BACKGROUND
steroids are central to zebra finch  neurobiology.
they are essential for early developmental organization of the song control system, and they continue to modulate brain and behavior throughout life  <cit> .
although some steroids are supplied to the brain from the periphery, others including estradiol can be synthesized within the brain, either de novo from cholesterol or by metabolism of precursor steroids that originate in the periphery, as shown by evidence from biochemical enzyme activity assays, explant and dissociated culture analysis, molecular identification and neuroanatomical mapping of steroidogenic factors, and in vivo steroid measurements  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
steroids synthesized within the brain, termed "neurosteroids," masculinize the song system and can be rapidly modulated by experience  <cit> .
steroids often act through nuclear receptor transcription factors, which can be abundant in the songbird brain, including in the song control system  <cit> .
thus the zebra finch brain has the capacity to both produce and respond to a variety of steroids that can alter neural function and song behavior.
steroids are small molecule hormones derived from cholesterol through a series of enzymatic conversions .
the first steroidogenic enzyme resides in the inner mitochondrial membrane, and the rate-limiting step of steroidogenesis is the transport of cholesterol across the outer mitochondrial membrane.
two major cholesterol transport proteins, which have been proposed to work in concert as part of a protein complex, are the steroidogenic acute regulatory protein  and the  <dig> kda cholesterol transport protein   <cit> .
steroid synthesis starts with the action of cytochrome p <dig> side chain cleavage , which produces pregnenolone.
pregnenolone can be converted to either progesterone or dehydroepiandrosterone via the action of cytochrome p <dig> 17α-hydroxylase/ <dig>  lyase  or 3β-hydroxsteroid dehydrogenase/Δ <dig> Δ <dig> isomerase , respectively.
androstenedione is produced from progesterone through the activity of hsd3b <dig> and from dehydroepiandrosterone through the activity of cyp <dig>  androstenedione can be converted to testosterone or an estrogen, estrone, via the activity of 17β-hydroxysteroid dehydrogenases  or cytochrome p <dig> aromatase , respectively.
hsd17b can also convert estrone to estradiol, and cyp <dig> metabolizes testosterone into estradiol.
of note is the fact that multiple hsd3b and hsd17b types exist in other animals, and that several of hsd17b enzymes can use androgens and estrogens as substrates  <cit> .
steroids most commonly act by binding to nuclear receptors that dimerize, translocate to the nucleus, and function as dna-binding transcription factors.
the steroid receptors belong to nuclear receptor class  <dig>  and maintain the typical four-domain structure of receptors in the nuclear receptor superfamily  <cit> .
there are four main nuclear receptors for the steroids produced along the estradiol-synthetic pathway: estrogens bind estrogen receptor alpha  and beta ; androgens bind androgen receptor , and progesterone binds progestin receptors .
with the assembly of the zebra finch genome sequence, we are now in position for the first time to assess the organization and regulation of the network of genes that control steroid synthesis and function in the songbird brain.
starting with the set of ensembl gene predictions for the zebra finch, we manually curated the assembled models for genes that encode the major components of the estradiol-synthetic pathway and related proteins, as well as four nuclear steroid receptors.
we characterized the structure, diversity, and evolutionary conservation of these genes.
this analysis led us to examine the neural expression patterns of a cholesterol transport protein in the context of song neurobiology.
RESULTS
characterization of steroidogenic genes in the zebra finch genome assembly
we used all available resources  and genbank cdna clone entries, and cross-species and functional domain homology searches) to identify the sequences of twenty steroid-related genes in the zebra finch genome assembly .
of these, two genes code for cholesterol transport proteins , five genes code for enzymes known to be active in the estradiol-synthetic pathway in songbirds , nine genes code for related hsd3b and hsd17b enzymes, some of which are likely to also be involved in the estrogen-synthetic pathway , and four genes code for the primary nuclear receptors for the steroids produced in the estradiol-synthetic pathway .
genomic information for all twenty genes examined.
ensembl gene identifiers are listed for the eighteen genes with official models .
chromosomal positions are also listed, as are the locations of either alternative mappings  or annotated promoter regions .
complete coding regions are present for the four receptor genes and for the hsd3b <dig>  cyp <dig>  hsd17b <dig>  hsd17b <dig>  hsd17b <dig> genes, but only partial gene sequences were identified for the other genes in the assembly.
in cases where full length cdna clone sequences were available, we could determine that incomplete gene models were due to assembly gaps.
some genome assembly gaps result in very small omissions , but others are large .
in the case of cyp <dig>  we identified a genome contig in the trace archive  that codes for exon  <dig> and a portion of the 3'utr that was not included in the current genome assembly .
the structures of each of the zebra finch genes are illustrated by their evolutionarily conserved regions when compared to chicken or human genes in zpicture format .
hsd3b is a family with up to seven enzymes whose members each derive from unique genes, almost all of which are located along the same chromosome in human and mouse  <cit> .
we used homology searches to try to identify zebra finch genes for all of the known hsd3b genes.
hsd3b type  <dig> is annotated in the genome on chromosome  <dig>  several other types of hsd3b genes from other species show homology to this same location, but blat analysis with the zebra finch hsd3b <dig> cdna clone sequence aligns only with this one position, suggesting that there is only one hsd3b gene in the zebra finch assembly.
homology searches with the conserved rossmann catalytic domain did not identify any additional putative hsd3b genes on chromosome  <dig>  further supporting the conclusion that the hsd3b <dig> gene is the only one on chromosome  <dig>  we did, however, identify an unannotated, incomplete gene for hsd3b type  <dig> on chromosome  <dig> by homology searches.
the hsd17b enzymes are also a family of similar enzymes.
fourteen hsd17b genes have been described to date in humans, and multiple hsd17b genes have also been cloned from or identified in the genomes of other mammals, fish, and chicken  <cit> .
we attempted to find all fourteen genes in the zebra finch genome assembly.
