Genomic and neural analysis of the estradiol-synthetic pathway in the zebra finch.

Abstract

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 3beta-hydroxysteroid dehydrogenase and 17beta-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 18 kDa cholesterol transport protein (TSPO) and steroidogenic acute regulatory protein (StAR). 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.

Full text

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Open Access
RESEARCH ARTICLE
Genomic and neural analysis of the
estradiol-synthetic pathway in the zebra finch
BioMed Central
© 2010 London and Clayton; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and repro-
duction in any medium, provided the original work is properly cited.
Research article
Sarah E London*1,2 and David F Clayton1,2,3
Abstract
Background: 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.
Results: 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 18 kDa cholesterol transport
protein (TSPO) and steroidogenic acute regulatory protein (StAR). 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.
Conclusions: 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.
Background
Steroids are central to zebra finch (Taeniopygia guttata)
neurobiology. They are essential for early developmental
organization of the song control system, and they con-
tinue to modulate brain and behavior throughout life
[1,2]. Although some steroids are supplied to the brain
from the periphery, others including estradiol can be syn-
thesized 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 [1,3-19] London, Itoh, Lance, Ekanayake,
Oyama, Arnold, Schlinger: Neural expression and post-
transcriptional dosage compensation of the steroid meta-
bolic enzyme 17β-HSD type 4: submitted. Steroids syn-
thesized within the brain, termed "neurosteroids,"
masculinize the song system and can be rapidly modu-
lated by experience [1,5,8]. Steroids often act through
nuclear receptor transcription factors, which can be
abundant in the songbird brain, including in the song
* Correspondence: slondon@illinois.edu
1 Institute for Genomic Biology, University of Illinois at Urbana-Champaign,
Urbana, IL, USA
Full list of author information is available at the end of the article

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control system [20-23]. Thus the zebra finch brain has the
capacity to both produce and respond to a variety of ste-
roids that can alter neural function and song behavior.
Steroids are small molecule hormones derived from
cholesterol through a series of enzymatic conversions
(Figure 1). 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 (StAR) and the 18
kDa cholesterol transport protein (TSPO; previously
named peripheral type benzodiazepine receptor) [24-29].
Steroid synthesis starts with the action of cytochrome
P450 side chain cleavage (CYP11A1), which produces
pregnenolone. Pregnenolone can be converted to either
progesterone or dehydroepiandrosterone via the action of
cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17)
or 3β-hydroxsteroid dehydrogenase/Δ5,Δ4 isomerase
(HSD3B1), respectively. Androstenedione is produced
from progesterone through the activity of HSD3B1 and
from dehydroepiandrosterone through the activity of
CYP17. Androstenedione can be converted to testoster-
one or an estrogen, estrone, via the activity of 17β-
hydroxysteroid dehydrogenases (HSD17B) or cyto-
chrome P450 aromatase (CYP19), respectively. HSD17B
can also convert estrone to estradiol, and CYP19 metabo-
lizes testosterone into estradiol. Of note is the fact that
multiple HSD3B and HSD17B types exist in other ani-
mals, and that several of HSD17B enzymes can use
androgens and estrogens as substrates [30-32].
Steroids most commonly act by binding to nuclear
receptors that dimerize, translocate to the nucleus, and
function as DNA-binding transcription factors. The ste-
roid receptors belong to nuclear receptor class 3, and
maintain the typical four-domain structure of receptors
in the nuclear receptor superfamily [33]. There are four
main nuclear receptors for the steroids produced along
the estradiol-synthetic pathway: estrogens bind estrogen
receptor alpha (ERα) and beta (ERβ); androgens bind
androgen receptor (AR), and progesterone binds proges-
tin receptors (PR).
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 (including Ensembl gene
models, alignments to brain expressed sequence tags
(ESTs) and GenBank cDNA clone entries, and cross-spe-
cies and functional domain homology searches) to iden-
tify the sequences of twenty steroid-related genes in the
zebra finch genome assembly (Table 1). Of these, two
genes code for cholesterol transport proteins (StAR and
TSPO), five genes code for enzymes known to be active in
Figure 1 The estradiol-synthetic pathway and nuclear receptors. Cholesterol is the universal steroid substrate. Initiation of steroidogenesis be-
gins with the transport of cholesterol, via the action of the 18 kDa cholesterol transport protein (TSPO) and/or the steroidogenic acute regulatory pro-
tein (StAR), to the first enzyme in the pathway, cytochrome P450 side chain cleavage (CYP11A1). From pregnenolone, four more enzymes are required
to produce estradiol: 3β-hydroxsteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17), 17β-hydroxysteroid dehy-
drogenases (HSD17B), and cytochrome P450 aromatase (CYP19). Cholesterol transport proteins and enzymes are in bold italics. Steroids are in plain
text. The four major nuclear receptors for the three classes of steroids produced along the estradiol-synthetic pathway are in italicized parentheses:
progesterone receptor (PR), estrogen receptor α (ERα), estrogen receptor β (ERβ), and androgen receptor (AR).

