BACKGROUND
sexual differences that arise in the brain during embryonic development underlie sex-specific reproductive behavior in adults of vertebrate species.
better knowledge of the mechanisms behind sexual differentiation of the nervous system can contribute to the understanding of the brain's functions and its susceptibility to disease  <cit> , and is critically needed in the context of developmental neurotoxicity and chemically induced disruption of the neuroendocrine system  <cit> .
for a long time the idea prevailed that somatic tissues in the embryo are gender neutral until the gonads become hormonally active following their sex-specific differentiation, a process presumed to be governed by cell-autonomous programs genetically determined by the sex chromosomes  <cit> .
however, several recent studies indicate that sex-specific neuronal characteristics may be influenced by genetic mechanisms preceding or occurring in parallel with the effects produced by the gonadal hormones  <cit> .
that the genetic sex of neurons contributes to the process of sexual differentiation is illustrated by studies of the neural song circuit in a gynandromorphic zebra finch, where genetically male and female brain cells develop differently in the presence of the same levels of circulating gonadal hormones  <cit> .
birds, in which males are homogametic  and females heterogametic , provide attractive models for the study of normal and disturbed sex-related neurobehavioral development  <cit> .
the recent advent of chicken expressed sequence tag  clone collections  <cit>  and the release of the first draft sequence of the chicken genome  <cit>  has paved the way for genome-wide studies of sexual differentiation in the chicken.
in the chicken, the indifferent embryonic gonad rudiments differentiate morphologically around embryonic day   <dig>   <cit> , whereas the expression of enzymes involved in steroidogenesis can be detected as early as ed <dig>  <cit> , and estrogen receptor  mrna is detectable in the male and female urogenital system from ed <dig>   <cit> .
the earliest signs of sexually dimorphic expression of genes related to gonadal hormones and steroidogenesis, however, have been detected at ed <dig> when mrna for anti-müllerian-hormone is more abundant in males  <cit> , and aromatase  <cit>  in females.
in the present study, we used cdna microarrays to investigate if there are gene expression differences between the male and female embryonic brain  that may be independent of morphological differentiation of the gonads.
we report evidence for sex-related gene expression differences from ed <dig>  through ed <dig>  for several genes located on sex chromosomes.
we propose that intrinsic genetic mechanisms are involved in sexual differentiation of the chicken brain, and that sex-linked genes may play key roles.
RESULTS
identification of gene expression differences between male and female embryonic chicken brains
to study sex-specific gene expression in the developing brain of chicken embryos during stages around morphological differentiation of the gonads, total rna extracted from male and female heads on ed <dig> and ed <dig>  and brains on ed <dig> and ed <dig>  was subjected to replicated microarray analysis using cdna microarrays containing approximately  <dig>  est clones.
for each sex, we generated a total of  <dig> biologically independent samples, comprising either individuals  or pools of four individuals .
these  <dig> samples were analyzed on  <dig> array slides, with technical replication as shown in figure  <dig>  using an empirical bayes moderated t-test  <cit> , and adjusted p-values according to benjamini and hochberg  <cit> , we found  <dig> clones with significant differential expression  across all investigated embryonic stages .
for most of these clones, the log <dig> female/male ratios were relatively similar across the studied developmental stages .
importantly, for a majority of these clones the expression differences between males and females were present at the earliest embryonic stage , i.e.
well before morphological gonadal differentiation.
characterization of differentially expressed genes
the  <dig> identified clones  were found to represent  <dig> genes,  <dig> of which were more highly expressed in females, and  <dig> in males .
among the 'female genes', three are located on the female-specific w chromosome , one on the z chromosome  and  <dig> on autosomes, whereas for four genes the location is unknown .
among the  <dig> 'male genes',  <dig> are located on the z chromosome  and  <dig> on autosomes, and for another  <dig> genes the location is unknown .
hence, given the fraction of z-linked clones present in the microarray , genes annotated as being located on the z chromosome appeared overrepresented among the differentially expressed genes .
as the chicken w chromosome is still poorly characterized, with many gaps in the sequence, the same conclusion is less straightforward to draw about w-linked genes .
