BACKGROUND
cross-species comparison of genome sequences and expressed sequence tag  data sets have given us enormous insight into the evolution of metazoan genomes.
in particular, the recent completion of a number of ecdysozoan and deuterostome genome projects as well as the collection of massive est data on lophotrochozoan and cnidarians enable us to identify orthologous groups of genes and gene losses in these  <dig> major metazoan clades.
research on acropora millepora  supports the view that a significant proportion of genes present only in the vertebrates and absent in model invertebrates is not a vertebrate-specific evolutionary change.
instead, these genes appear to have been lost in the specific metazoan lineages that branched from common ancestors during evolution  <cit> .
the same conclusion is reached by considering the fact that sea anemone nematostella vectensis contains many wnt subfamilies that were lost from drosophila and caenorhabditis  <cit> .
the cross-species comparison of aplysia californica est data sets with other metazoan genomes and with platynereis dumerilii demonstrate that gene loss and sequence divergence are most extensive in model ecdysozoans , and lophotrochozoans are less derived from the complex ancestral genome of urbilateria  <cit> .
urbilateria is a common ancestor of bilateria, and  <dig> major clades  subsequently branched off from it  <cit> .
honey bees belong to the hymenoptera, one of the  <dig> large holometabolous insect orders.
they are highly social insects and have been used as a model system to study complex animal behavior and cognitive ability comparable to those of some vertebrates  <cit> .
furthermore, caste differentiation  and sex determination via haplodiploidy  are specific to some hymenopterans such as honey bees, among the holometabolous insects.
the completion of genome sequencing of several species in the  <dig> major holometabolous insect orders , namely fruit flies   <cit> , mosquitoes , silk moth   <cit> , and honey bee   <cit>  and red flour beetle   <cit>  promises to provide new insights into how the genomes of holometabolous insects evolved in comparison to those of other metazoans.
a comparison of the above insect genomes has revealed that a number of genes are lost in specific insects, but they are present in the other insects and vertebrates.
these are the ancient genes of urbilateria, which were retained in some species but lost from others.
the loss and retention of these ancient genes in specific lineages are implicated in generating metazoan diversity; however, the causal relationships acting therein have not been studied extensively.
we, therefore, identified genes shared between vertebrates and honey bees but absent in the other sequenced insect and nematode genomes, and we characterized their expression profiles in various honey bee and mouse tissues as well as the mouse brain to gain insight into their present functions in honey bees and mice.
furthermore, we expected that this study would give us clues to ascertain whether these genes have been selectively or randomly retained in the specific species during evolution.
our results demonstrate that the ancient urbilaterian genes uniquely shared between honey bee and vertebrates are biased towards expression and functioning in the brain  and testis.
their possible functions and evolutionary mechanisms will be discussed further.
RESULTS
identification of genes specifically conserved between vertebrates and honey bee but lost from the other sequenced ecdysozoan genomes
we identified candidate genes shared between at least  <dig> vertebrate  and the honey bee but not with  <dig> dipterans  and a coleopteran  species by automatic large-scale sequence analysis, as described in methods  <cit> .
we, then, performed a tblastn analysis of these candidate genes with the genomic and gene model sequences of the above insects as well as those of b. mori  and c. elegans.
we discarded the ones that showed significant similarity with an e value < 1e- <dig>  this screening resulted in the identification of  <dig> honey bee genes shared with vertebrates, but not with other sequenced insect and nematode genomes.
we named them hvs  genes, and they are listed together with the mouse homologs in table  <dig>  twenty-one honey bee hvs genes encode proteins with known domains and/or functions.
these include, for example, dna methyltransferase dnmt <dig> , which has been reported previously  <cit> ; homeodomain only protein , which is highly expressed in the mouse brain  <cit>  and heart  <cit> ; tuba , which is a cdc42-specific guanine nucleotide exchange factor to regulate actin polymerization  <cit> ; and prenylcysteine oxidase  <dig> , a lysosomal enzyme degrading prenylcysteine  <cit> .
apis
the genes categorized as transcription and modification of dna as well as rna processing by go system are indicated with bold letters.
the expression domains of honey bee and mouse hvs genes are listed in the right column.
the genes ubiquitously and selectively expressed in the tissues are indicated as ubiquitous and selective, respectively.
the genes highly expressed in the brains of both honey bee and mouse are shown with bold letters.
the sequences of honey bee glean models are available in bcm database  and beebase .
distribution patterns of hvs genes in sequenced deuterostome genomes
we analyzed the copy numbers of hvs genes among the sequenced deuterostome  genomes, and the results are shown in table  <dig>  deuterostomes appear to retain most of the hvs genes, in contrast to ecdysozoans.
although each hvs gene is present as a single copy in the honey bee genome, multiple-copy hvs genes are often present in the deuterostome genomes.
all of the hvs genes, except dnmt3; tumor necrosis factor  receptor superfamily; tmem45; and f-box and leucine-rich repeat protein genes, are present as single copies in both sea urchin and ascidian genomes.
thus, the duplication of hvs genes is quite specific to vertebrate lineages.
