Cell-Autonomous Sex Determination Outside of the Gonad

Whittier College Poet Commons

Biology Faculty Publications & Research

2013

Cell-autonomous sex determination outside of the gonad

Jenny C. Link

Arthur P. Arnold

Xuqi Chen

Yuichiro Itoh

Karen Reue

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Part of the Biology Commons DEVELOPMENTAL DYNAMICS 242:371–379, 2013

SPECIAL ISSUE REVIEWS–A PEER REVIEW FORUM

a Cell-Autonomous Sex Determination Outside of the Gonad

Arthur P. Arnold,1* Xuqi Chen,1 Jenny C. Link,2 Yuichiro Itoh,1 and Karen Reue2,3

Background: The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell-autonomous sex-biasing action of X and Y genes. Results: Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high-fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex-biasing effects of gonadal hormones and sex chromosome genes. Conclusions: Other cell-autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell-autonomous sex determination in non-gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms. Developmental Dynamics 242:371–379, 2013. VC 2013 Wiley Periodicals, Inc.

Key words: sex chromosome; sex determination; X chromosome; Y chromosome; Z chromosome; W chromosome; sexual differentiation; androgens; estrogens

Accepted 16 January 2013

INTRODUCTION determination,’ which is the develop- that cause sex differences in the mental decision that directs the undif- gonads, external genitalia, internal The current dominant model of mam- ferentiated gonad to develop as a testis genitalia (structures derived from malian sex determination was recently or ovary and ‘sex differentiation,’ Mullerian€ and Wolffian ducts), and summarized as follows: “In mammals, which occurs once the gonad has much of sexual differentiation of the biological differences between males developed and is induced by the prod- mammalian brain that has been and females are determined geneti- ucts of the gonad to establish the phe- investigated. These sexual character- cally during embryonic development. notypicsex.Inmammals,sexistics have a large impact in deter- These differences have a significant determination equates to gonad devel- mining whether the individual will impact on the physical, reproductive, opment” (Eggers and Sinclair, 2012). reproduce as male or female, and are psychological, and social life of an indi- Based on the present evidence, this considered by many scientists and lay vidual. Sex development can be di- classical model of sex determination persons as the characteristics that vided into two processes, ‘sex adequately accounts for the forces define the sex of the individual.

1Department of Integrative Biology and Physiology, and Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles, California 2Molecular Biology Institute, University of California, Los Angeles, California 3Departments of Human Genetics and Medicine, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California Grant sponsor: NIH; Grant numbers: DK083561, NS043196, DC000217. *Correspondence to: Arthur P. Arnold, Department of Integrative Biology and Physiology, UCLA, 610 Charles Young Drive South, Los Angeles CA 90095-7239. E-mail: [email protected] DOI: 10.1002/dvdy.23936 Published online 30 January 2013 in Wiley Online Library (wileyonlinelibrary.com).

VC 2013 Wiley Periodicals, Inc. 372 ARNOLD ET AL.

