated with an array of physiological and behavioral deficits. Sleep is defined by changes in sensory gating that reduce sensory input to the brain, but little is known about the neural basis for interactions between sleep and sensory processing. Blind Mexican cavefish comprise an extant surface dwelling form and 29 cave morphs that have independently evolved increased numbers of mechanoreceptive lateral line neuromasts and convergent evolution of sleep loss. Ablation of the lateral line enhanced sleep in the Pachón cavefish population, suggesting that heightened sensory input underlies evolutionarily derived sleep loss. Targeted lateral line ablation and behavioral analysis localized the wake-promoting neuromasts in Pachón cavefish to superficial neuromasts of the trunk and cranial regions. Strikingly, lateral line ablation did not affect sleep in four other cavefish populations, suggesting that distinct neural mechanisms regulate the evolution of sleep loss in independently derived cavefish populations. Cavefish are subject to seasonal changes in food availability, raising the possibility that sensory modulation of sleep is influenced by metabolic state. We found that starvation promotes sleep in Pachón cavefish, and is not enhanced by lateral line ablation, suggesting that functional interactions occur between sensory and metabolic regulation of sleep. Taken together, these findings support a model where sensory processing contributes to evolutionarily derived changes in sleep that are modulated in accordance with food availability.
View details for DOI 10.1242/jeb.145128
View details for PubMedID 28100806
Sleep is characterized by extended periods of quiescence and reduced responsiveness to sensory stimuli. Animals ranging from insects to mammals adapt to environments with limited food by suppressing sleep and enhancing their response to food cues, yet little is known about the genetic and evolutionary relationship between these processes. The blind Mexican cavefish, Astyanax mexicanus is a powerful model for elucidating the genetic mechanisms underlying behavioral evolution. A. mexicanus comprises an extant ancestral-type surface dwelling morph and at least five independently evolved cave populations. Evolutionary convergence on sleep loss and vibration attraction behavior, which is involved in prey seeking, have been documented in cavefish raising the possibility that enhanced sensory responsiveness underlies changes in sleep.We established a system to study sleep and vibration attraction behavior in adult A. mexicanus and used high coverage quantitative trait loci (QTL) mapping to investigate the functional and evolutionary relationship between these traits. Analysis of surface-cave F2 hybrid fish and an outbred cave population indicates that independent genetic factors underlie changes in sleep/locomotor activity and vibration attraction behavior. High-coverage QTL mapping with genotyping-by-sequencing technology identify two novel QTL intervals that associate with locomotor activity and include the narcolepsy-associated tp53 regulating kinase. These QTLs represent the first genomic localization of locomotor activity in cavefish and are distinct from two QTLs previously identified as associating with vibration attraction behavior.Taken together, these results localize genomic regions underlying sleep/locomotor and sensory changes in cavefish populations and provide evidence that sleep loss evolved independently from enhanced sensory responsiveness.
View details for DOI 10.1186/s12915-015-0119-3
View details for PubMedID 25761998
View details for PubMedCentralID PMC4364459