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the estradiol-synthetic pathway in songbirds (CYP11A1,
HSD3B1, CYP17, CYP19, HSD17B4), nine genes code for
related HSD3B and HSD17B enzymes, some of which are
likely to also be involved in the estrogen-synthetic path-
way (HSD3B7, HSD17B1,
HSD17B6, HSD17B7,
HSD17B12), and four genes code for the primary nuclear
receptors for the steroids produced in the estradiol-syn-
thetic pathway (ERα, ERβ, AR, and PR).
Complete coding regions are present for the four recep-
tor genes and for the HSD3B1, CYP19, HSD17B4,
HSD17B6, HSD17B11 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 omis-
sions (e.g. StAR is missing ~100 bp of exon 1), but others
are large (e.g. CYP11A1 and CYP17 are missing 5 and 4
exons, respectively). In the case of CYP17, we identified a
HSD17B2,
HSD17B10,
HSD17B3,
HSD17B11,
genome contig in the trace archive (Contig 28.226) that
codes for exon 8 and a portion of the 3'UTR that was not
included in the current genome assembly (Figure 2). 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 (http://
zpicture.dcode.org/; Additional File 1, Figure S1).
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 [31,34-38]. We used homology searches to try
to identify zebra finch genes for all of the known HSD3B
genes. HSD3B type 1 is annotated in the genome on chro-
mosome 1. Several other types of HSD3B genes from
other species show homology to this same location, but
BLAT analysis with the zebra finch HSD3B1 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 Ross-
mann catalytic domain did not identify any additional
Table 1: Summary of steroid-related genes identified in the zebra finch genome assembly.
Ensembl model ID Chromosomal location Alternate location
StAR
ENSTGUG00000004778 chr22:2,790,291-2,803,402
TSPO
ENSTGUG00000012033chr1A:64,600,558-64,604,825
CYP11A1
ENSTGUG00000016385chrUn:111,323,270-111,327,067
HSD3B1
ENSTGUG00000013351chr1:90,752,924-90,765,922
HSD3B7
ENSTGUG00000004368 chr19:4700526-4724260
CYP17
ENSTGUG00000010219chr6:22,934,143-22,936,206
HSD17B1
ENSTGUG00000002682chr27:2,575,793-2,577,330
HSD17B2
ENSTGUG00000004388chr11:2,151,325-2,156,358 chrUn:30,861,771-30,869,026
HSD17B3
chrZ:9,425,091-9,449,191
HSD17B4
ENSTGUG00000001154chrZ:24,424,040-24,517,666
HSD17B6
chrUn:44,543,014-44,547,896
HSD17B7
ENSTGUG00000017081 chr8_random:518,775-526,426
HSD17B10
ENSTGUG00000015458 chrUn:14,031,915-14,032,480
HSD17B11
ENSTGUG00000002260chr4:8,273,553-8,281,927
HSD17B12
ENSTGUG00000010212chr5:19,883,132-19,948,030
CYP19
ENSTGUG00000006993chr10:9,056,446-9,074,753
promoter 1a
chr10:9,078,023-9,078,512
promoter 1b
chr10:9,052,768-9,083,661 chrUn:18,857,683-18,860,112
ERα
ENSTGUG00000011249 chr3:56,288,003-56,500,000
ERβ
ENSTGUG00000012942 chr5:54,900,184-54,948,467
PR
ENSTGUG00000012778chr1:77374938-77451302
AR
ENSTGUG00000002760chr4A: 6,416,086-6,447,982
Genomic information for all twenty genes examined. Ensembl gene identifiers are listed for the eighteen genes with official models (HSD17B3
and HSD17B6 do not have Ensembl gene models). Chromosomal positions are also listed, as are the locations of either alternative mappings
(HSD17B2) or annotated promoter regions (CYP19).