Moreover, the gender (social implica- sexual bias in tissues throughout the larger than females (at day 45, Fig. tions of sex) of the individual is often body (Arnold, 2011). Accordingly, in 1A). Adult gonadal males are about affected by and concordant with these this article we define “sex determi- 25% heavier than gonadal females, characteristics, so that these factors nation” to include any factors that whether they are XX or XY, confirm- indirectly impact the social environ- cause sex differences in cells, tissues, ing the importance of gonadal secre- ment of the individual. and individuals. The discrimination tions (at 75 days, or week 0 in Fig. The problem with the classical of separate processes of “sex determi- 1B). However, gonadally intact XX model is that it is unable to account nation”” and “sexual differentiation” mice are about 7% heavier than XY for an increasing number of sex differ- is unnecessary and counterproduc- mice, independent of their gonadal ences in important mammalian phe- tive. Factors that determine the sex of sex, indicating that the sexual pheno- notypes because these sex differences cells and individuals can be primary type also depends on the sex chromo- are not downstream of the develop- (encoded by the sex chromosomes of somes (at 75 days, or week 0 in Fig. mental decision to form testes or ova- the zygote) or secondary factors down- 1B). ries. The sex differences that are stream of the primary X and Y factors The role of sex chromosome comple- thereby defined as “outside” of sex (Arnold, 2011). In this view, Sry is a ment is revealed more dramatically determination are nevertheless quite primary sex-determining factor, and when the gonads are removed in important. At the top of the list is X testicular hormones secreted because adulthood (Fig. 1B). At first, the body inactivation, a process that is ubiqui- of the differentiation of testes are sec- weights of the four groups of FCG tous in all XX cells outside of the ondary sex determinants. mice converge, as mice that previ- germline. The process profoundly ously had ovaries increase body affects the transcriptional landscape weight and mice that previously had of the cell, involves significant epige- SEX CHROMOSOME testes plateau in their weight. Four netic changes and differential nuclear EFFECTS ON METABOLISM weeks after gonadectomy, there is no compartmentalization of chromo- difference in body weight among the AND OBESITY somes, and is tightly regulated and four groups. After this point, the body requires significant investment of the We focus first on recent evidence con- weight of XX mice increases faster cell’s resources (Heard and Disteche, cerning sex differences in body than that of XY mice, so that at 10 2006). X inactivation is fundamen- weight, adiposity, and metabolic dis- months after gonadectomy, XX mice tally female, never found in normal ease, because this example is particu- are 24% heavier than XY mice. The XY male cells. As such, it belongs larly instructive. In mice, adult males XX versus XY difference at this point within the framework of a general weigh more than females. Sex differ- is as great as the effect of gonadal theory of sex determination, not out- ences in body size have long been hormones prior to gonadectomy. The side of such a theory. Other sex differ- known to begin before implantation of greater weight of XX mice is attrib- ences are found prior to sexual the blastocyst, well before the embryo uted mostly to 88% greater fat mass differentiation of the gonads, indicat- has gonads, and continue into early in XX than XY (50% greater relative ing that the sex chromosomes encode post-natal life (Burgoyne et al., 1995, to body weight), although lean mass factors that act within and outside of 2002). Sex chromosome effects on is also greater in XX than XY. These the gonads (O et al., 1988; Burgoyne body weight are also found in adult large differences in adiposity are sur- et al., 1995; Bermejo-Alvarez et al., mice (Budefeld et al., 2008). These prising, considering that the mice are 2011a,b; Silversides et al., 2012; Nef findings establish the existence of sex eating a low fat (5% by weight) chow et al., 2005; Dewing et al., 2003). As is determinants not acknowledged by diet. Although XX and XY mice have discussed below, even after the the classical dogma. The sex determi- about the same level of physical activ- gonads differentiate, the number of X nants can be further demonstrated ity, XX mice eat more than XY mice chromosomes influences sex differen- using a mouse model—the four core during the daytime (their inactive ces in the phenotype of cells and indi- genotypes (FCG)—in which gonadal phase), independent of their gonadal viduals, independent of the gonadal sex and chromosomal sex are sex, which could contribute to their sex of the individual. The Y chromo- decoupled. FCG mice include XX and greater adiposity. some has effects outside of the gonad, XY mice with testes and XY and XX The differential role of the sex chro- and harbors genes that impact sexual mice with ovaries (De Vries et al., mosomes is illustrated further when phenotype, not just by determining 2002; Arnold and Chen, 2009). Analy- the mice are fed a high-fat diet. Diets the differentiation of the male’s sis of FCG mice revealed that both go- such as this are often used by investi- gonad. Thus, XX and XY individuals nadal secretions and sex chromosome gators studying metabolic diseases, with testes are not equivalent, and complement influence sex differences because they roughly mimic the high XX and XY individuals with ovaries in body weight and adiposity (Chen caloric diets consumed by people in are not equivalent. et al., 2012). Thus, at 3 weeks after developed countries. The high caloric The thesis of this article is that a birth, no difference in body weight is content provides a dietary stress that more accurate model of sex determi- evident in these mice (day 21, Fig. uncovers important pathophysiologi- nation recognizes multiple factors 1A). However, following the onset of cal effects in mice with varying me- encoded by the sex chromosomes, not puberty, sex differences in gonadal tabolism. On this diet, XX mice just those that determine gonadal secretions cause sex differences in gonadectomized as adults gain weight sex, which act in parallel to determine body size, with gonadal males being at an accelerated rate compared to XY CELL-AUTONOMOUS SEX DETERMINATION 373

