Sex Differences in the Neural Song Circuit and Its Relationship to Song Acoustic Complexity in House Wrens (Troglodytes aedon).

The song circuit in passerine birds is an outstanding model system for understanding the relationship between brain morphology and behavior, in part due to varying degrees of sex differences in structure and function across species. House wrens (Troglodytes aedon) offer a unique opportunity to advance our understanding of this relationship. Intermediate sex differences in song rate and complexity exist in this species compared to other passerines, and, among individual females, song complexity varies dramatically. Acoustic complexity in wild house wrens was quantified using a new machine learning approach. Volume, cell number, cell density, and neuron soma size were then measured for three song circuit regions, Area X, HVC (used as a proper name), and the robust nucleus of the arcopallium (RA), and one control region, the nucleus rotundus (Rt). For each song control area, males had a larger volume with more cells, larger somas, and lower cell density. Male songs had greater acoustic complexity than female songs, but these distributions overlapped. In females, increased acoustic complexity was correlated with larger volumes of and more cells in Area X and RA, as well as larger soma size in RA. In males, song complexity was unrelated to morphology, although our methods may underestimate male song complexity. This is the first study to identify song control regions in house wrens and one of few examining individual variation in both sexes. Parallels between morphology and the striking variability in female song in this species provide a new model for understanding relationships between neural structure and function.

Convergent origination of a Drosophila-like dosage compensation mechanism in a reptile lineage.

Sex chromosomes differentiated from different ancestral autosomes in various vertebrate lineages. Here, we trace the functional evolution of the XY Chromosomes of the green anole lizard (Anolis carolinensis), on the basis of extensive high-throughput genome, transcriptome and histone modification sequencing data and revisit dosage compensation evolution in representative mammals and birds with substantial new expression data. Our analyses show that Anolis sex chromosomes represent an ancient XY system that originated at least ≈160 million years ago in the ancestor of Iguania lizards, shortly after the separation from the snake lineage. The age of this system approximately coincides with the ages of the avian and two mammalian sex chromosomes systems. To compensate for the almost complete Y Chromosome degeneration, X-linked genes have become twofold up-regulated, restoring ancestral expression levels. The highly efficient dosage compensation mechanism of Anolis represents the only vertebrate case identified so far to fully support Ohno's original dosage compensation hypothesis. Further analyses reveal that X up-regulation occurs only in males and is mediated by a male-specific chromatin machinery that leads to global hyperacetylation of histone H4 at lysine 16 specifically on the X Chromosome. The green anole dosage compensation mechanism is highly reminiscent of that of the fruit fly, Drosophila melanogaster Altogether, our work unveils the convergent emergence of a Drosophila-like dosage compensation mechanism in an ancient reptilian sex chromosome system and highlights that the evolutionary pressures imposed by sex chromosome dosage reductions in different amniotes were resolved in fundamentally different ways.

The genome of the green anole lizard and a comparative analysis with birds and mammals.

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

Sexually dimorphic expression of CREB binding protein in the green anole brain.

Green anoles are seasonally breeding lizards in which male sexual behavior is primarily regulated by an annual increase in testosterone. This hormone activates stereotyped behaviors, as well as morphological and biochemical changes in the brain, with greater effect in the breeding season than in the non-breeding season. This study is the first description of CREB binding protein (CBP) in the reptilian brain, and investigates the possibility that changes in CBP, an androgen receptor coactivator, may facilitate differences in responsiveness to testosterone across seasons. A portion of this gene was cloned for the green anole, and in situ hybridization was performed to examine the expression of CBP in the brains of gonadally intact male and female green anoles across breeding states. Additionally, hormonal regulation of CBP was evaluated across sex and season in animals that were gonadectomized and treated with testosterone or a control. Similar to other vertebrates, CBP was expressed at relatively high levels in steroid-sensitive brain regions. In the anole ventromedial amygdala, CBP mRNA levels were nearly twice as high in gonadally intact females compared to males. In contrast, CBP expression did not differ across seasons or hormone manipulation in this brain region. No significant effects were detected in the preoptic area or ventromedial hypothalamus. This pattern suggests that CBP might influence female-biased functions controlled by the ventromedial amygdala, but is not consistent with a role in mediating seasonal differences in responsiveness to testosterone in these areas associated with reproductive function.

