Juli Wade

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.

Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch

In mammals and birds, sex differences in brain function and disease are thought to derive exclusively from sex differences in gonadal hormone secretions. For example, testosterone in male mammals acts during fetal and neonatal life to cause masculine neural development. However, male and female brain cells also differ in genetic sex; thus, sex chromosome genes acting within cells could contribute to sex differences in cell function. We analyzed the sexual phenotype of the brain of a rare gynandromorphic finch in which the right half of the brain was genetically male and the left half genetically female. The neural song circuit on the right had a more masculine phenotype than that on the left. Because both halves of the brain were exposed to a common gonadal hormone environment, the lateral differences indicate that the genetic sex of brain cells contributes to the process of sexual differentiation. Because both sides of the song circuit were more masculine than that of females, diffusible factors such as hormones of gonadal or neural origin also likely played a role in sexual differentiation.

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.

Differential expression of the immediate early genes FOS and ZENK following auditory stimulation in the juvenile male and female zebra finch.

The brains of adult zebra finches (Taeniopygia guttata) are tuned to the songs of conspecifics. In adult males, the caudomedial neostriatum (NCM) responds to zebra finch song, and in adult females the NCM and hippocampus (HP) are active following exposure to zebra finch song more than other auditory stimuli. The caudal hyperstriatum ventrale (cHV) in both sexes also responds to song, but in females not as selectively as the NCM and HP. While much is known about the adult perceptual circuit, less is known about its development. The present study exposed d30 male and female zebra finches to conspecific or heterospecific song, tones or silence, and examined the densities of FOS- and ZENK-immunoreactive nuclei in the NCM, cHV and HP. Significant interactions existed between sex and auditory stimulus condition for both immediate early genes, but they were in opposite directions. That is, across the three regions, FOS-immunoreactive neurons were increased in females following presentation of conspecific songs; males did not show an effect of stimulus exposure. In contrast, the density of ZENK-positive neurons was increased in males, but not females, following zebra finch song exposure. The FOS results demonstrate that some neural responses required for song perception may develop earlier in females than males; data on ZENK induction suggest the opposite. Overall, differences in juvenile immediate early gene activation suggest either that males and females employ divergent neural mechanisms for song perception or that the developmental trajectories leading to common neural responses differ.

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.

Aromatase activity and regulation of sexual behaviors in the green anole lizard

Sexual behaviors in green anoles are regulated by steroid hormones. Androgens activate the display of masculine courtship and copulatory behaviors, and estradiol activates feminine receptivity. Testosterone can also facilitate receptivity in females. The present study was conducted to test the role of converting testosterone to estradiol (aromatization) in the regulation of sexual and aggressive behaviors. Adult males and females were gonadectomized and implanted with a Silastic capsule containing either testosterone propionate (T) or estradiol benzoate (E) or with an empty (blank, BL) capsule. T- and BL-treated animals were then given injections of either Fadrozole (FAD, an aromatase inhibitor) or saline (SAL). E-treated animals received saline injections. Each individual was then tested alternately with male and female stimulus animals. Overall, T stimulated masculine sexual behaviors and receptivity, but the androgen had little effect on the display of aggressive behaviors. The inhibition of aromatase activity by treatment with Fadrozole eliminated the effect of T on receptivity. In contrast, Fadrozole treatment had no effect on mounting behavior or the frequency of courtship bouts. The inhibition of aromatase activity did increase the number of dewlap extensions (the display of a red throat fan) during courtship. These results suggest that the metabolism of testosterone to estradiol is a mechanism through which androgens can facilitate receptivity, and that such aromatization of testosterone is not required for the display of masculine sexual behaviors. In addition, females performed courtship and mounting behaviors less frequently than males, suggesting that an organizational component to steroid hormone regulation of sexual behaviors may exist in the anole.

Hormonal Control of Sex Differences in the Brain, Behavior and Accessory Sex Structures of Whiptail Lizards (Cnemidophorus Species)