we identified genes for hsd17b 1- <dig>   <dig>   <dig>  10- <dig> in the assembly .
we confirmed the available hsd17b <dig> genomic sequence .
two genes, hsd17b <dig> and hsd17b <dig>  were mapped to the z sex chromosome in the genome assembly; the hsd17b <dig> mapping is consistent with experimental data that show that this genes is localized to the z chromosome  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
the z chromosome position is of note because sex chromosome genes can be expressed at different levels in males and females and have been proposed to contribute to the sexual differentiation of the zebra finch song system  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
the hsd17b <dig> gene model is incomplete; there is one gap in the assembly that may remove several coding exons, but the assembly is uninterrupted for up to  <dig> kb outside of the boundaries of the gene model.
it was therefore possible that more gene coding sequence could be obtained through manual curation of this stretch of the assembly.
our attempts to identify additional exons through homology searches were, however, unsuccessful.
the hsd17b <dig> gene shares exon structure with mammalian and chicken hsd17b <dig> genes and contains only intronic assembly gaps; hsd17b <dig> was examined in more detail elsewhere .
several steroidogenic genes have the potential to form alternatively spliced transcripts or utilize alternate promoters.
star, tspo, cyp11a <dig>  erα, erβ, and ar are predicted to have alternative splice forms in other animals.
we identified splice sites in the zebra finch star and tspo genes that give rise to different length transcripts .
a model for cyp11a <dig> was not completed because of the large gap in genomic sequence, and we were unable to identify splice variants similar to those in mammals in the zebra finch erα, erβ, and ar genes.
the first exon of the cyp <dig> gene is untranslated and contains promoter elements that regulate tissue-specific transcription e.g.
<cit> .
in zebra finches as in mammals, there are several variants of exon 1; zebra finch brain cyp <dig> transcripts almost always use the "1a" exon  <cit> .
though not annotated as such, we identified the exon 1a sequence in the zebra finch genome assembly approximately  <dig> kb upstream of the first coding exon .
pcr confirmation of genomic sequence
the ensembl annotation of the genome assembly contains a predicted gene model for hsd17b <dig>  as way of validating the genomic assembly sequence one of the genes that had not been previously cloned, we used the gene model to design pcr primers and successfully amplified the predicted hsd17b <dig> sequence from zebra finch genomic dna .
however, we were still unable to obtain the sequence at the 3' end of the gene that is absent in the assembly due to a gap between contigs.
phylogeny and selection
cross-species multiple sequence alignments for each of the twenty genes demonstrated that functional domains were the most highly conserved regions of the genes and that the overall exon/intron structure of these genes is conserved across the fish, bird, and mammal species investigated .
to examine the relationship between genes in the three families examined here, hsd3b, hsd17b, and the nuclear receptors, we constructed unrooted phylogenetic trees of each of these groups, using predicted protein sequences.
phylogenetic analysis of mouse, human, zebrafish, chicken and zebra finch hsd3b proteins showed that the zebra finch hsd3b <dig> sequence is most similar to that of the chicken, and groups with the hsd3b1- <dig> protein sequences of the mouse and human .
the hsd3b <dig> zebra finch and chicken sequences are more similar to hsd3b <dig> proteins in the other species, but showed less homology compared to human than even the zebrafish sequence.
to look at the phylogenetic relationship among hsd17b predicted proteins, we used all annotated hsd17b genes in human, mouse, zebrafish, and chicken, plus those we identified in zebra finch .
in general, the zebra finch proteins segregated with the same enzyme type in chicken and the other species.
this analysis modeled hsd17b <dig> and hsd17b <dig> proteins as independent branches, not closely related to any other hsd17b enzyme.
several other protein types, however, showed more similarity to another.
for example, the type  <dig> and  <dig> proteins were clustered together on the tree, as were types  <dig> and  <dig>
we also examined the nuclear receptors .
the two types of er segregated together, with the zebra finch erα and erβ showing more similarity to their respective orthologs in other species than to each other.
similarly, the ar and pr predicted protein sequences were more similar to each other than to the ers.
for all four types of receptors, the zebra finch is most closely related to the chicken sequence.
the zebrafish is the most distantly related to the mammalian proteins, and birds in some cases  show more similarity to the mammalian proteins than does the platypus.
distribution of predicted transcription factor binding sites
multiple steroidogenic enzymes need to be present in close proximity to locally synthesize a variety of steroids, including estradiol.
the functional connection between these enzymes suggests the possibility that common transcriptional regulatory elements may be shared among the steroidogenic genes to direct their coordinated expression.
to pursue this hypothesis, we used a set of position weight matrix  predictions for specific transcription factor binding sites generated for the whole zebra finch genome as part of the primary analysis of the assembly  <cit> .
we then tested whether any pwm sites were more abundant in territories surrounding steroid related genes than in the genome as a whole.
when we used all  <dig> available ensembl models, one pwm, nr1h2-rxr, was identified as significantly  overrepresented in the gene set with the swan algorithm  <cit> .
no significantly overrepresented pwm sites were identified when subsets of the cholesterol transport and enzymes ensembl models were used.
however, when the four nuclear receptor genes were examined, nobox was shown to be significantly  overrepresented according to the sllr algorithm  <cit> .
two pairs of functionally related genes - star and tspo, and cyp <dig> and hsd17b <dig> - had complete coding regions in the assembly, and few intronic gaps.
we therefore inventoried and compared the pwm sites predicted to be in these genes to assess the potential for transcriptional co-regulation .
comparison of the pwm profiles in star and tspo showed that no sites were common to both genes in the 5'-focused region, and only 23% and 33% of them were shared when the whole gene was taken into account .
similarly, when pwm sites for cyp <dig> and hsd17b <dig> were compared, only one site was shared in the 5'-focused region, while 89% and 61%  were shared when the whole gene was considered.
all  <dig> jaspar pwms that were mapped onto the zebra finch genome assembly are listed.
blocks in the row denote the presence of the pwm either in the 5'-focused region  or across the entire gene territory  for star and tspo.
