Minimally invasive brain injections for viral-mediated transgenesis: New tools for behavioral genetics in sticklebacks.

Behavioral genetics in non-model organisms is currently gated by technological limitations. However, with the growing availability of genome editing and functional genomic tools, complex behavioral traits such as social behavior can now be explored in diverse organisms. Here we present a minimally invasive neurosurgical procedure for a classic behavioral, ecological and evolutionary system: threespine stickleback (Gasterosteus aculeatus). Direct brain injection enables viral-mediated transgenesis and pharmaceutical delivery which bypasses the blood-brain barrier. This method is flexible, fast, and amenable to statistically powerful within-subject experimental designs, making it well-suited for use in genetically diverse animals such as those collected from natural populations. Developing this minimally invasive neurosurgical protocol required 1) refining the anesthesia process, 2) building a custom surgical rig, and 3) determining the normal recovery pattern allowing us to clearly identify warning signs of failure to thrive. Our custom-built surgical rig (publicly available) and optimized anesthetization methods resulted in high (90%) survival rates and quick behavioral recovery. Using this method, we detected changes in aggression from the overexpression of either of two different genes, arginine vasopressin (AVP) and monoamine oxidase (MAOA), in outbred animals in less than one month. We successfully used multiple promoters to drive expression, allowing for tailored expression profiles through time. In addition, we demonstrate that widely available mammalian plasmids work with this method, lowering the barrier of entry to the technique. By using repeated measures of behavior on the same fish before and after transfection, we were able to drastically reduce the necessary sample size needed to detect significant changes in behavior, making this a viable approach for examining genetic mechanisms underlying complex social behaviors.

Nest construction and presence do not alter territorial aggression in male threespine stickleback.

Constructing a nest within a mating territory provides a clear benefit to the resident, particularly by improving the opportunity to mate. It is unclear whether animals who use nests exclusively for reproductive purposes account for either the effort invested or the resulting increase in mating potential when valuing their territories. We sought to explicitly reveal a nest's added subjective resource value through within-group comparison of aggressive behaviour before and after nest construction. An increase in aggression following construction would indicate that the resident perceives greater subjective value in his territory, and thus values the nest. Threespine stickleback, Gasterosteus aculeatus, fish demonstrate stereotypical aggressive behaviours during an easily induced territorial defence. The male's nest is used exclusively for reproductive purposes, avoiding any confound of shelter. Contrary to our hypothesis, neither nest presence, timing of construction, nor nesting outcome was associated with differences in behavioural measures of territorial aggression. Assessed behaviours were robust, repeatable and inter-correlated. We conclude that territorial aggression is neither predictive of nor altered by nesting in threespine stickleback fish. Our results suggest that nests used transiently for a portion of the mating season add negligible subjective resource value to a territory. We additionally demonstrate that examinations of territorial aggression in sticklebacks do not need to control for nest building, improving statistical power by decreasing dropout rates. These results dovetail with recent work in other fish species to suggest that assessment of territorial aggression absent a nest may be practicable for fish in general.

Neurogenomic insights into paternal care and its relation to territorial aggression.

Motherhood is characterized by dramatic changes in brain and behavior, but less is known about fatherhood. Here we report that male sticklebacks-a small fish in which fathers provide care-experience dramatic changes in neurogenomic state as they become fathers. Some genes are unique to different stages of paternal care, some genes are shared across stages, and some genes are added to the previously acquired neurogenomic state. Comparative genomic analysis suggests that some of these neurogenomic dynamics resemble changes associated with pregnancy and reproduction in mammalian mothers. Moreover, gene regulatory analysis identifies transcription factors that are regulated in opposite directions in response to a territorial challenge versus during paternal care. Altogether these results show that some of the molecular mechanisms of parental care might be deeply conserved and might not be sex-specific, and suggest that tradeoffs between opposing social behaviors are managed at the gene regulatory level.

Temporal dynamics of neurogenomic plasticity in response to social interactions in male threespined sticklebacks.

Animals exhibit dramatic immediate behavioral plasticity in response to social interactions, and brief social interactions can shape the future social landscape. However, the molecular mechanisms contributing to behavioral plasticity are unclear. Here, we show that the genome dynamically responds to social interactions with multiple waves of transcription associated with distinct molecular functions in the brain of male threespined sticklebacks, a species famous for its behavioral repertoire and evolution. Some biological functions (e.g., hormone activity) peaked soon after a brief territorial challenge and then declined, while others (e.g., immune response) peaked hours afterwards. We identify transcription factors that are predicted to coordinate waves of transcription associated with different components of behavioral plasticity. Next, using H3K27Ac as a marker of chromatin accessibility, we show that a brief territorial intrusion was sufficient to cause rapid and dramatic changes in the epigenome. Finally, we integrate the time course brain gene expression data with a transcriptional regulatory network, and link gene expression to changes in chromatin accessibility. This study reveals rapid and dramatic epigenomic plasticity in response to a brief, highly consequential social interaction.

A fluorescence in situ hybridization (FISH) protocol for stickleback tissue.

BACKGROUND: Threespine stickleback are an important model for behaviour and evolutionary studies. A growing number of quantitative trait loci (QTL) and gene expression studies are identifying genes related to ecologically important traits in sticklebacks. In order to visualize the expression of candidate genes, we developed a fluorescence in situ hybridization (FISH) protocol. METHODS: We present a protocol for FISH on fresh or flash-frozen dissected tissue, using either cryo- or paraffin embedding. The protocol covers probe design guidelines and synthesis, sample embedding, sectioning, and the hybridization process. The protocol is optimized for brain tissue. Key steps for modifying the protocol for other tissues are noted. RESULTS: The FISH protocol resulted in specific labelling under all combinations of dissection and embedding conditions. Paraffin embedding preserved morphology better than cryo-embedding. We provide representative results showing the expression of glial fibrillary acidic protein (GFAP), oxytocin receptor (OXTR), and tyrosine hydroxylase (TH) in the brain.

An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.