The effects of steroid hormones on sexual dimorphisms in the brain, behavior and accessory sex structures were investigated in two species of whiptail lizards. The studies were conducted both in adults and hatchlings of a sexually reproducing species (Cnemidophorus inornatus) and an all‐female species (C. uniparens) which displays ‘sexual’ behaviors typical of males and females. Adults were gonadectomized and approximately 3 months later given either a Silastic capsule filled with sex steroid or an empty capsule. Young animals of both species were left intact and given a capsule on the day of hatching. An additional group of C. uniparens was ovariectomized on the day of hatching. Following treatment, measures of oviduct (estrogen‐dependent), renal sex segment (androgen‐dependent) and wolffian duct (androgen‐dependent) hypertrophy were taken in some experiments. Animals were also tested for sexual behavior in some of the studies. The volumes of the anterior hypothalamus‐preoptic area and ventromedial hypothalamus were measured in each individual. Estrogen, testosterone and dihydrotestosterone stimulated peripheral structures at both time periods in both sexes and species. The hormones also stimulated courtship and copulatory behaviors in many of the adult animals. However, testosterone in the anterior hypothalamus‐preoptic area of male C. inornatus was the only treatment which produced parallel effects on the volume of a brain area and the behaviors which it controls. These data add whiptail lizards to the list of species in which steroid hormones affect the volume of brain regions in adulthood, but suggest that such changes in morphology are not necessarily predictive of functional differences.

Sex steroids in green anoles (Anolis carolinensis): uncoupled maternal plasma and yolking follicle concentrations, potential embryonic steroidogenesis, and evolutionary implications

The sex steroids testosterone (T) and estradiol-17beta (E2) play important roles in vertebrate reproduction and development. However, little is known about the relationship between plasma steroid levels (which can influence reproductive function) and yolk steroid levels (which can influence embryonic development) in oviparous species. Therefore, we examined the extent to which T and E2 are coupled in plasma and yolking follicles in adult females and explored the dynamics of yolk and embryo steroid content during egg incubation in green anoles (Anolis carolinensis). T and E2 levels were determined for the plasma and yolking follicles of breeding females and for whole embryos and yolks at several developmental stages by radioimmunoassay. Plasma and yolk concentrations of T and E2 were not correlated. On average, plasma T was only 30% that of plasma E2, but yolking follicle T was over 600% that of yolking follicle E2. Total yolk T and E2 content generally declined over the course of incubation. However, yolk T was an order of magnitude higher than yolk E2, and it showed a secondary peak in magnitude after approximately 75% of incubation was completed. Similarly, total embryonic T content rose by over 400% in the latter half of incubation whereas E2 did not change. These results demonstrate that plasma and yolking follicle steroid levels produced by breeding females can be uncoupled. Furthermore, embryos themselves may begin producing T, but likely not E2, during the latter stages of incubation. Thus, steroid exposure may be independently shaped by selection to serve both reproductive and developmental functions.

Distribution of androgen receptor mRNA expression and immunoreactivity in the brain of the green anole lizard.

Male courtship and copulation are androgen dependent in the green anole lizard, and female receptivity can be facilitated by testosterone. However, only a few, and relatively large, regions in the brain have been implicated in the control of these behaviours. In situ hybridization and immunohistochemistry were therefore used to determine in detail where androgens are likely to act in the brains of breeding males and females. A 697‐bp fragment of the anole androgen receptor (AR) was cloned from total RNA isolated from the kidney, which contains the highly androgen‐sensitive renal sex segment. The cloned fragment spanned part of the C, the entire D, and part of the E domains, and shared a high degree of similarity with the AR of various species. 35S‐labelled antisense and sense probes were generated from the 697‐bp fragment for use in in situ hybridization, and the AR antibody PG‐21 was used for immunohistochemistry. Both sexes consistently had AR mRNA expression and immunoreactivity in areas associated with vertebrate reproductive behaviours and in motor areas of the brainstem. Interestingly, the PG‐21 antibody produced labelling in both the nucleus and cytoplasm, including neuronal processes. The distribution of mRNA and immunoreactivity were comparable in males and females, and the amount of labelling was generally similar, although slightly greater in females. The expression pattern of AR in this species supports the idea that distribution is highly conserved among vertebrates, but that it probably does not dictate behavioural differences between the sexes in anoles.

Sexually Dimorphic Areas in the Brain of Whiptail Lizards

The whiptail lizard species Cnemidophorus inornatus exhibits sexually dimorphic mating behaviors. We report that complementary sexual dimorphisms exist in two hypothalamic regions in male and female C. inornatus. The anterior hypothalamus-preoptic area (AH-POA), which is involved in male-typical mounting and intromission behaviors, is larger in males. The ventromedial hypothalamus (VMH), which controls female-typical receptivity, is larger in females. The all-female whiptail lizard Cnemidophorus uniparens, a direct descendant of C. inornatus, regularly and reliably displays both male-like mounting and female-like receptive (pseudosexual) behaviors. Rather than having a male-like AH-POA, however, the AH-POA as well as the VMH of C. uniparens resemble that of female C. inornatus. The biological basis of pseudosexual behavior in the parthenogen appears to be due to an unusual sensitivity to ovarian progesterone rather than to a difference in brain morphology.