Platelet factors attenuate inflammation and rescue cognition in ageing.

Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.

Reinterpreting the work of Charles Breder: Sensory neuromasts and orbital skeleton variation in eyeless Astyanax cavefish.

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown "organizing force" likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this "evolutionary" model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia.

Feasibility of 10-minute arrival time to departure time metric for STEMI patients.

OBJECTIVES: Delays in reperfusion treatment in ST-elevation myocardial infarction (STEMI) patients leads to higher morbidity and mortality. Previous reports for Helicopter Emergency Medical Services (HEMS) suggests a 10-minute skid-to-skid (arrival to departure) time to achieve appropriate reperfusion times. However, there is no published data on whether this goal is achievable for a HEMS service.This study aims to see if a midwestern critical care service can consistently achieve a 10-minute helicopter skid-to-skid time or ground critical care service arrival to departure time. Further, comparing this metric between ground and helicopter transportations will help evaluate the ideal transportation method to optimize time to percutaneous intervention (PCI). METHODS: This was a retrospective chart review utilizing 10 years of data from our ground and HEMS program to assess whether a 10-minute arrival to departure time for STEMI patients could be achieved. Patients included were at least 18 years of age and were transported from the referring facility for further STEMI management. Wilcoxon rank sum test and Chi-square tests were used to evaluate data between helicopter and ground services. RESULTS: Included in the study were 686 patients, 608 by helicopter transport and 78 by ground transport. The median arrival to departure time was 14 minutes (IQR = 5) for helicopter patients and 13 minutes (IQR = 6) for ground patients. There was not a statistically significant difference in this metric for STEMI patients transported by helicopter versus ground. A statistically significant difference, though, existed between helicopter and ground transports among percent with times less than or equal to 10 minutes versus percent times greater than 10 minutes, X2 = 5.46, df = 1, p = 0.02. Two referring facilities had statistically significant differences in median arrival to departure times. CONCLUSIONS: Our study found that a median EMS arrival to departure time of 10 minutes to transport STEMI patients was not consistently achieved via either helicopter or ground transportation.

Phylogeographic relationships and morphological evolution between cave and surface Astyanax mexicanus populations (De Filippi 1853) (Actinopterygii, Characidae).

The Astyanax mexicanus complex includes two different morphs, a surface- and a cave-adapted ecotype, found at three mountain ranges in Northeastern Mexico: Sierra de El Abra, Sierra de Guatemala and Sierra de la Colmena (Micos). Since their discovery, multiple studies have attempted to characterize the timing and the number of events that gave rise to the evolution of these cave-adapted ecotypes. Here, using RADseq and genome-wide sequencing, we assessed the phylogenetic relationships, genetic structure and gene flow events between the cave and surface Astyanax mexicanus populations, to estimate the tempo and mode of evolution of the cave-adapted ecotypes. We also evaluated the body shape evolution across different cave lineages using geometric morphometrics to examine the role of phylogenetic signal versus environmental pressures. We found strong evidence of parallel evolution of cave-adapted ecotypes derived from two separate lineages of surface fish and hypothesize that there may be up to four independent invasions of caves from surface fish. Moreover, a strong congruence between the genetic structure and geographic distribution was observed across the cave populations, with the Sierra de Guatemala the region exhibiting most genetic drift among the cave populations analysed. Interestingly, we found no evidence of phylogenetic signal in body shape evolution, but we found support for parallel evolution in body shape across independent cave lineages, with cavefish from the Sierra de El Abra reflecting the most divergent morphology relative to surface and other cavefish populations.

Ventricular fibrillation in a 21-year-old after inhalation of an isobutyl nitrite "popper" product.

BACKGROUND: Alkyl nitrite analogs known as "poppers" have been inhaled recreationally for decades. They are available to be purchased from gas stations marketed as "nail polish remover not for human consumption". These rapid-onset, short-acting, vasodilators cause the user to experience euphoria, dizziness, tachycardia and flushing. While chronic use may lead to problems such as methemoglobinemia or neuropathy, nitrites rarely lead to acute life-threatening side effects such as ventricular dysrhythmias. CASE REPORT: We report a case of ventricular fibrillation cardiac arrest in a 21-year-old male after inhaling from a solution labeled to contain isobutyl nitrite, a rarely reported adverse effect of "popper" use. The product was analytically confirmed to contain mainly isobutyl alcohol, volatile hydrocarbons, and isobutyl nitrite, as well as smaller quantities of other substances. The patient was also prescribed escitalopram and hydroxyzine may have contributed. Return of spontaneous circulation was achieved in the field after initiation of CPR and defibrillation. He was found to have no clear predisposition to arrythmias during his care or on follow up. CONCLUSION: Alkyl nitrite "popper" users and clinicians should be aware that products labeled to contain nitrites may contain volatile hydrocarbons along with nitrites and have the potential to cause arrhythmia.

