BACKGROUND
a comparison of the genomes of five insects and five vertebrates revealed that there were approximately  <dig>  genes present in all the insects but absent in the vertebrates.
in contrast, there were approximately  <dig>  genes present in all the vertebrates but absent in the insects  <cit> .
the number of vertebrate-specific genes is five times larger than that of insect-specific genes, thus indicating that vertebrates have more complex gene pools than insects.
some of these genes have been acquired in order to support insect- and vertebrate-specific characteristics during evolution.
however, if some of these vertebrate genes are shared with lophotrochozoans , this would imply that they were present in the last common ancestor of bilateria  and have been lost from insects during evolution  <cit> .
a number of genes categorized to this group have already been reported and characterized  <cit> .
meanwhile, if some of these insect genes are shared with lophotrochozoans, this would suggest that they were specifically acquired by the last common ancestor of protostomes but not deuterostomes.
such genes have not been reported to date.
the origin and evolution of the metazoan central nervous system  have been intensively discussed.
large-scale expression analysis of neural genes in hemichordates has revealed that the mediolateral patterning genes  and neural differentiation markers are expressed around the circumference of the embryo  <cit> .
these results suggest that the centralization of a nervous system was acquired independently in deuterostomes and protostomes  <cit> .
meanwhile, several studies on the development of the cns in drosophila melanogaster and mouse have revealed common genetic patterning mechanisms in the formation of the insect and vertebrate brain.
in both insects and vertebrates, the correct regionalization and neuronal identity of the anterior brain region is regulated by the cephalic gap genes otd/otx and ems/emx, whereas patterning of the posterior brain involves members of the hox genes  <cit> .
a recent study on gene expression patterns in the brain of developing annelids  has demonstrated that the patterning mechanism of the cns is well conserved among chordates and annelids  <cit> .
these studies strongly indicate that urbilateria already had an anatomically complex cns.
furthermore, cross-species comparisons of genome sequences and expressed sequence tag data sets have demonstrated the presence of a common ancestral cns at the molecular level  <cit> .
these results suggest that urbilateria and the last common ancestor of protostomes were genetically complex, and have complex nervous systems  <cit> .
mushroom bodies  are lobed neuropils that comprise long and approximately parallel axons originating from clusters of minute basophilic cells located dorsally in the most anterior neuromere of the cns.
structures with these morphological properties are found in many marine annelids  and almost all arthropods  except crustaceans  <cit> .
mbs are higher multisensory centers  of the insect brain and are implicated in olfactory and other forms of associative learning  <cit> .
there are two possibilities regarding the presence of mbs in different animal lineages .
one possibility is that the ancient mb-like structure was present in the cns of the last common ancestor of all protostomes, and then some species have evolved the present mbs but the others have lost it secondarily during evolution.
another possibility is that mbs have independently evolved several times in different animal lineages by convergent evolution.
it was reported that several genes encoding transcription factors, eyeless , twin of eyeless , and dachsund , are necessary for the development of the drosophila mb  <cit> .
their homologs are present in various metazoan genomes, and pax <dig> , for example, also has essential roles for neural development  <cit> .
to prove the single origin of mbs, it will be necessary to demonstrate that the expression domains of the above genes are conserved in the arthropod and lophotrochozoan cns during development.
here, we report a novel gene, namely psgef , which is present in insect and lottia  genomes but absent in nematostella , deuterostome, and nematode genomes.
it is likely that psgef was specifically acquired by the last common ancestor of protostomes, and then lost in some species, for example nematodes.
intriguingly, the presence of psgef containing the c <dig>  pdz, and rhogef domains appears to coincide with the presence of mbs.
further, in drosophila, psgef functions as a gef for rac and is essential for axon development in mbs.
these results suggest that gain, retention, and loss of psgef are associated with some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians.
thus, psgef is one of candidate genes associated with the diversity of metazoan nervous systems.
RESULTS
identification of psgef gene uniquely shared between insects and limpets
a large-scale comparison of the genomes of five insects  and five vertebrates  revealed that there were approximately  <dig>  insect-specific orthologous genes  <cit> .
we searched among these genes for those that are highly expressed in the drosophila cns by screening transgenic lines in which gal <dig> was inserted in the promoter regions of candidate genes, in order to understand the genetic basis for the development and functions specific for the insect nervous system.
from the screening, we found one gene, the psgef gene .
drosophila psgef  encodes a protein with the c <dig>  pdz, and rhogef domains .
the c <dig> domain is a ca2+-dependent membrane-targeting module found in many proteins involved in signal transduction or membrane trafficking  <cit> .
it is thought to be involved in ca2+-dependent phospholipid binding and in membrane-targeting processes  <cit> .
the pdz domain mediates binding with other proteins and is found in many signaling proteins frequently associated with the plasma membrane  <cit> .
moreover, it is often associated with scaffolding proteins important for synaptic development  <cit> .
the rhogef domain activates rho family gtpases, namely, rho, rac, and cdc <dig>  through release of bound guanosine diphosphate and subsequent binding of guanosine triphosphate  <cit> .
thus, rhogef activity of psgef appears to require an increase in the intracellular ca2+ level as well as association of psgef with other proteins.
as shown in figures 1a and 1b, two different dmpsgef transcripts  are found.
the short mrna encodes a 786-amino acid protein containing the c <dig> and pdz domains, and the long mrna encodes a 1493-amino acid protein containing the c <dig>  pdz, and rhogef domains.
these two types of dmpsgef mrnas appear to be synthesized by alternative polyadenylation; polyadenylation at the 3' end of exons  <dig> and  <dig> results in the synthesis of short and long dmpsgef mrnas, respectively .
the short psgef with c <dig> and pdz domains exhibits significant similarity to the vertebrate rgs <dig>  which also contains c <dig> and pdz domains  <cit> .
however, psgef lacks amino acid sequences necessary for constituting the regulator of g-protein signaling  domain together with the c <dig> and pdz domains, thus suggesting that the short psgef does not function as an rgs.
dmpsgef orthologs are also present in the genomes of nasonia vitripenis , pediculus humanus corporis , and acyrthosiphon pisum .
the proteins encoded by these contain c <dig>  pdz, and rhogef domains, similar to the long version of the dmpsgef.
psgef was therefore present in the common ancestor of holometabolous and hemimetabolous insects.
further, we searched for insect psgef orthologs in the genomes of nematostella vectensis , strongylocentrotus purpuratus , ciona intestinalis , lottia gigantea , and caenorhabditis elegans.
we have found that only lottia contains an insect psgef ortholog coding for a protein containing the c <dig>  pdz, and rhogef domains.