all  <dig> jaspar predictions mapped onto the zebra finch genome assembly are listed.
blocks in the row denote the presence of the pwm either in the 5'-focused region  or across the entire gene territory  for cyp <dig> and hsd17b <dig>
neural expression of tspo
the finding that star and tspo shared few pwm sites suggested that these genes may have largely independent transcriptional regulatory mechanisms, which could result in distinct patterns of expression.
non-overlapping expression distributions would be inconsistent with recent models that propose that star and tspo work together in a complex that is required for transporting cholesterol across the mitochondrial membranes for the initiation of steroidogenesis  <cit> .
tspo is one of the few components of the estradiol-synthetic pathway not yet examined in the zebra finch brain.
we therefore utilized information from cdna and microarray resources developed alongside the genome assembly to confirm that tspo was expressed in the zebra finch brain, test whether or not it co-localized to brain areas that expressed star, and validate results from a zebra finch brain microarray that indicated that its mrna was rapidly regulated after experience  <cit> .
several tspo ests were identified in the zebra finch estima collection of brain cdnas indicating that it was transcribed in the brain  <cit> .
we used one of these clones to perform in situ hybridization to test if tspo expression was neuroanatomically distributed within two functional regions known to express star and other steroidogenic genes: the nuclei of the song control system in adult males, and the cells along the proliferative zone of lateral ventricle in posthatch day  <dig>  birds.
in situ hybridization showed that tspo is expressed in all four major song nuclei in adult male birds .
the intensity of labeling within these nuclei is not noticeably above that in the surrounding brain, and its hybridization distribution is widespread.
in p <dig> birds, tspo hybridization occurred at low levels throughout the brain but was strikingly absent from the cell-dense region surrounding the lateral ventricle .
hybridization of adjacent sections with sense riboprobe did not show any labeling in adult or p <dig> brains, suggesting that the observed hybridization patterns are specific for tspo.
we also measured tspo and star expression in the auditory forebrain lobule , a brain area required for song processing and learning  <cit> .
adult birds were either placed in silence or played  <dig> minutes of novel zebra finch song, and their brains were processed for in situ hybridization.
in the al, there was a strong but non-significant trend of song condition on the intensity of tspo labeling in cells  because labeling was less intense in the novel song condition compared to the silent condition .
hearing song had no effect on the total number of tspo-labeled cells in al .
in one set of sections, we detected a main effect of sex on the number of tspo-labeled cells , with males having more cells than females in the al, but this effect was not confirmed with a second, independent set of sections from other birds analyzed the same way.
sex had no effect on the average intensity of cell labeling , and there was no significant interaction between sex and song condition with respect to the number of tspo-labeled cells  or the intensity of cell labeling .
we found no significant effects of song condition , sex , or the song condition by sex interaction  on the level of star hybridization in al.
discussion
steroids exert powerful effects on many physiological systems across animals.
in the zebra finch, they are particularly relevant because they can be synthesized within the brain and may have a special role in the sexual differentiation of the developing song system and as rapid signaling molecules in the adult song system.
we took advantage of the newly-released zebra finch genome assembly to investigate the structures of the steroid-related genes.
we assess the diversity of their gene families and putative transcriptional regulatory elements, and contribute additional neuroanatomical expression data regarding the potential for regulated neurosteroidogenesis.
we first used homology searches to identify twenty steroid-related genes in the zebra finch genome assembly.
after comparing zebra finch genome sequence to gene sequences from other species and zebra finch cdna information, we determined that many of the genes were incomplete in the assembly, likely due to technical assembly gaps.
as expected, cross-species analysis of the gene sequences did generally show high homology to genes in other species, and the preservation of major enzyme active sites and overall domain structure of the receptors.
over half of the twenty genes we analyzed from the genome assembly belong to one of two enzyme families, hsd3b and hsd17b.
we were, however, unable to identify the full complement of genes in either of these families as described in mammals.
it is possible that zebra finches do have all of the hsd3b and hsd17b genes that mammals have and that their absence in the assembly simply reflects incomplete genome sequencing.
but based on evolutionary theories of the expansion of these gene families and the phylogenetic analysis of their predicted protein sequences performed here, it is likely that there is a biological, not technical, basis for this difference in gene family sizes.
for example, up to  <dig> different hsd3b genes have been discovered in other animals, each encoding a distinct enzyme.
in mammals, genes for hsd3b1- <dig> encode steroid-metabolizing enzymes that are believed to have arisen from duplication events, in part because they are localized to the same chromosome  <cit> .
hsd3b <dig> is considered primarily a bile-synthesizing enzyme and may have evolved independently from the other hsd3b proteins, as it is localized to a different chromosome as shown here and in mammals  <cit> .
previously in the zebra finch, a cdna for hsd3b <dig> had been cloned, and there was evidence for multiple hsd3b-like transcripts in both the gonads and the brain  <cit> .
the zebra finch hsd3b <dig> gene was mapped to chromosome  <dig>  but our homology searches were unable to identify any other hsd3b genes on this chromosome.
efforts to identify these genes in the chicken genome assembly were also unsuccessful.
our current findings therefore suggest that the hsd3b gene expansion that occurred in mammals did not occur in the avian lineage.
similarly, we could identify only nine of the fourteen mammalian hsd17b genes in the zebra finch genome assembly.
the enzymes of the hsd17b family catalyze reactions with different specificities and affinities for androgens and estrogens, as well as fatty acids, retinoids, and cholesterol  <cit> .
two hsd17b genes, hsd17b <dig> and hsd17b <dig>  were mapped to the z sex chromosome; other studies confirmed the z chromosome localization of hsd17b <dig>  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
this indicates the potential for different levels of expression or activity of these enzymes in males and females, as genes on the zebra finch z chromosome undergo incomplete dosage compensation  <cit> .