specific non-gonadal effects of Sry or other Y genes; Dewing et al., 2006). A more adequate theory recognizes several classes of factors on the sex chromosomes that make males and females different (Arnold, 2009b, 2011). These include Y genes, such as Sry, that act outside of the gonads, and X genes that escape inactivation or that receive a parental imprint. Candidate X genes include about 3% of X genes that escape inactivation in mice (Yang et al., 2010). Among these are genes that are consistently expressed at higher levels in mice with two X chromosomes compared to mice with one X chromosome (Lopes et al., 2010; Werler et al., 2011; Chen et al., 2012). A second group of X genes that are candidates for the X chromosome effect includes those that receive different parental imprints in XX and XY mice. XY mice experience Fig. 1. Cell-autonomous effects of sex chromosomes contribute to sex differences in body only a maternal imprint on X genes, weight and metabolism in mice. A: Four core genotypes (FCG) mice show little sex difference in but XX mice receive imprints from body weight at 21 days (weaning). After puberty at day 45, gonadal males weigh more than females. Ten months after gonadectomy (GDX, performed at 75 days of age), XX mice are 24% both parents. These factors all act heavier than XY mice, and an interaction (Int) between sex chromosome complement and go- cell-autonomously in the gonads and nadal sex is apparent because XX gonadal females are heavier than gonadal males, but XY go- throughout the body, to cause sex dif- nadal males and females are not different. yP < 0.0001, zP < 0.000001. B: Body weight in ferences in cell function. In addition, gonadally intact mice at day 75 and after GDX at day 75. The sex difference caused by gonadal secretions disappears in the first month after GDX, after which XX mice gain more weight than cells are also sexually differentiated XY mice. XXM, XX gonadal males; XYM, XY gonadal males; XXF, XX gonadal females; XYF, XY by hormonal factors from the gonads, gonadal females. C: Liver histology after eating a high-fat diet (beginning 4 weeks after gonad- which are the dominant factors caus- ectomy for a total of 16 weeks) shows that XX mice have dramatically greater accumulation of ing sex differences in phenotype triglycerides in the liver, relative to XY mice, irrespective of gonadal sex. D: Four types of prog- throughout the body. Thus, multiple eny of XY* fathers were compared to assess the effects of one versus two X chromosomes, or of the Y chromosome. After GDX at 75 days, mice with the equivalent of two X chromosomes parallel-acting factors encoded by the gain more weight and fat than mice with one X chromosome, and the presence of the Y chro- sex chromosomes have sex-determin- mosome has no apparent effect. From Chen et al. (2012). ing effects. Once several parallel pathways of sex determinants are recognized, it mice (Chen et al., 2012), and the liv- independently of each other (see Chen becomes possible to appreciate that ers of gonadectomized XX mice accu- et al., 2008, for description of the XY* the sexually differentiating role of one mulate dramatically higher levels of model). The analysis of the XY* model factor is conditioned by the effects of triglycerides, relative to XY mice, and shows that mice with two X chromo- others, and that sex-biasing factors independent of gonadal sex (Fig. 1C). somes have greater body weight and may counteract or reduce the effects Fatty liver is a major component of adiposity than mice with one X chro- of each other (De Vries, 2004). Neither metabolic syndrome, and a major risk mosome, whereas the presence or ab- of these important conclusions derives factor for liver cancer (Sun and Karin, sence of a Y chromosome has little from classic sex-determination theory. 2012). XX mice on a high-fat diet, rel- effect (Fig. 1D). Taken together with For example, the effect of X chromo- ative to XY, also showed signs of insu- data from the FCG model, the results some number on mouse body weight lin resistance (a twofold elevation in indicate that under hypogonadal con- is present more at some phases of life fasting insulin in plasma, despite sim- ditions, the number of X chromosomes than at others. In the weanling mouse ilar blood levels of glucose) (Chen in mice has dramatic effects on criti- when gonadal hormones are expected et al., 2012). Insulin resistance is cal variables that model human obe- to be low, the effect of X chromosome commonly associated with obesity in sity and metabolic disease. number on body weight is not appa- humans, and a key feature of the met- Classic sex determination theory, rent, for unknown reasons (day 21, abolic syndrome. although seductive in its simplicity, is Fig. 1A). It becomes obvious by adult- The role of sex chromosome comple- silent concerning the sexually differ- hood, when gonads are fully active, ment as a determinant of metabolism entiating role of the number of X chro- when it accounts for a 7% difference and obesity was demonstrated further mosomes, and of other possible cell- in body weight at 75 days of age using the XY* model, which varies autonomous effects of sex chromo- (week 0, Fig. 1B). When the gonads the number of X and Y chromosomes some complement (e.g., known male- are removed, the effects of X 374 ARNOLD ET AL. chromosome number slowly emerge adiposity of mice are profoundly influ- increasing number of mouse models over a period of months as a major enced by the number of X chromosomes of disease show sex chromosome factor, causing up to 24% difference in in the absence of gonads. An important effects, including studies of autoim- body weight and metabolic dysregula- goal of research investigating physio- mune diseases such as multiple scle- tion (Fig. 1AB), especially when mice logical