as psgef has a complex domain organization, it may be difficult to identify the orthologs if they contain very large introns in some cases.
psgef is present in insects  and limpet  but not sea anemone or deuterostomes, suggesting that psgef was specifically acquired by the last common ancestor of protostomes.
drosophila , tribolium , pediculus , apis , and lottia  psgefs  share the c <dig>  pdz, and rhogef domains as shown in figure 1c.
the alignment of amino acid sequences containing the above functional domains of five psgef proteins demonstrates that they show significant similarity only in the c <dig>  pdz, and rhogef domains .
this was also the case when the full-length amino acid sequences were analyzed .
the exon-intron organizations of the above five psgef genes are shown in figure 2a.
insect and limpet psgefs contain one and two introns in the c <dig> domain-coding regions, respectively.
intriguingly, the position of one out of two introns is conserved at the same phase in five species .
nevertheless, the size of this particular intron is varied, ranging from  <dig> to  <dig>  base pairs.
dmpsgef has no intron, tcpsgef and ampsgef have one intron, and phcpsgef and lgpsgef contain two introns in the pdz domain-coding regions .
tcpsgef, phcpsgef, and lgpsgef share one intron position as well as phases among them .
dmpsgef and lgpsgef contain one intron, ampsgef has two introns, and tcpsgef and phcpsgef contain four introns in the rhogef domain-coding regions .
among them, one intron position is conserved at the same phase in dmpsgef, tcpsgef, and phcpsgef.
in addition, the positions and phases of two introns are conserved in tcpsgef and phcpsgef.
thus, tcpsgef and phcpsgef share the same positions and phases of three out of four introns .
these results demonstrate that some but not all intron positions are conserved at the same phase in the c <dig>  pdz, and rhogef domain-coding regions of five psgefs from different species.
daphnia psgef lacks rhogef domain
we next analyzed the psgef ortholog in daphnia pulex, which belongs to crustacea, one of major arthropod groups.
the psgef ortholog is present in scaffold  <dig> of the daphnia pulex genome assembly; however, the exons encoding the rhogef domain are apparently missing .
according to the jgi database  <cit> , three predicted genes  are present in this genomic region.
a snap_ <dig> mrna was detected by using reverse transcriptase-polymerase chain reaction  and appears to encode the psgef protein containing only the c <dig> and pdz domains.
snap_ <dig>  but not snap_ <dig> mrna was detected by rt-pcr, and it encodes a novel protein with a ph-like domain .
the above results indicate that psgef with the c <dig>  pdz, and rhogef domains is specifically present in lophotrochozoan and insect genomes but not in crustacean genomes.
this distribution among metazoans accurately coincides with the presence of mbs, prominent neuropils involved in processing multiple sensory inputs as well as associative learning in the insect, platyhelminth, and annelid brains  <cit> .
we therefore tested whether psgef plays a role in the development or functions of mb in drosophila.
region-specific expression of dmpsgef in the embryonic and adult central nervous system
we identified several gal <dig> enhancer trap lines  located in the promoter region of dmpsgef.
these gal <dig> lines were crossed with uas-mcd8-gfp lines to detect the expression of dmpsgef in embryos and adults, and all of them exhibited the same expression patterns.
dmpsgef is expressed in the subsets of cells in the brain and ventral nerve cord as well as cells at the midgut fusion point in the stage- <dig> embryos .
dmpsgef is highly expressed in mbs, the antennal lobe, and the optic lobe of the adult brain.
in addition, there are several large discrete dmpsgef-positive cells surrounding the antennal lobe .
the presence of dmpsgef-positive lobes of mbs, as observed by staining for fasciclin ii , suggested that dmpsgef is expressed in the alpha/beta neurons of mbs .
ampsgef mrna is also highly expressed in the adult honey bee brain .
dmpsgef is necessary for the axon development of mushroom bodies
to understand the functions of dmpsgef, we generated dmpsgef loss-of-function mutants by imprecise excision of np <dig>  two deletion mutants were recovered.
the deletion mutants included dmpsgefΔ <dig>  in which  <dig>  kb genomic dna containing the promoter region is deleted, and dmpsgefΔ <dig>  in which  <dig>  kb genomic dna containing exons  <dig> and  <dig> is deleted .
both dmpsgefΔ <dig> and dmpsgefΔ <dig> are viable and fertile with no morphological defects.
the expression of dmpsgef mrna was examined in np <dig>  dmpsgefΔ <dig>  and dmpsgefΔ <dig> embryos by rt-pcr.
both short and long mrnas are expressed in the np <dig> embryos; however, they are absent in dmpsgefΔ <dig> and dmpsgefΔ <dig> embryos.
the adjacent cg <dig> mrna is equally expressed in all embryos .
these results suggest that both dmpsgefΔ <dig> and dmpsgefΔ <dig> are dmpsgef-null alleles.
since dmpsgef is highly expressed in the alpha/beta neurons of mbs , we analyzed the morphology of the alpha/beta lobes in dmpsgefΔ <dig> hemizygous males by immunostaining of fas ii.
the fas ii-positive alpha/beta lobes are thinner in dmpsgefΔ <dig> than in wild-type males .
furthermore, the alpha lobes of late-born alpha/beta neurons visualized by 201y-gal <dig> and uas-mcd8-gfp are thinner in dmpsgefΔ <dig> because the alpha lobes are often short, and their positions along the beta lobes vary .
the same results were obtained for dmpsgefΔ <dig> .
we also analyzed the morphology of the alpha'/beta' lobes in dmpsgefΔ <dig> and wild-type males by anti-trio antibody staining  <cit> .
no significant difference was observed .
for a more accurate analysis of the role of dmpsgef in the development of mbs, the morphology of alpha/beta neurons at a single-cell level was examined by using the mosaic analysis with a repressible cell marker  system  <cit> .
the wild-type alpha/beta neurons bifurcate their axons into the alpha and beta lobes ; however, 34% of late-born dmpsgefΔ <dig> alpha/beta neurons  have branching defects, and the axons toward the alpha lobe are missing .
these results demonstrate that dmpsgef is necessary for the correct axonal development of alpha/beta neurons in mbs.
dmpsgef genetically interacts with rac <dig> but not other rho family members for the axon development in mushroom bodies
as psgef is expected to function as a rhogef, we analyzed the genetic interactions of dmpsgef with rho family members.
we thus examined the morphology of the alpha/beta lobes in the mbs of dmpsgefΔ <dig> hemizygous males in a rho1e <dig> , rac1j <dig>  rac2Δ, and mtlΔ heterozygous background.