Reptilian heart development and the molecular basis of cardiac chamber evolution.

The emergence of terrestrial life witnessed the need for more sophisticated circulatory systems. This has evolved in birds, mammals and crocodilians into complete septation of the heart into left and right sides, allowing separate pulmonary and systemic circulatory systems, a key requirement for the evolution of endothermy. However, the evolution of the amniote heart is poorly understood. Reptilian hearts have been the subject of debate in the context of the evolution of cardiac septation: do they possess a single ventricular chamber or two incompletely septated ventricles? Here we examine heart development in the red-eared slider turtle, Trachemys scripta elegans (a chelonian), and the green anole, Anolis carolinensis (a squamate), focusing on gene expression in the developing ventricles. Both reptiles initially form a ventricular chamber that homogenously expresses the T-box transcription factor gene Tbx5. In contrast, in birds and mammals, Tbx5 is restricted to left ventricle precursors. In later stages, Tbx5 expression in the turtle (but not anole) heart is gradually restricted to a distinct left ventricle, forming a left-right gradient. This suggests that Tbx5 expression was refined during evolution to pattern the ventricles. In support of this hypothesis, we show that loss of Tbx5 in the mouse ventricle results in a single chamber lacking distinct identity, indicating a requirement for Tbx5 in septation. Importantly, misexpression of Tbx5 throughout the developing myocardium to mimic the reptilian expression pattern also results in a single mispatterned ventricular chamber lacking septation. Thus ventricular septation is established by a steep and correctly positioned Tbx5 gradient. Our findings provide a molecular mechanism for the evolution of the amniote ventricle, and support the concept that altered expression of developmental regulators is a key mechanism of vertebrate evolution.

Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

Relationships among sex, season and testosterone in the expression of androgen receptor mRNA and protein in the green anole forebrain.

Sexual behavior in male green anole lizards is regulated by a seasonal increase in testosterone (T). However, T is much more effective at activating behavioral, morphological and biochemical changes related to reproduction in the breeding season (BS; spring) compared to nonbreeding season (NBS; fall). An increase in androgen receptor (AR) during the BS is one potential mechanism for this differential responsiveness. AR expression has not been investigated in specific brain regions across seasons in anoles. The present studies were designed to determine relative AR expression in areas important for male (preoptic area, ventromedial amygdala) and female (ventromedial hypothalamus) sexual behavior, as well as whether T upregulates AR in the anole brain. In situ hybridization and Western blot analyses were performed in unmanipulated animals across sex and season, as well as in gonadectomized animals with and without T treatment. Among hormone-manipulated animals, more cells expressing AR mRNA were detected in females than males in the amygdala. T treatment increased the volume of the ventromedial hypothalamus of gonadectomized animals in the BS, but not the NBS. AR protein in dissections of the hypothalamus and preoptic area was increased in males compared to females specifically in the BS. Additionally, among females, it was increased in the NBS compared to the BS. Collectively, these results indicate that differences in central AR expression probably do not facilitate a seasonal responsiveness to T. However, they are consistent with a role for AR in regulating some differences between sexes in the display of reproductive behaviors.

Sex bias and dosage compensation in the zebra finch versus chicken genomes: general and specialized patterns among birds.

We compared global patterns of gene expression between two bird species, the chicken and zebra finch, with regard to sex bias of autosomal versus Z chromosome genes, dosage compensation, and evolution of sex bias. Both species appear to lack a Z chromosome-wide mechanism of dosage compensation, because both have a similar pattern of significantly higher expression of Z genes in males relative to females. Unlike the chicken Z chromosome, which has female-specific expression of the noncoding RNA MHM (male hypermethylated) and acetylation of histone 4 lysine 16 (H4K16) near MHM, the zebra finch Z chromosome appears to lack the MHM sequence and acetylation of H4K16. The zebra finch also does not show the reduced male-to-female (M:F) ratio of gene expression near MHM similar to that found in the chicken. Although the M:F ratios of Z chromosome gene expression are similar across tissues and ages within each species, they differ between the two species. Z genes showing the greatest species difference in M:F ratio were concentrated near the MHM region of the chicken Z chromosome. This study shows that the zebra finch differs from the chicken because it lacks a specialized region of greater dosage compensation along the Z chromosome, and shows other differences in sex bias. These patterns suggest that different avian taxa may have evolved specific compensatory mechanisms.