sex differences in disease is to rosis and lupus (Palaszynski et al., are eating a high-fat diet (Chen et al., identify sex-biasing factors that protect 2005; Smith-Bouvier et al., 2008; 2012). Because the sex chromosome one sex from disease, as part of a strat- Sasidhar et al., 2012), hypertension effect is larger in the absence of egy to uncover protective mechanisms (Ji et al., 2010; Caeiro et al., 2011; gonads than in their presence, it that might be targeted by novel thera- Ely et al., 2010), neural tube closure would appear that gonadal secretions pies. For example, mice with one X defects (Chen et al., 2008), cocksackie blunt or obscure the effects of sex chromosome are protected relative to viral infection (Robinson et al., 2011), chromosome complement. Inherent in mice with two X chromosomes from and behavioral tendencies related to that idea is that the effects of gonadal obesity and related metabolic dysregu- addiction and alcohol abuse (Quinn hormones differ in XX and XY cells. lation, including fatty liver and insulin et al., 2007; Barker et al., 2010). These ideas need further testing. The resistance. An improved model of sex Except for direct Sry effects on the data already suggest that the XX ver- determination has the advantage that brain (Dewing et al., 2006; Czech sus XY difference is in the “opposite” itfocusesattentiononmultiplenew et al., 2012) and possibly on the adre- direction of the hormone effects, at areas for research into such protective nal and kidney (Ely et al., 2010), it is least for body weight, because female mechanisms. not known which X or Y genes cause XX chromosome complement the sex chromosome effects. However, increases body weight, but male go- some studies have implicated the X nadal hormones produce greater body SEX CHROMOSOME chromosome as the origin of the sex- weight than female gonadal hormones EFFECTS ON DIVERSE biasing factor(s) (Chen et al., 2008, (Fig. 1A). 2009, 2012; Bonthuis et al., 2012). TISSUES The data in Figure 1 suggest other important conclusions about the The sex chromosome effects on body interaction of gonadal hormones and weight and metabolism, discussed X INACTIVATION AS A sex chromosome complement. At 10 above, illustrate the fascinating inter- DEFINING EVENT OF SEX months after gonadectomy, XX mice play of parallel sex-determining DETERMINATION that had ovaries have greater body mechanisms that operate independ- weight than XX mice that had testes ently and interdependently. These Above we suggested that X inactiva- (Fig. 1B). The phenotypic differences effects are among a growing list of XX tion, an event that happens in every (in body weight and adiposity) versus XY differences that are XX somatic (i.e., non-germline) cell, between these two groups is caused reported to influence a wide variety of but never in XY cells, is properly by the presence or absence of Sry, and phenotypes in numerous tissues. The viewed as a sex-determining event. is a gonadal hormonal effect that lasts effects are found in mice in which spe- Certainly, it represents a fundamen- long after the gonads are removed, cific Y genes are manipulated inde- tal phenotypic difference between and/or an effect of Sry outside of the pendent of gonadal effects (Dewing normal male and female cells, but gonads. Interestingly, this effect et al., 2006), in mice that never devel- large questions remain concerning depends on sex chromosome comple- oped gonads (Majdic and Tobet, 2011), the sex-biasing impact of this molecu- ment, because XY gonadal male and or in mice in which the XX versus XY lar phenotype. Several points are rele- female mice are not much different in comparison can be made in both go- vant. Firstly, X inactivation has large body weight. Because the two groups nadal sexes (Arnold, 2004, effects on gene expression, because it of factors (hormones and sex chromo- 2009a,b,2011; Arnold and Chen, 2009; effectively removes what would other- some complement) each condition the Abel and Rissman, 2011). XX versus wise be a large female bias in X gene effects of the other, the various factors XY effects are found in expression of expression (i.e., greater expression of (or their downstream effector path- important genes in the brain (De X genes in females than in males, Fig. ways) must interact directly. The na- Vries et al., 2002; Gatewood et al., 2). In other animal groups lacking ture of this interaction is completely 2006; Chen et al., 2009; Abel et al., similar chromosome-wide dosage com- unexplored at present. 2011; Xu et al., 2002, 2008a,b; Dewing pensation, in birds (see below) and in In gonadally intact young adult et al., 2006), in brain development mammalian blastocysts before X inac- mice, when all sex-biasing factors are (Carruth et al., 2002), and in behav- tivation is ubiquitous, the expected present, gonadal hormones rank first iors such as parental and aggressive female bias of X gene expression is as important determinants of the sex behaviors (Gatewood et al., 2006), present (Arnold et al., 2008; Bermejo- differences in body weight and compo- response to noxious stimuli (Gioiosa Alvarez et al., 2010). Thus, X inacti- sition, because the gonadal hormone et al., 2008a,b), sexual behaviors vation is seen as a female-specific effect is larger (25%) than the sex (Grgurevic et al., 2012; Bonthuis mechanism to adjust X gene dose (rel- chromosome effect (7%). More inter- et al., 2012), and social and investi- ative to autosome dose) to a level sim- esting, perhaps, to aging, post-meno- gative behaviors (McPhie-Lalman- ilar to that of the male. Although pausal, and other hypogonadal singh et al., 2008; Cox and Rissman, calling a factor that reduces sex bias humans, is that body weight and 2011; Grgurevic et al., 2008). An “sex determining” might seem CELL-AUTONOMOUS SEX DETERMINATION 375

