C. Elegans arthicle

1. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Seminars in Cell & Developmental Biology
j ourna l h o me page: www.elsevier.com/locate/semcdb
ReviewSexual modulation of neural circuits and behavior in CaenorhabditiselegansKelli A. Fagana,b, Douglas S. Portmanb,c,∗ aNeuroscience Graduate Program, University of Rochester, Rochester, NY 14642, United StatesbCenter for Neural Development and Disease and Department of Biomedical Genetics, University of Rochester, Rochester, NY 14642, United StatescDepartment of Biomedical Genetics, University of Rochester, Rochester, NY 14642, United Statesa r t i c l e i n f oArticle history: Available online xxxKeywords: C. elegansNeural circuitsSex differencesSexual differentiationBehavior geneticsa b s t r a c tSex differences in behavior—both sex-specific and shared behaviors—are fundamental to nearly all ani- mal species. One often overlooked mechanism by which these behavioral differences can be generated isthrough sex-specific modulation of shared circuitry (i.e., circuits present in both sexes). In vertebrates thismodulation is likely regulated by hormone-dependent mechanisms as well as by somatic sex itself; inver- tebrate models have particular promise for understanding the latter of these. Here we review molecularand behavioral evidence of sexual modulation of shared circuitry in the nematode Caenorhabditis elegans. Multiple behaviors in this species, both copulatory and not, are modulated by the genetic sex of sharedneurons and circuit. These studies are close to uncovering the molecular mechanisms by which somaticsex modulates neural function in the worm, mechanisms which may be well conserved in more complexorganisms. Improving our understanding of the modulation of neural circuit development and functionby somatic sex may lend important insight into sex differences in the mammalian nervous system which, in turn, may have important implications for sex biases in disease. © 2014 Published by Elsevier Ltd. Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Sex differences in the C. elegans nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Sex determination and sexual differentiation in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Sexual modulation of shared neurons and circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. Sexual modulation of neural circuitry: molecular evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. Sexual modulation of neural circuitry: behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.1. Odortaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.2. Exploration and food-seeking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.3. Response to sex pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.4. Motor behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2.5. The role of shared circuitry in reproductive behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00∗Corresponding author at: Center for Neural Development and Disease, University of Rochester, Rochester, NY 14642, United States. Tel.: +1 585 275 7414. E-mail address: douglas.portman@rochester.edu (D.S. Portman).
http://dx.doi.org/10.1016/j.semcdb.2014.06.007
1084-9521/© 2014 Published by Elsevier Ltd.

2. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
2 K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx
1. IntroductionSexual behaviors—and, by extension, sex-specificbehaviors—are essential for survival across the animal king- dom. Through both natural and sexual selection, males andfemales of nearly all species have evolved distinct features inmorphology and behavior [1]. In general, these sex differences canbe thought of as either sexually dimorphic or sexually modulated. While a clear distinction between these classes is not alwaysapparent, they provide a useful framework for thinking about theinfluence of sex on development and physiology. Sexually dimorphic morphology, where each sex possessesstructures that are absent in the other, is obvious in animal gonadsand genitalia. In many species, other body parts also feature promi- nent sexual dimorphism. Similarly, sexually dimorphic behaviorsare best known in courtship and copulation. In some cases, how- ever, this assignment can be arbitrary or even misleading: forexample, the mounting behavior exhibited by many male mam- mals is often considered a sex-specific behavior, though it can alsobe observed in females as a display of aggression or dominance. Regardless, the neural substrates for sexually dimorphic behaviorare an area of intense current interest, and the relative contrib- utions of shared vs. sex-specific neuroanatomy to these behaviorsremains unclear. In contrast to sexually dimorphic phenomena, sexually modu- lated features are morphological features and behavioral programsthat appear in both sexes but that display differences in one or moreaspects. For example, in many bird species, adults of both sexes pro- duce song, but the characteristics of these vocalizations differ bysex. Similarly, the morphological or physiological features of struc- tures present in both sexes may differ between males and females, illustrated in body size or pigmentation. Though just as pervasiveas sexual dimorphism, sexual modulation has received relativelyless attention. Understanding these processes holds great promisefor revealing the regulatory mechanisms that link sex determina- tion to sexual differentiation; moreover, it also has the potential toreveal key nodes in biological networks that can be “tuned” to giverise to adaptive variation in morphology and behavior. The nematode Caenorhabditis elegans provides an ideal model inwhich to address the neural basis for sex differences in behavior, particularly with regard to the role of the modulation of non- sex-specific (“shared”) neural circuits. This species produces twosexes, male and hermaphrodite, the latter of which are somaticfemales that produce a limited number of self-sperm late in larvaldevelopment. Thus, C. elegans hermaphrodites can produce bothself-progeny and, upon fertilization by a male, cross-progeny. Assuch, selection is likely to favor distinct behavioral repertoiresin hermaphrodites (which should favor the production of self- progeny) and in males (which need to locate and copulate with amate in order to reproduce). Consistent with this, C. elegans adultsexhibit numerous sex differences in behavior that are related bothdirectly and indirectly to reproduction. Here, we review recentwork to understand the means by which genetic sex (sometimescalled “sex chromosome content”) modulates the development andfunction of shared neurons in service of these behaviors. 