Chromatin-Based Encoding of Sex Differentiation in Instinctual Behavior Circuits

Abstract

Everything is about sex, except sex itself, which is about development. Animals of many species develop as two anatomic sexes that perform distinct and complex social behaviors. Though the social roles of males and females differ across organisms, sex-differentiation of innate social behaviors – courtship, aggression, and care of offspring – is nearly universal. Effectors of sex determination programs pattern differences in behavior by altering the underlying neural circuit structure during development and neuronal physiology throughout the lifespan. The genetic tractability and compact brain size of the fruit fly Drosophila melanogaster provides an experimental paradigm to untangle how sex differentiation influences the specification, maturation, and circuit connectivity of neurons to pattern these behaviors. In the insect nervous system, the male-specific BTB/POZ transcription factor Fruitless (FruM) regulates the development of male-specific courtship behavior. FruM shapes the final forms and circuit roles of post-mitotic neurons through the inhibition of cell death fates, alteration of axon and dendrite morphologies, and specification of synaptic connections. These effects are deployed in unique combinations across more than 60 classes of neurons which express fru. While the circuit and cellular changes made by FruM have been extensively characterized, the developmental and molecular mechanisms by which FruM exerts these effects are not understood. To ask how gene expression is organized by FruM across the courtship circuit, I identified genomic regulatory elements with specific chromatin accessibility states downstream of FruM using ATAC-seq. In the adult, I characterized these elements as bona-fide, cell-type specific enhancers with sexually differentiated activity in vivo. I mapped FruM-regulated enhancers to genes involved in axon guidance and synaptic connectivity and found that while the social behavior circuits re-use the same genes that are used to build other kinds of circuits, FruM controls their sex-specific patterns of expression via dedicated regulatory elements. Instead of acting on a common set of effector genes to coordinate connections across layers of the courtship circuit, I determined that FruM works by subtraction: FruM results in enhancer closing at its DNA-binding motifs, the genes near these enhancers are repressed, on average, and minimal repressor domains fused to FruM DNA-binding domains can rescue features of FruM-driven circuit architecture. The chromatin targets of FruM are indeed unique across cell types: Regions fated to be closed by FruM are opened up earlier in the neuronal lineage and have cell-type specific accessibility across fru neurons in the adult. Finally, while FruM’s impact on neurons ultimately patterns adult behavior, I find that it differentiates chromatin states within an early critical window of development; this early chromatin repression by FruM is maintained later. I provide a model case for how sex differentiation pathways intersect with other aspects of neuronal identity and development to generate subtly but critically different circuits. The work in this dissertation grapples with the definition of sex and asks how the genes we think of as sex-determining, namely fruitless in D. melanogaster, cause sexual differentiation of the brain and behavior.