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putative HSD3B genes on chromosome 1, further sup-
porting the conclusion that the HSD3B1 gene is the only
one on chromosome 1. We did, however, identify an
unannotated, incomplete gene for HSD3B type 7 on chro-
mosome 19 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 [32,36,39]. We attempted to
find all fourteen genes in the zebra finch genome assem-
bly. We identified genes for HSD17B 1-4, 6, 7, 10-12 in
the assembly (numbering follows that of the human
genes). We confirmed the available HSD17B1 genomic
sequence (below). Two genes, HSD17B3 and HSD17B4,
were mapped to the Z sex chromosome in the genome
assembly; the HSD17B4 mapping is consistent with
experimental data that show that this genes is localized to
the Z chromosome [40] London, Itoh, Lance, Ekanayake,
Oyama, Arnold, Schlinger: Neural expression and post-
transcriptional dosage compensation of the steroid meta-
bolic enzyme 17β-HSD type 4: submitted. The Z chromo-
some 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 dif-
ferentiation of the zebra finch song system [18,41-45]
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 HSD17B3 gene model is
incomplete; there is one gap in the assembly that may
remove several coding exons, but the assembly is uninter-
rupted for up to 65 kb outside of the boundaries of the
gene model. It was therefore possible that more gene cod-
ing sequence could be obtained through manual curation
of this stretch of the assembly. Our attempts to identify
additional exons through homology searches were, how-
ever, unsuccessful. The HSD17B4 gene shares exon struc-
ture with mammalian and chicken HSD17B4 genes and
contains only intronic assembly gaps; HSD17B4 was
examined in more detail elsewhere [London, Itoh, Lance,
Ekanayake, Oyama, Arnold, Schlinger: Neural expression
and post-transcriptional dosage compensation of the ste-
roid metabolic enzyme 17β-HSD type 4: submitted].
Several steroidogenic genes have the potential to form
alternatively spliced transcripts or utilize alternate pro-
moters. StAR, TSPO, CYP11A1, ERα, ERβ, and AR are
predicted to have alternative splice forms in other ani-
mals. We identified splice sites in the zebra finch StAR
and TSPO genes that give rise to different length tran-
scripts ([46]; Additional File 2, Figure S2). A model for
CYP11A1 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 CYP19
gene is untranslated and contains promoter elements that
regulate tissue-specific transcription e.g. [47-53]. In zebra
finches as in mammals, there are several variants of exon
1; zebra finch brain CYP19 transcripts almost always use
the "1a" exon [48]. Though not annotated as such, we
identified the exon 1a sequence in the zebra finch
genome assembly approximately 7 kb upstream of the
first coding exon (Additional File 2, Figure S2).
PCR confirmation of genomic sequence
The Ensembl annotation of the genome assembly con-
tains a predicted gene model for HSD17B1. As way of val-
idating 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 HSD17B1 sequence from zebra finch
genomic DNA (Additional File 3, Figure S3). 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
Figure 2 Alignments of chicken and zebra finch CYP17 sequenc-
es. Homology of chicken CYP17 gene (gDNA) and several zebra finch
CYP17 sequences, as depicted in zpicture. A) Comparison of chicken
gDNA to itself, to illustrate structure of the gene, B) comparison of a full
length zebra finch CYP17 cDNA clone sequence (Accession numbers
AY313844 and AY313845) to chicken gDNA, C) comparison of the
CYP17 gene sequence obtained from the zebra finch genomic assem-
bly to the chicken gDNA, showing substantial missing sequence, and
D) comparison of zebra finch Contig 28.226 sequence, a contig that
was not incorporated into the assembly, to the 3' end of the CYP17
gene. Note the scale of homology is from 50-100%, and that arrows in
top block denote the chicken gene sequence is oriented so that the 5'
end is on the right.

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that the overall exon/intron structure of these genes is
conserved across the fish, bird, and mammal species
investigated (see Methods for species; nucleotide align-
ments not shown, genomic structural homology to
chicken or human genes shown in Additional File 1, Fig-
ure S1). 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 HSD3B1 sequence is most similar to that of
the chicken, and groups with the HSD3B1-6 protein
sequences of the mouse and human (Figure 3). The
HSD3B7 zebra finch and chicken sequences are more
similar to HSD3B7 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 (Figure 4). In gen-
eral, the zebra finch proteins segregated with the same
enzyme type in chicken and the other species. This analy-
sis modeled HSD17B4 and HSD17B7 proteins as inde-
pendent branches, not closely related to any other
HSD17B enzyme. Several other protein types, however,
showed more similarity to another. For example, the type
3 and 12 proteins were clustered together on the tree, as
were types 2 and 6.
We also examined the nuclear receptors (Additional
File 4, Figure S4). 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 (ERβ and PR) 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 tran-
scriptional regulatory elements may be shared among the
steroidogenic genes to direct their coordinated expres-
sion. To pursue this hypothesis, we used a set of Position
Weight Matrix (PWM) predictions for specific transcrip-
tion factor binding sites generated for the whole zebra
finch genome as part of the primary analysis of the
assembly [54]. 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 18 available Ensembl models, one PWM,
NR1H2-RXR, was identified as significantly (p = 0.045)
overrepresented in the gene set with the SWAN algo-
rithm [54,55]. No significantly overrepresented PWM
sites were identified when subsets of the cholesterol
transport and enzymes Ensembl models were used. How-
ever, when the four nuclear receptor genes were exam-
ined, Nobox was shown to be significantly (p = 0.048)
overrepresented according to the SLLR algorithm [55].
Two pairs of functionally related genes - StAR and
TSPO, and CYP19 and HSD17B4 - had complete coding
regions in the assembly, and few intronic gaps. We there-
fore inventoried and compared the PWM sites predicted
to be in these genes to assess the potential for transcrip-
tional co-regulation (Tables 2 and 3). 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 (7/30 for StAR, 7/21 for
TSPO). Similarly, when PWM sites for CYP19 and
HSD17B4 were compared, only one site was shared in the
5'-focused region, while 89% and 61% (50/56 for CYP19,
50/81 for HSD17B4) were shared when the whole gene
was considered.
Figure 3 Unrooted phylogenetic tree of HSD3B predicted protein
sequences. The two HSD3B genes identified in the zebra finch ge-
nome assembly, HSD3B1 and HSD3B7, show the closest similarity to
the same HSD3B types in the chicken. The HSD3B1 protein sequence
is predicted to be more similar to the HSD3B1-6 mammalian proteins
than the HSD3B7 zebra finch protein is to the mammalian HSD3B7 pro-
tein. Bootstrap values are at branch points. Scale bar denotes substitu-
tion rate. zf = zebra finch, ch = chicken, h = human, m = mouse, dan =
zebrafish.