2. Sex differences in the C. elegans nervous systemA key advantage of C. elegans as an experimental model is theexceptional resolution with which its neuroanatomy is known. Likeother sex differences, those in the C. elegans nervous system can begrouped into two types [2] (Fig. 1). The more obvious of these is innumbers and types of cells: each sex possesses its own set of sex- specific neurons. Hermaphrodites have eight sex-specific neurons, two HSNs and six VCs, that enable egg-laying behavior; these are8 hermaphrodite-specific neurons (HSN, VC) AdultHermaphrodite(XX) AdultMale(XO) 294 shared (“core”) neurons87 male-specific neurons (CEM, CA/CP, tail neurons) Fig. 1. Sex differences in the C. elegans nervous system. The C. elegans nervoussystem contains both shared and sex-specific neurons. The shared component com- prises 294 neurons (represented in green) that are present in both sexes; thesecontrol behaviors that both sexes exhibit, including locomotion, chemosensation, and feeding. However, these neurons can be sexually modulated to produce subtledifferences in these behaviors. In addition, each sex contains sex-specific neurons, which are present only in the hermaphrodite (red) or male (blue). Sex-specific neu- rons are important for sex-specific behaviors like egg laying and mating. absent in males. In contrast, males possess 87 sex-specific neurons, most of which are located in the tail [3]. These cells provide the bulkof the circuitry that subserves copulation, a multistep, male-specificbehavior that is discussed elsewhere in this issue. The second type of sex difference found in the C. elegans ner- vous system is more subtle, and has only recently been appreciated: neurons and circuits that are present in both sexes can differ func- tionally. This shared (or “core”) component of the nervous systemcomprises 294 neurons that are lineally identical between thesexes [2]. However, behavioral and gene-expression studies frommultiple laboratories have demonstrated that shared neurons andcircuits can have sex-specific properties, suggesting that biologicalsex plays a much broader role in the nervous system than had beenpreviously thought. 3. Sex determination and sexual differentiation in C. elegansUltimately, all sex differences in biology arise from a primarysex-determining cue. In C. elegans, this is chromosomal status: hermaphrodite state is specified by two X chromosomes (XX), while males arise when only one is present (XO) [4,5]. This pri- mary cue is “read” by autosomal and X-chromosome signal elementgenes that converge on xol-1, a regulator of both sex determinationand dosage compensation [6,7]. Through a cryptic hedgehog-likepathway comprising her-1, tra-2 and the fem genes, xol-1 activitydetermines the state of the master regulator of C. elegans somaticsexual differentiation, tra-1 (Fig. 2). This gene’s active product, thetranscription factor TRA-1A [8], is stabilized in XX animals, allow- ing it to promote hermaphrodite development and repress maleDosagecompensationtra-3tra-1her-1xol-1tra-2fem-1fem-2fem-3sdc-1sdc-2sdc-3XAfox-1sex-1sea-1sea-2sea-3Fig. 2. The somatic sex determination pathway. tra-1 is the master regulator of sex- ual state in C. elegans: high tra-1 activity confers hermaphrodite state while lowactivity confers male state. tra-1 activity is controlled through a multistep genetichierarchy that is initiated by the ratio of X chromosomes to autosomes. By alteringtra-1 activity in a tissue- or cell-type-specific manner, animals with mosaic sex- ual states can be generated. Genetic feminization can be achieved by increasingtra-1 activity in males by expressing constitutively active forms of tra-2 or tra-1. Conversely, masculinization can be brought about by expression of fem-3, whichinhibits tra-1 function.

3. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx 3
development [9,10]. In XO animals, TRA-1A is degraded, allow- ing male development to occur. Importantly, tra-1 activity is bothnecessary and sufficient for all somatic sex differences. tra-1 XXloss-of-function mutants develop as fertile “pseudomales” thathave a completely masculinized soma and a nearly normal malegonad [9]. In contrast, tra-1 XO gain-of-function mutants developas somatic females [9]. Notably, the TRA-1A protein acts cell- autonomously to control sex differences in development, exceptin cases where it regulates sex-dependent signaling processeslike vulval induction [9,11]. Although the genetic functions oftra-1 as a master sexual regulator are well-established, its expres- sion pattern in the soma during development and in adulthoodhas not been carefully described, an important area for futurework. Downstream of tra-1 lie the developmental and physiologicaldifferences that characterize the male and hermaphrodite soma. Outside the nervous system, tra-1 directly regulates several tran- scription factors, particularly the doublesex-like genes mab-3 anddmd-3, to bring about sex differences in the intestine, tail tip, andelsewhere [12,13]. In the nervous system, one mechanism by whichtra-1 establishes sexual dimorphism is through transcriptional reg- ulation of cell death. Two direct tra-1 targets, egl-1 and ceh-30, control the sex-specific survival of the hermaphrodite-specific HSNneurons and the male-specific CEM neurons, respectively. egl-1encodes a BH3-domain activator of the cell death pathway. Inhermaphrodites, tra-1 acts through a cell-type-specific enhancerto repress egl-1 in the HSNs, allowing their survival; in males, tra- 1 inactivity permits egl-1 expression, leading to their death [14]. This genetic logic is reversed in the case of the CEMs, where tra-1represses ceh-30, a pro-survival homeodomain transcription factor, to cause cell death in hermaphrodites and survival in males [15,16]. In the case of other sex-specific neurons, tra-1 acts in more com- plex means to regulate the proliferation of neural precursors andspecification of cell fate [17,18]. In these cases, the specific mecha- nisms by which tra-1 brings about sex differences in neuroanatomyremain unclear. 