the differential expression of selected genes on ed <dig>  was investigated by real-time pcr of individual samples  and the results  are presented below in the context of the chromosomal locations of these genes.
w-linked genes
since the w chromosome is present only in female chickens, the expression of w-linked genes was reflected in the microarray analysis by high log <dig> female/male ratios , and in the real-time pcr analysis by even higher  ratios .
an exception from this was spinw, for which the exclusive expression in females was not indicated by similarly high log <dig> female/male ratios in the microarray analysis .
however, unlike what is known for the other w-linked genes reported here, spinw has a counterpart  on the z chromosome .
spinw was found by real-time pcr to be expressed in the female head on ed <dig> .
also the expression of wpkci- <dig>  for which female expression at all stages was detected by seven clones in the microarray analysis , was verified in the ed <dig> female head by real-time pcr.
the sequence of the est clone wla084d <dig> shows no similarity to any known gene or putative protein.
it is part of the unigene entry gga.
<dig> and the tigr gene index entry tc <dig> , located about  <dig> nucleotides from a chicken sequence similar  to the ube2r <dig> mouse sequence .
using primers designed to amplify this putative ube2r <dig> homolog, we did not detect any significant differential expression in embryonic chicken heads.
however, using primers amplifying the wla084d <dig> sequence, we found expression in heads from 4-day-old female embryos .
it remains to determine whether this est may represent a novel w-linked gene , which we provisionally suggest calling 'avian brain w-linked transcript' .
z-linked genes
whereas half of the genes  suggested from the microarray study to be more highly expressed in males are located on the z chromosome, only one clone with female-enhanced expression is known to be z-linked .
we found  <dig> nucleotides of this clone  to align with high similarity  to a partial sequence of the genbank entry with accession ab <dig> , representing a z-linked male hypermethylated  region.
this sequence is thought to be specifically transcribed into a non-coding nuclear rna in females  <cit> .
using primers designed to discriminate between the rja001b <dig> and ab <dig> sequences , we found female-specific transcription for both of these mhm genomic regions .
real-time pcr analysis confirmed the male-enhanced microarray expression for all z-linked genes examined .
we also found significantly higher expression in males  for two z-linked genes , but not for a third , that were not represented in the microarray but are functionally related  to other genes identified as differentially expressed .
a close to two-fold higher expression in males was observed for most of the z-linked genes in the real-time pcr analysis , suggestive of gene dosage.
exceptions were btf2-p <dig>  cdk <dig> and hmgcoa.
no differential expression was detected by real-time pcr for three genes  that were randomly selected among the non-differentially expressed z-linked genes in the microarray experiment , supporting that only certain z-linked genes are more highly expressed in males.
a fourth z-linked gene  randomly selected among genes that were not differentially expressed in the microarray analysis was revealed by real-time pcr to be more highly expressed in males .
however, examination of the corresponding microarray clone  by sequencing showed that the spotted cdna contained vector sequence only.
genes located on autosomes or having unknown chromosomal location
differential expression could be confirmed by real-time pcr for only three genes out of  <dig> analyzed genes with autosomal  or unknown location , all with higher expression in males .
errors in the microarray were uncovered for  <dig> of  <dig> genes that could not be confirmed by real-time pcr .
for example, the microarray spots thought to represent the male-enhanced autosomal genes aatf and acad <dig> were found to contain est clones representing the z-linked genes rps <dig> and plaa, respectively .
two of the microarray clones showing female-enhanced expression that could not be confirmed by real-time pcr were found to be erroneous and contain repetitive sequences , whereas two such clones  appeared to be correctly annotated .
the discrepancy between microarray and real-time pcr data for non-sex chromosomal gene expression remains unexplained.
unspecific cross-hybridization of sex chromosome-linked target cdna to non-sex chromosomal array probes may be one possibility.
because of this uncertainty we decided not to further follow up on non-sex chromosomal candidates.
only microarray data on sex-chromosomal genes are reported in table  <dig> .
we confirmed the absence of differential expression for the autosomal gene mat <dig>  a gene selected for pcr analysis because of its functional association with other genes identified as differentially expressed .