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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 [26]. 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 devel-
oped 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 rap-
idly regulated after experience [56,57].
Several TSPO ESTs were identified in the zebra finch
ESTIMA collection of brain cDNAs indicating that it was
transcribed in the brain [57]. We used one of these clones
to perform in situ hybridization to test if TSPO expres-
sion was neuroanatomically distributed within two func-
tional 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 1 (P1) birds. In situ
hybridization showed that TSPO is expressed in all four
major song nuclei in adult male birds (Figure 5). The
intensity of labeling within these nuclei is not noticeably
above that in the surrounding brain, and its hybridization
distribution is widespread. In P1 birds, TSPO hybridiza-
tion occurred at low levels throughout the brain but was
strikingly absent from the cell-dense region surrounding
the lateral ventricle (Figure 5). Hybridization of adjacent
sections with sense riboprobe did not show any labeling
in adult or P1 brains, suggesting that the observed
hybridization patterns are specific for TSPO.
We also measured TSPO and StAR expression in the
auditory forebrain lobule (AL; [58]), a brain area required
for song processing and learning [56,59-61]. Adult birds
were either placed in silence or played 30 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 (p = 0.057) because labeling was
less intense in the Novel song condition compared to the
Silent condition (Figure 6). Hearing song had no effect on
the total number of TSPO-labeled cells in AL (p = 0.202).
In one set of sections, we detected a main effect of sex on
the number of TSPO-labeled cells (p = 0.031), with males
having more cells than females in the AL, but this effect
was not confirmed with a second, independent set of sec-
Figure 4 Unrooted phylogenetic tree of HSD17B predicted pro-
tein sequences. We identified nine HSD17B genes in the zebra finch
assembly; these predicted proteins segregated with the same enzyme
type in chicken and the other species. The unrooted tree models some
enzymes (HSD17B4 and 7) as unique branches. Enzyme types predict-
ed to be evolutionarily related (HSD17B3 and 12, and HSD17B2 and 6),
are shown to be preserved in the zebra finch. Bootstrap support values
are at branch points. Scale bar denotes substitution rate. zf = zebra
finch, ch = chicken, h = human, m = mouse, dan = zebrafish, iso = iso-
form.

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Table 2: Qualitative listing of PWM predictions compiled from StAR and TSPO genes.
StARTSPOStARTSPOStARTSPOStARTSPO
ArNFYA
Arnt NHLH1
?
Arnt-Ahr
??
Nkx2-5
Bapx1
??
NKX3-1
??
cEBP
??
Nobox
?
CREB1
?
NR1H2-RXR
Ddit3-Cebpa
???
NR2F1
E2F1NR3C1
??
ELF5Pax2
ELK1 Pax4
En1Pax5
?
ESR1
??
Pax6
?
ETS1
??
Pbx
Evi1Pdx1
?
Fos
?
PPARG
?
Foxa2 PPARG-RXRA
FOXC1Prrx2
?
Foxd3 REL
FOXF2
??
RELA
??
FOXI1REST
??
FOXL1Roaz
??
Foxq1 RORA_1
GABPA
?
RORA_2
Gata1 RORA1
GATA2RREB1
??
GATA3RUNX1
??
GfiRUSH1-alfa
HAND1-TCF3
??
RXRA-VDR
HLF RXR-VDR
HNF1ASox17
??
HNF4Sox5
?
Hox11-CTF1SOX9
IRF1
???
SP1
??
IRF2SPI1
Klf4
??
SPIB
??
Lhx3
???
Spz1
MafB
??
SRF
MAXSRY
MEF2AStaf
?
MIZFSTAT1
MybT
MYC-MAXTAL1-TCF3

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tions from other birds analyzed the same way. Sex had no
effect on the average intensity of cell labeling (p = 0.190),
and there was no significant interaction between sex and
song condition with respect to the number of TSPO-
labeled cells (p = 0.387) or the intensity of cell labeling (p
= 0.173). We found no significant effects of song condi-
tion (cell number p = 0.341; labeling intensity p = 0.538),
sex (cell number p = 0.189; labeling intensity p = 0.099),
or the song condition by sex interaction (cell number p =
0.564; labeling intensity p = 0.421) on the level of StAR
hybridization in AL.
Discussion
Steroids exert powerful effects on many physiological sys-
tems across animals. In the zebra finch, they are particu-
larly relevant because they can be synthesized within the
brain and may have a special role in the sexual differentia-
tion 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 addi-
tional neuroanatomical expression data regarding the
potential for regulated neurosteroidogenesis.
We first used homology searches to identify twenty ste-
roid-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 assem-
bly 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 iden-
tify the full complement of genes in either of these fami-
lies 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 pre-
dicted 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 7 different
HSD3B genes have been discovered in other animals,
each encoding a distinct enzyme. In mammals, genes for
HSD3B1-6 encode steroid-metabolizing enzymes that are
believed to have arisen from duplication events, in part
because they are localized to the same chromosome [35-
37]. HSD3B7 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 [38]. Previ-
ously in the zebra finch, a cDNA for HSD3B1 had been
cloned, and there was evidence for multiple HSD3B-like
transcripts in both the gonads and the brain [3]. The
zebra finch HSD3B1 gene was mapped to chromosome 1,
but our homology searches were unable to identify any
other HSD3B genes on this chromosome. Efforts to iden-
tify these genes in the chicken genome assembly were
also unsuccessful. Our current findings therefore suggest
that the HSD3B gene expansion that occurred in mam-
mals 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 [30,39,62]. Two HSD17B genes,
HSD17B3 and HSD17B4, were mapped to the Z sex chro-
mosome; other studies confirmed the Z chromosome
localization of HSD17B4 [40] London, Itoh, Lance, Eka-
nayake, Oyama, Arnold, Schlinger: Neural expression
and post-transcriptional dosage compensation of the ste-
MycnTBP
Myf
?
TCF1
MZF1_1-4
??
TEAD
??
MZF1_5-13
??
TFAP2A
?
NFIL3TP53
???
NF-kappaB
?
USF1
NFKB1
?
YY1
NF-YZEB1
?
ZNF42_1-4
??
All 101 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 (filled in blocks) or across the entire gene territory (open blocks) for StAR and TSPO.
Table 2: Qualitative listing of PWM predictions compiled from StAR and TSPO genes. (Continued)