4. Sexual modulation of shared neurons and circuitsMultiple lines of evidence indicate that genetic sex has deepinfluences on the properties of nominally “shared” components ofthe nervous system. Several mechanisms can be imagined throughwhich this could come about. First, sex differences in synapticconnectivity between shared neurons could alter the flow andprocessing of information between hermaphrodites and males. Indeed, recent analysis of neural connectivity in the male tailhas identified several examples of this [3], pointing to interest- ing but unknown interactions between genetic sex and synapticspecification or stability. In contrast, though the detailed connec- tivity of the male head has not yet been reported, preliminaryevidence suggests that synaptic sex differences in this regionare relatively rare (S. Emmons and S. Cook, pers. comm.). Oneinterpretation of this is that shared neurons may play importantroles in copulatory behaviors by being incorporated into male- specific tail circuits, while sexually dimorphic connectivity maynot have a major role in generating sex differences in head cir- cuitry. Second, sexually dimorphic morphology could be importantfor establishing sex differences in neural function. Ultrastructuralstudies have revealed several examples of this, including the tailPHC sensory neurons, which harbor striated rootlets in the malebut not in the hermaphrodite [19], and the interneuron PDB, whose process exhibits elaborate male-specific branching [3]. Thedevelopmental underpinnings and functional significance of thesemorphological dimorphisms is not yet understood. Finally, sex-specific neuromodulation of shared neurons and cir- cuits could have important roles in regulating behavior. As theydo in other systems, neuromodulators regulate behavioral statesin C. elegans by altering neural excitability and modifying circuitcomposition. This effectively selects specific circuits to be activatedwhile forcing others to remain latent [20]. Therefore, sex differ- ences in neuromodulation could activate or inhibit shared circuitryto produce sexually dimorphic behaviors. Sex differences in neuro- modulation could arise from cell-autonomous regulation by tra-1of, for example, intracellular signaling components, sensory recep- tors, or receptors for neuromodulators themselves. Alternatively, tra-1 could regulate the production of neuromodulators, therebyhaving non-cell-autonomous effects on neurons that receive thesesignals. Indeed, neuromodulators are known to have importantroles in sex-specific behaviors—e.g., serotonin in hermaphroditeegg-laying and PDF neuropeptide signaling in male mate-searching[21,22]. Moreover, many sex-specific neurons produce modulatorycues, including monoamines and neuropeptides [23,24]. How- ever, the role of sexually dimorphic neuromodulation in regulatingshared circuits, particularly in the head, remains unclear. 4.1. Sexual modulation of neural circuitry: molecular evidenceAlthough focused, systematic studies have yet to be undertaken, several examples of sexually dimorphic gene expression in sharedC. elegans neurons have been identified. In most of these cases, the functional significance of these modifications is unknown; however, the observation that they emerge only upon adulthoodsuggests that they may have important roles in adult-specific sex- ual behavior. By examining the expression of reporter genes, at least fourexamples of sexually dimorphic gene expression in shared neuronshave been identified (Table 1A). The doublesex-family transcriptionfactor gene mab-3 exhibits male-specific expression in the sensoryneuron ADF [12], as does the candidate chemoreceptor srd-1 [25]. Asecond candidate chemoreceptor gene, srj-54, is male-specificallyexpressed in the AIM interneurons [26]; however, as these cells arenot exposed to the environment, the product of srj-54 may functionnot as a classical chemoreceptor but as a receptor for a neuromod- ulatory signal. A third chemoreceptor, the diacetyl receptor odr-10, shows sexually modulated expression in the AWA olfactory neu- rons, where it modulates food detection (see Section 4.2.2) [27]. Interestingly, mab-3 is a direct target of tra-1 in the intestine, whereit has an important role in repressing yolk expression [12]. Thus, itsmale-specific expression in ADF might be important for maintain- ing a male-like gene expression profile in this neuron; however, mab-3 does not appear to regulate srd-1 in this cell [12]. In addition to these gene-by-gene approaches, microarray anddeep RNA sequencing studies have also been undertaken to char- acterize sex differences in gene expression [28–31] (K.A.F. andD.S.P., unpublished). Using total worm RNA, these studies haveidentified numerous genes with differential expression betweenmales and hermaphrodites. However, many or even most of thegenes identified using these approaches may be expressed in sex- specific somatic (or in some cases, germline) tissues. More focusedstudies—in particular, those using methods to specifically iso- late RNA from the nervous system or even from specific neurontypes—will be necessary to detect sex differences in genes that areonly expressed in a handful of neurons. These concerns aside, genome-wide approaches have iden- tified members of three neuropeptide families—insulin-likepeptides, FMRFamide-related peptides, and the neuropeptide-likeproteins—as having sexually dimorphic expression. Some of thesegenes are known to be expressed in sex-specific neuron types [23], but recent evidence suggests that some insulin-like peptides mayshow sex differences in expression in shared circuitry [32]. Further

4. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
4 K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx
Table 1
Genetic and functional sex differences in non-sex-specific C. elegans neurons.