for the interaction with cdc <dig>  dmpsgefΔ <dig> homozygous females with a cdc <dig> heterozygous background were analyzed.
it was observed that reducing the gene dosages of cdc <dig>  rho <dig>  rac <dig>  and mtl does not affect the phenotypes of the alpha/beta lobes as observed with dmpsgefΔ <dig> .
however, reduction in the rac <dig> gene dosage dramatically influences the phenotypes: a pair of alpha/beta lobes was missing in 90% of the animals examined .
it was observed that 65% of rac1j11/+ heterozygotes have normal alpha/beta lobes , and 35% have the branching defects as previously reported  <cit> .
these results demonstrate that dmpsgef genetically interacts with rac <dig>  however, dmpsgef does not exhibit genetic interaction with pak, which is a downstream effector of rac <dig> .
this suggests that dmpsgef activates rac1; the active gtp·rac <dig> is involved in the axon development in mb neurons via a pak-independent signaling pathway.
dmpsgef functions as a gef for rac in cultured cells
to test whether psgef is a gef for rac, the rhogef domain of dmpsgef was expressed in hela cells, and the f-actin of these cells was visualized by using fluorescein isothiocyanate -phalloidin.
the rhogef activity of psgef is likely to be affected by the intracellular ca2+ level as well as its interaction with other proteins through the c <dig> and pdz domains.
thus, only the rhogef domain of dmpsgef was expressed in the hela cells.
actin polymerization in membranes resulting in membrane ruffling is specifically observed in the cells expressing the rhogef domain of dmpsgef .
these phenotypes are similar to those obtained with the expression of active forms of rac but not cdc <dig> or rho  <cit> .
the active forms of cdc <dig> and rho are known to induce filopodia and stress fibers, respectively  <cit> .
these results therefore suggest that the rhogef domain of dmpsgef activates rac but not cdc <dig> or rho in cultured cells.
this finding is consistent with the genetic interaction of dmpsgef with rac <dig> but not cdc <dig> or rho <dig>  as described earlier.
discussion
genes specifically necessary for the development and/or functions of the insect nervous system
we are interested in discovering the specific molecular features underlying the functions of insect nervous systems.
there are several properties specific to the insect nervous system.
one of the characteristics of the holometabolous insect brain is the development of the brain from the larval to the adult stages during metamorphosis.
although the overall brain organization is preserved during metamorphosis, dramatic remodeling of neuronal circuits occurs.
some embryonic and early larval neurons die, and others undergo axon pruning and regrowth.
furthermore, many neurons are born during metamorphosis  <cit> .
the genes involved in neuronal remodeling and metamorphosis might be specific to the insect genomes.
with regard to the structural characteristics of the insect brain, mbs are specific neuropil structures found in many marine annelids and almost all arthropod groups, except crustaceans  <cit> .
mbs are lobed neuropils that comprise long and approximately parallel axons originating from clusters of minute basophilic cells located dorsally in the most anterior neuromere of the cns.
mbs are higher olfactory and multisensory centers of the insect brain and are implicated in odor discrimination and in olfactory and other forms of associative learning  <cit> .
it is not known how mbs evolved in different taxa.
there may be some common genes among annelids and insects, and they may have a role in the evolution of mbs.
psgef may be one of these genes.
origin and evolution of psgef
we first identified psgef as an insect-specific gene, which is highly expressed in the drosophila cns.
it is present in the sequenced genomes of insects but not vertebrates.
a search for psgef orthologs in other sequenced genomes revealed that psgef is present in the parasitic wasp, the human body louse, and, surprisingly, the limpet lottia.
due to the phylogenetic distance between insects and limpets, it is unlikely that psgef independently evolved in these taxa.
in fact, a domain-by-domain phylogenetic analysis of rhogefs from different species demonstrates that the c <dig>  pdz, and rhogef domains of lgpsgef cluster with those of insect psgefs, and do not branch with those of different families .
thus, it is unlikely that psgefs have been independently generated by exon shuffling  in the lophotrochozoan and insect lineages.
the exon-intron structures of five psgefs from different species also support this conclusion since the positions as well as phases of some introns are conserved in the c <dig>  pdz, and rhogef domain-coding regions .
this may suggest that the last common ancestor of protostomes gained the intron-rich ancestor of psgef, and some of these ancient introns have been lost, and some new introns have been gained in each species, as demonstrated with, for example, nuclear oxphos genes  <cit> .
apparently, intron loss has been most extensive in dmpsgef.
psgef was absent in urbilateria, and then specifically acquired by the common ancestor of all protostomes but not deuterostomes or cnidarians.
how was the ancient psgef originated?
since vertebrates possess rgs <dig> with c <dig> and pdz domains similar to the short form of dmpsgef, this gene was present in urbilateria.
it then acquired the exons encoding the rhogef domain by exon shuffling or exon capture from the different families to generate psgef  <cit> .
intriguingly, psgef is absent in nematodes, suggesting that it has been secondarily lost from some ecdysozoan species.
since it has been demonstrated that the evolutionary rates of insects and nematodes are fast and comparable  <cit> , it is difficult to imagine that nematodes evolved an alternative pathway or paralogs of psgef to compensate for the absence of psgef and insects failed to evolve such systems.
a more plausible explanation is that psgef has been selectively retained in the insect and limpet genomes because it continues to play an important role in these animals but not in nematodes.
an analysis of the daphnia genome revealed that the daphnia psgef contains only the c <dig> and pdz domains, and it corresponds to the short form of dmpsgef.
the exons encoding rhogef domains are apparently missing in the daphnia genome .
this demonstrates that the exons encoding the rhogef domain were specifically deleted in crustaceans after split from a common ancestor of crustaceans and insects  <cit> .
these results suggest that daphnia psgef cannot function as a rhogef, which is critical for mb axon development .
thus, psgef containing the c <dig>  pdz, and rhogef domains appears to be present only in the limpet lottia and insects but not in crustaceans.
although it is not known whether mb-like structures are present in the limpet brain, this distribution pattern among metazoans coincides with the presence of mbs in the brains of certain platyhelminthes, marine annelids, and insects but not crustaceans  <cit> .
these results suggest that psgef may be associated with the evolution of mb-like brain structures and is, in fact, necessary for mb axon development in drosophila .
molecular functions of psgef
alternative splicing of dmpsgef determines the presence or absence of the rhogef domain in the protein .
the activation of rac by psgef is therefore directly regulated by alternative splicing.
what are the functions of the short version of psgef with only the c <dig> and pdz domains, which is also present in daphnia?