discussion
relatively little is known about the genes that are involved in neuronal sex differentiation in birds, whether influenced by gonadal steroid hormones or not.
in the present study we used cdna microarrays to identify genes that are differentially expressed between males and females in the developing chicken brain  before  and during  morphological differentiation of the gonads.
we found statistical significance  for differential expression of  <dig> microarray clones across the embryonic stages and types of tissue sample .
these clones correspond to ~ <dig> % of the ~ <dig>  clones included in the analysis.
using real-time pcr we were able to confirm female expression for all three w-linked genes identified, and the differential expression of all ten z-linked genes tested and three genes with autosomal or unknown location.
the real-time pcr analysis furthermore revealed one false negative among z-linked genes , and some false positives among genes located on autosomal or unknown chromosomes.
in many cases, however, this could be traced to errors in the microarrays.
a major finding was that sex-specific differential expression could be detected for numerous genes already at the earliest studied embryonic stage , before the stage  at which the earliest sex differences in gonadal gene expression with regard to steroidogenesis have been found  <cit> .
expression of several genes for steroidogenic enzymes  in chicken gonads already on ed <dig> has been documented, but no sex-dependent expression differences  <cit> .
even if we cannot rule out the possibility that hormonal differences between males and females may exist on ed <dig>  the early sex-specific expression of the genes reported in the present study may be primarily regulated by other mechanisms than hormonal control.
in favor of this notion, we found no apparent relationship between the expected differences in hormonal levels associated with gonadal differentiation  <cit>  and the magnitude of the expression differences between the sexes for these genes .
moreover, our preliminary data from chicken embryos exposed to ethinyl estradiol in ovo on ed <dig> show that the expression in the brain on ed <dig>   for the majority of these genes is virtually unaffected by treatment in either sex .
gene expression differences independent of gonadal hormones have previously been suggested to play critical roles in the sexual differentiation of the brain in birds  <cit>  and mice  <cit> .
why an apparently different set of genes should be involved in the chicken compared to the mouse  <cit>  is unclear.
that such a large proportion of genes located on the sex chromosomes  were identified as differentially expressed in the chicken is therefore intriguing considering the independent evolution and different gene content of the sex chromosomes in birds and mammals  <cit> .
a majority of the genes indicated to be differentially expressed from the microarray analysis were more highly expressed in males than in females, and about half of these genes are located on the z chromosome.
this raises the issue of dosage compensation versus biallelic expression of z-linked genes in males  <cit> .
although biallelic expression has previously been demonstrated  <cit> , and might be inferred from the close to two-fold higher expression in males of many z-linked genes in our study, the expression levels of some z-linked genes were found both by microarray and pcr analysis not to differ significantly between the sexes.
such a lack of sex differences in the expression of certain genes located on the z chromosome would be in line with previous evidence for dosage compensation  <cit> .
taken together, our results suggest that if dosage compensation does occur in chicken it is unlikely to involve a widespread inactivation of the z chromosome similar to that of the mammalian x chromosome.
studies by mcqueen and coworkers  <cit>  in chicken embryos have shown that at least six genes on the z chromosome are dosage compensated.
however, kuroda and coworkers  <cit>  found that transcription of five genes, including two of the genes studied by mcqueen and coworkers  <cit> , is taking place on both z chromosomes of male chicken.
this provides further support that dosage compensation in birds does not involve inactivation of a large majority of genes on the z chromosome.
that epigenetic mechanisms such as methylation and acetylation may be involved in the sex-specific expression of genes located on the z chromosome has previously been implicated by the female-specific expression of the z-linked mhm region  <cit> .
in agreement with this, our microarray results showed high female/male ratios suggestive of expression in females only of two distinctive transcripts containing an mhm region.
whether these transcripts, represented by microarray clone rja001b <dig>  and genbank entry ab <dig> <cit> , respectively, are expressed from the same or independent genomic regions is unclear.
their highly similar mhm regions  and their female-specific expression indicate some common function, possibly the repression of the adjacent gene dmrt <dig>  suggested as a conserved sex determining gene  <cit> , through the accumulation of a non-coding rna  <cit> .