A. Sexually dimorphic gene expression in shared neurons
Gene Site of sexually dimorphic expression Description
odr-10 AWA High expression in adult hermaphrodites; low in adult males [27]
srj-54 AIM Male-specific expression in adults [26]
mab-3 ADF Male-specific expression [12]
srd-1 ADF Male-specific expression [25]
B. Behaviors controlled by sex-specific modulation of shared neurons/circuits
Sexually modulated behavior Site of sexual modulation Evidence
Diacetyl attraction/food-seeking AWA Genetic sex-reversal of AWA [27]
Ascaroside attraction ASK, ADL and others Laser ablation [42,45] and genetic sex-reversala
Non-ascaroside attraction AWA, AWC and many additional candidates Laser ablation and genetic sex reversal [47]
Locomotion Sensory neurons Genetic sex-reversal [35]
Sex-specific behavior
Male Mating Sensory neurons Genetic sex-reversalb
Male Food-leaving Sensory neurons Genetic sex-reversalb
Hermaphrodite egg laying Sensory neurons Genetic sex-reversalb
aK.A.F. and D.S.P., unpublished data. bRenee M. Miller and D.S.P., unpublished data. studies of the expression patterns and functions of these mod- ulators could reveal important roles for peptidergic signaling ingenerating sex differences in behavior. 4.2. Sexual modulation of neural circuitry: behaviorIn contrast to the “bottom-up” approach of characterizingsex-dependent gene-expression differences in shared neurons, “top-down” studies have focused on the role of shared circuits ingenerating sex differences in behavior. These have identified sexdifferences in a variety of behaviors, including chemosensory andlocomotor behaviors (Table 1B). They have also provided evidencethat shared neurons and circuits have key roles in sex-specificreproductive behaviors. An approach that has proven particularly powerful in these stud- ies takes advantage of the ability to manipulate tra-1 function in acell-type-specific manner. By expressing the tra-1 inhibitor fem- 3 under the control of cell-type-specific promoters, particular celltypes can be genetically “masculinized” [26,33]. Conversely, cell- type-specific expression of a tra-1(gf) allele is able to genetically“feminize” cells [34]; expression of a constitutively active form oftra-2 called tra-2(IC) has similar effects [35,36] (Fig. 2). By driv- ing the expression of these sex-reversal transgenes with promotersthat are active only postmitotically, it is possible to flip the sexual“state” of a cell without altering the cell lineages that give rise tosex-specific neurons. 4.2.1. OdortaxisChemosensation plays a critical role in the life of a worm. C. elegans relies on chemosensory cues to identify food, avoid tox- ins and predators, sense developmental cues, and mate [37]. Muchof the worm’s chemosensory apparatus is in the head and, withthe exception of the male-specific CEM (cephalic companion) sen- sory neurons, is sexually isomorphic. Nevertheless, studies haverevealed that these shared structures differ functionally by sex. Earlier work from our group identified sex differences in olfac- tory function, such that hermaphrodites are more attracted tosome canonical odorants (particularly diacetyl and benzaldehyde), while males may be more attracted to others (e.g., pyrazine) [26]. Using cell-type-specific genetic masculinization, we found thatthe genetic sex of sensory neurons themselves, rather than ofdownstream circuit components, was critical for these sex differ- ences [26]. Subsequent work has demonstrated that a single olfactory neu- ron, AWA, is an important focus of sexual modulation. Specificgenetic sex-reversal of this cell is sufficient to “flip” sex-typicalpatterns of diacetyl-pyrazine preference in both sexes. Moreover, these studies have found that the sex difference in diacetyl attrac- tion results from sex-specific regulation of the diacetyl receptorodr-10: hermaphrodites express high levels of this receptor, whileexpression is low or even absent in males [27]. Though thisregulation depends on the cell-autonomous action of tra-1, themechanism(s) whereby tra-1 modulates odr-10 expression remainsunknown. 4.2.2. Exploration and food-seekingSex differences also exist in exploratory and food-seekingbehavior. When individual animals are placed on a small patch offood, hermaphrodites will remain on the patch to feed while maleswill leave to explore their surroundings. The observation that thepresence of a mate will efficiently inhibit male exploration indi- cates that it reflects a sexually motivated mate-searching drive[38]. Interestingly, males will not explore if they are food-deprived, indicating the existence of active mechanisms that prioritize thesebehaviors by sex and feeding state. Male exploration is promotedby male-specific ray neurons and PDF neuropeptide signaling, asdiscussed elsewhere in this issue [21,39]. In addition to these inputs from male-specific neurons, recentwork has indicated that the sexual state of the shared chemosen- sory neuron AWA is important for setting the relative priority ofexploratory and feeding behaviors [27]. Interestingly, these stud- ies have indicated that odr-10, whose expression is significantlylower in males than in hermaphrodites, is important for efficientfood detection. When odr-10 levels are low, males are less attractedto food and instead tend to explore their environment. Tran- sient food-deprivation upregulates odr-10 expression in AWA toincrease food detection, accounting for the temporary suppressionof exploratory behavior upon starvation. This dynamic regulationof odr-10, and the ability to switch between feeding-oriented andmating-oriented states, is critical for male reproductive fitness: well-fed males that are unable to repress odr-10 are less efficientat finding mates, reducing their reproductive success [27].

5. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx 5
4.2.3. Response to sex pheromonesAside from copulatory behavior per se, one of the most sexuallydimorphic behaviors in C. elegans is its response to pheromonescalled ascarosides, glycosides of the dideoxysugar ascarylose. Bothsexes secrete a number of ascaroside variants that vary by sex, developmental stage and environmental condition [40–44]. Thesemolecules have been proposed to serve as a modular chemical“language” that animals use to exchange information about pop- ulation density, nutritional conditions and the presence of toxins, pathogens and predators [41]. One important function of certainascarosides—particularly ascr#2 (also called “C6”), ascr#3 (“C9”) and ascr#8—is that they are potent, synergistic, sex-specific attrac- tors of males and are produced preferentially by hermaphrodites[40,42]. Hermaphrodites are weakly repelled by this same mix- ture; thus, these compounds are considered sex pheromones[42]. Recent work from several groups has begun to shed light on themechanisms that regulate ascaroside attraction in C. elegans males. Interestingly, ascarosides are sensed by male-specific head sen- sory neurons (the CEMs) as well as non-sex-specific “shared” headsensory neurons [42,45]. Removal of the CEM neurons from malesstill leaves substantial pheromone attraction intact [45], indicatingthat the sexual state of shared circuitry is likely to be importantfor making pheromone response sexually dimorphic. Preliminarystudies from our group using genetic sex-reversal approaches haveconfirmed this idea, highlighting roles for sex differences in sen- sory function (K.A.F. and D.S.P., unpublished). Ablation studies haveshown that the sensory neuron ASK is important for pheromoneattraction in males [42], and that the amphid neuron ADL, whichmay have a role in ascr#3 repulsion in hermaphrodites, is less activein males [45]. One interesting possibility is that sex differences inneuromodulatory signaling impinge on sensory or interneurons inthe pheromone circuit to differentially regulate its activity. Alongthese lines, reducing the activity of the neuropeptide receptor NPR- 1 can bring about some ascr#3 attraction in hermaphrodites [46]. However, male pheromone attraction does not result from sex dif- ferences in NPR-1 signaling [45], indicating that biological sex usesother unknown mechanisms to tune neural circuit function. Under- standing these mechanisms is an important goal for further studies. C. elegans hermaphrodites also secrete non-ascaroside com- pounds that attract males, the response to which also depends onthe sexual state of shared circuitry [33,47]. The molecular identityof these non-ascaroside pheromones is unknown, and the circuitrythat controls the responses that they elicit seems to overlap onlypartially with the ascaroside response circuitry. Genetic masculin- ization of the hermaphrodite nervous system is sufficient to conferattraction to these cues, indicating a central role for the sex-specificmodulation of shared neurons [33]. Interestingly, recent work hasindicated that TGFß signaling plays an important role in generatingsex-specific attraction to these compounds [47]. Hermaphrodite-derived pheromones are also important for sex- specifically modulating olfactory plasticity [34]. Although salt isnormally a moderate attractant to C. elegans, pairing salt expo- sure with starvation reverses the valence of this cue, makingit a repellant [48]. This “salt chemotaxis learning” occurs inboth males and hermaphrodites. However, if males are salt- conditioned under starvation conditions but in the presence ofother hermaphrodites, males will remain attracted to salt. Malesrequire both a hermaphrodite-specific contact cue as well ashermaphrodite-secreted pheromones to block the induction of saltaversion. Interestingly, genetic feminization of the nervous sys- tem is sufficient to restore salt avoidance in males, indicating animportant role for the genetic sex of shared neurons [34]. However, masculinizing the hermaphrodite nervous system is not sufficientto prevent salt avoidance in the presence of other hermaphrodites, suggesting that both male-specific and shared circuitry areimportant for male olfactory plasticity. While the specific focus ofgenetic sex in the salt chemotaxis learning has yet to be identified, this work demonstrates an important role for sexual modulation ofneural function that allows males to use past mating experiencesto tune their olfactory system to promote reproductive success. 4.2.4. Motor behaviorC. elegans adults also show sex differences in locomotion. Specif- ically, adult males move at a higher velocity and with a higheramplitude compared to adult hermaphrodites [38,49]. These differ- ences are thought to contribute to increased exploratory activity, thereby promoting males’ ability to locate mates. Recent workon the mechanisms underlying this have shown that the higherbody bend frequency of males and differences in the shape ofmale and hermaphrodite body waves contribute to the increasedspeed observed in males [35]. Furthermore, genetic sex-reversalexperiments revealed an important role for the sexual state ofthe shared nervous system in generating the sex difference inbody bend frequency. Surprisingly, sensory neurons, rather thancommand interneurons or motor neurons, appear to be an impor- tant site through which genetic sex acts to modulate locomotion[35]. Though the means through which this modulation occurs isunknown, there is precedent for sensory regulation of locomotorfrequency through the mechanical sensing of food [50] and it islikely that this extends to other stimuli. Further work should helpreveal the specific modulatory mechanisms that are targeted by sexto alter body-bend frequency. In contrast to body-bend frequency, other aspects of locomo- tor behavior, particularly body wave geometry, are not primarilydetermined by the sexual state of the nervous system. Interest- ingly, sex-specific properties of muscle appear to have importantroles in modulating wave propagation, indicating that distributedsexual modifications act to coordinate sex-typical locomotion[35]. 