BACKGROUND
the actin multigene family
actin is a cytoskeletal protein that is ubiquitously expressed in many eukaryotic cells.
examples for actin functions include maintenance of the cytoskeleton, cell motility and muscle contraction.
several studies have further shown the importance of actin in a number of cellular processes, e.g.
gene transcription and chromosome morphology  <cit> , control of the cell cycle  <cit> , modulation of a variety of membrane responses  <cit> , translation of several mrna species  <cit>  and modulation of enzyme activity and localization within the cell  <cit> .
six primary actin isoforms have been identified in higher vertebrates, being alpha-skeletal , alpha-cardiac , alpha-smooth muscle , gamma-smooth muscle , beta-cytoplasmic  and gamma-cytoplasmic isoactin   <cit>  .
actins can be classified in three pairs: two isoforms expressed in striated muscle , two isoforms from smooth muscle  and two cytoplasmic isoforms  <cit> .
later studies have reported additional actin isoforms in higher vertebrates  <cit> .
the evolution of the actin multigene family
cytoplasmic actins in vertebrates resemble actins present in various amoebas, yeast and slime molds  <cit> .
invertebrate muscle actins are more closely related to vertebrate cytoplasmic actins than to vertebrate muscle actin isoforms  <cit> .
actin isoforms specific for striated muscle tissue first evolved in primitive chordates  <cit> .
urochordates and lampreys still express an alpha-cardiac-like isoform in their primitive muscles.
at the level of early amphibians or stem reptiles this gene probably duplicated, which resulted in an alpha-skeletal and a modern alpha-cardiac isoactin  <cit> .
the smooth muscle isoactins are believed to have evolved during later development of warm-blooded vertebrates  <cit>  and likely originated from an early skeletal muscle actin  <cit> .
altogether, over  <dig> different actins have been characterized from various muscle sources, some of them having a very specialized role  <cit> .
the actin multigene family is highly conserved.
the sequences of alpha-skeletal and alpha-cardiac actin differ by only four amino acids over a total of  <dig> residues in cow  <cit> .
this is among the highest conservation found in vertebrate actins  <cit> .
skeletal alpha-actin differs eight amino acids from the alpha-smooth muscle isoform and six amino acids from the γ-smooth muscle isoform in bos taurus.
in comparison to the non-muscle actin isoforms,  <dig> and  <dig> differences in the amino acid sequence are found for γ- and β-cytoplasmic actin respectively  <cit> .
these differences are mainly located at the amino terminus of the protein  <cit> .
being the site of most actin-myosin interactions, it has an important role in cytoarchitecture and other protein-protein interactions.
the high sequence conservation between the two striated muscle isoactins is also apparent in their gene structures.
skeletal alpha-actin and alpha-cardiac actin are interrupted by five introns on corresponding locations  <cit> .
even non-coding regions show striking similarity between the two genes and between different species  <cit> .
two large segments that constitute most of the 3' untranslated regions  show a similarity of 92% and 85% between the human and the rat alpha-skeletal actin gene  <cit> .
on average, sequences of rat/human gene pairs show only 37–45% similarity in the 3' utr.
based on the early separation of the two genes, this high similarity indicates a high selective pressure to conserve the sequence.
these conserved utr's may affect the stability, translational capacity and the intracellular location of the transcripts, thereby further regulating the expression of the protein.
the expression of actin isoforms
the expression of the different actin isoforms is developmentally regulated in a temporal and tissue-specific manner.
in general, the cytoplasmic actins are ubiquitously expressed in all cell types and throughout development.
the other two pairs of actin isoforms have been shown to be expressed in striated  and smooth muscle   <cit> .
earlier studies showed that the striated and smooth muscle pairs are coexpressed during development  <cit> .
this coexpression is not very surprising, since there is also a high conservation in the regulatory sequences of these genes  <cit> .
cardiac alpha-actin, which is the main actin isoform in the adult heart, has also been shown to be the predominant form in early muscle development   <cit> .
it has been hypothesized that this coordinated co-expression occurs to facilitate rapid accumulation of these proteins  <cit> .