if both short and long psgefs are present in a single neuron, the short protein may exert dominant negative effects on the long protein.
the short and long proteins may compete for binding with ca2+ and certain proteins through the c <dig> and pdz domains, respectively.
this, in turn, regulates the rhogef activity of the long psgef.
if their expression is mutually exclusive, they may have independent functions in different neurons.
since it is difficult to distinguish the expression patterns of short and long dmpsgef mrnas in drosophila, an examination of the functions of short psgef in daphnia may provide an answer to the abovementioned question.
although alternative splicing of psgef occurs in the fruit fly and honey bee , it remains to be established whether the same alternative splicing occurs in other insects and limpets.
loss of dmpsgef results in thinner alpha lobes than those of wild-type .
furthermore, short and multiple alpha lobes derived from late-born 201y-gal4-expressing alpha/beta neurons were observed in mbs of dmpsgefΔ <dig> flies, as shown in figures 6c to 6e.
these results suggest that the alpha/beta neurons bifurcate their axons at more random positions in dmpsgefΔ <dig> flies.
further, the dmpsgefΔ <dig> alpha/beta neurons may not respond well to the signals provided by guidance neurons .
moreover, some alpha/beta neurons fail to extend their alpha-axonal branches .
meanwhile, gamma neurons appear to be normal.
alpha'/beta' neurons are generally born during the late third instar, and their axons  remain intact during metamorphosis  <cit> .
these lobes may serve as the guiding axons for the bifurcation of the axons of alpha/beta neurons.
we therefore examined the alpha'/beta' lobes in dmpsgefΔ <dig> flies by immunostaining with anti-trio antibody  <cit> .
their morphology was found to be normal, thus suggesting that dmpsgef is specifically necessary for the axonal development of alpha/beta neurons in mbs.
to identify rho family members activated by psgef, we first tested the genetic interaction of dmpsgef with five rho family members.
the results indicated that dmpsgef genetically interacts with rac <dig> but not other family members .
consistent with this observation, the ectopic expression of the rhogef domain of dmpsgef induces actin polymerization in the membrane of hela cells .
these results thus suggest that psgef activates rac but not cdc <dig> or rho in vivo.
intriguingly, dmpsgef does not genetically interact with pak, one of the downstream effectors of rac .
trio was shown to activate rac and promote lim kinase activity via pak to induce axon growth inhibition  <cit> .
thus, dmpsgef induces rac activation and may stimulate axon growth via a pak-independent pathway along with still life   <cit> .
three gefs for rac  appear to function for the morphogenesis of mbs in drosophila.
since each gef protein has specific functional domains  in addition to the rhogef domain, the mechanism to activate individual gefs should be different.
specifically, the presence of the c <dig> domain in psgef suggests that the increase of intracellular ca2+ level is essential for the localization of psgef at the plasma membrane where rac proteins are anchored.
the function of dmpsgef is therefore dependent on the local increase of cytosolic ca2+ level which occurs during the axonal development of alpha/beta neurons in mbs.
dmpsgef is expressed not only in the mbs but also in the antennal and optic lobes in adult drosophila brain.
we analyzed the axonal projection patterns of several olfactory neurons into the antennal lobes in dmpsgefΔ <dig> flies; however, no significant difference was observed relative to the patterns observed in the wild-type .
the glomerular structures of the antennal lobes visualized by immunostaining with anti-nc <dig> antibody were found to be normal .
although we did not observe gross morphological defects in the antennal and optic lobes in dmpsgefΔ <dig> flies, it is possible that there were subtle defects in these brain regions.
no other proteins with c <dig> and pdz domains are present besides dmpsgef in drosophila.
however, other gefs for rac  could be partially redundant with dmpsgef.
this may explain the lack of phenotypes associated with the antennal lobes in dmpsgefΔ <dig> flies.
we are also testing the olfactory and visual behaviors of adult dmpsgefΔ <dig> flies at present.
association of psgef with structural and/or functional features common between insect and lophotrochozoan nervous systems
the specific acquisition of psgef by the last common ancestor of protostomes followed by the retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians.
we would like to propose that psgef may be associated with the presence of mbs, specific brain structures in insects, annelids, and platyhelminthes  <cit> .
the fact that psgef containing the c <dig>  pdz, and rhogef domains is specifically retained in animals having mbs  in addition to functioning as a gef for rac, which is essential for the correct axonal development of mbs in drosophila, suggests that the last common ancestor of protostomes might have possessed an ancient mb-like structure in the nervous system, and the mbs found in present animals may have evolved into specific neuropil structures in insect and lophotrochozoan brains supported in part by the retention of psgef in their genomes during evolution.
nematodes and crustaceans lost the full length psgef, and mb-like structures have disappeared as a result .
however, the functions of psgef do not appear to be limited to mbs because it is also expressed in the optic and antennal lobes of adult drosophila brain.
although mb-like structures are found in mollusks such as achatina  <cit>  and octopus  <cit> , it is not known whether limpets also have mb-like structures in their brains.
it is thus possible that the conservation of psgef could be for other functional and developmental constraints.
recent studies have demonstrated that the expression profile and roles of genes patterning the nervous system in the embryos of vertebrates and annelids are quite similar  <cit> .
since it is unlikely that this remarkable similarity arose from convergent evolution, they suggest that urbilateria may have already had a quite complex cns, which is also supported by comparative genomics  <cit> .
it is therefore not surprising that the origin of mbs could be traced back to the last common ancestor of protostomes, in which ancient mb-like structures might have played a role in multimodal sensory integration and even learning and memory.
to prove a single origin of mbs, it will be necessary to demonstrate that the same gene sets  act similarly for the development of mbs in insects and lophotrochozoans.
the homologs of these genes are also present in the vertebrate genomes, and they function in the early development of nervous systems.
as these genes encode transcription factors, they could function as a common 'genetic code' to specify the brain region, giving rise to mbs and its vertebrate equivalents.
then, as a next level, a different set of genes such as psgef is necessary to generate the specific structures of mbs, which are present in the brains of insects and lophotrochozoans but not vertebrates.
in this regard, psgef is one of genes involved in generating the diversity of metazoan nervous systems.
CONCLUSIONS
the specific acquisition of psgef by the last common ancestor of protostomes followed by the retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems , which are absent in all deuterostomes and cnidarians.
psgef is therefore one of genes associated with the diversity of metazoan nervous systems.