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Table 3: Qualitative listing of PWM predictions compiled from CYP19 and HSD17B4 genes.
CYP19 HSD17B4CYP19 HSD17B4CYP19 HSD17B4 CYP19 HSD17B4
Ar NFYA
???
Arnt
??
NHLH1
??
Arnt-Ahr
???
Nkx2-5
??
Bapx1
??
NKX3-1
?
cEBP
??
Nobox
?
CREB1
??
NR1H2-RXR
???
Ddit3-Cebpa
???
NR2F1
?
E2F1
??
NR3C1
??
ELF5
?
Pax2
?
ELK1
??
Pax4
??
En1
??
Pax5
??
ESR1
??
Pax6
??
ETS1
???
Pbx
??
Evi1
??
Pdx1
??
Fos
?
PPARG
??
Foxa2
?
PPARG-
RXRA
???
FOXC1
?
Prrx2
??
Foxd3REL
??
FOXF2
???
RELA
?
FOXI1
??
REST
??
FOXL1
??
Roaz
???
Foxq1
??
RORA_1
???
GABPA
???
RORA_2
??
Gata1
??
RORA1
?
GATA2
??
RREB1
?
GATA3
??
RUNX1
???
Gfi
??
RUSH1-alfa
?
HAND1-TCF3
??
RXRA-VDR
???
HLF
?
RXR-VDR
???
HNF1A
?
Sox17
?
HNF4
?
Sox5
?
Hox11-CTF1
?
SOX9
??
IRF1
???
SP1
??
IRF2
?
SPI1
?
Klf4
?
SPIB
??
Lhx3
???
Spz1
???
MafB
??
SRF
???
MAXSRY
??
MEF2A
?
Staf
MIZF
?
STAT1
??
Myb
???
T
??
MYC-MAX TAL1-TCF3
??

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roid 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 [18,41]. 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 [18,41-45] London, Itoh, Lance,
Ekanayake, Oyama, Arnold, Schlinger: Neural expression
and post-transcriptional dosage compensation of the ste-
roid metabolic enzyme 17β-HSD type 4: submitted.
Interestingly, the zebra finch HSD17B3 gene may be dis-
tinct from HSD17B3 in other species because we were
unable to build a complete gene model based on cross-
species sequence homology. The HSD17B4 gene and pro-
tein was further investigated elsewhere to examine the
potential for sex differences in neural expression and
activity [45] London, Itoh, Lance, Ekanayake, Oyama,
Arnold, Schlinger: Neural expression and post-transcrip-
tional dosage compensation of the steroid metabolic
enzyme 17β-HSD type 4: submitted. Our inability to
identify the five other HSD17B genes described in mam-
mals may simply reflect low sequence homology across
species [30,39,62]. 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 [30,39,62,63]; 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
[32,39,63-66].
Phylogenetic analysis of the HSD3B and HSD17B pre-
dicted protein sequences suggested that the HSD17B
zebra finch and mammalian isoforms are similar to each
other, but that the bird HSD3B7 enzyme may have a
slightly different function than that in mammals. For
example, in the HSD17B family, HSD17B types 4 and 7
were placed on their own branches of the phylogenetic
tree. This is consistent with the fact that the HSD17B4
enzyme has a unique set of catalytic structures and that
HSD17B7 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 [32,67-70]. Further, HSD17B types 3
and 12 were closely associated on the tree, as were types 2
and 6. HSD17B3 and HSD17B12 show higher sequence
similarity in humans, too, and it has been previously pos-
tulated that HSD17B2 and HSD17B6 are derived from a
common ancestral invertebrate enzyme [71-74]. On the
other hand, phylogenetic modeling of the HSD3B pre-
dicted amino acid sequences suggested that the chicken
and zebra finch HSD3B type 7 proteins are distinct from
those in other species. If all of the steroidogenic conver-
sions performed by the isozymes in mammals are neces-
sary in birds, too, it may be that one of the two avian
HSD3B enzymes can catalyze several reactions that are
divided amongst HSD3B types 2-6 in other animals.
The extent to which different components in the ste-
roidogenic 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 overrepre-
sented 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 signal-
ing in the zebra finch [75,76]. A comparison of predicted
PWM sites in CYP19 and HSD17B4, genes that code for
estradiol synthesizing and metabolizing enzymes, sug-
gested that many regulatory elements are common
between the two genes and indeed, some transcription
factors have been identified that regulate expression of
Mycn
?
TBP
???
Myf
?
TCF1
?
MZF1_1-4
??
TEAD
MZF1_5-13
??
TFAP2A
??
NFIL3
??
TP53
NF-kappaB
?
USF1
???
NFKB1YY1
???
NF-Y
???
ZEB1
ZNF42_1-4
??
All 101 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 (filled in blocks) or across the entire gene territory (open blocks) for CYP19 and HSD17B4.
Table 3: Qualitative listing of PWM predictions compiled from CYP19 and HSD17B4 genes. (Continued)