the vertebrates have 4– <dig> copies of bcl <dig>  one of the key regulators of apoptosis  <cit> , and 4– <dig> copies of tnf receptor superfamily genes.
analysis of hvs genes by go terms
we predicted hvs gene functions by assigning gene ontology  terms  and found that  <dig> out of  <dig> gene products were proposed to be involved in transcription and modification of dna as well as rna processing in the nucleus.
these include dnmt3a/3b , hop , rad <dig> , cdca7/cdca7like, pnrc1/ <dig>  snapc <dig>  and gemin <dig>  gemin <dig> appears to be involved in rna processing.
the others are associated with the intracellular signaling cascade , regulation of apoptosis , actin filament organization , receptor activity , and ubiquitin cycle .
in drosophila, proteins associated with the regulation of gene expression represent only 10% of the total proteins  <cit> .
considering that the total number of honey bee hvs genes is  <dig>  it appears that hvs genes are biased to contain genes associated with these categories of cellular functions .
expression profiles of hvs genes in honey bee and mouse tissues
to confirm mrna expression and to determine the functional implications of hvs genes, we carried out semiquantitative rt-pcr analysis for each hvs gene with various honey bee and mouse tissues.
all hvs mrnas were expressed at various levels in the honey bee brain, thorax, and abdomen .
ubiquitous expression was observed in the cases of gb <dig>  gb <dig>  gb <dig>  gb <dig>  and gb <dig> .
expressions of gb <dig>  gb <dig>  gb <dig>  and gb <dig> mrna were at least  <dig>  times higher in the brain and abdomen than that in the thorax .
gb <dig> and gb <dig> mrnas expression was  <dig> times higher in the brain and thorax than that in the abdomen, and the expression of gb <dig> mrna was weak in the abdomen  .
the remaining 60% of the honey bee hvs genes  were expressed at a higher level in the brain than in the abdomen and thorax.
in particular, the expressions of gb <dig>  gb <dig>  and gb <dig> were exclusively detected in the brain .
gb <dig>  gb <dig>  gb <dig>  gb <dig>  and gb <dig> mrna expressions were  <dig> ,  <dig>   <dig>   <dig>  and  <dig> times higher in the brain than in the abdomen, respectively .
expressions of gb <dig>  gb <dig>  gb <dig>  gb <dig>  gb <dig>  gb <dig>  gb <dig>  gb <dig>  gb <dig>  and gb <dig> mrna were  <dig> ,  <dig>   <dig> ,  <dig> ,  <dig>   <dig>   <dig> ,  <dig>   <dig> , and  <dig> times higher in the brain than in the thorax, respectively .
the expression of mouse hvs genes in various tissues was also analyzed.
for mouse hvs genes with more than  <dig> copies in the genome, the expression of  <dig> genes sharing the highest similarity with the honey bee gene was analyzed.
thus, we performed semiquantitative rt-pcr analysis with  <dig> mouse hvs genes .
most of the hvs genes showed tissue-selective expression patterns in mouse .
ubiquitous expression was observed with rhbdd <dig>  dnmt3a, mtfr <dig>  armc <dig>  pnrc <dig>  1300010m03rik, fbxlike <dig>  gemin <dig>  siva <dig>  loc <dig>  and fbxo <dig>  .
the expressions of pcyoxl and pnrc <dig> were primarily detected in the brain .
c530028i08rik and rad <dig> were primarily expressed in the testis .
the expression levels of 4933429f08rik, dnmt3b, cdca <dig>  loc <dig>  1700012p22rik, 2610016c23rik, and armc <dig> mrnas were high in both the brain and testis .
thus, mouse hvs genes expressed in either or both the brain and testis accounted for a total of  <dig>  genes.
loc <dig> and 5430433g21rik mrnas were highly expressed in the lung .
the expression of snx <dig> was primarily detected in the spleen .
cdca7like and 2010001m09rik mrnas were expressed in the brain, lung, spleen, kidney, and testis but to a lesser extent in the muscle, heart, and liver .
snapc <dig> and bcl2like <dig> were expressed in all tissues examined, except in the muscle .
all other genes  showed different tissue-selective expression patterns .
expression profiles of hvs genes in mouse brain
next, we characterized the spatial expression patterns of hvs genes in the mouse brain by searching data in the allen brain atlas  <cit> .
the atlas provides 2- and 3-dimensional images of the spatial expression patterns of ~ <dig>  genes in the adult mouse brain.
in situ hybridization data are available for  <dig> mouse hvs genes.
among them,  <dig> genes exhibited only weak expression and, therefore, their expression could not be attributed to the specific cell types or brain regions.
neuron-enriched expression was detected for tuba, 4933429f08rik, bcl2like <dig>  pcyox <dig>  pcyox1like, hop, armc <dig>  armc <dig>  pnrc <dig>  1300010m03rik, 1810015c04rik, snapc <dig>  and rad <dig> .
only fbxo <dig> was expression in both neurons and glial cells .