this leads to a male-bias in expression of z-linked genes that may be relevant to the organization or function of the steroid-sensitive sexually dimorphic song system  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
interestingly, the zebra finch hsd17b <dig> gene may be distinct from hsd17b <dig> in other species because we were unable to build a complete gene model based on cross-species sequence homology.
the hsd17b <dig> gene and protein was further investigated elsewhere to examine the potential for sex differences in neural expression and activity  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
our inability to identify the five other hsd17b genes described in mammals may simply reflect low sequence homology across species  <cit> .
alternatively, it may be that some of the hsd17b types have evolved in mammals but not birds.
most hsd17b genes are thought to have evolved independently  <cit> ; some of the missing enzymes display some functional redundancies with hsd17b enzymes we could identify in the zebra finch.
perhaps then the complement of hsd17b enzymes in zebra finches can perform all of the necessary reactions represented in the fourteen mammalian enzymes  <cit> .
phylogenetic analysis of the hsd3b and hsd17b predicted protein sequences suggested that the hsd17b zebra finch and mammalian isoforms are similar to each other, but that the bird hsd3b <dig> enzyme may have a slightly different function than that in mammals.
for example, in the hsd17b family, hsd17b types  <dig> and  <dig> were placed on their own branches of the phylogenetic tree.
this is consistent with the fact that the hsd17b <dig> enzyme has a unique set of catalytic structures and that hsd17b <dig> may have sequence structure that is optimized for cholesterol synthesis rather than the steroid or fatty acid metabolism more commonly performed by the other hsd17b enzymes  <cit> .
further, hsd17b types  <dig> and  <dig> were closely associated on the tree, as were types  <dig> and  <dig>  hsd17b <dig> and hsd17b <dig> show higher sequence similarity in humans, too, and it has been previously postulated that hsd17b <dig> and hsd17b <dig> are derived from a common ancestral invertebrate enzyme  <cit> .
on the other hand, phylogenetic modeling of the hsd3b predicted amino acid sequences suggested that the chicken and zebra finch hsd3b type  <dig> proteins are distinct from those in other species.
if all of the steroidogenic conversions performed by the isozymes in mammals are necessary in birds, too, it may be that one of the two avian hsd3b enzymes can catalyze several reactions that are divided amongst hsd3b types 2- <dig> in other animals.
the extent to which different components in the steroidogenic pathway are co-expressed can influence the local steroid concentration and mixture, and the pattern of receptor expression can directly affect the signaling efficacy of those steroids.
we therefore examined, both quantitatively and qualitatively, the profile of pwms in these genes.
using ensembl models and the genome-wide mapping of pwm sites, we identified two transcription factors, nr1h2-rxr and nobox, that were overrepresented either in the entire set of genes or within the nuclear receptor genes, respectively.
both nr1h2-rxr and nobox have known relationships with steroids and may therefore be relevant to steroid synthesis and signaling in the zebra finch  <cit> .
a comparison of predicted pwm sites in cyp <dig> and hsd17b <dig>  genes that code for estradiol synthesizing and metabolizing enzymes, suggested that many regulatory elements are common between the two genes and indeed, some transcription factors have been identified that regulate expression of both genes in other systems such as steroidogenic cell lines  <cit> .
although accurately predicting active transcription factor binding sites is complicated and certainly not definitive, this analysis suggests some transcription factors to target in future experiments that investigate mechanisms of steroid regulation in the zebra finch.
the zebra finch brain produces estradiol; there is ample evidence from a variety of methods that demonstrate that steroidogenic enzymes are present and active within the brain, and that brain slices, which cannot receive steroid precursors from the periphery, synthesize estradiol de novo  <cit>  london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted.
however, the precise pathways and locations of steroid synthesis in the brain are still uncertain.
according to one recent proposal based on in vitro experiments, the star and tspo proteins may work together in a complex that controls the transport of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, effectively controlling the initiation of steroidogenesis  <cit> .
however, we observed minimal overlap in pwm sites associated with these two genes, and only partial neuroanatomical colocalization of the two mrnas when our tspo results here were compared with previous studies of star brain expression  <cit> .
notably, only one of the two transcripts is found in two brain regions relevant to the development and function of the steroid-sensitive song system: area x of the adult male song circuit and the region surrounding the lateral ventricle in p <dig> birds.
these results suggest either that star and tspo coexpression are not absolutely necessary for steroidogenesis in brain tissue, or that neurosteroid synthesis is occurring at sites other than the current foci of research.
as further evidence that transcription of these two genes is controlled differently, tspo showed a strong trend towards regulated transcription in the al after birds experienced song playbacks, consistent with a previous report, but levels of star mrna were unchanged across the same conditions  <cit> .
the current findings emphasize the importance of investigating these genes in the tissue where they function, and indicate that more experiments that delve into the cholesterol transport mechanisms for steroidogenesis in the brain are required.
CONCLUSIONS
we took advantage of the zebra finch genome assembly to integrate genomic and neural investigation of the enzymes and receptors of the estradiol-synthetic pathway.
while mechanisms of steroid synthesis and action are largely conserved across phylogeny, the zebra finch and other birds may have evolved several unique features.
notably, genomic and molecular analysis of two major cholesterol transport proteins suggests that the regulation of steroidogenic initiation may be even more complex than previously.
believed.