in later development the expression of alpha-cardiac actin is downregulated and alpha-skeletal actin becomes the dominant isoform in the adult tissue, accounting for more than 95% of the total striated muscle actin isoforms  <cit> .
skeletal alpha-actin is also expressed in the mammalian heart tissue  <cit> .
during the late stage of fetal development alpha-cardiac actin becomes the main isoform, but alpha-skeletal actin might still make up for almost half of the total striated muscle actin content  <cit> .
similar results have been shown in the chicken heart  <cit> .
in somite development of the amphibian xenopus laevis embryos also both alpha-skeletal and alpha-cardiac actin increase in content in a coordinate manner  <cit> .
however, in amphibians there appear to be at least three distinct isoforms expressed in striated muscle <cit>  and only one isoform expressed in smooth muscle  <cit> .
this developmental paradigm has been further complicated by studies which have demonstrated that alpha-smooth muscle isoactin is also expressed during early cardiac and skeletal muscle development in a variety of myofibroblast-like cells  <cit> .
so far no significant evidence has been found to answer the controversial question whether this co-expression reflects a truly regulated expression or rather is a result from a persistent "leakage" from the initial high expression during early myogenesis.
there might also be "leakage" because of partially diverged regulatory elements, resulting in cross-responsiveness on cellular or environmental stimuli.
based on the high sequence similarity and the absence of pathology in a mutant mouse model that expresses high alpha-skeletal actin in the heart, it is hypothesized that at least these two actin isoforms are functionally redundant  <cit> .
the function of these two actins is likely more related to their differential regulation in space and time than to their protein coding information  <cit> .
although several studies in the past have elucidated the specific expression pattern of alpha-skeletal actin in a model organism, no comparison of different species has been made so far.
in this study we compare alpha-skeletal actin expression of five vertebrate species, in at least two distinct developmental.
we identified here several novel non-muscular expression domains of alpha-skeletal actin.
furthermore, we compared the sequences, gene architectures and 3d protein models of several species to shed light on the evolutionary history of alpha-skeletal actin.
RESULTS
phylogenetic analysis
the sequences of all known actins of selected vertebrate species and of muscle-like actins of a number of other species, covering a broad range of evolutionary history, were extracted from genbank .
for our phylogenetic analysis, we selected alpha-skeletal actins in several different species, or where that was not possible, an isoform most similar to alpha-skeletal actin by direct sequence comparison.
a non-rooted tree is shown in figure  <dig> and an additional phylogenetic analysis using yeast actin  <dig>  as an outgroup is shown in supplemental figure  <dig>  invertebrate muscle actin genes cluster with human cytoplasmic actins, as was also the case in previous phylogenetic studies on actins  <cit> .
the first muscle actin is found in deuterostomes, illustrated by the clustering of the sea urchin s. purpuratus with the other muscle actins.
actins specific for striated muscle cells are first expressed in early chordates, confirming findings in earlier studies  <cit> .
the alpha-skeletal actins of vertebrates group together, except for the alpha-skeletal actin of zebrafish, which clusters with the actin of the urochordate ciona savignyi.
in earlier studies it was already shown that the real alpha-skeletal actins evolved in early amphibians or stem reptiles, explaining why the zebrafish actin is not found on the same branch as the other vertebrates  <cit> .
interestingly, the sequence of the urochordate c. savignyi is closer related to the vertebrate alpha-skeletal actins than the sequence of the branchiostoma b. belcheri.
a possible explanation might be that the used branchiostoma sequence is not the expected alpha-skeletal actin, although it was identified as the most homologues gene when searched with alpha-skeletal actin sequences of different species.
sequence comparison and evolution of genomic architectures
the alignment of the amino acid sequence shows extremely high conservation of the protein in a broad range of species .
most of the differences are located in the n-terminus of the protein, as has already been shown in a previous study  <cit> .
the shading shows the mode of conservation, the darker blocks being more conserved.