4.2.5. The role of shared circuitry in reproductive behaviorsA variety of studies have shown that the circuits for sex-specificreproductive behaviors comprise shared as well as sex-specificneurons. This appears to be especially true in males, as sex-specificcircuitry is well connected to shared circuitry [3]. One way thisrecruitment occurs is through modulation of shared behavioralprograms by sex-specific circuitry to increase mating efficiency. For example, male-specific circuitry in the tail can modulate gen- eral locomotion programs to generate reversals when requiredduring mating [51]. This is achieved through male-specific cholin- ergic innervation of AVA, a shared command interneuron thatplays a major role in switching from forward to backward loco- motion. Conversely, shared circuitry can also modulate activity insex-specific circuitry to ensure proper execution of reproductivebehavior. For instance, shared touch receptor neurons release neu- ropeptides that likely act on downstream male-specific circuitry toprevent repetitive turning behavior during mating [52]. Further- more, unpublished work from our group has found that geneticsex-reversal of shared circuitry compromises male mating, mate- searching, and hermaphrodite egg-laying behaviors (R.M. Millerand D.S.P., unpublished data). Together, these data suggest that thesexual properties of recruited shared circuitry are important forsex-specific reproductive behaviors. 5. OutlookIn most species, the extent to which neural circuits differanatomically (i.e., the extent to which they are sexually dimorphic) is not known, though it is thought that these differences are gener- ally subtle and highly specific. Similarly, the degree to which sharedcircuits are differentially modulated by sex is unclear. Studies of

6. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
6 K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx
the sexual modulation of gene expression and behavior in C. ele- gans have highlighted an important, active role for shared neuronsand circuits. One recurring theme of these studies is that sensoryfunction itself is highly sexually modulated: the importance of thiscan be seen in odortaxis and food sensation (e.g., regulation of odr- 10 in AWA), in pheromone response (e.g., sex differences in thepheromone-response properties of ADL), and in locomotion (theimportance of the sexual state of sensory neurons in setting body- bend frequency). Interestingly, this parallels a number of recentstudies from other systems in which modulated sensory functionhas been shown to be an important determinant of behavioral plas- ticity [53]. Understanding the mechanisms through which sensoryfunction is sexually modulated, and its significance for generatingadaptive sex differences in behavior, will be important areas forfuture work. One aspect that has not yet been addressed concerns the devel- opmental point at which tra-1 activity establishes sex differencesin shared circuitry. Interestingly, while some sex differences (e.g. the survival of sex-specific neurons) appear in the embryo, oth- ers do not become apparent until adulthood (e.g. odr-10 and srj-54expression, pheromone attraction and locomotion). Furthermore, it is largely unknown where tra-1 is expressed and whether itsactivity is constant throughout development and adulthood. Onepossibility is that tra-1 functions only during early development toestablish sex differences that then later become activated. Anotheris that tra-1 is dynamically regulated in each cell depending ondevelopmental stage. In both instances, convergence with otherdevelopmental signals could be important for activating sex dif- ferences in older animals or regulating tra-1. These issues warrantfurther investigation. In more complex animals, sex differences in the nervous sys- tem have important roles in development and behavior; in humans, such differences are likely to be related to sex differences in suscep- tibility to a variety of neurological and neuropsychiatric disorders. While a model based on the organizational and activational func- tions of gonadal steroids has guided the field for many years[54], numerous recent studies have shown that the genetic sexof the nervous system itself is a key determinant of some sexdifferences in development and physiology. As a result, a morenuanced view has emerged, in which multiple cues from the gonadand nervous system itself interact to specify sex differences andalso to compensate for their undesired effects [55,56]. Impor- tantly, however, the molecular mechanisms through which somaticsex modulates neural development and function are almost com- pletely unknown. Genetically tractable invertebrate models arelikely to be able to make important contributions to this. Althoughupstream sex-determination mechanisms vary widely betweenspecies, downstream regulatory processes that connect the out- put of sex determination to the modulation of developmental andphysiological processes may be conserved [57]. Because studies inC. elegans are now approaching complete descriptions of the mech- anisms that link genetic sex to modulation of neural function andbehavior, they may shed important light onto similar processes thatoperate in much more complex systems. AcknowledgmentsWe thank members of the C. elegans research community andof the Portman laboratory for ongoing discussion and intellectualexchange, and are