Sensory evolution in a cavefish radiation: patterns of neuromast distribution and associated behaviour in Sinocyclocheilus (Cypriniformes: Cyprinidae).

The genus Sinocyclocheilus, comprising a large radiation of freshwater cavefishes, are well known for their presence of regressive features (e.g. variable eye reduction). Fewer constructive features are known, such as the expansion of the lateral line system (LLS), which is involved in detecting water movements. The precise relationship between LLS expansion and cave adaptation is not well understood. Here, we examine morphology and LLS-mediated behaviour in Sinocyclocheilus species characterized by broad variation in eye size, habitat and geographical distribution. Using live-staining techniques and automated behavioural analyses, we examined 26 Sinocyclocheilus species and quantified neuromast organ number, density and asymmetry within a phylogenetic context. We then examined how these morphological features may relate to wall-following, an established cave-associated behaviour mediated by the lateral line. We show that most species demonstrated laterality (i.e. asymmetry) in neuromast organs on the head, often biased to the right. We also found that wall-following behaviour was distinctive, particularly among eyeless species. Patterns of variation in LLS appear to correlate with the degree of eye loss, as well as geographical distribution. This work reveals that constructive LLS evolution is convergent across distant cavefish taxa and may mediate asymmetric behavioural features that enable survival in stark subterranean microenvironments.

The stability of tastant detection by mouse lingual chemosensory tissue requires Regulator of G protein Signaling-21 (RGS21).

The T1R and T2R families of G protein-coupled receptors (GPCRs) initiate tastant perception by signaling via guanine nucleotide exchange and hydrolysis performed by associated heterotrimeric G proteins (Gαßγ). Heterotrimeric G protein signal termination is sped up by Gα-directed GTPase-accelerating proteins (GAPs) known as the Regulators of G protein Signaling (RGS proteins). Of this family, RGS21 is highly expressed in lingual epithelial cells and we have shown it acting in vitro to decrease the potency of bitterants on cultured cells. However, constitutive RGS21 loss in mice reduces organismal response to GPCR-mediated tastants-opposite to expectations arising from observed in vitro activity of RGS21 as a GAP and inhibitor of T2R signaling. Here, we show reduced quinine aversion and reduced sucrose preference by mice lacking RGS21 does not result from post-ingestive effects, as taste-salient brief-access tests confirm the reduced bitterant aversion and reduced sweetener preference seen using two-bottle choice testing. Eliminating Rgs21 expression after chemosensory system development, via tamoxifen-induced Cre recombination in eight week-old mice, led to a reduction in quinine aversive behavior that advanced over time, suggesting that RGS21 functions as a negative regulator to sustain stable bitter tastant reception. Consistent with this notion, we observed downregulation of multiple T2R proteins in the lingual tissue of Rgs21-deficient mice. Reduced tastant-mediated responses exhibited by mice lacking Rgs21 expression either since birth or in adulthood has highlighted the potential requirement for a GPCR GAP to maintain the full character of tastant signaling, likely at the level of mitigating receptor downregulation.

Potential for Kappa-Opioid Receptor Agonists to Engineer Nonaddictive Analgesics: A Narrative Review.

A serious adverse effect of prescription opioid analgesics is addiction, both to these analgesics and to illicit drugs like heroin that also activate the µ-opioid receptor (MOR). Opioid use disorder (OUD) and opioid overdose deaths represent a current American health crisis, and the prescription of opioid analgesics has contributed significantly to this crisis. While prescription opioids are highly effective analgesics, there currently exists no facile way to use them for extended periods without the risk of addiction. If addiction caused by MOR-targeting analgesics could be blocked by blending in a new "antiaddiction" ingredient that does not diminish analgesia and does not introduce its own therapeutically limiting side effects, then continued clinical use of prescription opioids for treating pain could be maintained (or even enhanced) instead of curtailed. In this narrative review, we contextualize this hypothesis, first with a brief overview of the current American opioid addiction crisis. The neurobiology of 2 key receptors in OUD development, MOR and the κ-opioid receptor (KOR), is then discussed to highlight the neuroanatomical features and circuitry in which signal transduction from these receptors lie in opposition-creating opportunities for pharmacological intervention in curtailing the addictive potential of MOR agonism. Prior findings with mixed MOR/KOR agonists are considered before exploring new potential avenues such as biased KOR agonists. New preclinical data are highlighted, demonstrating that the G protein-biased KOR agonist nalfurafine reduces the rewarding properties of MOR-targeting analgesics and enhances MOR-targeting analgesic-induced antinociception. Finally, we discuss the recent discovery that a regulator of G protein signaling (namely, RGS12) is a key component of signaling bias at KOR, presenting another drug discovery target toward identifying a single agent or adjuvant to be added to traditional opioid analgesics that could reduce or eliminate the addictive potential of the latter drug.