in detail black background, conserved in 100% of sequences , dark grey background conserved in more than 70% of sequences, light grey background conserved in more than 60% of sequences and white background below 60% of sequences.
we have identified here the intron-exon boundaries of the alpha-skeletal actin genes in several vertebrates , chordates , nematodes , insects , plants  and yeast .
we compare the genomic organization of these alpha-skeletal actin genes and derive an evolutionary model for the development of their gene architecture .
the yeast gene consists of one exon, which is also the case for the studied insect actin genes.
in plants and nematodes the gene consists of four exons, but only the first exon shares the same boundaries in these two groups.
surprisingly, the intron between the second and third exon in plants, is located on the same site as the intron in the ciona species.
the other exon boundaries differ from plants and nematodes.
the last exon in ciona is split in two separate exons in the vertebrates.
the zebrafish gene shows another split of the second exon in three separate pieces.
the gene architectures of the other vertebrate alpha-skeletal actin genes are even more conserved.
the genes in human, mouse, chicken, snake and frog have the same intron-exon boundaries, differing only in the length of the untranslated regions.
in summary, we can show here that in plants, like arabidopsis thaliana,oryza sativa and populus trichocarpa,  <dig> exons were present.
in the course of evolution some species like the studied insect species and also yeast have lost all introns within their alpha-skeletal actin gene.
in the nematodes,caenorhabditis elegans and caenorhabditis briggsae, the second intron was lost and exon  <dig> and  <dig> fused.
however this intron as seen in our evolutionary model in figure  <dig> has been conserved from plants to higher chordates.
these results show that the genomic organization of the alpha-skeletal actin differs significantly between species.
molecular modeling
we modelled here the  <dig> dimensional protein structures of  <dig> different actins  and show the  <dig> actins for which structural differences were found .
the molecular models of all vertebrate species, s. purpuratus, d. melanogaster and c. elegans do not show a structural difference when compared to each other .
additionally, we have found no visible difference between the models of the six different human actins .
for each of the organisms that were used here, we list the expression domains found in this study in the upper row.
newly in this study discovered expression domains are shown in bold.
expressions domains that have been described in previous studies are listed in the second row.
however, we found important structural differences between the other species investigated .
the major changes between the other muscle actins in secondary structures are pointed out by arrows.
these sites are also mapped on the alignment of the primary protein sequences .
the b. belcheri model shows an additional coil in the alpha helix in subdomain  <dig> .
this is also the case for yeast, which also shows a difference in the structure on the right in subdomain  <dig>  <cit> .
in c. savignyi the beta sheets in the middle of the molecule in subdomain  <dig> are a little shorter .
in the a. thaliana model the secondary structures in subdomain  <dig> are shorter , whereas the alpha helix in subdomain  <dig> is longer .
the structure in subdomain  <dig>  <cit>  and the beta sheet in subdomain  <dig>  are also longer.
besides, the a. thaliana protein seems to miss the short structure in the middle of the molecule in subdomain  <dig> , which is present in all the other models.
this structure might as well be fused with the beta sheet in subdomain  <dig> , explaining why this structure is significantly longer in a. thaliana.
not all differences in the protein model can easily be recognized in the amino acid sequence.
the corresponding regions in the alignment are highlighted by colored arrows .
the ultimate configuration of the protein depends on several regions and their allosteric interactions, sometimes a change in the amino acid sequence can have an effect on a distant part of the protein, where the respective amino acid is actually not located.
this explains why some regions, which show differences in the model, do not show clear differences in the alignment.
at the same time, the alignment shows less conserved regions, which are not detectable as such in the 3d model.
the structural differences we describe here can point to important functional differences or affect specific molecular interactions of actins.
it is important to note, that the structural models presented here, are based on predictions and that the subtle changes in structure we observe here, are not likely to have an influence on actins major structural functions e.g.
filament assembly or myosin binding.
further studies are necessary to delineate these structure/function relationships and to identify novel functions of actin which could be related to them.
here, molecular approaches  or bioinformatic analysis of potential interactors for example, could give better insights on the significance of our findings.