 a comparison of the genomes of five insects and five vertebrates revealed that there were approximately genes present in all the insects but absent in the vertebrates. in contrast, there were approximately genes present in all the vertebrates but absent in the insects. the number of vertebrate-specific genes is five times larger than that of insect-specific genes, thus indicating that vertebrates have more complex gene pools than insects. some of these genes have been acquired in order to support insect- and vertebrate-specific characteristics during evolution. however, if some of these vertebrate genes are shared with lophotrochozoans, this would imply that they were present in the last common ancestor of bilateria and have been lost from insects during evolution. a number of genes categorized to this group have already been reported and characterized. meanwhile, if some of these insect genes are shared with lophotrochozoans, this would suggest that they were specifically acquired by the last common ancestor of protostomes but not deuterostomes. such genes have not been reported to date. the origin and evolution of the metazoan central nervous system have been intensively discussed. large-scale expression analysis of neural genes in hemichordates has revealed that the mediolateral patterning genes and neural differentiation markers are expressed around the circumference of the embryo. these results suggest that the centralization of a nervous system was acquired independently in deuterostomes and protostomes. meanwhile, several studies on the development of the cns in drosophila melanogaster and mouse have revealed common genetic patterning mechanisms in the formation of the insect and vertebrate brain. in both insects and vertebrates, the correct regionalization and neuronal identity of the anterior brain region is regulated by the cephalic gap genes otd/otx and ems/emx, whereas patterning of the posterior brain involves members of the hox genes. a recent study on gene expression patterns in the brain of developing annelids has demonstrated that the patterning mechanism of the cns is well conserved among chordates and annelids. these studies strongly indicate that urbilateria already had an anatomically complex cns. furthermore, cross-species comparisons of genome sequences and expressed sequence tag data sets have demonstrated the presence of a common ancestral cns at the molecular level. these results suggest that urbilateria and the last common ancestor of protostomes were genetically complex, and have complex nervous systems. mushroom bodies are lobed neuropils that comprise long and approximately parallel axons originating from clusters of minute basophilic cells located dorsally in the most anterior neuromere of the cns. structures with these morphological properties are found in many marine annelids and almost all arthropods except crustaceans. mbs are higher multisensory centers of the insect brain and are implicated in olfactory and other forms of associative learning. there are two possibilities regarding the presence of mbs in different animal lineages. one possibility is that the ancient mb-like structure was present in the cns of the last common ancestor of all protostomes, and then some species have evolved the present mbs but the others have lost it secondarily during evolution. another possibility is that mbs have independently evolved several times in different animal lineages by convergent evolution. it was reported that several genes encoding transcription factors, eyeless, twin of eyeless, and dachsund, are necessary for the development of the drosophila mb. their homologs are present in various metazoan genomes, and pax, for example, also has essential roles for neural development. to prove the single origin of mbs, it will be necessary to demonstrate that the expression domains of the above genes are conserved in the arthropod and lophotrochozoan cns during development. here, we report a novel gene, namely psgef, which is present in insect and lottia genomes but absent in nematostella, deuterostome, and nematode genomes. it is likely that psgef was specifically acquired by the last common ancestor of protostomes, and then lost in some species, for example nematodes. intriguingly, the presence of psgef containing the c pdz, and rhogef domains appears to coincide with the presence of mbs. further, in drosophila, psgef functions as a gef for rac and is essential for axon development in mbs. these results suggest that gain, retention, and loss of psgef are associated with some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians. thus, psgef is one of candidate genes associated with the diversity of metazoan nervous systems.  identification of psgef gene uniquely shared between insects and limpets a large-scale comparison of the genomes of five insects and five vertebrates revealed that there were approximately insect-specific orthologous genes. we searched among these genes for those that are highly expressed in the drosophila cns by screening transgenic lines in which gal was inserted in the promoter regions of candidate genes, in order to understand the genetic basis for the development and functions specific for the insect nervous system. from the screening, we found one gene, the psgef gene. drosophila psgef encodes a protein with the c pdz, and rhogef domains. the c domain is a ca2+-dependent membrane-targeting module found in many proteins involved in signal transduction or membrane trafficking. it is thought to be involved in ca2+-dependent phospholipid binding and in membrane-targeting processes. the pdz domain mediates binding with other proteins and is found in many signaling proteins frequently associated with the plasma membrane. moreover, it is often associated with scaffolding proteins important for synaptic development. the rhogef domain activates rho family gtpases, namely, rho, rac, and cdc through release of bound guanosine diphosphate and subsequent binding of guanosine triphosphate. thus, rhogef activity of psgef appears to require an increase in the intracellular ca2+ level as well as association of psgef with other proteins. as shown in figures 1a and 1b, two different dmpsgef transcripts are found. the short mrna encodes a 786-amino acid protein containing the c and pdz domains, and the long mrna encodes a 1493-amino acid protein containing the c pdz, and rhogef domains. these two types of dmpsgef mrnas appear to be synthesized by alternative polyadenylation; polyadenylation at the 3' end of exons and results in the synthesis of short and long dmpsgef mrnas, respectively. the short psgef with c and pdz domains exhibits significant similarity to the vertebrate rgs which also contains c and pdz domains. however, psgef lacks amino acid sequences necessary for constituting the regulator of g-protein signaling domain together with the c and pdz domains, thus suggesting that the short psgef does not function as an rgs. dmpsgef orthologs are also present in the genomes of nasonia vitripenis, pediculus humanus corporis, and acyrthosiphon pisum. the proteins encoded by these contain c pdz, and rhogef domains, similar to the long version of the dmpsgef. psgef was therefore present in the common ancestor of holometabolous and hemimetabolous insects. further, we searched for insect psgef orthologs in the genomes of nematostella vectensis, strongylocentrotus purpuratus, ciona intestinalis, lottia gigantea, and caenorhabditis elegans. we have found that only lottia contains an insect psgef ortholog coding for a protein containing the c pdz, and rhogef domains. as psgef has a complex domain organization, it may be difficult to identify the orthologs if they contain very large introns in some cases. psgef is present in insects and limpet but not sea anemone or deuterostomes, suggesting that psgef was specifically acquired by the last common ancestor of protostomes. drosophila, tribolium, pediculus, apis, and lottia psgefs share the c pdz, and rhogef domains as shown in figure 