tuba mrna was expressed relatively well in the anterior part of the brain ; its duplicated gene 4933429f08rik was, however, expressed in the cerebellum, particularly in purkinje cells .
the expression of hop was primarily detected in cerebellar purkinje cells .
expression of 1810015c04rik mrna was abundant in the mitral layer of the olfactory bulb and in cerebellar purkinje cells .
the expression of armc <dig> was ubiquitous throughout the entire brain; however, it was relatively well expressed in the hippocampus .
pcyox <dig>  bcl2like <dig>  and pnrc <dig> mrnas were ubiquitously expressed in the brain, except for an increased hippocampal expression .
pcyox1like, armc <dig>  1300010m03rik, rad <dig>  and snapc <dig> mrnas were uniformly expressed throughout the brain .
identification of genes specifically conserved between vertebrates and red flour beetle but lost from the other sequenced ecdysozoan genomes
as controls, we used urbilaterian genes that were uniquely shared between t. castaneum and vertebrate  identified in the same method as that used for hvs genes.
in contrast to the  <dig> honey bee hvs genes, red flour beetle tvs genes were only  <dig> .
we also analyzed the expression patterns of  <dig> mouse tvs genes in various tissues as described above .
nearly ubiquitous expression was observed with gm2a, naif <dig>  parp <dig>  parp <dig>  and art <dig> genes.
art2b was predominantly expressed in the lung, spleen, and liver; rsad <dig>  in the lung, spleen, heart, and kidney; and crlf <dig>  in the lung and spleen.
thus, none of the mouse tvs genes were expressed in either or both the brain and testis, unlike mouse hvs genes.
moreover, only  <dig> tvs gene, the poly  polymerase family was suggested to have been associated with gene expression in go terms.
since only  <dig> mouse tvs genes were identified, it was not possible to statistically compare them with  <dig> mouse hvs genes.
nevertheless, these results support that honey bees retain more ancient urbilaterian genes than red flour beetle.
tribolium glean
the sequences of tribolium glean models are available in bcm database .
discussion
evolution of hvs genes
searching for hvs genes uniquely shared between vertebrates and honey bee resulted in the identification of  <dig> honey bee and  <dig> mouse genes.
there are more hvs genes in mice than honey bees since multiple-copy hvs genes are often present in the mouse genomes as against single copy genes in the honey bee genome .
this finding suggested that urbilateria had a relatively complex genome and that hvs genes have been lost from various ecdysozoans, which were shown to have more derived genomes than lophotrochozoan and deuterostome  <cit> .
the loss and retention of these ancient genes in specific lineages has been implicated in generating metazoan diversity  <cit> ; however, the causal relationships involved therein have not been extensively studied.
there are  <dig> possibilities explaining the lineage-specific retention and loss of urbilaterian genes.
one possibility is that some genes are retained in specific lineages because during evolution, they may have had vital roles in these lineages but not in others.
the other possibility is that many lineages evolved, for example, distant paralogs or gene network interactions, to compensate for the functions of urbilaterian genes retained in the specific lineages, and the lineages eventually lost these genes.
if the latter case is considered, some urbilaterian genes must have been stochastically retained in specific lineages.
we, thus, characterized hvs genes as a model to gain insights into the evolutionary mechanisms resulting in the presence of urbilaterian genes only in specific lineages.
during the course of the identification of honey bee hvs genes, we first identified  <dig> genes shared between vertebrates and honey bee but not other sequenced insect  genomes.
furthermore, the nematode genome also lost  <dig> but retained  <dig> genes, demonstrating that its evolutionary rate was comparable to that of fruit fly and mosquito, as previously reported  <cit> .
ten genes shared between honey bee and nematode included a malonyl-coa decarboxylase responsible for the conversion of malonyl-coa to acetyl-coa, which is used in fatty acid biosynthesis as well as energy production.
these functions should be very critical since this gene is conserved in bacteria and plants.
the apparent lack of this enzyme in  <dig> insect species suggests that these species must have developed an alternative pathway for fatty acid metabolism.
the functions of other genes shared between honey bee and nematode have not been reported.
both sea urchin and ascidian contain a comparable number of hvs genes ; however, amino acid substitutions are more extensive in ascidian than in sea urchin.
when the ascidian genome was searched for hvs genes by tblastn analysis using honey bee hvs genes as queries, only  <dig> genes with significant similarity were recovered.
consistent with previous reports  <cit> , this result demonstrates that although ascidian is more closely related to the vertebrate lineage, it has a more derived genome than sea urchin.
honey bees contain a single copy for each hvs gene; however, vertebrates have multiple copies for  <dig> out of  <dig>  honey bee hvs genes.
this ratio is lower than the average percentage  of genes having multiple copies in human and mouse.
this may suggest that the loss of gene copy occurred more frequently with hvs genes after whole- or local-genome duplication events in vertebrates  <cit>  .
the tissue expression patterns of hvs gene pairs are different in many cases , demonstrating that they either adopted a new function  or partitioned old functions .
among hvs genes, the copy-number expansions of genes encoding bcl <dig> and tnf receptor superfamilies were detected in vertebrate genomes.