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

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.

Parallel evolution of regressive and constructive craniofacial traits across distinct populations of Astyanax mexicanus cavefish.

Life in complete darkness has driven the evolution of a suite of troglobitic features in the blind Mexican cavefish Astyanax mexicanus, such as eye and pigmentation loss. While regressive evolution is a hallmark of obligate cave-dwelling organisms, constructive (or augmented) traits commonly arise as well. The cavefish cranium has undergone extensive changes compared with closely-related surface fish. These alterations are rooted in both cranial bones and surrounding sensory tissues such as enhancements in the gustatory and lateral line systems. Cavefish also harbor numerous cranial bone asymmetries: fluctuating asymmetry of individual bones and directional asymmetry in a dorsal bend of the skull. This asymmetry is mirrored by the asymmetrical patterning of mechanosensory neuromasts. We explored the relationship between facial bones and neuromasts using in vivo fluorescent colabeling and microcomputed tomography. We found an increase in neuromast density within dermal bone boundaries across three distinct populations of cavefish compared to surface-dwelling fish. We also show that eye loss disrupts early neuromast patterning, which in turn impacts the development of dermal bones. While cavefish exhibit alterations in cranial bone and neuromast patterning, each population varied in the severity. This variation may reflect observed differences in behavior across populations. For instance, a bend in the dorsal region of the skull may expose neuromasts to water flow on the opposite side of the face, enhancing sensory input and spatial mapping in the dark.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

Canal neuromast position prefigures developmental patterning of the suborbital bone series in Astyanax cave- and surface-dwelling fish.

Developmental patterning is a complex biological phenomenon, involving integrated cellular and molecular signaling across diverse tissues. In Astyanax cavefish, the lateral line sensory system is dramatically expanded in a region of the cranium marked by significant bone abnormalities. This system provides the opportunity to understand how facial bone patterning can become altered through sensory system changes. Here we investigate a classic postulation that mechanosensory receptor neuromasts seed intramembranous facial bones in aquatic vertebrates. Using an in vivo staining procedure across individual life history, we observed infraorbital canal neuromasts serving as sites of ossification for suborbital bones. The manner in which cavefish departed from the stereotypical and symmetrical canal neuromast patterns of closely-related surface-dwelling fish were associated with specific changes to the suborbital bone complex. For instance, bony fusion, rarely observed in surface fish, was associated with shorter distances between canal neuromasts in cavefish, suggesting that closer canal neuromasts result in bony fusions. Additionally, cavefish lacking the sixth suborbital bone (SO6) uniformly lacked the associated (sixth) canal neuromast. This study suggests that patterning of canal neuromasts may impact spatial position of suborbital bones across development. The absence of an eye and subsequent orbital collapse in cavefish appears to influence positional information normally inherent to the infraorbital canal. These alterations result in coordinated changes to adult neuromast and bone structures. This work highlights complex interactions between visual, sensory and bony tissues during development that explain certain abnormal craniofacial features in cavefish.

Preclinical Testing of Nalfurafine as an Opioid-sparing Adjuvant that Potentiates Analgesia by the Mu Opioid Receptor-targeting Agonist Morphine.

Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria). Recent data suggest that some functionally selective, or biased, KOR-targeting agonists might retain the therapeutic effects of KOR activation without inducing undesirable side effects. Nalfurafine, used safely in Japan since 2009 for uremic pruritus, is one such functionally selective KOR-targeting agonist. Here, we quantify the bias of nalfurafine and several other KOR agonists relative to an unbiased reference standard (U50,488) and show that nalfurafine and EOM-salvinorin-B demonstrate marked G protein-signaling bias. While nalfurafine (0.015 mg/kg) and EOM-salvinorin-B (1 mg/kg) produced spinal antinociception equivalent to 5 mg/kg U50,488, only nalfurafine significantly enhanced the supraspinal analgesic effect of 5 mg/kg morphine. In addition, 0.015 mg/kg nalfurafine did not produce significant conditioned place aversion, yet retained the ability to reduce morphine-induced conditioned place preference in C57BL/6J mice. Nalfurafine and EOM-salvinorin-B each produced robust inhibition of both spontaneous and morphine-stimulated locomotor behavior, suggesting a persistence of sedative effects when coadministered with morphine. Taken together, these findings suggest that nalfurafine produces analgesic augmentation, while also reducing opioid-induced reward with less risk of dysphoria. Thus, adjuvant administration of G protein-biased KOR agonists like nalfurafine may be beneficial in enhancing the therapeutic potential of MOR-targeting analgesics, such as morphine.