expression in mouse
in the early stage mouse embryo  expression seems to be present in several brain areas .
also the heart, branchial arches , lens, hind limb bud, forelimb bud, liver and somites are clearly stained.
the strongest staining in the limb bud is located at the most distal part, the distal progress zone and the apical ectodermal ridge .
the mouse at stage e <dig>  shows staining in the heart, branchial arches, lens, hind limb bud, forelimb bud, liver and somites .
in addition, the otic vesicle and distinct parts of the brain  show staining.
detailed pictures of the atrium are shown for the early stage embryo  and the late stage embryo .
expression in chicken
in the early stage of the chicken embryo  the hind limb bud and forelimb bud are strongly stained .
the heart, branchial arches , otic vesicle, nasal pituitary, lens, and somites are also stained.
in addition, parts of the brain  show expression.
at the older stage of the chicken-embryo  there is clear staining in the heart , branchial arches, otic vesicle, liver, forelimb bud and hind limb bud .
details of the atrium are shown for the embryos of both stages .
expression in snake
the youngest snake embryo  shows strong expression in the proliferative cell layer .
furthermore, various brain regions  and cephalic musculature are stained.
expression in the heart, branchial arches, nasal pituitary and somites is visible as well.
within the heart the strongest staining is located in the primary atrial septum.
in the older snake embryo  the proliferative cell layer, heart , brain regions , nasal pituitary and somites show clear expression .
detailed pictures of the heart are shown for the two embryos .
expression in salamander
the youngest salamander embryo  only shows staining in the somites .
in the older embryo   there is also expression in the heart.
pigmentation is visible throughout the embryo.
this is clearly visible in the detailed picture of the head and heart region .
expression in zebrafish
as early as the 4cell stage , there is staining in the blastomeres.
in the  <dig> k stage the embryo shows expression in the germinal plate .
at the shield stage  and the tail bud stage  the staining focuses at the area where the somites will develop.
at prim- <dig>  there is clear expression at the somites, at the lens and in the proliferative cell layer.
there is also some expression in the head region.
a detail from the head  additionally shows staining in the hindbrain, cerebellum, epiphysis and telencephalon.
at the prim- <dig> stage  the somites are strongly stained.
the lens and proliferative cell layer are a bit weaker stained.
staining is also visible in the midbrain, hindbrain and heart.
at the next stage, long-pec  there is high expression in the somites, heart, hindbrain, lens, proliferative cell layer, branchial arches, cephalic musculature, midbrain, hindbrain and the midcerebral vein.
the last stage,  <dig> dpf  shows a strong staining, especially in the branchial arches, jaw musculature, lens, midbrain, liver and somites.
comparison of expression patterns
in this study all used embryos displayed clear staining in the somites.
except for the early stage salamander embryo , all embryos  showed also expression in the heart.
table  <dig> gives an overview of expression domains of alpha-skeletal actin in several vertebrate species found in this study.
in addition we summarize in table  <dig> the findings of other studies on alpha-skeletal actin gene expression.
in general, staining is detected in regions which are bound to give rise to skeletal muscle.
this is illustrated by the strong staining in the somites and limb buds.
we also show here staining in a number of muscles in the head and neck, which also has been described by other studies in rodents and zebrafish  <cit> .
in addition we show that the expression is specifically strong in the branchial arches, which will give rise to a number of facial muscles and muscles that are associated with the larynx and pharynx.
all current studies in mouse or chicken have detected expression in the heart as well but expression we show here in the zebrafish heart has not been described before.
here, we also discover alpha-skeletal actin expression in the liver of all species except the salamander.
furthermore, staining in the eye, otic vesicle, nasal pituitary and brain regions has not previously been described.
we can not entirely rule out that there was no crossreactivity of the probe with other isoactins.
however, hybridization conditions were very stringent and the fact that most other actins are expressed ubiquitously and that we do not see ubiquitous staining, argues against this.
still, crossreactivity with cardiac actin is not ruled out by this argument but here other studies also have shown that alpha-skeletal actin is weakly expressed in the vertebrate heart.