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both genes in other systems such as steroidogenic cell
lines [24,77-79]. Although accurately predicting active
transcription factor binding sites is complicated and cer-
tainly not definitive, this analysis suggests some tran-
scription 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
Figure 5 TSPO in situ hybridization in adult song system and along P1 lateral ventricles. In situ hybridization with antisense-configured ribo-
probes showed that TSPO is expressed within all four major song nuclei - LMAN, Area X, HVC, and RA - in adult male brain. Antisense-configured ribo-
probes do not label the cells surrounding the lateral ventricle in P1 brains. Song nuclei and region of the lateral ventricle are identified with arrows.
Hybridization of adjacent brain sections with negative control sense-configured riboprobes shows very low levels of label, suggesting specific labeling
with antisense probes. High magnification images of each region of interest show cellular labeling. Scale bars for whole brain images = 1 mm; scale
bar for high magnification images = 500 μm.

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brain, and that brain slices, which cannot receive steroid
precursors from the periphery, synthesize estradiol de
novo [1,3-15,18,19,45] London, Itoh, Lance, Ekanayake,
Oyama, Arnold, Schlinger: Neural expression and post-
transcriptional dosage compensation of the steroid meta-
bolic 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 pro-
posal 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, effec-
tively controlling the initiation of steroidogenesis [26].
However, we observed minimal overlap in PWM sites
associated with these two genes, and only partial neuro-
anatomical colocalization of the two mRNAs when our
TSPO results here were compared with previous studies
of StAR brain expression [3,19]. 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 P1 birds.
These results suggest either that StAR and TSPO coex-
pression 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 con-
trolled 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 [56]. 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 path-
way. 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 regula-
tion of steroidogenic initiation may be even more com-
plex than previously. believed.
Methods
All procedures that involve animals were approved by the
University of Illinois, Urbana-Champaign Institutional
Animal Care and Use Committee.
Identification of steroidogenic genes
Whenever possible (StAR, TSPO, CYP11A1, HSD3B1,
CYP17, CYP19, HSD17B4, ERα, ERβ, AR, PR), we used
existing zebra finch cDNA sequence to do homology
searches of the zebra finch genome trace archive http://
www.ncbi.nlm.nih.gov/genome/seq/BlastGen/Blast-
Gen.cgi?taxid=59729 and the genome assembly http://
genome.ucsc.edu. When no zebra finch sequence was
available, we used homology searches of the assembly to
identify genes with available chicken sequences
(HSD3B7, HSD17B2), or human sequence if a chicken
gene model was not available (HSD17B1, HSD17B3,
HSD17B6, HSD17B7,
HSD17B12). We also used sequence from specific con-
served functional domains for homology searches to
attempt to identify enzyme genes. All genomic sequences
were corrected using cDNA and protein information
from zebra finch, chicken, mouse, and human in Apollo
Genome Annotation Curation Tool [80]. We used zpic-
ture http://zpicture.dcode.org/ to visualize conservation
of gene structures and completeness of genomic
sequences.
HSD17B10, HSD17B11,
PCR with genomic DNA
We used PCR with genomic DNA to validate one of the
Ensembl gene models for a gene that had not been cloned
previously. DNA was extracted from zebra finch tissue
samples and purified with DNeasy kit using manufac-
ture's instructions (Qiagen, Valencia, CA). PCR was per-
formed with HotStarTaq (Qiagen) for 55 cycles, products
were gel extracted (Gel Extraction, Qiagen), and ligated
into PCRScript Amp plamsid (Stratagene, La Jolla, CA).
Clones were sequenced on both strands and their identi-
ties were confirmed by BLAST homology searches. We
Figure 6 TSPO in situ hybridization in the adult AL after song
playback experience. A) Representative images of AL (teardrop-
shaped brain area in center of images) from males and females that
heard either Silence or Novel song. B) The intensity of TSPO labeling
showed a non-significant trend (p = 0.057) towards a decrease in birds
that heard novel song. AL = auditory forebrain lobule, HP = hippocam-
pus. Scale bar = 500 μm.