 steroids are central to zebra finch neurobiology. they are essential for early developmental organization of the song control system, and they continue to modulate brain and behavior throughout life. although some steroids are supplied to the brain from the periphery, others including estradiol can be synthesized within the brain, either de novo from cholesterol or by metabolism of precursor steroids that originate in the periphery, as shown by evidence from biochemical enzyme activity assays, explant and dissociated culture analysis, molecular identification and neuroanatomical mapping of steroidogenic factors, and in vivo steroid measurements london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. steroids synthesized within the brain, termed "neurosteroids," masculinize the song system and can be rapidly modulated by experience. steroids often act through nuclear receptor transcription factors, which can be abundant in the songbird brain, including in the song control system. thus the zebra finch brain has the capacity to both produce and respond to a variety of steroids that can alter neural function and song behavior. steroids are small molecule hormones derived from cholesterol through a series of enzymatic conversions. the first steroidogenic enzyme resides in the inner mitochondrial membrane, and the rate-limiting step of steroidogenesis is the transport of cholesterol across the outer mitochondrial membrane. two major cholesterol transport proteins, which have been proposed to work in concert as part of a protein complex, are the steroidogenic acute regulatory protein and the kda cholesterol transport protein. steroid synthesis starts with the action of cytochrome p side chain cleavage, which produces pregnenolone. pregnenolone can be converted to either progesterone or dehydroepiandrosterone via the action of cytochrome p 17α-hydroxylase/ lyase or 3β-hydroxsteroid dehydrogenase/Δ Δ isomerase, respectively. androstenedione is produced from progesterone through the activity of hsd3b and from dehydroepiandrosterone through the activity of cyp androstenedione can be converted to testosterone or an estrogen, estrone, via the activity of 17β-hydroxysteroid dehydrogenases or cytochrome p aromatase, respectively. hsd17b can also convert estrone to estradiol, and cyp metabolizes testosterone into estradiol. of note is the fact that multiple hsd3b and hsd17b types exist in other animals, and that several of hsd17b enzymes can use androgens and estrogens as substrates. steroids most commonly act by binding to nuclear receptors that dimerize, translocate to the nucleus, and function as dna-binding transcription factors. the steroid receptors belong to nuclear receptor class and maintain the typical four-domain structure of receptors in the nuclear receptor superfamily. there are four main nuclear receptors for the steroids produced along the estradiol-synthetic pathway: estrogens bind estrogen receptor alpha and beta; androgens bind androgen receptor, and progesterone binds progestin receptors. with the assembly of the zebra finch genome sequence, we are now in position for the first time to assess the organization and regulation of the network of genes that control steroid synthesis and function in the songbird brain. starting with the set of ensembl gene predictions for the zebra finch, we manually curated the assembled models for genes that encode the major components of the estradiol-synthetic pathway and related proteins, as well as four nuclear steroid receptors. we characterized the structure, diversity, and evolutionary conservation of these genes. this analysis led us to examine the neural expression patterns of a cholesterol transport protein in the context of song neurobiology.  characterization of steroidogenic genes in the zebra finch genome assembly we used all available resources and genbank cdna clone entries, and cross-species and functional domain homology searches) to identify the sequences of twenty steroid-related genes in the zebra finch genome assembly. of these, two genes code for cholesterol transport proteins, five genes code for enzymes known to be active in the estradiol-synthetic pathway in songbirds, nine genes code for related hsd3b and hsd17b enzymes, some of which are likely to also be involved in the estrogen-synthetic pathway, and four genes code for the primary nuclear receptors for the steroids produced in the estradiol-synthetic pathway. genomic information for all twenty genes examined. ensembl gene identifiers are listed for the eighteen genes with official models. chromosomal positions are also listed, as are the locations of either alternative mappings or annotated promoter regions. complete coding regions are present for the four receptor genes and for the hsd3b cyp hsd17b hsd17b hsd17b genes, but only partial gene sequences were identified for the other genes in the assembly. in cases where full length cdna clone sequences were available, we could determine that incomplete gene models were due to assembly gaps. some genome assembly gaps result in very small omissions, but others are large. in the case of cyp we identified a genome contig in the trace archive that codes for exon and a portion of the 3'utr that was not included in the current genome assembly. the structures of each of the zebra finch genes are illustrated by their evolutionarily conserved regions when compared to chicken or human genes in zpicture format. hsd3b is a family with up to seven enzymes whose members each derive from unique genes, almost all of which are located along the same chromosome in human and mouse. we used homology searches to try to identify zebra finch genes for all of the known hsd3b genes. hsd3b type is annotated in the genome on chromosome several other types of hsd3b genes from other species show homology to this same location, but blat analysis with the zebra finch hsd3b cdna clone sequence aligns only with this one position, suggesting that there is only one hsd3b gene in the zebra finch assembly. homology searches with the conserved rossmann catalytic domain did not identify any additional putative hsd3b genes on chromosome further supporting the conclusion that the hsd3b gene is the only one on chromosome we did, however, identify an unannotated, incomplete gene for hsd3b type on chromosome by homology searches. the hsd17b enzymes are also a family of similar enzymes. fourteen hsd17b genes have been described to date in humans, and multiple hsd17b genes have also been cloned from or identified in the genomes of other mammals, fish, and chicken. we attempted to find all fourteen genes in the zebra finch genome assembly. we identified genes for hsd17b 1- 10- in the assembly. we confirmed the available hsd17b genomic sequence. two genes, hsd17b and hsd17b were mapped to the z sex chromosome in the genome assembly; the hsd17b mapping is consistent with experimental data that show that this genes is localized to the z chromosome london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. the z chromosome position is of note because sex chromosome genes can be expressed at different levels in males and females and have been proposed to contribute to the sexual differentiation of the zebra finch song system london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. the hsd17b gene model is incomplete; there is one gap in the assembly that may remove several coding exons, but the assembly is uninterrupted for up to kb outside of the boundaries of the gene model. it was therefore possible that more gene coding sequence could be obtained through manual curation of this stretch of the assembly. our attempts to identify additional exons through homology searches were, however, unsuccessful. the hsd17b gene shares exon structure with mammalian and chicken hsd17b genes and contains only intronic assembly