Telehealth Impact on Primary Care Related Ambulance Transports.

Introduction: Telehealth has been used nominally for trauma, neurological, and cardiovascular incidents in prehospital emergency medical services (EMS). Yet, much less is known about the use of telehealth for low-acuity primary care. We examine the development of one telehealth program and its impact on unnecessary ambulance transports. Objective: The objective of this study is to describe the development and impact of a large-scale telehealth program on ambulance transports. Methods: We describe the patient characteristics and results from a cohort of patients in Houston, Texas who received a prehospital telehealth consultation from an emergency medicine physician. Inclusion criteria were adults and pediatric patients with complaints considered to be non-urgent, primary care related. Data were analyzed for 36 months, from January 2015 through December 2017. Our primary dependent variable was the percentage of patients transported by ambulance. We used descriptive statistics to describe patient demographics, chi-square to examine differences between groups, and logistic regression to explore the effects with multivariate controls including age, gender, race, and chief complaint. Results: A total of 15,067 patients were enrolled (53% female; average age 44 years ± 19 years) over the three-year period. The 3 primary chief complaints were based on abdominal pains (13% of cases), nausea/vomiting/diarrhea (NVD) (9.4%), and back pain (9.3%). Ambulance transports represented 11.2% of all transports in the program, while alternative taxi transportation was used in 75.6%, and the remainder were self- or no-transports. Taxi transportation to an alternate, affiliated clinic (versus ED) was utilized in 5% of incidents. After multivariate controls, older age patients presenting with low-risk, non-acute chest pain, shortness of breath, and dizziness were much more likely to use ambulance transport. Race and gender were not significant predictors of ambulance transport. Conclusions: We found telehealth offers a technology strategy to address potentially unnecessary ambulance transports. Based on prior cost-effectiveness analyses, the reduction of unnecessary ambulance transports translates to an overall reduction in EMS agency costs. Telehealth programs offer a viable solution to support alternate destination and alternate transport programs.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

A local duplication of the Melanocortin receptor 1 locus in Astyanax.

In this study, we report evidence of a novel duplication of Melanocortin receptor 1 (Mc1r) in the cavefish genome. This locus was discovered following the observation of excessive allelic diversity in a ∼820 bp fragment of Mc1r amplified via degenerate PCR from a natural population of Astyanax aeneus fish from Guerrero, Mexico. The cavefish genome reveals the presence of two closely related Mc1r open reading frames separated by a 1.46 kb intergenic region. One open reading frame corresponds to the previously reported Mc1r receptor, and the other open reading frame (duplicate copy) is 975 bp in length, encoding a receptor of 325 amino acids. Sequence similarity analyses position both copies in the syntenic region of the single Mc1r locus in 16 representative craniate genomes spanning bony fish (including Astyanax) to mammals, suggesting we discovered tandem duplicates of this important gene. The two Mc1r copies share ∼89% sequence similarity and, within Astyanax, are more similar to one another compared to other melanocortin family members. Future studies will inform the precise functional significance of the duplicated Mc1r locus and if this novel copy number variant may have adaptive significance for the Astyanax lineage.

A Comparative Transcriptomic Analysis of Development in Two Astyanax Cavefish Populations.

Organisms that are isolated into extreme environments often evolve extreme phenotypes. However, global patterns of dynamic gene expression changes that accompany dramatic environmental changes remain largely unknown. The blind Mexican cavefish, Astyanax mexicanus, has evolved a number of severe cave-associated phenotypes including loss of vision and pigmentation, craniofacial bone fusions, increased fat storage, reduced sleep, and amplified nonvisual sensory systems. Interestingly, surface-dwelling forms have repeatedly entered different caves throughout Mexico, providing a natural set of "replicate" instances of cave isolation. These surrogate "ancestral" surface-dwelling forms persist in nearby rivers, enabling direct comparisons to the "derived" cave-dwelling form. We evaluated changes associated with subterranean isolation by measuring differential gene expression in two geographically distinct cave-dwelling populations (Pachón and Tinaja). To understand the impact of these expression changes on development, we performed RNA-sequencing across four critical stages during which troglomorphic traits first appear in cavefish embryos. Gene ontology (GO) studies revealed similar functional profiles evolved in both independent cave lineages. However, enrichment studies indicated that similar GO profiles were occasionally mediated by different genes. Certain "master" regulators, such as Otx2 and Mitf, appear to be important loci for cave adaptation, as remarkably similar patterns of expression were identified in both independent cave lineages. This work reveals that adaptation to an extreme environment, in two distinct cavefish lineages, evolves through a combination of unique and shared gene expression patterns. Shared expression profiles reflect common environmental pressures, while unique expression likely reflects the fact that similar adaptive traits evolve through diverse genetic mechanisms.