both bcl <dig> and tnf receptor superfamilies control cell death and survival  <cit> , a more extensive feature in vertebrates than in honey bee, sea urchin, and ascidian.
interestingly, among the vertebrates examined, the honey bee gb <dig> orthologs were only present in mouse and human .
this result suggests that gene loss also occurs in vertebrates, as reported previously  <cit> .
to find the paralogs of hvs genes in the sequenced insect genomes, we first identified functional domains in each hvs protein by interproscan  and collected these functional domain sequences from ensembl.
we, then, constructed the hmm models by hmmer  <dig> programs.
insect protein sets of ensembl, baylor, and the aedes genome sequencing consortium were finally searched for the paralogs of hvs proteins by these hmms with an e-value cutoff level of  <dig> .
an example indicates that the functions of hvs genes were compensated by the paralogs and/or gene network interactions.
both honey bees and vertebrate express dnmt <dig>   <dig>  and 3; however, members of the genus drosophila express only dnmt <dig>  while those of tribolium express dnmt <dig> and  <dig>  nonexpression of dnmt <dig> and  <dig> is consistent with the lack of cpg methylated dna in drosophila  <cit> , and dnmt <dig> expression is associated with the presence of cpg methylated dna in honey bee  <cit> .
it is well established that dnmt <dig> is de novo dna methyltransferase, and dnmt <dig> is necessary for maintaining dna methylation in vertebrates  <cit> .
if cpg methylated dna is present in the tribolium genome, it can be inferred that dnmt <dig>  the paralog of dnmt <dig>  might also carry out de novo dna methylation.
meanwhile, the epigenetic repression of gene transcription is functional in drosophila through histone modifications, suggesting that drosophila evolves the epigenetic control system independent of the methylated dna by mechanisms such as modification of gene network interactions.
present functions of hvs genes appear to be biased towards expression in the brain and gonads
most honey bee hvs genes  were expressed at different levels in the brain, thorax, and abdomen .
among them, 72%  show a higher level of expression in the brain than in the thorax and abdomen, suggesting that their mrnas appear to be enriched in the neurons.
most mouse hvs genes  show tissue-specific or selective expression patterns .
among them, 32%  are highly expressed in either or both the brain and testis.
intriguingly,  <dig> genes are highly expressed in both brain and testis.
hvs genes are not essential for the survival of all animals since they are absent in many insect species other than honey bee.
these genes are unlikely to have major roles for cell viability and maintenance and, thus, they were easily co-opted or recruited to function in the specific tissues, which in this case were mouse brain and testis.
both tissues contain a significant population of stem cells and, thus, the  <dig> hvs genes may have specific roles in regulating the proliferation and/or differentiation of these stem cells.
many genes associated with neuronal excitation and transmission are highly expressed in the brain but not in the testis.
meanwhile, genes involved in spermatogenesis are expressed exclusively in the testis and not in the brain.
genes highly expressed in both tissues are, therefore, not very common in mouse.
it was reported that the total number of genes predominantly expressed in the human brain and testis is  <dig> out of the  <dig> examined, accounting for only  <dig> %  <cit> .
thus, the ancient urbilaterian genes selectively retained between vertebrates and honey bee appear to be biased toward playing major roles in brain and gonad functions in mouse.
in agreement with this finding, only  <dig> red flour beetle tvs genes  were identified, and no mouse tvs gene exhibited predominant expression either in the brain or testis .
we, therefore, would like to propose that at least some hvs genes might have been selectively maintained in order to support honey bee-specific brain and, possibly, gonad functions.
it is very likely that brain functions involved in supporting social behavior and cognitive ability of honey bees and vertebrates evolve independently, without the sharing of any genetic components, on account of their evolutionary distance and the difference in their brain structures; however, selective genes of urbilateria could be a part of the genetic bases underlying the evolution of advanced brain functions.
we found that  <dig> mouse hvs genes were highly expressed in the neurons of the brain.
among them,  <dig> genes exhibited brain-region selective expression patterns.
most of them were highly expressed in the cerebellar and in the forebrain area, including the olfactory bulb, cerebral cortex, and hippocampus.
these brain regions are responsible for the integration and processing of various sensory information as well as various forms of learning and memory.
the roles of many other hvs genes in brain functions in both honey bees and mice remain to be determined.
the number of honey bee hvs genes is only  <dig>  and this is consistent with the fact that gene loss and acquisition events are relatively rare as compared to amino acid substitution, cis-regulatory mutation, dna rearrangement, and duplication during evolution.
a vast majority of honey bee genes are, indeed, conserved in all solitary ecdysozoan genomes  <cit> , suggesting that advanced honey bee brain functions are primarily driven by increasing complexity in the network of gene interactions.
since  <dig> out of  <dig> hvs gene products were predicted, by using go terms, to be associated with transcription and modification of dna as well as rna processing in the nucleus, they could participate in such processes by regulating dna transcription, splicing, and non-coding rna synthesis.
these hvs genes would provide an important resource for future studies on gene function.