gaps; hsd17b was examined in more detail elsewhere. several steroidogenic genes have the potential to form alternatively spliced transcripts or utilize alternate promoters. star, tspo, cyp11a erα, erβ, and ar are predicted to have alternative splice forms in other animals. we identified splice sites in the zebra finch star and tspo genes that give rise to different length transcripts. a model for cyp11a was not completed because of the large gap in genomic sequence, and we were unable to identify splice variants similar to those in mammals in the zebra finch erα, erβ, and ar genes. the first exon of the cyp gene is untranslated and contains promoter elements that regulate tissue-specific transcription e.g.. in zebra finches as in mammals, there are several variants of exon 1; zebra finch brain cyp transcripts almost always use the "1a" exon. though not annotated as such, we identified the exon 1a sequence in the zebra finch genome assembly approximately kb upstream of the first coding exon. pcr confirmation of genomic sequence the ensembl annotation of the genome assembly contains a predicted gene model for hsd17b as way of validating the genomic assembly sequence one of the genes that had not been previously cloned, we used the gene model to design pcr primers and successfully amplified the predicted hsd17b sequence from zebra finch genomic dna. however, we were still unable to obtain the sequence at the 3' end of the gene that is absent in the assembly due to a gap between contigs. phylogeny and selection cross-species multiple sequence alignments for each of the twenty genes demonstrated that functional domains were the most highly conserved regions of the genes and that the overall exon/intron structure of these genes is conserved across the fish, bird, and mammal species investigated. to examine the relationship between genes in the three families examined here, hsd3b, hsd17b, and the nuclear receptors, we constructed unrooted phylogenetic trees of each of these groups, using predicted protein sequences. phylogenetic analysis of mouse, human, zebrafish, chicken and zebra finch hsd3b proteins showed that the zebra finch hsd3b sequence is most similar to that of the chicken, and groups with the hsd3b1- protein sequences of the mouse and human. the hsd3b zebra finch and chicken sequences are more similar to hsd3b proteins in the other species, but showed less homology compared to human than even the zebrafish sequence. to look at the phylogenetic relationship among hsd17b predicted proteins, we used all annotated hsd17b genes in human, mouse, zebrafish, and chicken, plus those we identified in zebra finch. in general, the zebra finch proteins segregated with the same enzyme type in chicken and the other species. this analysis modeled hsd17b and hsd17b proteins as independent branches, not closely related to any other hsd17b enzyme. several other protein types, however, showed more similarity to another. for example, the type and proteins were clustered together on the tree, as were types and we also examined the nuclear receptors. the two types of er segregated together, with the zebra finch erα and erβ showing more similarity to their respective orthologs in other species than to each other. similarly, the ar and pr predicted protein sequences were more similar to each other than to the ers. for all four types of receptors, the zebra finch is most closely related to the chicken sequence. the zebrafish is the most distantly related to the mammalian proteins, and birds in some cases show more similarity to the mammalian proteins than does the platypus. distribution of predicted transcription factor binding sites multiple steroidogenic enzymes need to be present in close proximity to locally synthesize a variety of steroids, including estradiol. the functional connection between these enzymes suggests the possibility that common transcriptional regulatory elements may be shared among the steroidogenic genes to direct their coordinated expression. to pursue this hypothesis, we used a set of position weight matrix predictions for specific transcription factor binding sites generated for the whole zebra finch genome as part of the primary analysis of the assembly. we then tested whether any pwm sites were more abundant in territories surrounding steroid related genes than in the genome as a whole. when we used all available ensembl models, one pwm, nr1h2-rxr, was identified as significantly overrepresented in the gene set with the swan algorithm. no significantly overrepresented pwm sites were identified when subsets of the cholesterol transport and enzymes ensembl models were used. however, when the four nuclear receptor genes were examined, nobox was shown to be significantly overrepresented according to the sllr algorithm. two pairs of functionally related genes - star and tspo, and cyp and hsd17b - had complete coding regions in the assembly, and few intronic gaps. we therefore inventoried and compared the pwm sites predicted to be in these genes to assess the potential for transcriptional co-regulation. comparison of the pwm profiles in star and tspo showed that no sites were common to both genes in the 5'-focused region, and only 23% and 33% of them were shared when the whole gene was taken into account. similarly, when pwm sites for cyp and hsd17b were compared, only one site was shared in the 5'-focused region, while 89% and 61% were shared when the whole gene was considered. all jaspar pwms that were mapped onto the zebra finch genome assembly are listed. blocks in the row denote the presence of the pwm either in the 5'-focused region or across the entire gene territory for star and tspo. all jaspar predictions mapped onto the zebra finch genome assembly are listed. blocks in the row denote the presence of the pwm either in the 5'-focused region or across the entire gene territory for cyp and hsd17b neural expression of tspo the finding that star and tspo shared few pwm sites suggested that these genes may have largely independent transcriptional regulatory mechanisms, which could result in distinct patterns of expression. non-overlapping expression distributions would be inconsistent with recent models that propose that star and tspo work together in a complex that is required for transporting cholesterol across the mitochondrial membranes for the initiation of steroidogenesis. tspo is one of the few components of the estradiol-synthetic pathway not yet examined in the zebra finch brain. we therefore utilized information from cdna and microarray resources developed alongside the genome assembly to confirm that tspo was expressed in the zebra finch brain, test whether or not it co-localized to brain areas that expressed star, and validate results from a zebra finch brain microarray that indicated that its mrna was rapidly regulated after experience. several tspo ests were identified in the zebra finch estima collection of brain cdnas indicating that it was transcribed in the brain. we used one of these clones to perform in situ hybridization to test if tspo expression was neuroanatomically distributed within two functional regions known to express star and other steroidogenic genes: the nuclei of the song control system in adult males, and the cells along the proliferative zone of lateral ventricle in posthatch day birds. in situ hybridization showed that tspo is expressed in all four major song nuclei in adult male birds. the intensity of labeling within these nuclei is not noticeably above that in the surrounding brain, and its hybridization distribution is widespread. in p birds, tspo hybridization occurred at low levels throughout the brain but was strikingly absent from the cell-dense region surrounding the lateral ventricle. hybridization of adjacent sections with sense riboprobe did not show any labeling in adult or p