however, unlike in male gonads before and during gonadal differentiation  <cit> , we found no evidence of male-enhanced expression of dmrt <dig> in the embryonic chicken brain.
we found high female/male expression ratios on ed <dig> for three genes/clones  located on the female-specific w chromosome, indicating significant early expression in the female embryonic brain.
because of its early expression  in the developing female gonads  <cit> , wpkci- <dig>  has been suggested to be involved in avian sex determination  <cit> .
early wpkci- <dig> expression  has also been detected in the spinal cord, spinal ganglion, and myotomes  <cit> .
the wpkci- <dig> gene, which is reiterated approximately  <dig> times on the w chromosome  <cit> , and its single-copy homolog on the z chromosome hint  <cit>  both encode proteins belonging to the hint family of dimeric nucleotide hydrolases  <cit> .
the wpkci- <dig> protein, however, lacks the histidine triad  motif  <cit>  and appears to act as a dominant suppressor of hint activity, possibly by conferring signals for mislocalization or degradation of the hint/wpkci- <dig> heterodimers  <cit>  .
it is an interesting possibility that suppressed levels of active hint in the brain of female chicken embryos, resulting from both lower expression of the hint gene itself and inhibition by expressed wpkci- <dig>  could contribute to sexual differentiation of the nervous system.
female expression of wpkci- <dig> and male-enhanced expression of hint has previously been detected in the telencephalon of juvenile zebra finches  <cit> .
the hint protein  is well conserved between species  <cit> , but whether this protein is involved in sex determination or sexual differentiation in various species is unclear.
we found male-enhanced expression for two z-linked genes  known to encode subunits of the cyclin-dependent kinase activating kinase  complex, and a third one  encoding a subunit of the transcription factor ii h  core complex.
together these complexes constitute the basal transcription complex tfiih, which is involved in transcription, dna repair, and cell-cycle control  <cit> .
among the genes encoding the nine proteins  in this complex  <cit> , two  appear to be less conserved in the chicken genome .
the implications of the male-enhanced expression of some but not all components of cak  and the tfiih core complex remains to be elucidated.
however, it is intriguing that both cak and tfiih are involved in estrogen receptor α  and androgen receptor  transactivation in mammals  <cit> , and that a functional cak is important for both the mitotic and meiotic cell cycle  <cit> .
the proteins encoded by the z-linked genes hint and aptx, are both members of the hit protein superfamily  <cit> .
aptx, which in the microarrays is more highly expressed in males, encodes aprataxin, a protein involved in single-strand break repair  <cit>  and hint seems to function as a positive regulator of components of tfiih  <cit> , including the ccnh homolog ccl <dig>  <cit> .
considering the role of tfiih in dna-repair, a sex specific control of dna repair seems possible.
in addition to tfiih components, three other z-linked genes associated with dna repair  were found in the microarray result to be more highly expressed in males.
analogous to the sex-dependent expression of wpkci- <dig> and hint, the w-linked gene spinw was expressed in females while the z-linked homolog spinz showed male-enhanced expression.
spinw, which is prominently expressed in ovarian and somatic tissues, has been suggested to be involved in gonadal differentiation  <cit> , but the early  sex-specific expression of these genes in the brain may indicate a more widespread involvement in sexual differentiation.
we also identified a w-linked transcript, abwt, which in addition to being expressed in the early female embryonic brain has also been found in cdna libraries from chicken ovaries and heart .
abwt maps to the genome in the vicinity of the homolog to mouse ube2r <dig> , a gene belonging to the e <dig> family of ubiquitin-conjugating enzymes  <cit> .
as we were unable to detect any differential expression of ube2r <dig> transcripts, however, the detected abwt transcripts appear to be distinct from these.
it remains to be determined whether abwt is a splice variant of a ube2r <dig> transcript, a novel gene encoding a yet unidentified protein or a non-coding rna.