particularly grateful to S. Cook and S. Emmons(Albert Einstein College of Medicine) for sharing data on male headconnectivity prior to publication. We thank Deborah A. Ryan andRenee M. Miller for their critical review of this manuscript. Currentwork in the Portman laboratory is supported by NIH grants F31NS086283 (K.A.F.) and R21 NS082849 (D.S.P.). References[1] Lande R. Sexual dimorphism, sexual selection, and adapta- tion in polygenic characters. Evolution (NY) 1980;34:292–305, http://dx.doi.org/10.2307/2407393. [2] Portman DS. Genetic control of sex differences in C. ele- gans neurobiology and behavior. Adv Genet 2007;59:1–37, http://dx.doi.org/10.1016/S0065-2660(07)59001-2. [3] Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Nichol Thomson J, et al. Theconnectome of a decision-making neural network. Science 2012;337:437–44, http://dx.doi.org/10.1126/science.1221762. [4] Brenner S. The genetics of Caenorhabditis elegans. Genetics 1974;77: 71–94. [5] Nigon V, Dougherty EC. Reproductive patterns and attempts at reciprocalcrossing of Rhabditis elegans Maupas, 1900, and Rhabditis briggsae Doughertyand nigon, 1949 (Nematoda: Rhabditidae). J Exp Zool 1949;112:485–503, http://dx.doi.org/10.1002/jez.1401120307. [6] Meyer BJ. X-Chromosome dosage compensation. WormBook 2005:1–14, http://dx.doi.org/10.1895/wormbook.1.8.1. [7] Zarkower D. Somatic sex determination. WormBook 2006:1–12, http://dx.doi.org/10.1895/wormbook.1.84.1. [8] Zarkower D, Hodgkin J. Molecular analysis of the C. elegans sex-determininggene tra-1: a gene encoding two zinc finger proteins. Cell 1992;70: 237–49. [9] Hodgkin J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1987;1:731–45, http://dx.doi.org/10.1101/gad.1.7.731. [10] Schvarzstein M, Spence AM. The C. elegans sex-determining GLI proteinTRA-1A is regulated by sex-specific proteolysis. Dev Cell 2006;11:733–40, http://dx.doi.org/10.1016/j.devcel.2006.09.017. [11] Hunter C, Wood W. The tra-1 gene determines sexualphenotype cell-autonomously in C. elegans. Cell 1990;63: 1193–204. [12] Yi W, Ross JM, Zarkower D. Mab-3 is a direct tra-1 target gene regulating diverseaspects of C. elegans male sexual development and behavior. Development2000;127:4469–80. [13] Mason DA, Rabinowitz JS, Portman DS. dmd-3, a doublesex-related gene regu- lated by tra-1, governs sex-specific morphogenesis in C. elegans. Development2008;135:2373–82, http://dx.doi.org/10.1242/dev.017046. [14] Conradt B, Horvitz HR. The TRA-1A sex determination protein of C. elegans regu- lates sexually dimorphic cell deaths by repressing the egl-1 cell death activatorgene. Cell 1999;98:317–27. [15] Schwartz H, Horvitz H. The C. elegans protein CEH-30 protects male-specificneurons from apoptosis independently of the Bcl-2 homolog CED-9. Genes Dev2007:3181–94, http://dx.doi.org/10.1101/gad.1607007. [16] Peden E, Kimberly E, Gengyo-Ando K, Mitani S, Xue D. Control of sex-specificapoptosis in C. elegans by the BarH homeodomain protein CEH-30 and thetranscriptional repressor UNC-37/Groucho. Genes Dev 2007;21:3195–207, http://dx.doi.org/10.1101/gad.1607807. [17] Ross JM, Kalis AK, Murphy MW, Zarkower D. The DM domain protein MAB-3promotes sex-specific neurogenesis in C. elegans by regulating bHLH proteins. Dev Cell 2005;8:881–92, http://dx.doi.org/10.1016/j.devcel.2005.03.017. [18] Kalis AK, Kissiov DU, Kolenbrander ES, Palchick Z, Raghavan S, et al. Patterning ofsexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord byHox and TALE homeodomain transcription factors. Dev Dyn 2014;243:159–71, http://dx.doi.org/10.1002/dvdy.24064. [19] Sulston JE, Albertson DG, Thomson JN. The Caenorhabditis eiegansmale: postembryonic development of nongonadal structures. Dev Biol1980;78:542–76. [20] Bargmann CI. Beyond the connectome: how neuromodulators shape neural cir- cuits. Bioessays 2012;34:458–65, http://dx.doi.org/10.1002/bies.201100185. [21] Barrios A, Ghosh R, Fang C, Emmons SW, Barr MM. PDF-1 neuropeptidesignaling modulates a neural circuit for mate-searching behavior in C. elegans. Nat Neurosci 2012;15, http://dx.doi.org/10.1038/nn.3253. [22] Horvitz HR, Chalfie M, Trent C, Sulston JE, Evans PD. Serotonin andoctopamine in the nematode Caenorhabditis elegans. Science 1982;216: 1012–4. [23] Li C, Kim K. Neuropeptides. WormBook 2008:1–36, http://dx.doi.org/10.1895/wormbook.1.142.1. [24] Sulston J, Dew M, Brenner S. Dopaminergic neurons in the nema- tode Caenorhabditis elegans. J Comp Neurol 1975;163:215–26, http://dx.doi.org/10.1002/cne.901630207. [25] Troemel ER, Chou JH, Dwyer ND, Colbert H, Bargmann CI. Divergent seven trans- membrane receptors are candidate chemosensory receptors in C. elegans. Cell1995;83:207–18. [26] Lee K, Portman DS. Neural sex modifies the function of a C. elegans sensory cir- cuit. Curr Biol 2007;17:1858–63, http://dx.doi.org/10.1016/j.cub.2007.10.015. [27] Ryan, D.A., K.H. Lee, R.M. Miller, S. Neal, P. Sengupta, and D.S. Portman. Sex, ageand hunger regulate behavioral prioritization through dynamic modulation ofchemoreceptor expression. 2014, Submitted. [28] Jiang M, Ryu J, Kiraly M, Duke K, Reinke V, Kim SK. Genome-wideanalysis of developmental and sex-regulated gene expression profiles

7. Please cite this article in press as: Fagan KA, Portman DS. Sexual modulation of neural circuits and behavior in Caenorhabditis elegans.
Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.06.007
ARTICLE IN PRESS G Model
YSCDB-1616; No. of Pages 7
K.A. Fagan, D.S. Portman / Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx 7