The value of comparative approaches to our understanding of puberty as illustrated by investigations in birds and reptiles.

This article is part of a Special Issue "Puberty and Adolescence". Studies of birds and reptiles have provided many basic insights into the neuroendocrine control of reproductive processes. This research has elucidated mechanisms regulating both early development, including sexual differentiation, and adult neuroendocrine function and behavior. However, phenomena associated with the transition into sexual maturation (puberty) have not been a focus of investigators working on species in these taxonomic classes. Research is complicated in birds and reptiles by a variety of factors, including what can be extended times to maturation, the need to reach particular body size regardless of age, and environmental conditions that can support or inhibit endocrine responses. However, careful selection of model systems, particularly those with available genetic tools, will lead to important comparative studies that can elucidate both generalizability and diversity of mechanisms regulating the onset of reproductive maturity.

Seasonal and sexual dimorphisms in expression of androgen receptor and its coactivators in brain and peripheral copulatory tissues of the green anole.

Green anoles are seasonally breeding lizards, with an annual rise in testosterone (T) being the primary activator of male sexual behaviors. Responsiveness to T is decreased in the non-breeding season (NBS) compared to breeding season (BS) on a variety of levels, including displays of reproductive behavior and the morphology and biochemistry of associated tissues. To evaluate the possibility that seasonal changes in responsiveness to T are regulated by androgen receptors (AR) and/or two of its coactivators, CREB binding protein (CBP) and steroid receptor coactivator-1 (SRC-1), we tested whether they differ in expression across season in brains of both sexes and in peripheral copulatory tissues of males (hemipenis and retractor penis magnus muscle). AR mRNA was increased in the brains of males compared to females and in copulatory muscle in the BS compared to NBS. In the hemipenis, transcriptional activity appeared generally diminished in the NBS. T-treatment increased AR mRNA in the copulatory muscle and AR protein in the hemipenis, the latter to a greater extent in the BS than the NBS. T also decreased SRC-1 protein in hemipenis. Interpretations are complicated, in part because levels of mRNA and protein expression were not correlated and multiple sizes of the AR and CBP proteins were detected, with some tissue specificity. However, the results are consistent with the idea that differences in receptor and coactivator expression at central and peripheral levels may play roles in regulating sex and seasonal differences in the motivation or physical ability to engage in sexual behavior.

Sculpting reproductive circuits: relationships among hormones, morphology and behavior in anole lizards.

Morphology parallels function on a variety of levels in reproductive circuits in anole lizards, as in many vertebrate groups. For example, across species within the anole genus the muscle fibers regulating extension of a throat fan used in courtship are larger in males than females. Endocrine factors controlling behavior and morphology have been studied in detail in one species, the green anole (Anolis carolinensis). This review briefly describes the results that have been obtained and highlights key areas for future investigation that will provide insights on mechanisms from a comparative perspective.

Aromatase and 5α-reductase type 2 mRNA in the green anole forebrain: an investigation of the effects of sex, season and testosterone manipulation.

Aromatase and 5α-reductase (5αR) catalyze the synthesis of testosterone (T) metabolites: estradiol and 5α-dihydrotestosterone, respectively. These enzymes are important in controlling sexual behaviors in male and female vertebrates. To investigate factors contributing to their regulation in reptiles, male and female green anole lizards were gonadectomized during the breeding and non-breeding seasons and treated with a T-filled or blank capsule. In situ hybridization was used to examine main effects of and interactions among sex, season, and T on expression of aromatase and one isozyme of 5αR (5αR2) in three brain regions that control reproductive behaviors: the preoptic area, ventromedial nucleus of the amygdala and ventromedial hypothalamus (VMH). Patterns of mRNA generally paralleled previous evaluations of intact animals. Although no main effects of T were detected, interactions were present in the VMH. Specifically, the density of 5αR2 expressing cells was greater in T-treated than control females in this region, regardless of season. Among breeding males, blank-treated males had a denser population of 5αR2 positive cells than T-treated males. Overall, T appears to have less of a role in the regulation of these enzymes than in other vertebrate groups, which is consistent with the primary role of T (rather than its metabolites) in regulation of reproductive behaviors in lizards. However, further investigation of protein and enzyme activity levels are needed before specific conclusions can be drawn.