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then aligned the clone sequence to that of the genome
assembly to test for sequence matches.
Phylogenetic analysis
Predicted amino acid sequences for zebra finch genes
were aligned with a combination of human, mouse,
chicken, platypus, and zebrafish in MAFFT [81,82], and
edited when necessary
www.mbio.ncsu.edu/BioEdit/bioedit.html.
neighbor-joining phylogenic trees of three gene families,
HSD3B, HSD17B, and the nuclear receptors were con-
structed with MEGA4 [83,84]. Bootstrap support was
performed with 1000 replications; consensus trees are
shown.
in BioEdit http://
Unrooted
Distribution of transcription factor binding motifs
We performed a statistical test for overrepresented PWM
sites within the territories of the Ensembl gene models
using the JASPAR http://jaspar.cgb.ki.se/ set of non-
redundant and curated PWMs [54]. This tested if any of
the JASPAR PWM sites were more abundant in the ste-
roid-related gene models compared to their distribution
across the whole genome assembly. We investigated sev-
eral sets of genes: all of the 18 genes from our whole set of
20 that had Ensembl models, just StAR and TSPO, just
CYP19 and HSD17B4, all of the predicted components of
the estradiol-synthetic pathway (StAR, TSPO, CYP11A1,
CYP17, HSD3B1, HSD17B1, HSD17B2, HSD17B4,
CYP19), and the four nuclear receptors (ERα, ERβ, AR,
and PR).
We also utilized the JASPAR PWM predictions across
the whole genome assembly to perform a purely qualita-
tive description of two pairs of functionally related genes
that had complete coding regions and few gaps in the
assembly: StAR and TSPO, and CYP19 and HSD17B4
[54]. We performed this inventory on two regions of the
gene. One, focused on the putative 5' proximal regulatory
region, captured the PWMs that were 5 kb upstream and
2 kb downstream of the 5'-most exon; the second,
designed to identify regulatory elements scattered across
the entire gene region, captured the PWMs within the
boundaries of the entire gene plus those contained within
5 kb both 5' and 3' to the predicted gene model. This
whole gene analysis was done with two caveats. To be
conservative about what we included in this analysis, we
did not extend the PWM characterization across an
assembly gap unless there was cDNA evidence that the
gene indeed spanned the gap, nor did we include PWMs
that were within 5 kb of the gene of interest if an adjacent
gene was predicted to fall within that boundary. In those
cases, we only describe PWM that are non-overlapping
with the adjacent gene.
In situ hybridization
The region along the lateral ventricles at P1 and the song
control nuclei of adult males were previously identified to
be brain areas that express genes in the estradiol-syn-
thetic pathway [3,9,19,85]. Here, we performed in situ
hybridization for TSPO on P1 (n = 3 males, n = 3 females)
and adult male (n = 3) brains to determine whether or not
it was also expressed in these two regions. P1 birds were
removed from their nests in a breeding aviary and adult
males were removed from single-sex holding aviaries
located in a room that housed both males and females.
Within 3 minutes of removal from their housing environ-
ment, birds were sacrificed and brains were flash frozen
and sectioned to 20 μm in the coronal plane. Sex of the P1
birds was confirmed by visual inspection of the gonads.
The TSPO DIG-labeled riboprobe was in vitro tran-
scribed using a zebra finch brain EST from the Songbird
Neurogenomic Initiative's ESTIMA collection (GenBank
Accession number DV952129) as the template [57]. This
EST is predicted to contain the entire open reading from
of the TSPO transcript and is highly specific for TSPO (e
score = 1e-180). Sense riboprobes were also synthesized
as negative hybridization controls. For every slide hybrid-
ized with the antisense probe, an adjacent slide was
hybridized with the sense riboprobe for control. Labeling
patterns on brain sections hybridized with antisense or
sense probes were compared to evaluate if specific
hybridization occurred along the lateral ventricles and in
major song nuclei.
In situ hybridization was performed for all slides as fol-
lows [86]. The tissue was postfixed for 15 minutes in 4%
paraformaldehyde (pH 7.4), then washed 4 × 5 minutes in
0.02 M KPBS (pH 7.4). The slides were equilibrated in
TEA and treated with 0.25% acetic anhydride in TEA for
10 minutes, then rinsed in 2× SSC. Finally, the sections
were dehydrated through increasing concentrations of
ethanol. Hybridization with 500 ng of riboprobe (hybrid-
ization solution: 50% formamide, 2× SSPE [pH 7.4], 2 mg/
ml tRNA, 1 mg/ml bovine serum albumin, 300 ng/ml
polyadenylic acid, 0.1 M dithiothreitol) proceeded for 3
hours at 65°C. After hybridization, slides were rinsed in
2× SCC at room temperature, then washed in 50% forma-
mide/1× SSC for 10 minutes at 65°C with 2 × 20 minute
final high stringency washes in 0.1 X SSC, all at 65°C.
Slides were then placed in blocking buffer (1% blocking
reagent (Roche Applied Science, Indianapolis, IN) in buf-
fer A (100 mM Tris [pH 7.5], 150 mM NaCl, and 0.05%
Triton X-100) at 4°C overnight. The next day, slides were
rinsed in buffer A and incubated for 3 hours at room tem-
perature with alkaline phosphatase conjugated anti-DIG
antibody (Roche Applied Science) diluted 1:5000 in
blocking buffer. Slides were washed 4 × 5 minutes in buf-
fer A, then in buffer B (100 mM Tris [pH 9.5], 100 mM
NaCl, and 50 mM MgCl2) for 10 min. Color detection was
performed with BCIP/NBT alkaline phosphatate sub-
strate (Sigma-Aldrich, St. Louis, MO).
A previous study reported that TSPO mRNA showed a
relatively rapid (within 30 minutes) decrease in levels in

