Decoding Sex Differences in the Brain,One Worm at a Time

Abstract

Sex differences in the brain are prominent features across the animal kingdom. Understanding the anatomical and regulatory mechanisms behind these differences is critical for both explaining sexually dimorphic behaviors and developing sex-targeted treatments for neurological disorders. Clinical studies considering sex biases and basic research on animal models have provided much evidence for the existence of sex differences in the brain and, in a larger sense, sexual dimorphisms in the nervous system. However, due to the complexity of structure and dimorphic behaviors, it is yet unclear precisely how neuronal sexual dimorphisms are regulated on a molecular or cellular level. This commentary reviews available tools for investigating sexual dimorphisms using a simple model organism, the roundworm Caenorhabditis elegans (C. elegans), which enables one to study gene regulation at single-cell resolution with a number of cutting-edge molecular and genetic technologies. I highlight the doublesex/mab-3 family of transcription factors, first discovered in invertebrates, and their roles in a potentially universal regulatory mechanism underlying neuronal sexual dimorphisms. Studies of these transcription factors using C. elegans, fruit flies, and vertebrates will promote our understanding of fundamental mechanisms behind sex differences in the brain.

The Nervous System Is Sexually Dimorphic Across the Animal Kingdom

Apart from environmental and cultural influences, biological sexual dimorphisms in the nervous system are responsible for a wide array of sex-biased behaviors in humans. ^1,2 During normal development, brains of males and females differ in structure, causing differentiated brain activity between the two sexes; ^3,4 under disease states, men and women are susceptible to pain, depression, and neurodegenerative disorders to differing degrees, leading to an increasingly well-accepted recognition that sex should be considered as a variable in treating neurological diseases. ^{5 –7}

Neuronal sexual dimorphisms are not unique to humans. For years, sex-biased behaviors in various animal species and studies of mechanisms underlying these behaviors have provided strong evidence that biological neuronal dimorphisms determine sex-specific behavioral differences. ^{8 –13} One of the best examples of decoding behavioral dimorphisms through underlying brain differences focused on songbirds’ vocal control. A series of studies in the 1970s showed that multiple brain regions in male zebra finches are larger than those in females, and this size difference directly correlates with the birds’ singing behavior. ¹⁴ Further, studies in other vertebrates and invertebrates established a correlation between anatomical neuronal dimorphisms and sex differences in behaviors that do not require prior social experience or training, such as mating and aggression. ^{10,15 –18} From these animal studies, as well as recent human brain imaging data that showed sexual dimorphisms in brain structure, ^3,4 neurobiologists have come to the consensus that human behavioral differences are not only consequences of environmental variation and cultural diversity, but rather they are also closely correlated with inherent biological differences in the nervous system. Thus, it is critical to identify the regulatory mechanisms controlling such sexual dimorphisms to improve sex-specific therapeutic approaches. ¹⁹ This review will highlight the utility of Caenorhabditis elegans (C. elegans), a roundworm whose nervous system is sexually dimorphic, for parsing these mechanisms and describe a candidate gene family that provides a molecular entry point for understanding sex differences in the nervous system.

C. elegans Is a Unique Tool for Deciphering Molecular and Cellular Mechanisms of Neuronal Sexual Dimorphisms

The adult human brain contains billions of neurons and glial cells, as well as trillions of synapses. ²⁰ To study such a complex organ is a daunting task, and it is both technically and ethically challenging to tease out the precise molecular and cellular mechanisms during normal human development. Therefore, mechanistic studies of the brain are often worked out using animal models.

The use of model organisms with simple anatomical structures has been successful for answering fundamental biological questions and developing novel technologies that yield significant implications for human therapies. Notable examples include the discovery of molecular mechanisms controlling the circadian rhythm with studies in fruit flies (2017 Nobel Prize), ²¹ the development of the groundbreaking gene-editing tool Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) that originated from work in archaea and bacteria, ^{22 –24} the discovery of green fluorescent protein in jellyfish and its adaptation in C. elegans (2008 Nobel Prize), ²⁵ and, again using C. elegans, the findings of RNA interference by small regulatory RNAs (2006 Nobel Prize) ²⁶ and the genetic programs that control development and cell death (2002 Nobel Prize). ²⁷

C. elegans is a nonparasitic roundworm that was adapted as a model organism for developmental neurobiology in the 1960s due to its simple neuronal structure (a few hundred neurons), susceptibility to genetic manipulation, and short life cycle. ^28,29 In the several decades that followed its debut in scientific research, this anatomically simple-yet-scientifically powerful worm has fostered the establishment of hundreds of research laboratories worldwide, tens of thousands of publications in basic and biomedical sciences, and most importantly, it has tremendously enhanced our understanding of the molecular and cellular regulatory mechanisms that are fundamental and universal across animal species.

As the first multicellular animal whose genome was completely sequenced and cell lineage fully mapped, C. elegans provides unprecedented molecular and cellular precision for characterizing gene expression and regulation at single-neuron resolution in a developing nervous system. ^{30 –32} The complete C. elegans connectome—the synaptic wiring diagram of all neurons in its nervous system—was constructed from electron microscopic data for both sexes and is the only existing example of its kind for an entire organism. ^29,33,34 Comparing the connectomes of the C. elegans male and hermaphrodite (a somatic female) reveals that, although the majority of neurons are born in both sexes of the animal and are identical in morphology, position, and lineage, synaptic connections of the two sexes display sexual dimorphisms. ^29,33,34 Interestingly, recent studies of the human brain also showed that although structural differences in the male and female brains do not fully explain sex differences in human behaviors, there exist unique sexual dimorphisms in brain connectivity. In supratentorial regions, men’s brains are structured to facilitate more intrahemispheric connectivity, whereas women’s brains are prone to function predominantly interhemispherically. ⁴ Thus, although sex differences of cellular components may vary from one animal species to another, dimorphisms on connections, either structural or synaptic, are potential contributors to behavioral differences.