1c. the alignment of amino acid sequences containing the above functional domains of five psgef proteins demonstrates that they show significant similarity only in the c pdz, and rhogef domains. this was also the case when the full-length amino acid sequences were analyzed. the exon-intron organizations of the above five psgef genes are shown in figure 2a. insect and limpet psgefs contain one and two introns in the c domain-coding regions, respectively. intriguingly, the position of one out of two introns is conserved at the same phase in five species. nevertheless, the size of this particular intron is varied, ranging from to base pairs. dmpsgef has no intron, tcpsgef and ampsgef have one intron, and phcpsgef and lgpsgef contain two introns in the pdz domain-coding regions. tcpsgef, phcpsgef, and lgpsgef share one intron position as well as phases among them. dmpsgef and lgpsgef contain one intron, ampsgef has two introns, and tcpsgef and phcpsgef contain four introns in the rhogef domain-coding regions. among them, one intron position is conserved at the same phase in dmpsgef, tcpsgef, and phcpsgef. in addition, the positions and phases of two introns are conserved in tcpsgef and phcpsgef. thus, tcpsgef and phcpsgef share the same positions and phases of three out of four introns. these results demonstrate that some but not all intron positions are conserved at the same phase in the c pdz, and rhogef domain-coding regions of five psgefs from different species. daphnia psgef lacks rhogef domain we next analyzed the psgef ortholog in daphnia pulex, which belongs to crustacea, one of major arthropod groups. the psgef ortholog is present in scaffold of the daphnia pulex genome assembly; however, the exons encoding the rhogef domain are apparently missing. according to the jgi database, three predicted genes are present in this genomic region. a snap_ mrna was detected by using reverse transcriptase-polymerase chain reaction and appears to encode the psgef protein containing only the c and pdz domains. snap_ but not snap_ mrna was detected by rt-pcr, and it encodes a novel protein with a ph-like domain. the above results indicate that psgef with the c pdz, and rhogef domains is specifically present in lophotrochozoan and insect genomes but not in crustacean genomes. this distribution among metazoans accurately coincides with the presence of mbs, prominent neuropils involved in processing multiple sensory inputs as well as associative learning in the insect, platyhelminth, and annelid brains. we therefore tested whether psgef plays a role in the development or functions of mb in drosophila. region-specific expression of dmpsgef in the embryonic and adult central nervous system we identified several gal enhancer trap lines located in the promoter region of dmpsgef. these gal lines were crossed with uas-mcd8-gfp lines to detect the expression of dmpsgef in embryos and adults, and all of them exhibited the same expression patterns. dmpsgef is expressed in the subsets of cells in the brain and ventral nerve cord as well as cells at the midgut fusion point in the stage- embryos. dmpsgef is highly expressed in mbs, the antennal lobe, and the optic lobe of the adult brain. in addition, there are several large discrete dmpsgef-positive cells surrounding the antennal lobe. the presence of dmpsgef-positive lobes of mbs, as observed by staining for fasciclin ii, suggested that dmpsgef is expressed in the alpha/beta neurons of mbs. ampsgef mrna is also highly expressed in the adult honey bee brain. dmpsgef is necessary for the axon development of mushroom bodies to understand the functions of dmpsgef, we generated dmpsgef loss-of-function mutants by imprecise excision of np two deletion mutants were recovered. the deletion mutants included dmpsgefΔ in which kb genomic dna containing the promoter region is deleted, and dmpsgefΔ in which kb genomic dna containing exons and is deleted. both dmpsgefΔ and dmpsgefΔ are viable and fertile with no morphological defects. the expression of dmpsgef mrna was examined in np dmpsgefΔ and dmpsgefΔ embryos by rt-pcr. both short and long mrnas are expressed in the np embryos; however, they are absent in dmpsgefΔ and dmpsgefΔ embryos. the adjacent cg mrna is equally expressed in all embryos. these results suggest that both dmpsgefΔ and dmpsgefΔ are dmpsgef-null alleles. since dmpsgef is highly expressed in the alpha/beta neurons of mbs, we analyzed the morphology of the alpha/beta lobes in dmpsgefΔ hemizygous males by immunostaining of fas ii. the fas ii-positive alpha/beta lobes are thinner in dmpsgefΔ than in wild-type males. furthermore, the alpha lobes of late-born alpha/beta neurons visualized by 201y-gal and uas-mcd8-gfp are thinner in dmpsgefΔ because the alpha lobes are often short, and their positions along the beta lobes vary. the same results were obtained for dmpsgefΔ. we also analyzed the morphology of the alpha'/beta' lobes in dmpsgefΔ and wild-type males by anti-trio antibody staining. no significant difference was observed. for a more accurate analysis of the role of dmpsgef in the development of mbs, the morphology of alpha/beta neurons at a single-cell level was examined by using the mosaic analysis with a repressible cell marker system. the wild-type alpha/beta neurons bifurcate their axons into the alpha and beta lobes; however, 34% of late-born dmpsgefΔ alpha/beta neurons have branching defects, and the axons toward the alpha lobe are missing. these results demonstrate that dmpsgef is necessary for the correct axonal development of alpha/beta neurons in mbs. dmpsgef genetically interacts with rac but not other rho family members for the axon development in mushroom bodies as psgef is expected to function as a rhogef, we analyzed the genetic interactions of dmpsgef with rho family members. we thus examined the morphology of the alpha/beta lobes in the mbs of dmpsgefΔ hemizygous males in a rho1e, rac1j rac2Δ, and mtlΔ heterozygous background. for the interaction with cdc dmpsgefΔ homozygous females with a cdc heterozygous background were analyzed. it was observed that reducing the gene dosages of cdc rho rac and mtl does not affect the phenotypes of the alpha/beta lobes as observed with dmpsgefΔ. however, reduction in the rac gene dosage dramatically influences the phenotypes: a pair of alpha/beta lobes was missing in 90% of the animals examined. it was observed that 65% of rac1j11/+ heterozygotes have normal alpha/beta lobes, and 35% have the branching defects as previously reported. these results demonstrate that dmpsgef genetically interacts with rac however, dmpsgef does not exhibit genetic interaction with pak, which is a downstream effector of rac. this suggests that dmpsgef activates rac1; the active gtp·rac is involved in the axon development in mb neurons via a pak-independent signaling pathway. dmpsgef functions as a gef for rac in cultured cells to test whether psgef is a gef for rac, the rhogef domain of dmpsgef was expressed in hela cells, and the f-actin of these cells was visualized by using fluorescein isothiocyanate -phalloidin. the rhogef activity of psgef is likely to be affected by the intracellular ca2+ level as well as its interaction with other proteins through the c and pdz domains. thus, only the rhogef domain of dmpsgef was expressed in the hela cells. actin polymerization in membranes resulting in membrane ruffling is specifically observed in the cells expressing the rhogef domain of dmpsgef. these phenotypes are similar to those obtained with the expression of active forms of rac but not cdc or rho. the active forms of cdc and rho are known to induce filopodia and stress fibers, respectively. these results therefore suggest that the rhogef domain of dmpsgef activates rac but not cdc or rho in cultured cells. this finding is consistent with the genetic interaction of dmpsgef with rac but not cdc or rho as described earlier. discussion genes specifically necessary for the development and/or functions of the insect nervous system we are interested