CONCLUSIONS
large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of urbilateria is lost in each animal lineage.
this event could be one of the underlying mechanisms responsible for metazoan diversity.
we have found that hvs genes are biased towards expression in the brains and gonads of honey bees and mice, and 35% of hvs genes are associated with dna transcription and rna processing by go analysis.
these results suggest that hvs genes include genes that are biased towards expression in the brain and gonad.
it also demonstrates that at least some of urbilaterian genes are selectively retained in the specific animal lineage to support the species-specific phenotypes.

 cross-species comparison of genome sequences and expressed sequence tag data sets have given us enormous insight into the evolution of metazoan genomes. in particular, the recent completion of a number of ecdysozoan and deuterostome genome projects as well as the collection of massive est data on lophotrochozoan and cnidarians enable us to identify orthologous groups of genes and gene losses in these major metazoan clades. research on acropora millepora supports the view that a significant proportion of genes present only in the vertebrates and absent in model invertebrates is not a vertebrate-specific evolutionary change. instead, these genes appear to have been lost in the specific metazoan lineages that branched from common ancestors during evolution. the same conclusion is reached by considering the fact that sea anemone nematostella vectensis contains many wnt subfamilies that were lost from drosophila and caenorhabditis. the cross-species comparison of aplysia californica est data sets with other metazoan genomes and with platynereis dumerilii demonstrate that gene loss and sequence divergence are most extensive in model ecdysozoans, and lophotrochozoans are less derived from the complex ancestral genome of urbilateria. urbilateria is a common ancestor of bilateria, and major clades subsequently branched off from it. honey bees belong to the hymenoptera, one of the large holometabolous insect orders. they are highly social insects and have been used as a model system to study complex animal behavior and cognitive ability comparable to those of some vertebrates. furthermore, caste differentiation and sex determination via haplodiploidy are specific to some hymenopterans such as honey bees, among the holometabolous insects. the completion of genome sequencing of several species in the major holometabolous insect orders, namely fruit flies, mosquitoes, silk moth, and honey bee and red flour beetle promises to provide new insights into how the genomes of holometabolous insects evolved in comparison to those of other metazoans. a comparison of the above insect genomes has revealed that a number of genes are lost in specific insects, but they are present in the other insects and vertebrates. these are the ancient genes of urbilateria, which were retained in some species but lost from others. the loss and retention of these ancient genes in specific lineages are implicated in generating metazoan diversity; however, the causal relationships acting therein have not been studied extensively. we, therefore, identified genes shared between vertebrates and honey bees but absent in the other sequenced insect and nematode genomes, and we characterized their expression profiles in various honey bee and mouse tissues as well as the mouse brain to gain insight into their present functions in honey bees and mice. furthermore, we expected that this study would give us clues to ascertain whether these genes have been selectively or randomly retained in the specific species during evolution. our results demonstrate that the ancient urbilaterian genes uniquely shared between honey bee and vertebrates are biased towards expression and functioning in the brain and testis. their possible functions and evolutionary mechanisms will be discussed further.  identification of genes specifically conserved between vertebrates and honey bee but lost from the other sequenced ecdysozoan genomes we identified candidate genes shared between at least vertebrate and the honey bee but not with dipterans and a coleopteran species by automatic large-scale sequence analysis, as described in methods. we, then, performed a tblastn analysis of these candidate genes with the genomic and gene model sequences of the above insects as well as those of b. mori and c. elegans. we discarded the ones that showed significant similarity with an e value < 1e- this screening resulted in the identification of honey bee genes shared with vertebrates, but not with other sequenced insect and nematode genomes. we named them hvs genes, and they are listed together with the mouse homologs in table twenty-one honey bee hvs genes encode proteins with known domains and/or functions. these include, for example, dna methyltransferase dnmt, which has been reported previously; homeodomain only protein, which is highly expressed in the mouse brain and heart; tuba, which is a cdc42-specific guanine nucleotide exchange factor to regulate actin polymerization; and prenylcysteine oxidase, a lysosomal enzyme degrading prenylcysteine. apis the genes categorized as transcription and modification of dna as well as rna processing by go system are indicated with bold letters. the expression domains of honey bee and mouse hvs genes are listed in the right column. the genes ubiquitously and selectively expressed in the tissues are indicated as ubiquitous and selective, respectively. the genes highly expressed in the brains of both honey bee and mouse are shown with bold letters. the sequences of honey bee glean models are available in bcm database and beebase. distribution patterns of hvs genes in sequenced deuterostome genomes we analyzed the copy numbers of hvs genes among the sequenced deuterostome genomes, and the results are shown in table deuterostomes appear to retain most of the hvs genes, in contrast to ecdysozoans. although each hvs gene is present as a single copy in the honey bee genome, multiple-copy hvs genes are often present in the deuterostome genomes. all of the hvs genes, except dnmt3; tumor necrosis factor receptor superfamily; tmem45; and f-box and leucine-rich repeat