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the AL after an adult male bird heard novel conspecific
song [56]. Therefore, we also used in situ hybridization to
confirm this finding and extend the investigation of
TSPO expression in the AL to females. We also per-
formed in situ hybridization for StAR using a clone previ-
ously described to compare TSPO and StAR mRNA
changes after song playback experience [3]. We used
adult male and adult female birds (n = 3 each sex) and
exposed them to a standard song playback paradigm.
Birds, who had been group-housed in single sex aviares in
a room that housed both males and females, were
removed from this communal setting and placed individ-
ually in a sound isolation chamber overnight. The next
morning, birds were either played a zebra finch song
unfamiliar to the bird for 30 minutes (one song bout
every 10 seconds, for a total of 180 bouts; "Novel song")
or left in silence ("Silence"), and sacrificed immediately
after song playback ceased (or, for the Silence birds,
within 30 minutes of the Novel song birds). To ensure
that the song used for playback was unfamiliar to the
experimental birds, we used a song recorded more than
10 years ago from a bird that was no longer present in the
aviary population. Brains were extracted, flash frozen,
and sectioned in the sagittal plane to 12 μm. In situ
hybridization was performed on three AL sections ~340
μm apart to represent the medial-lateral extent of the
auditory forebrain for each bird. Sections were hybridized
with the TSPO and StAR riboprobes under the condi-
tions described above [86].
Low magnification images of the adult male coronal
brain sections were captured with a Nikon slide scanner
(Super Coolscan 8000ED; Nikon Inc., Melville, NY).
Images of P1 brains, individual song nuclei, and AL-con-
taining brain sections were digitally captured on an Axio-
Imager A1 Microscope (Carl Zeiss Microimaging,
Thornwood, NJ) with a CCD camera (Microfire; Optron-
ics, Goleta, CA). Representative in situ hybridization
images shown were modified for contrast to highlight lev-
els and areas of gene expression; modifications were per-
formed equally for all images of a set.
The number and intensity of labeled cells in the AL was
quantified within the entire AL and the adjacent non-
auditory hippocampus (HP) for control purposes using
ImageProPlus 4.5.1 (MediaCybernetics; Bethesda, MD)
[87]. To remove potential differences in AL staining pat-
tern across slides or from non-specific background stain-
ing, each AL value was normalized to the HP value on the
same section. We then calculated a "total AL" value by
summing the normalized values for all the three AL sec-
tions obtained in each bird. Normalized AL values were
analyzed with two-way ANOVA (SPSS, Chicago, IL; α =
0.05) to test for effects of sex, song condition, and the sex
by song condition interaction.
Additional material
Authors' contributions
SEL designed, performed, and interpreted the experiments and analyses, and
wrote the manuscript. DFC participated in the design of experiments and anal-
yses and contributed to manuscript writing. Both authors read and approved
this manuscript.
Acknowledgements
We thank Chris Balakrishnan and James Lee for help with the phylogenetic
analysis, Lisa Stubbs and Saurabh Sinha for consultations on the transcription
factor binding analysis and access to the PWM statistical comparison program,
and Barney Schlinger for helpful comments on the manuscript. This study was
supported by NIH RO1 NS045264 to DFC and NINDS Postdoctoral NRSA
NS554132 to SEL.
Author Details
1Institute for Genomic Biology, University of Illinois at Urbana-Champaign,
Urbana, IL, USA, 2Beckman Institute of Advanced Science and Technology,
University of Illinois at Urbana-Champaign, Urbana, IL, USA and 3Department
of Cell and Developmental Biology, University of Illinois at Urbana-Champaign,
Urbana, IL, USA
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system. Ann N Y Acad Sci 2004, 1016:540-559.
3. London SE, Monks DA, Wade J, Schlinger BA: Widespread capacity for
steroid synthesis in the avian brain and song system. Endocrinology
2006, 147:5975-5987.
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Additional file 1 Figure S1 - Conservation of gene structure for nine-
teen zebra finch genes, represented by zpictures. Zebra finch
sequences from the genomic assembly, and when available, full length
cDNA clone sequences, aligned to annotated genes to show evolutionarily
conserved regions. When annotated, chicken genes were used as the base
gene, otherwise the human gene was used. Percent sequence homology
of regions above 50% similarity is depicted by the height of the colored
bars. Direction of arrows at the top of each gene's alignment indicates the
5' to 3'direction of the base sequence.
Additional file 2 Figure S2 - Gene models showing alternative tran-
scripts. Images of gene models corrected in Apollo Genome Annotation
Curation Tool, showing predicted alternative transcripts based on evidence
in other species. Position on the chromosome is depicted by the scale
across the bottom of each gene. Dark blue portion denotes coding
sequence, light blue denotes untranslated regions, green and red stripes
represent position of translational start and stop codons, respectively.
Additional file 3 Figure S3 - Alignment of the HSD17B1 sequence
from the zebra finch assembly and from the clone obtained from PCR
amplification from zebra finch genomic DNA. Sequence alignment of
the genomic HSD17B1 gene from the assembly aligned in ClustalX to the
clone amplified directly from zebra finch DNA, validating a portion of this
sequence.
Additional file 4 Figure S4 -Unrooted phylogenetic tree of nuclear
receptor predicted protein sequences. Unrooted phylogenetic trees of
the four nuclear receptor types examined model the zebra finch ER recep-
tor subtypes as more similar to each other than AR and PR, which are more
closely related to each other. Cross-species positioning within each recep-
tor type show evolutionary changes between birds and mammals. Boot-
strap support values are at branch points. Scale bar denotes substitution
rate. zf = zebra finch, ch = chicken, h = human, m = mouse, dan = zebrafish,
platy = platypus, iso = isoform.
Received: 16 December 2009 Accepted: 1 April 2010
Published: 1 April 2010
This article is available from: http://www.biomedcentral.com/1471-2202/11/46© 2010 London and Clayton; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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