A pleiotropic interaction between vision loss and hypermelanism in Astyanax mexicanus cave x surface hybrids.

BACKGROUND: Cave-dwelling animals evolve various traits as a consequence of life in darkness. Constructive traits (e.g., enhanced non-visual sensory systems) presumably arise under strong selective pressures. The mechanism(s) driving regression of features, however, are not well understood. Quantitative trait locus (QTL) analyses in Astyanax mexicanus Pachón cave x surface hybrids revealed phenotypic effects associated with vision and pigmentation loss. Vision QTL were uniformly associated with reductions in the homozygous cave condition, however pigmentation QTL demonstrated mixed phenotypic effects. This implied pigmentation might be lost through both selective and neutral forces. Alternatively, in this report, we examined if a pleiotropic interaction may exist between vision and pigmentation since vision loss has been shown to result in darker skin in other fish and amphibian model systems. RESULTS: We discovered that certain members of Pachón x surface pedigrees are significantly darker than surface-dwelling fish. All of these "hypermelanic" individuals demonstrated severe visual system malformations suggesting they may be blind. A vision-mediated behavioral assay revealed that these fish, in stark contrast to surface fish, behaved the same as blind cavefish. Further, hypermelanic melanophores were larger and more dendritic in morphology compared to surface fish melanophores. However, hypermelanic melanophores responded normally to melanin-concentrating hormone suggesting darkening stemmed from vision loss, rather than a defect in pigment cell function. Finally, a number of genomic regions were coordinately associated with both reduced vision and increased pigmentation. CONCLUSIONS: This work suggests hypermelanism in hybrid Astyanax results from blindness. This finding provides an alternative explanation for phenotypic effect studies of pigmentation QTL as stemming (at least in part) from environmental, rather than exclusively genetic, interactions between two regressive phenotypes. Further, this analysis reveals persistence of background adaptation in Astyanax. As the eye was lost in cave-dwelling forms, enhanced pigmentation resulted. Given the extreme cave environment, which is often devoid of nutrition, enhanced pigmentation may impose an energetic cost. Such an energetic cost would be selected against, as a means of energy conservation. Thus, the pleiotropic interaction between vision loss and pigmentation may reveal an additional selective pressure favoring the loss of pigmentation in cave-dwelling animals.

Natural bone fragmentation in the blind cave-dwelling fish, Astyanax mexicanus: candidate gene identification through integrative comparative genomics.

Animals that colonize dark and nutrient-poor subterranean environments evolve numerous extreme phenotypes. These include dramatic changes to the craniofacial complex, many of which are under genetic control. These phenotypes can demonstrate asymmetric genetic signals wherein a QTL is detected on one side of the face but not the other. The causative gene(s) underlying QTL are difficult to identify with limited genomic resources. We approached this task by searching for candidate genes mediating fragmentation of the third suborbital bone (SO3) directly inferior to the orbit of the eye. We integrated positional genomic information using emerging Astyanax resources, and linked these intervals to homologous (syntenic) regions of the Danio rerio genome. We identified a discrete, approximately 6 Mb, conserved region wherein the gene causing SO3 fragmentation likely resides. We interrogated this interval for genes demonstrating significant differential expression using mRNA-seq analysis of cave and surface morphs across life history. We then assessed genes with known roles in craniofacial evolution and development based on GO term annotation. Finally, we screened coding sequence alterations in this region, identifying two key genes: transforming growth factor ß3 (tgfb3) and bone morphogenetic protein 4 (bmp4). Of these candidates, tgfb3 is most promising as it demonstrates significant differential expression across multiple stages of development, maps close (<1 Mb) to the fragmentation critical locus, and is implicated in a variety of other animal systems (including humans) in non-syndromic clefting and malformations of the cranial sutures. Both abnormalities are analogous to the failure-to-fuse phenotype that we observe in SO3 fragmentation. This integrative approach will enable discovery of the causative genetic lesions leading to complex craniofacial features analogous to human craniofacial disorders. This work underscores the value of cave-dwelling fish as a powerful evolutionary model of craniofacial disease, and demonstrates the power of integrative system-level studies for informing the genetic basis of craniofacial aberrations in nature.