protein genes, are present as single copies in both sea urchin and ascidian genomes. thus, the duplication of hvs genes is quite specific to vertebrate lineages. the vertebrates have 4– copies of bcl one of the key regulators of apoptosis, and 4– copies of tnf receptor superfamily genes. analysis of hvs genes by go terms we predicted hvs gene functions by assigning gene ontology terms and found that out of gene products were proposed to be involved in transcription and modification of dna as well as rna processing in the nucleus. these include dnmt3a/3b, hop, rad, cdca7/cdca7like, pnrc1/ snapc and gemin gemin appears to be involved in rna processing. the others are associated with the intracellular signaling cascade, regulation of apoptosis, actin filament organization, receptor activity, and ubiquitin cycle. in drosophila, proteins associated with the regulation of gene expression represent only 10% of the total proteins. considering that the total number of honey bee hvs genes is it appears that hvs genes are biased to contain genes associated with these categories of cellular functions. expression profiles of hvs genes in honey bee and mouse tissues to confirm mrna expression and to determine the functional implications of hvs genes, we carried out semiquantitative rt-pcr analysis for each hvs gene with various honey bee and mouse tissues. all hvs mrnas were expressed at various levels in the honey bee brain, thorax, and abdomen. ubiquitous expression was observed in the cases of gb gb gb gb and gb. expressions of gb gb gb and gb mrna were at least times higher in the brain and abdomen than that in the thorax. gb and gb mrnas expression was times higher in the brain and thorax than that in the abdomen, and the expression of gb mrna was weak in the abdomen. the remaining 60% of the honey bee hvs genes were expressed at a higher level in the brain than in the abdomen and thorax. in particular, the expressions of gb gb and gb were exclusively detected in the brain. gb gb gb gb and gb mrna expressions were, and times higher in the brain than in the abdomen, respectively. expressions of gb gb gb gb gb gb gb gb gb and gb mrna were,,,,, and times higher in the brain than in the thorax, respectively. the expression of mouse hvs genes in various tissues was also analyzed. for mouse hvs genes with more than copies in the genome, the expression of genes sharing the highest similarity with the honey bee gene was analyzed. thus, we performed semiquantitative rt-pcr analysis with mouse hvs genes. most of the hvs genes showed tissue-selective expression patterns in mouse. ubiquitous expression was observed with rhbdd dnmt3a, mtfr armc pnrc 1300010m03rik, fbxlike gemin siva loc and fbxo. the expressions of pcyoxl and pnrc were primarily detected in the brain. c530028i08rik and rad were primarily expressed in the testis. the expression levels of 4933429f08rik, dnmt3b, cdca loc 1700012p22rik, 2610016c23rik, and armc mrnas were high in both the brain and testis. thus, mouse hvs genes expressed in either or both the brain and testis accounted for a total of genes. loc and 5430433g21rik mrnas were highly expressed in the lung. the expression of snx was primarily detected in the spleen. cdca7like and 2010001m09rik mrnas were expressed in the brain, lung, spleen, kidney, and testis but to a lesser extent in the muscle, heart, and liver. snapc and bcl2like were expressed in all tissues examined, except in the muscle. all other genes showed different tissue-selective expression patterns. expression profiles of hvs genes in mouse brain next, we characterized the spatial expression patterns of hvs genes in the mouse brain by searching data in the allen brain atlas. the atlas provides 2- and 3-dimensional images of the spatial expression patterns of ~ genes in the adult mouse brain. in situ hybridization data are available for mouse hvs genes. among them, genes exhibited only weak expression and, therefore, their expression could not be attributed to the specific cell types or brain regions. neuron-enriched expression was detected for tuba, 4933429f08rik, bcl2like pcyox pcyox1like, hop, armc armc pnrc 1300010m03rik, 1810015c04rik, snapc and rad. only fbxo was expression in both neurons and glial cells. tuba mrna was expressed relatively well in the anterior part of the brain; its duplicated gene 4933429f08rik was, however, expressed in the cerebellum, particularly in purkinje cells. the expression of hop was primarily detected in cerebellar purkinje cells. expression of 1810015c04rik mrna was abundant in the mitral layer of the olfactory bulb and in cerebellar purkinje cells. the expression of armc was ubiquitous throughout the entire brain; however, it was relatively well expressed in the hippocampus. pcyox bcl2like and pnrc mrnas were ubiquitously expressed in the brain, except for an increased hippocampal expression. pcyox1like, armc 1300010m03rik, rad and snapc mrnas were uniformly expressed throughout the brain. identification of genes specifically conserved between vertebrates and red flour beetle but lost from the other sequenced ecdysozoan genomes as controls, we used urbilaterian genes that were uniquely shared between t. castaneum and vertebrate identified in the same method as that used for hvs genes. in contrast to the honey bee hvs genes, red flour beetle tvs genes were only. we also analyzed the expression patterns of mouse tvs genes in various tissues as described above. nearly ubiquitous expression was observed with gm2a, naif parp parp and art genes. art2b was predominantly expressed in the lung, spleen, and liver; rsad in the lung, spleen, heart, and kidney; and crlf in the lung and spleen. thus, none of the mouse tvs genes were expressed in either or both the brain and testis, unlike mouse hvs genes. moreover, only tvs gene, the poly polymerase family was suggested to have been associated with gene expression in go terms. since only mouse tvs genes were identified, it was not possible to statistically compare them with mouse hvs genes. nevertheless, these results support that honey bees retain more ancient urbilaterian genes than red flour beetle. tribolium glean the sequences of tribolium glean models are available in bcm database. discussion evolution of hvs genes searching