Developing a community-based genetic nomenclature for anole lizards.

BACKGROUND: Comparative studies of amniotes have been hindered by a dearth of reptilian molecular sequences. With the genomic assembly of the green anole, Anolis carolinensis available, non-avian reptilian genes can now be compared to mammalian, avian, and amphibian homologs. Furthermore, with more than 350 extant species in the genus Anolis, anoles are an unparalleled example of tetrapod genetic diversity and divergence. As an important ecological, genetic and now genomic reference, it is imperative to develop a standardized Anolis gene nomenclature alongside associated vocabularies and other useful metrics. RESULTS: Here we report the formation of the Anolis Gene Nomenclature Committee (AGNC) and propose a standardized evolutionary characterization code that will help researchers to define gene orthology and paralogy with tetrapod homologs, provide a system for naming novel genes in Anolis and other reptiles, furnish abbreviations to facilitate comparative studies among the Anolis species and related iguanid squamates, and classify the geographical origins of Anolis subpopulations. CONCLUSIONS: This report has been generated in close consultation with members of the Anolis and genomic research communities, and using public database resources including NCBI and Ensembl. Updates will continue to be regularly posted to new research community websites such as lizardbase. We anticipate that this standardized gene nomenclature will facilitate the accessibility of reptilian sequences for comparative studies among tetrapods and will further serve as a template for other communities in their sequencing and annotation initiatives.

Relationships among hormones, brain and motivated behaviors in lizards.

Lizards provide a rich opportunity for investigating the mechanisms associated with arousal and the display of motivated behaviors. They exhibit diverse mating strategies and modes of conspecific communication. This review focuses on anole lizards, of which green anoles (Anolis carolinensis) have been most extensively studied. Research from other species is discussed in that context. By considering mechanisms collectively, we can begin to piece together neural and endocrine factors mediating the stimulation of sexual and aggressive behaviors in this group of vertebrates.

Behavioural display systems across nine Anolis lizard species: sexual dimorphisms in structure and function.

Relationships between structure and function are a primary focus in biology, yet they are most often considered within individual species. Sexually dimorphic communication behaviours and the morphology of associated structures can vary widely, even among closely related species, and these traits provide an ideal opportunity to investigate the evolution of structure-function patterns. Using nine Anolis lizard species, we addressed a series of questions regarding sex differences in and the evolution of relationships between extension of the throat fan (dewlap) and morphology of the muscles and cartilage controlling it. The main results indicated that within species, males displayed the dewlap more often than females and consistently exhibited larger associated structures. These data are consistent with work in other vertebrates in which corresponding sex differences in reproductive morphology and behaviour have been documented. Across species, however, we found no evidence that the rate of dewlap extension evolved in association with dewlap morphology. Thus, we provide an example of traits that, when considered in a phylogenetic framework, exhibited limited associations between behaviour and morphology, perhaps as the result of constraints imposed by the ecological contexts in which different species occur.

Distribution of two isozymes of 5α-reductase in the brains of adult male and female green anole lizards.

The 5α-reductase (5αR) enzyme converts testosterone to 5α-dihydrotestosterone. This local metabolism within the brain is important for the full expression of male sexual behavior in many species, including green anole lizards. Two isozymes of 5αR exist and little is known about their specific distributions. We conducted in situ hybridization for both isozymes in intact male and female green anole brains during the breeding (BS) and non-breeding (NBS) seasons. 5αR1 mRNA was only detected in the brainstem, while 5αR2 was expressed in specific areas throughout the brain. As our primary interest was evaluating the potential role of 5αR in forebrain regulation of reproductive behavior, we quantified 5αR2 expression in the preoptic area, amygdala (AMY), and ventromedial hypothalamus (VMH). More 5αR2 cells were detected during the NBS than BS in the AMY, and the density of these cells was greater in females than males. In the VMH, the right side contained more 5αR2 cells than the left, an effect driven by a lateralized increase in the NBS. These data expand understanding of the distribution and potential roles of both isozymes in the adult brain, and differences in expression patterns between mammals and birds suggest that they may have been co-opted for different functions later in evolution.