In recent years, advancement of genetic and molecular tools has brought new opportunities for studying neuronal sexual dimorphisms on a cellular basis with even more accuracy and on a larger scale than before. These tools, for example, CRISPR, which enables fast and inexpensive genome editing, and GFP Reconstitution Across Synaptic Partners (GRASP), which allows transsynaptic labeling for closely studying synaptic connectivity, have been readily adapted to C. elegans research. ^35,36 In contrast to human studies involving genetic manipulation that would inevitably raise debates on ethical and feasibility issues, C. elegans research has been making steady progress in revealing molecular mechanisms controlling neuronal sexual dimorphisms with a combination of genetic, molecular, and cellular techniques. To name a few examples, with transgenic labeling of a G protein-coupled receptor, one study characterized the sexually dimorphic regulation of a sensory neuron that functions in the C. elegans olfactory circuit; ³⁷ with GRASP, another study demonstrated that sex-specific synaptic connectivity is the result of sexually dimorphic pruning of specific synapses in C. elegans; ³⁸ and with transgenic labeling of a glutamate transporter, a third study identified a sexually dimorphic transcriptional scaling mechanism that regulates sex-specific neuronal functions. ³⁹ It is with no doubt that decades after its adaptation to scientific research, C. elegans will continue to serve as a key animal model in the forefront of studies on sex differences in the nervous system.

A Conserved Gene Family Is a Critical Molecular Entry Point for Decoding Neuronal Sexual Dimorphisms

A central task for characterizing regulatory mechanisms of sexual dimorphisms in the nervous system is identifying genes in molecular pathways that lead to the determination of one sex or the other. Work in mammals, nonmammalian vertebrates, and invertebrates has thus far revealed diverse pathways that highly depend on the organism. ^{10,15,16,40,41} For example, in mice and most other mammals, the presence or absence of a Y chromosome-linked Sry gene induces the development of the testis or ovary, which further secretes sex-specific hormones that drive somatic sexual traits. However, in fruit flies, a cascade of alternative splicing events leads to sex-specific protein expression and behaviors; in C. elegans, the TRA-1 transcription factor serves as a master regulator of somatic sexual traits. ^{17,42 –47}

Despite great diversity in sex determination and differentiation mechanisms, various species use the doublesex/mab-3 (DM) family of transcription factors and their homologs downstream of initial sex-biased signals. ^{16,17,48 –50} Originally characterized by the molecular and functional conservation between the fruit fly doublesex (dsx) gene and its C. elegans homolog, ^51,52 11 DM genes have been thus far identified in C elegans. Of these genes, some have been reported to regulate neurogenesis of male-specific neurons ^{53 –56} and sexually dimorphic synaptic connectivity. ^38,39 Subsequently, homologs of DM genes (termed “DMRTs”) have also been found in almost all vertebrate and mammalian animals. ^{49,50,57 –61} However, in contrast to studies of neuronal sexual dimorphisms involving C. elegans DM genes and the fruit fly dsx, whose sex-specific isoforms have been shown to display an array of sex-specific functions in the nervous system, ^{62 –72} vertebrate DMRTs have been mainly studied in the context of male gonadal development. ^{59 –61} Although a few recent reports indicated their roles in neurogenesis, ^{73 –76} very few studies have investigated precisely how DMRTs may function in regulating vertebrate neuronal sexual dimorphisms. This is in line with the enormous sex bias in molecular neuroscience studies, which are mostly conducted on male animals. ⁷⁷ Recently, using the transsynaptic labeling technique GRASP, one study in C. elegans showed the roles of two DM factors in regulating sexually dimorphic synaptic pruning and male-specific mating behaviors. ³⁸ Another study demonstrated that a third DM factor controls sexually dimorphic transcriptional scaling. ³⁹ Based on these important roles of DM family genes on regulating neuronal sexual dimorphisms in anatomically simple model organisms, in particular C. elegans and fruit flies, I propose that continuing studies of these genes on their expression, regulation, and potential contribution in sexually dimorphic synaptic connectivity in more animal models will tremendously enhance our understanding of sex differences in the brain.

Concluding Remarks

Sex differences in the brain result in distinct human behaviors between men and women. Because of the anatomical complexity of the human brain, understanding the molecular and cellular mechanisms underlying its sex-related differences calls for the use of anatomically simple animal models. Studies from multiple animals, including the roundworm C. elegans, have shown that although sex determination and differentiation pathways vary among species, the use of the DM family transcription factors is universal. Detailed analyses with molecular, cellular, and genetic tools developed for C. elegans and other invertebrates have suggested that these transcription factors may contribute to sexual dimorphisms in the nervous system on the levels of both cellular gene expression and synaptic connectivity. Further investigation on the DM family genes in vertebrate and mammalian animal models will help shed light on fundamental and potentially universal mechanisms underlying sex differences in the brain.