for hvs genes uniquely shared between vertebrates and honey bee resulted in the identification of honey bee and mouse genes. there are more hvs genes in mice than honey bees since multiple-copy hvs genes are often present in the mouse genomes as against single copy genes in the honey bee genome. this finding suggested that urbilateria had a relatively complex genome and that hvs genes have been lost from various ecdysozoans, which were shown to have more derived genomes than lophotrochozoan and deuterostome. the loss and retention of these ancient genes in specific lineages has been implicated in generating metazoan diversity; however, the causal relationships involved therein have not been extensively studied. there are possibilities explaining the lineage-specific retention and loss of urbilaterian genes. one possibility is that some genes are retained in specific lineages because during evolution, they may have had vital roles in these lineages but not in others. the other possibility is that many lineages evolved, for example, distant paralogs or gene network interactions, to compensate for the functions of urbilaterian genes retained in the specific lineages, and the lineages eventually lost these genes. if the latter case is considered, some urbilaterian genes must have been stochastically retained in specific lineages. we, thus, characterized hvs genes as a model to gain insights into the evolutionary mechanisms resulting in the presence of urbilaterian genes only in specific lineages. during the course of the identification of honey bee hvs genes, we first identified genes shared between vertebrates and honey bee but not other sequenced insect genomes. furthermore, the nematode genome also lost but retained genes, demonstrating that its evolutionary rate was comparable to that of fruit fly and mosquito, as previously reported. ten genes shared between honey bee and nematode included a malonyl-coa decarboxylase responsible for the conversion of malonyl-coa to acetyl-coa, which is used in fatty acid biosynthesis as well as energy production. these functions should be very critical since this gene is conserved in bacteria and plants. the apparent lack of this enzyme in insect species suggests that these species must have developed an alternative pathway for fatty acid metabolism. the functions of other genes shared between honey bee and nematode have not been reported. both sea urchin and ascidian contain a comparable number of hvs genes; however, amino acid substitutions are more extensive in ascidian than in sea urchin. when the ascidian genome was searched for hvs genes by tblastn analysis using honey bee hvs genes as queries, only genes with significant similarity were recovered. consistent with previous reports, this result demonstrates that although ascidian is more closely related to the vertebrate lineage, it has a more derived genome than sea urchin. honey bees contain a single copy for each hvs gene; however, vertebrates have multiple copies for out of honey bee hvs genes. this ratio is lower than the average percentage of genes having multiple copies in human and mouse. this may suggest that the loss of gene copy occurred more frequently with hvs genes after whole- or local-genome duplication events in vertebrates. the tissue expression patterns of hvs gene pairs are different in many cases, demonstrating that they either adopted a new function or partitioned old functions. among hvs genes, the copy-number expansions of genes encoding bcl and tnf receptor superfamilies were detected in vertebrate genomes. both bcl and tnf receptor superfamilies control cell death and survival, a more extensive feature in vertebrates than in honey bee, sea urchin, and ascidian. interestingly, among the vertebrates examined, the honey bee gb orthologs were only present in mouse and human. this result suggests that gene loss also occurs in vertebrates, as reported previously. to find the paralogs of hvs genes in the sequenced insect genomes, we first identified functional domains in each hvs protein by interproscan and collected these functional domain sequences from ensembl. we, then, constructed the hmm models by hmmer programs. insect protein sets of ensembl, baylor, and the aedes genome sequencing consortium were finally searched for the paralogs of hvs proteins by these hmms with an e-value cutoff level of. an example indicates that the functions of hvs genes were compensated by the paralogs and/or gene network interactions. both honey bees and vertebrate express dnmt and 3; however, members of the genus drosophila express only dnmt while those of tribolium express dnmt and nonexpression of dnmt and is consistent with the lack of cpg methylated dna in drosophila, and dnmt expression is associated with the presence of cpg methylated dna in honey bee. it is well established that dnmt is de novo dna methyltransferase, and dnmt is necessary for maintaining dna methylation in vertebrates. if cpg methylated dna is present in the tribolium genome, it can be inferred that dnmt the paralog of dnmt might also carry out de novo dna methylation. meanwhile, the epigenetic repression of gene transcription is functional in drosophila through histone modifications, suggesting that drosophila evolves the epigenetic control system independent of the methylated dna by mechanisms such as modification of gene network interactions. present functions of hvs genes appear to be biased towards expression in the brain and gonads most honey bee hvs genes were expressed at different levels in the brain, thorax, and abdomen. among them, 72% show a higher level of expression in the brain than in the thorax and abdomen, suggesting that their mrnas appear to be enriched in the neurons. most mouse hvs genes show tissue-specific or selective expression patterns. among them, 32% are highly expressed in either or both the brain and testis. intriguingly, genes are highly expressed in both brain and testis. hvs genes are not essential for the survival of all animals since they are absent in many insect species other than honey bee. these genes are unlikely to have major roles for cell viability and maintenance and, thus, they were easily co-opted or recruited to function in the specific tissues, which in this