CONCLUSIONS
in conclusion, this study provides hints to mechanisms behind sexual differentiation of the nervous system in birds and raises several new questions.
the expression of w-linked genes supports the presence of 'sex-control genes' similar to the mammalian sry gene, in addition to the possible gene dosage effects from genes on the z chromosomes.
future studies will reveal if the w- and z-linked genes are differentially expressed in the early embryonic brain of different bird species and if differential expression of these genes is present also in other tissues.
it should be noted, however, that wpkci- <dig>  an apparent key player in sexual differentiation  <cit> , is not reiterated on  <cit> , and may even not be linked to  <cit> , the w chromosome of the primitive ratites .
the crucial importance of sex steroids for organization of the brain and behavior in birds has been shown in several studies , but the results presented in this paper point toward the possible presence also of a genetic component in sex-specific neuronal differentiation.
consequently, it seems likely that genetic and hormonal control interact to organize the avian brain dimorphism during differentiation.

 sexual differences that arise in the brain during embryonic development underlie sex-specific reproductive behavior in adults of vertebrate species. better knowledge of the mechanisms behind sexual differentiation of the nervous system can contribute to the understanding of the brain's functions and its susceptibility to disease, and is critically needed in the context of developmental neurotoxicity and chemically induced disruption of the neuroendocrine system. for a long time the idea prevailed that somatic tissues in the embryo are gender neutral until the gonads become hormonally active following their sex-specific differentiation, a process presumed to be governed by cell-autonomous programs genetically determined by the sex chromosomes. however, several recent studies indicate that sex-specific neuronal characteristics may be influenced by genetic mechanisms preceding or occurring in parallel with the effects produced by the gonadal hormones. that the genetic sex of neurons contributes to the process of sexual differentiation is illustrated by studies of the neural song circuit in a gynandromorphic zebra finch, where genetically male and female brain cells develop differently in the presence of the same levels of circulating gonadal hormones. birds, in which males are homogametic and females heterogametic, provide attractive models for the study of normal and disturbed sex-related neurobehavioral development. the recent advent of chicken expressed sequence tag clone collections and the release of the first draft sequence of the chicken genome has paved the way for genome-wide studies of sexual differentiation in the chicken. in the chicken, the indifferent embryonic gonad rudiments differentiate morphologically around embryonic day, whereas the expression of enzymes involved in steroidogenesis can be detected as early as ed, and estrogen receptor mrna is detectable in the male and female urogenital system from ed. the earliest signs of sexually dimorphic expression of genes related to gonadal hormones and steroidogenesis, however, have been detected at ed when mrna for anti-müllerian-hormone is more abundant in males, and aromatase in females. in the present study, we used cdna microarrays to investigate if there are gene expression differences between the male and female embryonic brain that may be independent of morphological differentiation of the gonads. we report evidence for sex-related gene expression differences from ed through ed for several genes located on sex chromosomes. we propose that intrinsic genetic mechanisms are involved in sexual differentiation of the chicken brain, and that sex-linked genes may play key roles.  identification of gene expression differences between male and female embryonic chicken brains to study sex-specific gene expression in the developing brain of chicken embryos during stages around morphological differentiation of the gonads, total rna extracted from male and female heads on ed and ed and brains on ed and ed was subjected to replicated microarray analysis using cdna microarrays containing approximately est clones. for each sex, we generated a total of biologically independent samples, comprising either individuals or pools of four individuals. these samples were analyzed on array slides, with technical replication as shown in figure using an empirical bayes moderated t-test, and adjusted p-values according to benjamini and hochberg, we found clones with significant differential expression across all investigated embryonic stages. for most of these clones, the log female/male ratios were relatively similar across the studied developmental stages. importantly, for a majority of these clones the expression differences between males and females were present at the earliest embryonic stage, i.e. well before morphological gonadal differentiation. characterization of differentially expressed genes the identified clones were found to represent genes, of which were more highly expressed in females, and in males. among the'female genes', three are located on the female-specific w chromosome, one on the z chromosome and on autosomes, whereas for four genes the location is unknown. among the'male genes', are located on the z chromosome and on autosomes, and for another genes the location is unknown. hence, given the fraction of