brains, suggesting that the observed hybridization patterns are specific for tspo. we also measured tspo and star expression in the auditory forebrain lobule, a brain area required for song processing and learning. adult birds were either placed in silence or played minutes of novel zebra finch song, and their brains were processed for in situ hybridization. in the al, there was a strong but non-significant trend of song condition on the intensity of tspo labeling in cells because labeling was less intense in the novel song condition compared to the silent condition. hearing song had no effect on the total number of tspo-labeled cells in al. in one set of sections, we detected a main effect of sex on the number of tspo-labeled cells, with males having more cells than females in the al, but this effect was not confirmed with a second, independent set of sections from other birds analyzed the same way. sex had no effect on the average intensity of cell labeling, and there was no significant interaction between sex and song condition with respect to the number of tspo-labeled cells or the intensity of cell labeling. we found no significant effects of song condition, sex, or the song condition by sex interaction on the level of star hybridization in al. discussion steroids exert powerful effects on many physiological systems across animals. in the zebra finch, they are particularly relevant because they can be synthesized within the brain and may have a special role in the sexual differentiation of the developing song system and as rapid signaling molecules in the adult song system. we took advantage of the newly-released zebra finch genome assembly to investigate the structures of the steroid-related genes. we assess the diversity of their gene families and putative transcriptional regulatory elements, and contribute additional neuroanatomical expression data regarding the potential for regulated neurosteroidogenesis. we first used homology searches to identify twenty steroid-related genes in the zebra finch genome assembly. after comparing zebra finch genome sequence to gene sequences from other species and zebra finch cdna information, we determined that many of the genes were incomplete in the assembly, likely due to technical assembly gaps. as expected, cross-species analysis of the gene sequences did generally show high homology to genes in other species, and the preservation of major enzyme active sites and overall domain structure of the receptors. over half of the twenty genes we analyzed from the genome assembly belong to one of two enzyme families, hsd3b and hsd17b. we were, however, unable to identify the full complement of genes in either of these families as described in mammals. it is possible that zebra finches do have all of the hsd3b and hsd17b genes that mammals have and that their absence in the assembly simply reflects incomplete genome sequencing. but based on evolutionary theories of the expansion of these gene families and the phylogenetic analysis of their predicted protein sequences performed here, it is likely that there is a biological, not technical, basis for this difference in gene family sizes. for example, up to different hsd3b genes have been discovered in other animals, each encoding a distinct enzyme. in mammals, genes for hsd3b1- encode steroid-metabolizing enzymes that are believed to have arisen from duplication events, in part because they are localized to the same chromosome. hsd3b is considered primarily a bile-synthesizing enzyme and may have evolved independently from the other hsd3b proteins, as it is localized to a different chromosome as shown here and in mammals. previously in the zebra finch, a cdna for hsd3b had been cloned, and there was evidence for multiple hsd3b-like transcripts in both the gonads and the brain. the zebra finch hsd3b gene was mapped to chromosome but our homology searches were unable to identify any other hsd3b genes on this chromosome. efforts to identify these genes in the chicken genome assembly were also unsuccessful. our current findings therefore suggest that the hsd3b gene expansion that occurred in mammals did not occur in the avian lineage. similarly, we could identify only nine of the fourteen mammalian hsd17b genes in the zebra finch genome assembly. the enzymes of the hsd17b family catalyze reactions with different specificities and affinities for androgens and estrogens, as well as fatty acids, retinoids, and cholesterol. two hsd17b genes, hsd17b and hsd17b were mapped to the z sex chromosome; other studies confirmed the z chromosome localization of hsd17b london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. this indicates the potential for different levels of expression or activity of these enzymes in males and females, as genes on the zebra finch z chromosome undergo incomplete dosage compensation. this leads to a male-bias in expression of z-linked genes that may be relevant to the organization or function of the steroid-sensitive sexually dimorphic song system london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. interestingly, the zebra finch hsd17b gene may be distinct from hsd17b in other species because we were unable to build a complete gene model based on cross-species sequence homology. the hsd17b gene and protein was further investigated elsewhere to examine the potential for sex differences in neural expression and activity london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. our inability to identify the five other hsd17b genes described in mammals may simply reflect low sequence homology across species. alternatively, it may be that some of the hsd17b types have evolved in mammals but not birds. most hsd17b genes are thought to have evolved independently; some of the missing enzymes display some functional redundancies with hsd17b enzymes we could identify in the zebra finch. perhaps then the complement of hsd17b enzymes in zebra finches can perform all of the necessary reactions represented in the fourteen mammalian enzymes. phylogenetic analysis of the hsd3b and hsd17b predicted protein sequences suggested that the hsd17b zebra finch and mammalian isoforms are similar to each other, but that the bird hsd3b enzyme may have a slightly different function than that in mammals. for example, in the hsd17b family, hsd17b types and were placed on their own branches of the phylogenetic tree. this is consistent with the fact that the hsd17b enzyme has a unique set of catalytic structures and that hsd17b may have sequence structure that is optimized for cholesterol synthesis rather than the steroid or fatty acid metabolism more commonly performed by the other hsd17b enzymes. further, hsd17b types and were closely associated on the tree, as were types and hsd17b and hsd17b show higher sequence similarity in humans, too, and it has been previously postulated that hsd17b and hsd17b are derived from a common ancestral invertebrate enzyme. on the other hand, phylogenetic modeling of the hsd3b predicted amino acid sequences suggested that the chicken and zebra finch hsd3b type proteins are distinct from those in other species. if all of the steroidogenic conversions performed by the isozymes in mammals are necessary in birds, too, it may be that one of the two avian hsd3b enzymes can catalyze several reactions that are divided amongst hsd3b types 2- in other animals. the extent to which different components in the steroidogenic pathway are co-expressed can influence the local steroid concentration and mixture, and the pattern of receptor expression can directly affect the signaling efficacy of those steroids. we therefore examined, both quantitatively and qualitatively, the profile of pwms in these genes. using ensembl models and the genome-wide mapping of pwm sites, we identified two transcription factors, nr1h2-rxr and nobox, that were