Characterizing the genetic basis of trait evolution in the Mexican cavefish.

Evolution in response to a change in ecology often coincides with various morphological, physiological, and behavioral traits. For most organisms little is known about the genetic and functional relationship between evolutionarily derived traits, representing a critical gap in our understanding of adaptation. The Mexican tetra, Astyanax mexicanus, consists of largely independent populations of fish that inhabit at least 30 caves in Northeast Mexico, and a surface fish population, that inhabit the rivers of Mexico and Southern Texas. The recent application of molecular genetic approaches combined with behavioral phenotyping have established A. mexicanus as a model for studying the evolution of complex traits. Cave populations of A. mexicanus are interfertile with surface populations and have evolved numerous traits including eye degeneration, insomnia, albinism, and enhanced mechanosensory function. The interfertility of different populations from the same species provides a unique opportunity to define the genetic relationship between evolved traits and assess the co-evolution of behavioral and morphological traits with one another. To define the relationships between morphological and behavioral traits, we developed a pipeline to test individual fish for multiple traits. This pipeline confirmed differences in locomotor activity, prey capture, and startle reflex between surface and cavefish populations. To measure the relationship between traits, individual F2 hybrid fish were characterized for locomotor behavior, prey-capture behavior, startle reflex, and morphological attributes. Analysis revealed an association between body length and slower escape reflex, suggesting a trade-off between increased size and predator avoidance in cavefish. Overall, there were few associations between individual behavioral traits, or behavioral and morphological traits, suggesting independent genetic changes underlie the evolution of the measured behavioral and morphological traits. Taken together, this approach provides a novel system to identify genetic underpinnings of naturally occurring variation in morphological and behavioral traits.

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 that 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. Furthermore, 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.

Evolution of the acoustic startle response of Mexican cavefish.

The ability to detect threatening stimuli and initiate an escape response is essential for survival and under stringent evolutionary pressure. In diverse fish species, acoustic stimuli activate Mauthner neurons, which initiate a C-start escape response. This reflexive behavior is highly conserved across aquatic species and provides a model for investigating the neural mechanism underlying the evolution of escape behavior. Here, we characterize evolved differences in the C-start response between populations of the Mexican cavefish, Astyanax mexicanus. Cave populations of A. mexicanus inhabit an environment devoid of light and macroscopic predators, resulting in evolved differences in various morphological and behavioral traits. We find that the C-start is present in river-dwelling surface fish and multiple populations of cavefish, but that response kinematics and probability differ between populations. The Pachón population of cavefish exhibits an increased response probability, a slower response latency and speed, and reduction of the maximum bend angle, revealing evolved differences between surface and cave populations. Analysis of the responses of two other independently evolved populations of cavefish, revealed the repeated evolution of reduced angular speed. Investigation of surface-cave hybrids reveals a correlation between angular speed and peak angle, suggesting these two kinematic characteristics are related at the genetic or functional levels. Together, these findings provide support for the use of A. mexicanus as a model to investigate the evolution of escape behavior.

Genetic Increases in Olfactory Bulb BDNF Do Not Enhance Survival of Adult-Born Granule Cells.

Adult-born neurons produced in the dentate gyrus subgranular zone (SGZ) develop as excitatory hippocampal granule cells (GCs), while those from the subventricular zone (SVZ) migrate to the olfactory bulb (OB), where most develop as GABAergic olfactory GCs. Both types of neurons express TrkB as they mature. Normally ~50% of new olfactory GCs survive, but survival declines if sensory drive is reduced. Increases in endogenous brain-derived neurotrophic factor (BDNF) in hippocampus, particularly with wheel running, enhance dentate GC survival. Whether survival of new olfactory GCs is impacted by augmenting BDNF in the OB, where they mature and integrate, is not known. Here, we determined if increasing OB BDNF expression enhances survival of new GCs, and if it counters their loss under conditions of reduced sensory activity. Neurogenesis was assessed under normal conditions, and following unilateral naris occlusion, in mice overexpressing BDNF in the granule cell layer (GCL). OB BDNF levels were significantly higher in transgenic mice compared to controls, and this was maintained following sensory deprivation. Bromodeoxyuridine (BrdU) cell birth dating showed that at 12-14 days post-BrdU, numbers of new GCs did not differ between genotypes, indicating normal recruitment to the OB. At later intervals, transgenic and control mice showed levels of GC loss in deprived and nondeprived animals that were indistinguishable, as was the incidence of apoptotic cells in the GCL. These results demonstrate that, in contrast to new dentate GCs, elevations in endogenous BDNF do not enhance survival of adult-born olfactory GCs.

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Increased Olfactory Bulb BDNF Expression Does Not Rescue Deficits in Olfactory Neurogenesis in the Huntington's Disease R6/2 Mouse.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expansion of CAG trinucleotide repeats in the huntingtin gene. Mutant huntingtin protein (mhtt) interferes with the actions of brain-derived neurotrophic factor (BDNF), and BDNF signaling is reduced in the diseased striatum. Loss of this trophic support is thought to contribute to loss of striatal medium spiny neurons in HD. Increasing BDNF in the adult striatum or ventricular ependyma slows disease progression in HD mouse models, and diverts subventricular zone (SVZ)-derived neuroblasts from their normal destination, the olfactory bulb, to the striatum, where some survive and develop features of mature neurons. Most neuroblasts that migrate to the olfactory bulb differentiate as granule cells, with approximately half surviving whereas others undergo apoptosis. In the R6/2 HD mouse model, survival of adult-born granule cells is reduced. Newly maturing cells express the BDNF receptor TrkB, suggesting that mhtt may interfere with normal BDNF trophic activity, increasing their loss. To determine if augmenting BDNF counteracts this, we examined granule cell survival in R6/2 mice that overexpress BDNF in olfactory bulb. Although we detected a decline in apoptosis, increased BDNF was not sufficient to normalize granule cell survival within their normal target in R6/2 mice.