and Festenstein 2011; Wijchers et al., reversed relative to mammals, with 2010). Moreover, the presence of an the male being homogametic (ZZ) and inactive X chromosome correlates with the female heterogametic (ZW). More reduced methylation of the genome of importantly in the present context, embryonic stem cells, and XX cells (rel- birds appear to lack a chromosome- ative to XY) show reduced expression wide mechanism of Z gene dosage ofthedenovoDNAmethyltransferases compensation comparable to X inacti- Dnmt3a and Dnmt3b (Zvetkova et al., vation (Arnold et al., 2008). In every 2005). The question of genome-wide bird species studied to date, most Z effects of the inactive X chromosome, genes are expressed higher in males not mediated by dosage of the X genes, than in females in numerous tissues Fig. 2. Effective sex chromosome dosage is ripe for further analysis. before and after gonadal differentia- compensation in the mouse compared with tion (Fig. 2) (Itoh et al., 2007, 2010; ineffective dosage compensation in the chick Ellegren et al., 2007; Zhang et al., embryo. Microarray mRNA expression profil- THE CONTRAST OF 2010; Mank, 2009; Chue and Smith, ing was conducted in adult mice and chick MARSUPIAL (METATHER- embryos of both sexes. The graphs show the 2011; Wolf and Bryk, 2011; Naurin distribution of M/F ratios of expression of IAN) AND EUTHERIAN et al., 2011). Birds, more than euther- autosomal genes and X or Z chromosome MAMMALS ian mammals, may easily evolve genes. Autosomal genes have modal M/F diverse cell-autonomous sex-determi- ratios near 1 (log of zero), but some genes are Like eutherian mammals, marsupials nation mechanisms because of the expressed higher in males or in females. Log 2 have an XX-XY sex chromosome sys- availability of many more sex-biased M/F ratios are rarely greater that 1 (twofold tem. The Y chromosome contains the higher in males) or less than 1 (twofold (Z-linked) cell-autonomous signals higher in females). In mammals such as the testis-determining gene Sry, and many throughout their body, which can mice, M/F ratios for X genes show a distribu- steps of gonadal differentiation are evolve a role to control sex differences tion closely matched to autosomal genes, de- comparable to those in eutherians. in diverse phenotypes. In species with spite the presence of two X chromosomes in However, three major reproductive tis- females compared to one of males. In birds effective dosage compensation such as such as the chick, the distribution of M/F sues,pouch,scrotum,andmammary eutherian mammals, most sex chro- ratios for autosomal genes is similar to that in tissue, are sexually differentiated inde- mosome (in this case, X chromosome) mammals, but in the absence of chromo- pendent of gonadal differentiation. In genes are expressed at similar levels some-wide dosage compensation of the Z the tammar wallaby, for example, the in the two sexes (Fig. 2), and thus are chromosome, ZZ males have higher expres- anlage of the scrotum, pouch, and sion of Z genes compared to ZW females, for not likely to evolve a primary role in most Z genes. From Itoh et al. (2007). mammary tissue begin to form prior to controlling different developmental gonadal differentiation, and sex-spe- programs in the two sexes. Perhaps counterintuitive, X inactivation cer- cific patterns of development are not because of the lack of effective Z chro- tainly ranks as one of the most pro- influenced by changes in gonadal hor- mosome dosage compensation that found sex-specific forces in the mone levels. The sex-determining fac- may have favored more cell-autono- mammalian genome. Furthermore, tors appear to be X-linked, because an mous sex determination, the small its role is predominantly to counteract XXY marsupial (with testes and a number of studies of birds has had a another female bias, in the number of penis) also has a pouch and mammary major catalytic role in changing atti- X chromosomes, which underscores glands, whereas an XO marsupial tudes about the gonad-first classic the idea that different sex-determin- (with ovaries and uteri) has neither a model of sex determination. ing factors can oppose each other. In pouch nor a mammary gland (reviewed In the last half of the 20th century, the end, the process