in discovering the specific molecular features underlying the functions of insect nervous systems. there are several properties specific to the insect nervous system. one of the characteristics of the holometabolous insect brain is the development of the brain from the larval to the adult stages during metamorphosis. although the overall brain organization is preserved during metamorphosis, dramatic remodeling of neuronal circuits occurs. some embryonic and early larval neurons die, and others undergo axon pruning and regrowth. furthermore, many neurons are born during metamorphosis. the genes involved in neuronal remodeling and metamorphosis might be specific to the insect genomes. with regard to the structural characteristics of the insect brain, mbs are specific neuropil structures found in many marine annelids and almost all arthropod groups, except crustaceans. mbs are lobed neuropils that comprise long and approximately parallel axons originating from clusters of minute basophilic cells located dorsally in the most anterior neuromere of the cns. mbs are higher olfactory and multisensory centers of the insect brain and are implicated in odor discrimination and in olfactory and other forms of associative learning. it is not known how mbs evolved in different taxa. there may be some common genes among annelids and insects, and they may have a role in the evolution of mbs. psgef may be one of these genes. origin and evolution of psgef we first identified psgef as an insect-specific gene, which is highly expressed in the drosophila cns. it is present in the sequenced genomes of insects but not vertebrates. a search for psgef orthologs in other sequenced genomes revealed that psgef is present in the parasitic wasp, the human body louse, and, surprisingly, the limpet lottia. due to the phylogenetic distance between insects and limpets, it is unlikely that psgef independently evolved in these taxa. in fact, a domain-by-domain phylogenetic analysis of rhogefs from different species demonstrates that the c pdz, and rhogef domains of lgpsgef cluster with those of insect psgefs, and do not branch with those of different families. thus, it is unlikely that psgefs have been independently generated by exon shuffling in the lophotrochozoan and insect lineages. the exon-intron structures of five psgefs from different species also support this conclusion since the positions as well as phases of some introns are conserved in the c pdz, and rhogef domain-coding regions. this may suggest that the last common ancestor of protostomes gained the intron-rich ancestor of psgef, and some of these ancient introns have been lost, and some new introns have been gained in each species, as demonstrated with, for example, nuclear oxphos genes. apparently, intron loss has been most extensive in dmpsgef. psgef was absent in urbilateria, and then specifically acquired by the common ancestor of all protostomes but not deuterostomes or cnidarians. how was the ancient psgef originated? since vertebrates possess rgs with c and pdz domains similar to the short form of dmpsgef, this gene was present in urbilateria. it then acquired the exons encoding the rhogef domain by exon shuffling or exon capture from the different families to generate psgef. intriguingly, psgef is absent in nematodes, suggesting that it has been secondarily lost from some ecdysozoan species. since it has been demonstrated that the evolutionary rates of insects and nematodes are fast and comparable, it is difficult to imagine that nematodes evolved an alternative pathway or paralogs of psgef to compensate for the absence of psgef and insects failed to evolve such systems. a more plausible explanation is that psgef has been selectively retained in the insect and limpet genomes because it continues to play an important role in these animals but not in nematodes. an analysis of the daphnia genome revealed that the daphnia psgef contains only the c and pdz domains, and it corresponds to the short form of dmpsgef. the exons encoding rhogef domains are apparently missing in the daphnia genome. this demonstrates that the exons encoding the rhogef domain were specifically deleted in crustaceans after split from a common ancestor of crustaceans and insects. these results suggest that daphnia psgef cannot function as a rhogef, which is critical for mb axon development. thus, psgef containing the c pdz, and rhogef domains appears to be present only in the limpet lottia and insects but not in crustaceans. although it is not known whether mb-like structures are present in the limpet brain, this distribution pattern among metazoans coincides with the presence of mbs in the brains of certain platyhelminthes, marine annelids, and insects but not crustaceans. these results suggest that psgef may be associated with the evolution of mb-like brain structures and is, in fact, necessary for mb axon development in drosophila. molecular functions of psgef alternative splicing of dmpsgef determines the presence or absence of the rhogef domain in the protein. the activation of rac by psgef is therefore directly regulated by alternative splicing. what are the functions of the short version of psgef with only the c and pdz domains, which is also present in daphnia? if both short and long psgefs are present in a single neuron, the short protein may exert dominant negative effects on the long protein. the short and long proteins may compete for binding with ca2+ and certain proteins through the c and pdz domains, respectively. this, in turn, regulates the rhogef activity of the long psgef. if their expression is mutually exclusive, they may have independent functions in different neurons. since it is difficult to distinguish the expression patterns of short and long dmpsgef mrnas in drosophila, an examination of the functions of short psgef in daphnia may provide an answer to the abovementioned question. although alternative splicing of psgef occurs in the fruit fly and honey bee, it remains to be established whether the same alternative splicing occurs in other insects and limpets. loss of dmpsgef results in thinner alpha lobes than those of wild-type. furthermore, short and multiple alpha lobes derived from late-born 201y-gal4-expressing alpha/beta neurons were observed in mbs of dmpsgefΔ flies, as shown in figures 6c to 6e. these results suggest that the alpha/beta neurons bifurcate their axons at more random positions in dmpsgefΔ flies. further, the dmpsgefΔ alpha/beta neurons may not respond well to the signals provided by guidance neurons. moreover, some alpha/beta neurons fail to extend their alpha-axonal branches. meanwhile, gamma neurons appear to be normal. alpha'/beta' neurons are generally born during the late third instar, and their axons remain intact during metamorphosis. these lobes may serve as the guiding axons for the bifurcation of the axons of alpha/beta neurons. we therefore examined the alpha'/beta' lobes in dmpsgefΔ flies by immunostaining with anti-trio antibody. their morphology was found to be normal, thus suggesting that dmpsgef is specifically necessary for the axonal development of alpha/beta neurons in mbs. to identify rho family members activated by psgef, we first tested the genetic interaction of dmpsgef with five rho family members. the results indicated that dmpsgef genetically interacts with rac but not other family members. consistent with this observation, the ectopic expression of the rhogef domain of dmpsgef induces actin polymerization in the membrane of hela cells. these results thus suggest that psgef activates rac but not cdc or rho in vivo. intriguingly, dmpsgef does not genetically interact with pak, one of the downstream effectors of rac. trio was shown to activate rac and promote lim kinase activity via pak to induce axon growth inhibition. thus, dmpsgef induces rac activation and may stimulate axon growth via a pak-independent pathway along with still life. three gefs for rac appear to function for the morphogenesis of mbs in drosophila. since