discussion
in this study, we have compared the gene expression patterns of alpha-skeletal actin in five different vertebrate species and describe several novel expression domains for this functional and structural important gene.
because of the high conservation of the actin multigene family, a single probe could be used for the in situ hybridization.
this proved to be a useful method when comparing expression patterns of the alpha-skeletal actin gene in these vertebrate species.
an important question remains still, how the expression patterns of alpha-skeletal actin and the other actin isoforms are regulated.
here, results from experiments on differentiated, cultured cells have often differed from in vivo studies  <cit> .
factors such as innervation, growth factors and mitogens, oncogenes, cell density, hormones, mechanical and biological stress and cell-cell interactions may all contribute to the regulation of this multigene family  <cit> .
promoter regions of different actins have been extensively analyzed.
so far it is clear that interaction of multiple cis-acting and trans-acting regulatory elements are involved  <cit> .
however, the sequences and factors that interact with these regions and confer to positive and/or negative transcriptional regulation are poorly understood.
additionally, a variety of post-transcriptional modifications contribute to the regulation of actin protein expression  <cit> .
future studies should shed more light on these regulatory mechanisms and actin functions.
here, a comparative evolutionary analysis of regulatory elements should help in unraveling some of the signaling pathways and transcriptional networks responsible.
CONCLUSIONS
the phylogenetic analysis, the alignment, the comparison of the genomic architecture and the 3d protein models we show here reveal extreme high conservation of alpha-skeletal actins.
the high homology between higher vertebrate actins and muscle-like actins in plant and yeast is striking when looked at the evolutionary distance.
furthermore, alpha-skeletal actin expression patterns show strong conservation in evolution and are very similar in all tested vertebrate species.
interestingly, we find many novel expression domains of alpha-skeletal actin including several in non-muscular tissues and organs.
despite all observed similarities, we show here also important structural and other differences between alpha-skeletal actins of different species.
our evolutionary model illustrates that the genomic organization of the alpha-skeletal actin differs significantly between species.
insects and yeast for example have lost all introns within their alpha-skeletallike actin genes.

 the actin multigene family actin is a cytoskeletal protein that is ubiquitously expressed in many eukaryotic cells. examples for actin functions include maintenance of the cytoskeleton, cell motility and muscle contraction. several studies have further shown the importance of actin in a number of cellular processes, e.g. gene transcription and chromosome morphology, control of the cell cycle, modulation of a variety of membrane responses, translation of several mrna species and modulation of enzyme activity and localization within the cell. six primary actin isoforms have been identified in higher vertebrates, being alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. actins can be classified in three pairs: two isoforms expressed in striated muscle, two isoforms from smooth muscle and two cytoplasmic isoforms. later studies have reported additional actin isoforms in higher vertebrates. the evolution of the actin multigene family cytoplasmic actins in vertebrates resemble actins present in various amoebas, yeast and slime molds. invertebrate muscle actins are more closely related to vertebrate cytoplasmic actins than to vertebrate muscle actin isoforms. actin isoforms specific for striated muscle tissue first evolved in primitive chordates. urochordates and lampreys still express an alpha-cardiac-like isoform in their primitive muscles. at the level of early amphibians or stem reptiles this gene probably duplicated, which resulted in an alpha-skeletal and a modern alpha-cardiac isoactin. the smooth muscle isoactins are believed to have evolved during later development of warm-blooded vertebrates and likely originated from an early skeletal muscle actin. altogether, over different actins have been characterized from various muscle sources, some of them having a very specialized role. the actin multigene family is highly conserved. the sequences of alpha-skeletal and alpha-cardiac actin differ by only four amino acids over a total of residues in cow. this is among the highest conservation found in vertebrate actins. skeletal alpha-actin differs eight amino acids from the alpha-smooth muscle isoform and six amino acids from the γ-smooth muscle isoform in bos taurus. in comparison to the non-muscle actin isoforms, and differences in the amino acid sequence are found for γ- and β-cytoplasmic actin respectively. these differences are mainly located at the amino terminus of the protein. being the site of most actin-myosin