overrepresented either in the entire set of genes or within the nuclear receptor genes, respectively. both nr1h2-rxr and nobox have known relationships with steroids and may therefore be relevant to steroid synthesis and signaling in the zebra finch. a comparison of predicted pwm sites in cyp and hsd17b genes that code for estradiol synthesizing and metabolizing enzymes, suggested that many regulatory elements are common between the two genes and indeed, some transcription factors have been identified that regulate expression of both genes in other systems such as steroidogenic cell lines. although accurately predicting active transcription factor binding sites is complicated and certainly not definitive, this analysis suggests some transcription factors to target in future experiments that investigate mechanisms of steroid regulation in the zebra finch. the zebra finch brain produces estradiol; there is ample evidence from a variety of methods that demonstrate that steroidogenic enzymes are present and active within the brain, and that brain slices, which cannot receive steroid precursors from the periphery, synthesize estradiol de novo london, itoh, lance, ekanayake, oyama, arnold, schlinger: neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-hsd type 4: submitted. however, the precise pathways and locations of steroid synthesis in the brain are still uncertain. according to one recent proposal based on in vitro experiments, the star and tspo proteins may work together in a complex that controls the transport of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, effectively controlling the initiation of steroidogenesis. however, we observed minimal overlap in pwm sites associated with these two genes, and only partial neuroanatomical colocalization of the two mrnas when our tspo results here were compared with previous studies of star brain expression. notably, only one of the two transcripts is found in two brain regions relevant to the development and function of the steroid-sensitive song system: area x of the adult male song circuit and the region surrounding the lateral ventricle in p birds. these results suggest either that star and tspo coexpression are not absolutely necessary for steroidogenesis in brain tissue, or that neurosteroid synthesis is occurring at sites other than the current foci of research. as further evidence that transcription of these two genes is controlled differently, tspo showed a strong trend towards regulated transcription in the al after birds experienced song playbacks, consistent with a previous report, but levels of star mrna were unchanged across the same conditions. the current findings emphasize the importance of investigating these genes in the tissue where they function, and indicate that more experiments that delve into the cholesterol transport mechanisms for steroidogenesis in the brain are required.  we took advantage of the zebra finch genome assembly to integrate genomic and neural investigation of the enzymes and receptors of the estradiol-synthetic pathway. while mechanisms of steroid synthesis and action are largely conserved across phylogeny, the zebra finch and other birds may have evolved several unique features. notably, genomic and molecular analysis of two major cholesterol transport proteins suggests that the regulation of steroidogenic initiation may be even more complex than previously. believed. 
we found very little qualitative overlap in predicted transcription factor binding sites in the genes for two cholesterol transport proteins, the kda cholesterol transport protein and steroidogenic acute regulatory protein. here, we analyzed the zebra finch genome assembly to assess the content, conservation, and organization of genes that code for components of the estrogen-synthetic pathway and steroid nuclear receptors. we present sequence-based analysis of twenty steroid-related genes using the genome assembly and other resources. the diversity of steroidogenic enzymes and receptors may be lower in songbirds than in mammals; we were unable to identify all known mammalian isoforms of the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase families in the zebra finch genome assembly, and not all splice sites described in mammals were identified in the corresponding zebra finch genes. the genes required for estradiol synthesis and action are represented in the zebra finch genome assembly, though the complement of steroidogenic genes may be smaller in birds than in mammals. based on these analyses, we also investigated neural expression of a cholesterol transport protein gene in the context of song neurobiology. coordinated transcription of multiple steroidogenic genes is possible, but results were inconsistent with the hypothesis that star and tspo mrnas are co-regulated. in the zebra finch, estrogenic steroids are particularly interesting because they masculinize the neural circuit that controls singing and their synthesis in the brain is modulated by experience. generally, zebra finch genes showed high homology to genes in other species. we therefore performed in situ hybridization for tspo and found that its mrna was not always detected in brain regions where star and steroidogenic enzymes were previously shown to be expressed. integration of genomic and neuroanatomical analyses will continue to provide insights into the evolution and function of steroidogenesis in the songbird brain. steroids are small molecule hormones derived from cholesterol.
 steroids are small molecule hormones derived from cholesterol. steroids affect many tissues, including the brain. in the zebra finch, estrogenic steroids are particularly interesting because they masculinize the neural circuit that controls singing and their synthesis in the brain is modulated by experience. here, we analyzed the zebra finch genome assembly to assess the content, conservation, and organization of genes that code for components of the estrogen-synthetic pathway and steroid nuclear receptors. based on these analyses, we also investigated neural expression of a cholesterol transport protein gene in the context of song neurobiology.  we present sequence-based analysis of twenty steroid-related genes using the genome assembly and other resources. generally, zebra finch genes showed high homology to genes in other species. the diversity of steroidogenic enzymes and receptors may be lower in songbirds than in mammals; we were unable to identify all known mammalian isoforms of the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase families in the zebra finch genome assembly, and not all splice sites described in mammals were identified in the corresponding zebra finch genes. we did identify two factors, nobox and nr1h2-rxr, that may be important for coordinated transcription of multiple steroid-related genes. we found very little qualitative overlap in predicted transcription factor binding sites in the genes for two cholesterol transport proteins, the kda cholesterol transport protein and steroidogenic acute regulatory protein. we therefore performed in situ hybridization for tspo and found that its mrna was not always detected in brain regions where star and steroidogenic enzymes were previously shown to be expressed. also, transcription of tspo, but not star, may be regulated by the experience of hearing song.  the genes required for estradiol synthesis and action are represented in the zebra finch genome assembly, though the complement of steroidogenic genes may be smaller in birds than in mammals. coordinated transcription of multiple steroidogenic genes is possible, but results were inconsistent with the hypothesis that star and tspo mrnas are co-regulated. integration of genomic and neuroanatomical analyses will continue to provide insights into the evolution and function of steroidogenesis in the songbird brain. 