Testosterone selectively affects aromatase and 5alpha-reductase activities in the green anole lizard brain.

Testosterone (T) and its metabolites are important in the regulation of reproductive behavior in males of a variety of vertebrate species. Aromatase converts T to estradiol and 5alpha-reductase converts T to 5alpha-dihydrotestosterone (DHT). Male green anole reproduction depends on androgens, yet 5alpha-reductase in the brain is not sexually dimorphic and does not vary with season. In contrast, aromatase activity in the male brain is increased during the breeding compared to non-breeding season, and males have higher levels than females during the breeding season. Aromatase is important for female, but not male, sexual behaviors. The present experiment was conducted to determine whether 5alpha-reductase and aromatase are regulated by T. Enzyme activity was quantified in whole brain homogenates in both the breeding and non-breeding seasons in males and females that had been treated with either a T or blank implant. In males only, T increased 5alpha-reductase activity regardless of season and up-regulated aromatase during the breeding season specifically. Thus, regulation of both enzymes occurs in males, whereas females do not show parallel sensitivity to T. When considered with previous results, the data suggest that aromatase might influence a male function associated with the breeding season other than sexual behavior. 5alpha-Reductase can be mediated by T availability, but this regulation may not serve a sex- or season-specific purpose.

Sexual differentiation of the zebra finch song system: potential roles for sex chromosome genes.

BACKGROUND: Recent evidence suggests that some sex differences in brain and behavior might result from direct genetic effects, and not solely the result of the organizational effects of steroid hormones. The present study examined the potential role for sex-biased gene expression during development of sexually dimorphic singing behavior and associated song nuclei in juvenile zebra finches. RESULTS: A microarray screen revealed more than 2400 putative genes (with a false discovery rate less than 0.05) exhibiting sex differences in the telencephalon of developing zebra finches. Increased expression in males was confirmed in 12 of 20 by qPCR using cDNA from the whole telencephalon; all of these appeared to be located on the Z sex chromosome. Six of the genes also showed increased expression in one or more of the song control nuclei of males at post-hatching day 25. Although the function of half of the genes is presently unknown, we have identified three as: 17-beta-hydroxysteroid dehydrogenase type IV, methylcrotonyl-CoA carboxylase, and sorting nexin 2. CONCLUSION: The data suggest potential influences of these genes in song learning and/or masculinization of song system morphology, both of which are occurring at this developmental stage.

Sexually dimorphic estrogen receptor alpha mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards.

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERalpha) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERalpha mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.

Neural activity associated with rhythmicity of song in juvenile male and female zebra finches.

Rhythm is an important aspect of both human speech and birdsong. Adult zebra finches show increased neural activity following exposure to arrhythmic compared to rhythmic song in regions similar to the mammalian auditory association cortex and amygdala. This pattern may indicate that birds are detecting errors in the arrhythmic song relative to their learned song template or to more general expectations of song structure. Here we exposed juvenile zebra finches to natural conspecific song (rhythmic) or song with altered inter-syllable intervals (arrhythmic) prior to or during template formation, or afterward as males are matching vocal production to a memorized song template (sensorimotor integration). Before template formation, expression of the immediate early gene ZENK was increased in the caudomedial nidopallium (NCM) of birds exposed to rhythmic relative to arrhythmic song. During template formation, ZENK expression was increased in the caudomedial mesopallium (CMM) of birds exposed to arrhythmic relative to rhythmic song. These results suggest that the youngest birds may be predisposed to respond to a more natural stimulus, and a template may be required for arrhythmic song to elicit increased neural activity. It also appears that functional development across the brain regions investigated continues to maturity.