in Caenorhabditis elegans. Proc Natl Acad Sci USA 2001;98:218–23, http://dx.doi.org/10.1073/pnas.011520898. [29] Thoemke K, Yi W, Ross JM, Kim S, Reinke V, et al. Genome-wide analysis ofsex-enriched gene expression during C. elegans larval development. Dev Biol2005;284:500–8, http://dx.doi.org/10.1016/j.ydbio.2005.05.017. [30] Thomas CG, Li R, Smith HE, Woodruff GC, Oliver B, et al. Simplificationand desexualization of gene expression in self-fertile nematodes. Curr Biol2012;22:2167–72, http://dx.doi.org/10.1016/j.cub.2012.09.038. [31] Reinke V, Gil IS, Ward S, Kazmer K. Genome-wide germline-enrichedand sex-biased expression profiles in Caenorhabditis elegans. Development2004;131:311–23, http://dx.doi.org/10.1242/dev.00914. [32] Ritter AD, Shen Y, Bass JF, Jeyaraj S, Deplancke B, Mukhopadhyay A, et al. Complex expression dynamics and robustness in C. elegans insulin networks. Genome Res 2013:1–12, http://dx.doi.org/10.1101/gr.150466.112.23. [33] White JQ, Nicholas TJ, Gritton J, Truong L, Davidson ER, et al. The sensory cir- cuitry for sexual attraction in C. elegans males. Curr Biol 2007;17:1847–57, http://dx.doi.org/10.1016/j.cub.2007.09.011. [34] Sakai N, Iwata R, Yokoi S, Butcher RA, Clardy J, Tomioka M, et al. A sexu- ally conditioned switch of chemosensory behavior in C. elegans. PLoS ONE2013;8:e68676, http://dx.doi.org/10.1371/journal.pone.0068676. [35] Mowrey WR, Bennett JR, Portman DS. Distributed effects of biological sexdefine sex-typical motor behavior in Caenorhabditis elegans. J Neurosci2014;34:1579–91, http://dx.doi.org/10.1523/JNEUROSCI.4352-13.2014. [36] Lum D, Kuwabara PE, Zarkower D, Spence AM. Direct protein–proteininteraction between the intracellular domain of TRA-2 and the transcrip- tion factor TRA-1A modulates feminizing activity in C. elegans. Genes Dev2000;14:3153–65. [37] Bargmann CI. Chemosensation in C. elegans. WormBook 2006:1–29, http://dx.doi.org/10.1895/wormbook.1.123.1. [38] Lipton J, Kleemann G, Ghosh R, Lints R, Emmons SW. Matesearching in Caenorhabditis elegans: a genetic model for sexdrive in a simple invertebrate. J Neurosci 2004;24:7427–34, http://dx.doi.org/10.1523/JNEUROSCI.1746-04.2004. [39] Barrios A, Nurrish S, Emmons SW. Sensory regulation of C. ele- gans male mate-searching behavior. Curr Biol 2008;18:1865–71, http://dx.doi.org/10.1016/j.cub.2008.10.050. [40] Izrayelit Y, Srinivasan J, Campbell SL, Jo Y, von Reuss SH, Genoff MC, et al. Targeted metabolomics reveals a male pheromone and sex-specific ascaro- side biosynthesis in Caenorhabditis elegans. ACS Chem Biol 2012;7:1321–5, http://dx.doi.org/10.1021/cb300169c. [41] Srinivasan J, von Reuss SH, Bose N, Zaslaver A, Mahanti P, et al. A modular libraryof small molecule signals regulates social behaviors in Caenorhabditis elegans. PLoS Biol 2012;10:e1001237, http://dx.doi.org/10.1371/journal.pbio.1001237. [42] Srinivasan J, Kaplan F, Ajredini R, Zachariah C, Alborn HT, Teal PE, et al. A blendof small molecules regulates both mating and development in Caenorhabditiselegans. Nature 2008;454:1115–8, http://dx.doi.org/10.1038/nature07168. [43] Butcher RA, Fujita M, Schroeder FC, Clardy J. Small-molecule pheromonesthat control dauer development in Caenorhabditis elegans. Nat Chem Biol2007;3:420–2, http://dx.doi.org/10.1038/nchembio.2007.3. [44] Artyukhin AB, Yim JJ, Srinivasan J, Izrayelit Y, Bose N, von Reuss SH, et al. Succinylated octopamine ascarosides and a new pathway of biogenicamine metabolism in Caenorhabditis elegans. J Biol Chem 2013;288:18778–83, http://dx.doi.org/10.1074/jbc.C113.477000. [45] Jang H, Kim K, Neal SJ, Macosko E, Kim D, Butcher RA, et al. Neu- romodulatory state and sex specify alternative behaviors throughantagonistic synaptic pathways in C. elegans. Neuron 2012;75:585–92, http://dx.doi.org/10.1016/j.neuron.2012.06.034. [46] Macosko EZ, Pokala N, Feinberg EH, Chalasani SH, Butcher RA, Clardy J, et al. Ahub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 2009;458:1171–5, http://dx.doi.org/10.1038/nature07886. [47] White JQ, Jorgensen EM. Sensation in a single neuron pair repressesmale behavior in hermaphrodites. Neuron 2012;75:593–600, http://dx.doi.org/10.1016/j.neuron.2012.03.044. [48] Saeki S, Yamamoto M, Iino Y. Plasticity of chemotaxis revealed by paired pre- sentation of a chemoattractant and starvation in the nematode Caenorhabditiselegans. J Exp Biol 2001;204:1757–64. [49] Hodgkin J (PhD thesis) Genetic and anatomical aspects of the Caenorhabditiselegans male. University of Cambridge; 1974. [50] Sawin ER, Ranganathan R, Horvitz HR. C. elegans locomotory rate is modu- lated by the environment through a dopaminergic pathway and by experiencethrough a serotonergic pathway. Neuron 2000;26:619–31. [51] Sherlekar AL, Janssen A, Siehr MS, Koo PK, Caflisch L, et al. The C. ele- gans male exercises directional control during mating through cholinergicregulation of sex-shared command interneurons. PLoS ONE 2013;8:e60597, http://dx.doi.org/10.1371/journal.pone.0060597. [52] Liu T, Kim K, Li C, Barr MM. FMRFamide-like neuropeptides andmechanosensory touch receptor neurons regulate male sexual tur- ning behavior in Caenorhabditis elegans. J Neurosci 2007;27:7174–82, http://dx.doi.org/10.1523/JNEUROSCI.1405-07.2007. [53] Sengupta P. The belly rules the nose: feeding state-dependent modula- tion of peripheral chemosensory responses. Curr Opin Neurobiol 2012:1–8, http://dx.doi.org/10.1016/j.conb.2012.08.001. [54] Breedlove SM, Cooke BM, Jordan CL. The orthodox view of brain sexual differen- tiation. Brain Behav Evol 1999;54:8–14, http://dx.doi.org/10.1159/000006607. [55] McCarthy MM, Arnold AP. Reframing sexual differentiation of the brain. NatNeurosci 2011;14:677–83, http://dx.doi.org/10.1038/nn.2834. [56] McCarthy MM, Arnold AP, Ball GF, Blaustein JD, De Vries GJ. Sex differ- ences in the brain: the not so inconvenient truth. J Neurosci 2012;32:2241–7, http://dx.doi.org/10.1523/JNEUROSCI.5372-11.2012. [57] Wilkins AS. Moving up the hierarchy: a hypothesis on the evolu- tion of a genetic sex determination pathway. Bioessays 1995;17:71–7, http://dx.doi.org/10.1002/bies.950170113.