of shutting down by Renfree and Short 1988; Renfree many studies of birds showed that a large chromosome would reasonably et al., 2002; Glickman et al., 2005). sexual phenotype is controlled by go- be thought to leave XX cells distinctly Thus, marsupials represent an early nadal hormones (e.g., Balthazart different from XY cells, but at present and iconic example of important sex- et al., 2009; Balthazart and Adkins- there is only a small amount of evi- determining factors that are not down- Regan, 2002). One informative model dence that the presence of a large het- stream of gonadal differentiation. To system has been the neural circuit erochromatic chromosome has effects develop a comprehensive understand- controlling song in Passerine birds elsewhere in the genome. For example, ing of sex determination in marsupials, (Wade and Arnold, 2004). Male zebra mice with two X chromosomes, com- it is important to discover the X-linked finches sing a courtship song that pared to mice with one X chromosome, sex determinants that operate in paral- females do not sing, and the brain show greater expression of an autoso- lel with sex determinants that control regions controlling song are 5–6 times mal transgene that is a barometer of sexual differentiation of the gonads. larger in males (Nottebohm and the status of nearby heterochromatin. Arnold, 1976). At first, the search for The results suggest that the presence BIRDS AS MODELS OF SEX the sex-determining factors focused of a large heterochromatic inactive X on the classical model, and manipu- CHROMOSOME EFFECTS chromosome in XX cells may reduce lated gonadal hormone levels in the availability of heterochromatizing Birds offer another important con- females and males. Females treated factors generally, affecting gene expres- trast to eutherian mammals. Their with estradiol at hatching were per- sion throughout the genome (Wijchers sex chromosome complement is manently masculinized, both in their 376 ARNOLD ET AL. brain and behavior (Gurney and uniformly but modestly higher on the zebra finch song system (Agate et al., Konishi, 1980). The result was inter- right side compared to the left, com- 2003; Kim et al., 2004; Chen et al., preted to support the idea that males patible with ZZ genotype on the right 2005). Hormonal manipulations show normally secrete testosterone, which and a single Z on the left. Because go- hormonal effects, and genetic manip- is converted to estradiol in the brain nadal hormones would be expected to ulations show genetic effects, and of- where it has a permanent masculiniz- affect both sides of the brain and ten both are important. That ing effect, a finding similar to previ- body, the greater masculinization of conclusion would seem to apply to ous results in mammals. However, the brain on the right side suggested both birds and mammals, although several observations were at odds that it was controlled in a cell-autono- the difficulty of performing the with the hormonal theory. Manipula- mous fashion by sex chromosome genetic manipulations means that tions of gonadal hormones in males complement of brain cells. there is much less information about (for example, blocking estradiol Analysis of several gynandromor- that type of sex-biasing factor. action) did not block masculine brain phic chickens, using BAC probes to Above we proposed a revised defini- development (Arnold and Schlinger, determine the number of Z and W tion and framework for thinking 1993). When genetic females were chromosomes in specific cells, proved about sex determination. This frame- injected early in development with a that the birds had a mixture of ZZ work undermines the classic distinc- drug that caused differentiation of and ZW cells on the two sides of the tion between two distinct processes, testes, those females were not mascu- body, with ZZ predominating on one sex determination and sexual differ- linized by the presence of functional side and ZW on the other (Zhao et al., entiation, which was originally testicular tissue (Wade and Arnold, 2010). On the ZZ side, the phenotypes rationalized based on the order of 1996). When these studies raised sig- of numerous tissues were more mas- events (sex determination preceding nificant doubt about the gonadal hor- culine