case were mouse brain and testis. both tissues contain a significant population of stem cells and, thus, the hvs genes may have specific roles in regulating the proliferation and/or differentiation of these stem cells. many genes associated with neuronal excitation and transmission are highly expressed in the brain but not in the testis. meanwhile, genes involved in spermatogenesis are expressed exclusively in the testis and not in the brain. genes highly expressed in both tissues are, therefore, not very common in mouse. it was reported that the total number of genes predominantly expressed in the human brain and testis is out of the examined, accounting for only %. thus, the ancient urbilaterian genes selectively retained between vertebrates and honey bee appear to be biased toward playing major roles in brain and gonad functions in mouse. in agreement with this finding, only red flour beetle tvs genes were identified, and no mouse tvs gene exhibited predominant expression either in the brain or testis. we, therefore, would like to propose that at least some hvs genes might have been selectively maintained in order to support honey bee-specific brain and, possibly, gonad functions. it is very likely that brain functions involved in supporting social behavior and cognitive ability of honey bees and vertebrates evolve independently, without the sharing of any genetic components, on account of their evolutionary distance and the difference in their brain structures; however, selective genes of urbilateria could be a part of the genetic bases underlying the evolution of advanced brain functions. we found that mouse hvs genes were highly expressed in the neurons of the brain. among them, genes exhibited brain-region selective expression patterns. most of them were highly expressed in the cerebellar and in the forebrain area, including the olfactory bulb, cerebral cortex, and hippocampus. these brain regions are responsible for the integration and processing of various sensory information as well as various forms of learning and memory. the roles of many other hvs genes in brain functions in both honey bees and mice remain to be determined. the number of honey bee hvs genes is only and this is consistent with the fact that gene loss and acquisition events are relatively rare as compared to amino acid substitution, cis-regulatory mutation, dna rearrangement, and duplication during evolution. a vast majority of honey bee genes are, indeed, conserved in all solitary ecdysozoan genomes, suggesting that advanced honey bee brain functions are primarily driven by increasing complexity in the network of gene interactions. since out of hvs gene products were predicted, by using go terms, to be associated with transcription and modification of dna as well as rna processing in the nucleus, they could participate in such processes by regulating dna transcription, splicing, and non-coding rna synthesis. these hvs genes would provide an important resource for future studies on gene function.  large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of urbilateria is lost in each animal lineage. this event could be one of the underlying mechanisms responsible for metazoan diversity. we have found that hvs genes are biased towards expression in the brains and gonads of honey bees and mice, and 35% of hvs genes are associated with dna transcription and rna processing by go analysis. these results suggest that hvs genes include genes that are biased towards expression in the brain and gonad. it also demonstrates that at least some of urbilaterian genes are selectively retained in the specific animal lineage to support the species-specific phenotypes. 
many hvs genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee hvs genes was found to be brain enriched, and 24% of mouse hvs genes were highly expressed in either or both the brain and testis. moreover, a minimum of 38% of mouse hvs genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of bilateria is lost in each animal lineage. these results suggest that hvs genes include genes that are biased towards expression in the brain and gonads. they also demonstrate that at least some of urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes. to understand the functions and evolutionary mechanisms of such ancient urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes as a model. however, the present functions of these ancient genes have not been addressed extensively. this event could be one of the underlying mechanisms involved in generating metazoan diversity. we identified honey bee and mouse hvs genes. furthermore, gene ontology analysis of hvs genes predicted that 35% of genes are associated with dna transcription and rna processing.
 large-scale comparison of metazoan genomes has revealed that a significant fraction of genes of the last common ancestor of bilateria is lost in each animal lineage. this event could be one of the underlying mechanisms involved in generating metazoan diversity. however, the present functions of these ancient genes have not been addressed extensively. to understand the functions and evolutionary mechanisms of such ancient urbilaterian genes, we carried out comprehensive expression profile analysis of genes shared between vertebrates and honey bees but not with the other sequenced ecdysozoan genomes as a model.  we identified honey bee and mouse hvs genes. many hvs genes exhibited tissue-selective expression patterns; intriguingly, the expression of 60% of honey bee hvs genes was found to be brain enriched, and 24% of mouse hvs genes were highly expressed in either or both the brain and testis. moreover, a minimum of 38% of mouse hvs genes demonstrated neuron-enriched expression patterns, and 62% of them exhibited expression in selective brain areas, particularly the forebrain and cerebellum. furthermore, gene ontology analysis of hvs genes predicted that 35% of genes are associated with dna transcription and rna processing.  these results suggest that hvs genes include genes that are biased towards expression in the brain and gonads. they also demonstrate that at least some of urbilaterian genes retained in the specific animal lineage may be selectively maintained to support the species-specific phenotypes. 