z-linked clones present in the microarray, genes annotated as being located on the z chromosome appeared overrepresented among the differentially expressed genes. as the chicken w chromosome is still poorly characterized, with many gaps in the sequence, the same conclusion is less straightforward to draw about w-linked genes. the differential expression of selected genes on ed was investigated by real-time pcr of individual samples and the results are presented below in the context of the chromosomal locations of these genes. w-linked genes since the w chromosome is present only in female chickens, the expression of w-linked genes was reflected in the microarray analysis by high log female/male ratios, and in the real-time pcr analysis by even higher ratios. an exception from this was spinw, for which the exclusive expression in females was not indicated by similarly high log female/male ratios in the microarray analysis. however, unlike what is known for the other w-linked genes reported here, spinw has a counterpart on the z chromosome. spinw was found by real-time pcr to be expressed in the female head on ed. also the expression of wpkci- for which female expression at all stages was detected by seven clones in the microarray analysis, was verified in the ed female head by real-time pcr. the sequence of the est clone wla084d shows no similarity to any known gene or putative protein. it is part of the unigene entry gga. and the tigr gene index entry tc, located about nucleotides from a chicken sequence similar to the ube2r mouse sequence. using primers designed to amplify this putative ube2r homolog, we did not detect any significant differential expression in embryonic chicken heads. however, using primers amplifying the wla084d sequence, we found expression in heads from 4-day-old female embryos. it remains to determine whether this est may represent a novel w-linked gene, which we provisionally suggest calling'avian brain w-linked transcript'. z-linked genes whereas half of the genes suggested from the microarray study to be more highly expressed in males are located on the z chromosome, only one clone with female-enhanced expression is known to be z-linked. we found nucleotides of this clone to align with high similarity to a partial sequence of the genbank entry with accession ab, representing a z-linked male hypermethylated region. this sequence is thought to be specifically transcribed into a non-coding nuclear rna in females. using primers designed to discriminate between the rja001b and ab sequences, we found female-specific transcription for both of these mhm genomic regions. real-time pcr analysis confirmed the male-enhanced microarray expression for all z-linked genes examined. we also found significantly higher expression in males for two z-linked genes, but not for a third, that were not represented in the microarray but are functionally related to other genes identified as differentially expressed. a close to two-fold higher expression in males was observed for most of the z-linked genes in the real-time pcr analysis, suggestive of gene dosage. exceptions were btf2-p cdk and hmgcoa. no differential expression was detected by real-time pcr for three genes that were randomly selected among the non-differentially expressed z-linked genes in the microarray experiment, supporting that only certain z-linked genes are more highly expressed in males. a fourth z-linked gene randomly selected among genes that were not differentially expressed in the microarray analysis was revealed by real-time pcr to be more highly expressed in males. however, examination of the corresponding microarray clone by sequencing showed that the spotted cdna contained vector sequence only. genes located on autosomes or having unknown chromosomal location differential expression could be confirmed by real-time pcr for only three genes out of analyzed genes with autosomal or unknown location, all with higher expression in males. errors in the microarray were uncovered for of genes that could not be confirmed by real-time pcr. for example, the microarray spots thought to represent the male-enhanced autosomal genes aatf and acad were found to contain est clones representing the z-linked genes rps and plaa, respectively. two of the microarray clones showing female-enhanced expression that could not be confirmed by real-time pcr were found to be erroneous and contain repetitive sequences, whereas two such clones appeared to be correctly annotated. the discrepancy between microarray and real-time pcr data for non-sex chromosomal gene expression remains unexplained. unspecific cross-hybridization of sex chromosome-linked target cdna to non-sex chromosomal array probes may be one possibility. because of this uncertainty we decided not to further follow up on non-sex chromosomal candidates. only microarray data on sex-chromosomal genes are reported in table. we confirmed the absence of differential expression for the autosomal gene mat a gene selected for pcr analysis because of its functional association with other genes identified as differentially expressed. discussion relatively little is known about the genes that are involved in neuronal sex differentiation in birds, whether influenced by gonadal steroid hormones or not. in the present study we used cdna microarrays to identify genes that are differentially expressed between males and females in the developing chicken brain before and during morphological differentiation of the gonads. we found statistical significance