than on the ZW side, including and causing sexual differentiation), monal origin of the sex differences, it muscle, bone, leg, plumage, and wat- and on different mechanisms (genetic became reasonable to consider the tles. Moreover, when ZZ cells were sex determination versus hormonal idea that cell-autonomous factors, implanted into ZW embryos or vice sexual differentiation). In a revision encoded by the sex chromosomes, versa, the cells retained sexual pheno- of the classic model (Fig. 3), it is might be the primary cause of the sex types consistent with their own sex appropriate to recognize multiple sex difference in brain circuits and the chromosome complement after the chromosome mechanisms, acting reproductive behaviors that they con- cells took up residence within the so- along parallel mechanistic pathways, trol (Arnold, 1996). matic component of the recipient that are inherently different in vari- Rare avian gynandromorphs are gonads (Zhao et al., 2010), confirming ous types of male versus female cells, dramatic intersex animals, sometimes that the genetic sex of somatic go- which lead to downstream sex differ- with a sharp line at the animal’s mid- nadal cells plays a significant role in ences. In many phenotypes, we can line separating male plumage on one determining their sexual phenotype. expect an interaction of the parallel side from female plumage on the Cell-autonomous sex determination pathways, so that both hormonal and other side. Other sexual characters probably explains the development of sex chromosome factors modify the are also lateralized, such as type of a larger number of sexually dimorphic effect of the other. A new concept of gonad and body size. As early as phenotypes in birds than in eutherian sex determination frees us from inac- 1939, based on analysis of gynandro- mammals, because of the lack of Z- curate and misleading over-simplifi- morphs, Witschi concluded that chromosome-wide dosage compensa- cations such as “in mammals, sex is “many of the secondary sex characters tion that results in male bias in determined genetically,” or “in birds, of ... birds are not under hormonal expression of most Z-gene expression sex is cell-autonomous.” Instead, we but direct intracellular control” throughout the body. Nevertheless, can recognize that even eutherian (Witschi, 1939). The use of modern most of the phenotypes that show a mammals have significant cell-auton- molecular reagents to analyze gynan- sex chromosome effect in birds (differ- omous sex determinants, and in birds dromorphs has confirmed this conclu- ent phenotype in ZZ versus ZW) are hormonal and genetic factors interact. sion. In a gynandromorphic zebra often also influenced by gonadal hor- We propose that taxonomic differen- finch, the right side had male mone levels. A salient example is the ces in the importance of cell-autono- plumage, masculine brain character- chicken’s comb and wattle, large in mous factors may be related to the istics, and a testis, whereas the left males and small in females. These effectiveness of sex-chromosome dos- side had female plumage, less mascu- structures have long been known to age compensation, which affects the line brain, and an ovary (Agate et al., be sensitive to androgens, so that the number of inherently sexually dimor- 2003). The W chromosome, normally sex difference was thought to be phic signals within each male and only found in ZW females, was downstream of gonadal determina- female cell that can evolve a sex- enriched in the genomic DNA of the tion. Yet, the chicken gynandromorph determining function. animal’s left side (ruling out a ZO ge- has a larger comb and wattle on the notype). Expression of W-linked genes male side, indicating that sex chromo- FUTURE DIRECTIONS was very high on the entire left side some complement also contributes to of the brain, but nearly absent (except the sex difference (Zhao et al., 2010). Numerous mouse models are now for a small number of cells) on the Similar roles for both factors (hormo- widely available for the routine mea- right. Expression of Z genes was also nal and genetic) are proposed in the surement of the importance of sex CELL-AUTONOMOUS SEX DETERMINATION 377

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