each gef protein has specific functional domains in addition to the rhogef domain, the mechanism to activate individual gefs should be different. specifically, the presence of the c domain in psgef suggests that the increase of intracellular ca2+ level is essential for the localization of psgef at the plasma membrane where rac proteins are anchored. the function of dmpsgef is therefore dependent on the local increase of cytosolic ca2+ level which occurs during the axonal development of alpha/beta neurons in mbs. dmpsgef is expressed not only in the mbs but also in the antennal and optic lobes in adult drosophila brain. we analyzed the axonal projection patterns of several olfactory neurons into the antennal lobes in dmpsgefΔ flies; however, no significant difference was observed relative to the patterns observed in the wild-type. the glomerular structures of the antennal lobes visualized by immunostaining with anti-nc antibody were found to be normal. although we did not observe gross morphological defects in the antennal and optic lobes in dmpsgefΔ flies, it is possible that there were subtle defects in these brain regions. no other proteins with c and pdz domains are present besides dmpsgef in drosophila. however, other gefs for rac could be partially redundant with dmpsgef. this may explain the lack of phenotypes associated with the antennal lobes in dmpsgefΔ flies. we are also testing the olfactory and visual behaviors of adult dmpsgefΔ flies at present. association of psgef with structural and/or functional features common between insect and lophotrochozoan nervous systems the specific acquisition of psgef by the last common ancestor of protostomes followed by the retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians. we would like to propose that psgef may be associated with the presence of mbs, specific brain structures in insects, annelids, and platyhelminthes. the fact that psgef containing the c pdz, and rhogef domains is specifically retained in animals having mbs in addition to functioning as a gef for rac, which is essential for the correct axonal development of mbs in drosophila, suggests that the last common ancestor of protostomes might have possessed an ancient mb-like structure in the nervous system, and the mbs found in present animals may have evolved into specific neuropil structures in insect and lophotrochozoan brains supported in part by the retention of psgef in their genomes during evolution. nematodes and crustaceans lost the full length psgef, and mb-like structures have disappeared as a result. however, the functions of psgef do not appear to be limited to mbs because it is also expressed in the optic and antennal lobes of adult drosophila brain. although mb-like structures are found in mollusks such as achatina and octopus, it is not known whether limpets also have mb-like structures in their brains. it is thus possible that the conservation of psgef could be for other functional and developmental constraints. recent studies have demonstrated that the expression profile and roles of genes patterning the nervous system in the embryos of vertebrates and annelids are quite similar. since it is unlikely that this remarkable similarity arose from convergent evolution, they suggest that urbilateria may have already had a quite complex cns, which is also supported by comparative genomics. it is therefore not surprising that the origin of mbs could be traced back to the last common ancestor of protostomes, in which ancient mb-like structures might have played a role in multimodal sensory integration and even learning and memory. to prove a single origin of mbs, it will be necessary to demonstrate that the same gene sets act similarly for the development of mbs in insects and lophotrochozoans. the homologs of these genes are also present in the vertebrate genomes, and they function in the early development of nervous systems. as these genes encode transcription factors, they could function as a common'genetic code' to specify the brain region, giving rise to mbs and its vertebrate equivalents. then, as a next level, a different set of genes such as psgef is necessary to generate the specific structures of mbs, which are present in the brains of insects and lophotrochozoans but not vertebrates. in this regard, psgef is one of genes involved in generating the diversity of metazoan nervous systems.  the specific acquisition of psgef by the last common ancestor of protostomes followed by the retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians. psgef is therefore one of genes associated with the diversity of metazoan nervous systems. 
in drosophila melanogaster, psgef encodes a short version of a protein with the c and pdz domains, as well as a long version with the c pdz, and rhogef domains through alternative splicing. mushroom bodies are prominent neuropils involved in the processing of multiple sensory inputs as well as associative learning in the insect, platyhelminth, and annelid brains. intriguingly, the exons encoding the rhogef domain are specifically deleted in the daphnia pulex genome, suggesting that daphnia psgef contains only the c and pdz domains. gene gain and subsequent retention or loss during evolution may be one of the underlying mechanisms involved in generating the diversity of metazoan nervous systems. psgef is therefore one of genes associated with the diversity of metazoan nervous systems. the specific acquisition of psgef by the last common ancestor of protostomes followed by its retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians. in the adult drosophila brain, psgef is expressed in mushroom bodies, antennal lobe, and optic lobe, where it is necessary for the correct axon branch formation of alpha/beta neurons in mushroom bodies. psgef genetically interacts with rac but not other rho family members, and the rhogef domain of psgef induces actin polymerization in the membrane, thus resulting in the membrane ruffling that is observed in cultured cells with activated forms of rac. thus, the distribution of psgef containing the c pdz, and rhogef domains among metazoans appears to coincide with the presence of mushroom bodies.
 gene gain and subsequent retention or loss during evolution may be one of the underlying mechanisms involved in generating the diversity of metazoan nervous systems. however, the causal relationships acting therein have not been studied extensively.  we identified the gene psgef, which is present in all the sequenced genomes of insects and limpet but absent in those of sea anemones, deuterostomes, and nematodes. in drosophila melanogaster, psgef encodes a short version of a protein with the c and pdz domains, as well as a long version with the c pdz, and rhogef domains through alternative splicing. intriguingly, the exons encoding the rhogef domain are specifically deleted in the daphnia pulex genome, suggesting that daphnia psgef contains only the c and pdz domains. thus, the distribution of psgef containing the c pdz, and rhogef domains among metazoans appears to coincide with the presence of mushroom bodies. mushroom bodies are prominent neuropils involved in the processing of multiple sensory inputs as well as associative learning in the insect, platyhelminth, and annelid brains. in the adult drosophila brain, psgef is expressed in mushroom bodies, antennal lobe, and optic lobe, where it is necessary for the correct axon branch formation of alpha/beta neurons in mushroom bodies. psgef genetically interacts with rac but not other rho family members, and the rhogef domain of psgef induces actin polymerization in the membrane, thus resulting in the membrane ruffling that is observed in cultured cells with activated forms of rac.  the specific acquisition of psgef by the last common ancestor of protostomes followed by its retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems, which are absent in all deuterostomes and cnidarians. psgef is therefore one of genes associated with the diversity of metazoan nervous systems. 