for differential expression of microarray clones across the embryonic stages and types of tissue sample. these clones correspond to ~ % of the ~ clones included in the analysis. using real-time pcr we were able to confirm female expression for all three w-linked genes identified, and the differential expression of all ten z-linked genes tested and three genes with autosomal or unknown location. the real-time pcr analysis furthermore revealed one false negative among z-linked genes, and some false positives among genes located on autosomal or unknown chromosomes. in many cases, however, this could be traced to errors in the microarrays. a major finding was that sex-specific differential expression could be detected for numerous genes already at the earliest studied embryonic stage, before the stage at which the earliest sex differences in gonadal gene expression with regard to steroidogenesis have been found. expression of several genes for steroidogenic enzymes in chicken gonads already on ed has been documented, but no sex-dependent expression differences. even if we cannot rule out the possibility that hormonal differences between males and females may exist on ed the early sex-specific expression of the genes reported in the present study may be primarily regulated by other mechanisms than hormonal control. in favor of this notion, we found no apparent relationship between the expected differences in hormonal levels associated with gonadal differentiation and the magnitude of the expression differences between the sexes for these genes. moreover, our preliminary data from chicken embryos exposed to ethinyl estradiol in ovo on ed show that the expression in the brain on ed for the majority of these genes is virtually unaffected by treatment in either sex. gene expression differences independent of gonadal hormones have previously been suggested to play critical roles in the sexual differentiation of the brain in birds and mice. why an apparently different set of genes should be involved in the chicken compared to the mouse is unclear. that such a large proportion of genes located on the sex chromosomes were identified as differentially expressed in the chicken is therefore intriguing considering the independent evolution and different gene content of the sex chromosomes in birds and mammals. a majority of the genes indicated to be differentially expressed from the microarray analysis were more highly expressed in males than in females, and about half of these genes are located on the z chromosome. this raises the issue of dosage compensation versus biallelic expression of z-linked genes in males. although biallelic expression has previously been demonstrated, and might be inferred from the close to two-fold higher expression in males of many z-linked genes in our study, the expression levels of some z-linked genes were found both by microarray and pcr analysis not to differ significantly between the sexes. such a lack of sex differences in the expression of certain genes located on the z chromosome would be in line with previous evidence for dosage compensation. taken together, our results suggest that if dosage compensation does occur in chicken it is unlikely to involve a widespread inactivation of the z chromosome similar to that of the mammalian x chromosome. studies by mcqueen and coworkers in chicken embryos have shown that at least six genes on the z chromosome are dosage compensated. however, kuroda and coworkers found that transcription of five genes, including two of the genes studied by mcqueen and coworkers, is taking place on both z chromosomes of male chicken. this provides further support that dosage compensation in birds does not involve inactivation of a large majority of genes on the z chromosome. that epigenetic mechanisms such as methylation and acetylation may be involved in the sex-specific expression of genes located on the z chromosome has previously been implicated by the female-specific expression of the z-linked mhm region. in agreement with this, our microarray results showed high female/male ratios suggestive of expression in females only of two distinctive transcripts containing an mhm region. whether these transcripts, represented by microarray clone rja001b and genbank entry ab, respectively, are expressed from the same or independent genomic regions is unclear. their highly similar mhm regions and their female-specific expression indicate some common function, possibly the repression of the adjacent gene dmrt suggested as a conserved sex determining gene, through the accumulation of a non-coding rna. however, unlike in male gonads before and during gonadal differentiation, we found no evidence of male-enhanced expression of dmrt in the embryonic chicken brain. we found high female/male expression ratios on ed for three genes/clones located on the female-specific w chromosome, indicating significant early expression in the female embryonic brain. because of its early expression in the developing female gonads, wpkci- has been suggested to be involved in avian sex determination. early wpkci- expression has also been detected in the spinal cord, spinal ganglion, and myotomes. the wpkci- gene, which is reiterated approximately times on the w chromosome, and its single-copy homolog on the z chromosome hint both encode proteins belonging to the hint family of dimeric nucleotide hydrolases. the wpkci- protein, however, lacks the histidine triad motif and appears to act as a dominant suppressor of hint activity, possibly by conferring signals for mislocalization or degradation of the hint/wpkci- heterodimers. it is an interesting possibility that suppressed levels of active hint in the brain of female chicken embryos, resulting from both lower expression of the hint gene itself and inhibition by expressed wpkci- could contribute to sexual differentiation of the nervous system. female expression of wpkci- and male-enhanced expression of hint has previously been detected in the telencephalon of juvenile zebra finches. the hint protein is well conserved between species, but whether this protein is involved in sex determination or sexual differentiation in various species is unclear. we found male-enhanced expression for two z-linked genes known to encode subunits of the cyclin-dependent kinase activating kinase complex, and a third one encoding a subunit of the transcription factor ii h core complex. together these complexes constitute the basal transcription complex tfiih, which is involved in transcription, dna repair, and cell-cycle control. among the genes encoding the nine proteins in this complex, two appear to be less conserved in the chicken genome. the implications of the male-enhanced expression of some but not all components of cak and the tfiih core complex remains to be elucidated. however, it is intriguing that both cak and tfiih are involved in estrogen receptor α and androgen receptor transactivation in mammals, and that a functional cak is important for both the mitotic and meiotic cell cycle. the proteins encoded by the z-linked genes hint and aptx, are both members of the hit protein superfamily. aptx, which in the microarrays is more highly expressed in males, encodes aprataxin, a protein involved in single-strand break repair and hint seems to function as a positive regulator of components of tfiih, including the ccnh homolog ccl. considering the role of tfiih in dna-repair, a sex specific control of dna repair seems possible. in addition to tfiih components, three other z-linked genes associated with dna repair were found in the microarray result to be more highly expressed in males. analogous to the sex-dependent expression of wpkci- and hint, the w-linked gene spinw was expressed in females while the z-linked homolog spinz showed male-enhanced expression. spinw, which is prominently expressed in ovarian and somatic tissues, has been suggested to be involved in gonadal differentiation, but the early sex-specific expression of these genes in the brain may indicate a more widespread involvement in sexual differentiation. we also identified a w-linked transcript, abwt, which in addition to being expressed in the early female embryonic brain has also been found in cdna libraries from chicken ovaries and heart. abwt maps to the genome in the vicinity of the homolog to mouse ube2r, a gene belonging to the e family of ubiquitin-conjugating enzymes. as we were unable to detect any differential expression of ube2r transcripts, however, the detected abwt transcripts appear to be distinct from these. it remains to be determined whether abwt is a splice variant of a ube2r transcript, a novel gene encoding a yet unidentified protein or a non-coding rna.  in conclusion, this study provides hints to mechanisms behind sexual differentiation of the nervous system in birds and raises several new questions. the expression of w-linked genes supports the presence of'sex-control genes' similar to the mammalian sry gene, in addition to the possible gene dosage effects from genes on the z chromosomes. future studies will reveal if the w- and z-linked genes are differentially expressed in the early embryonic brain of different bird species and if differential expression of these genes is present also in other tissues. it should be noted, however, that wpkci- an apparent key player in sexual differentiation, is not reiterated on, and may even not be linked to, the w chromosome of the primitive ratites. the crucial importance of sex steroids for organization of the brain and behavior in birds has been shown in several studies, but the results presented in this paper point toward the possible presence also of a genetic component in sex-specific neuronal differentiation. consequently, it seems likely that genetic and hormonal control interact to organize the avian brain dimorphism during differentiation. 
using cdna microarrays and real-time pcr we found gene expression differences between the male and female embryonic brain that may be independent of morphological differentiation of the gonads. in conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds. genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms. genes located on the sex chromosomes were common among the differentially expressed genes, several of which have previously been implicated in avian sex determination.
 differentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms.  using cdna microarrays and real-time pcr we found gene expression differences between the male and female embryonic brain that may be independent of morphological differentiation of the gonads. genes located on the sex chromosomes were common among the differentially expressed genes, several of which have previously been implicated in avian sex determination. a majority of the identified genes were more highly expressed in males. three of these genes encode subunits of the transcription factor iih complex, indicating a role for this complex in neuronal differentiation.  in conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds. 