interactions, it has an important role in cytoarchitecture and other protein-protein interactions. the high sequence conservation between the two striated muscle isoactins is also apparent in their gene structures. skeletal alpha-actin and alpha-cardiac actin are interrupted by five introns on corresponding locations. even non-coding regions show striking similarity between the two genes and between different species. two large segments that constitute most of the 3' untranslated regions show a similarity of 92% and 85% between the human and the rat alpha-skeletal actin gene. on average, sequences of rat/human gene pairs show only 37–45% similarity in the 3' utr. based on the early separation of the two genes, this high similarity indicates a high selective pressure to conserve the sequence. these conserved utr's may affect the stability, translational capacity and the intracellular location of the transcripts, thereby further regulating the expression of the protein. the expression of actin isoforms the expression of the different actin isoforms is developmentally regulated in a temporal and tissue-specific manner. in general, the cytoplasmic actins are ubiquitously expressed in all cell types and throughout development. the other two pairs of actin isoforms have been shown to be expressed in striated and smooth muscle. earlier studies showed that the striated and smooth muscle pairs are coexpressed during development. this coexpression is not very surprising, since there is also a high conservation in the regulatory sequences of these genes. cardiac alpha-actin, which is the main actin isoform in the adult heart, has also been shown to be the predominant form in early muscle development. it has been hypothesized that this coordinated co-expression occurs to facilitate rapid accumulation of these proteins. in later development the expression of alpha-cardiac actin is downregulated and alpha-skeletal actin becomes the dominant isoform in the adult tissue, accounting for more than 95% of the total striated muscle actin isoforms. skeletal alpha-actin is also expressed in the mammalian heart tissue. during the late stage of fetal development alpha-cardiac actin becomes the main isoform, but alpha-skeletal actin might still make up for almost half of the total striated muscle actin content. similar results have been shown in the chicken heart. in somite development of the amphibian xenopus laevis embryos also both alpha-skeletal and alpha-cardiac actin increase in content in a coordinate manner. however, in amphibians there appear to be at least three distinct isoforms expressed in striated muscle and only one isoform expressed in smooth muscle. this developmental paradigm has been further complicated by studies which have demonstrated that alpha-smooth muscle isoactin is also expressed during early cardiac and skeletal muscle development in a variety of myofibroblast-like cells. so far no significant evidence has been found to answer the controversial question whether this co-expression reflects a truly regulated expression or rather is a result from a persistent "leakage" from the initial high expression during early myogenesis. there might also be "leakage" because of partially diverged regulatory elements, resulting in cross-responsiveness on cellular or environmental stimuli. based on the high sequence similarity and the absence of pathology in a mutant mouse model that expresses high alpha-skeletal actin in the heart, it is hypothesized that at least these two actin isoforms are functionally redundant. the function of these two actins is likely more related to their differential regulation in space and time than to their protein coding information. although several studies in the past have elucidated the specific expression pattern of alpha-skeletal actin in a model organism, no comparison of different species has been made so far. in this study we compare alpha-skeletal actin expression of five vertebrate species, in at least two distinct developmental. we identified here several novel non-muscular expression domains of alpha-skeletal actin. furthermore, we compared the sequences, gene architectures and 3d protein models of several species to shed light on the evolutionary history of alpha-skeletal actin.  phylogenetic analysis the sequences of all known actins of selected vertebrate species and of muscle-like actins of a number of other species, covering a broad range of evolutionary history, were extracted from genbank. for our phylogenetic analysis, we selected alpha-skeletal actins in several different species, or where that was not possible, an isoform most similar to alpha-skeletal actin by direct sequence comparison. a non-rooted tree is shown in figure and an additional phylogenetic analysis using yeast actin as an outgroup is shown in supplemental figure invertebrate muscle actin genes cluster with human cytoplasmic actins, as was also the case in previous phylogenetic studies on actins. the first muscle actin is found in deuterostomes, illustrated by the clustering of the sea urchin s. purpuratus with the other muscle actins. actins specific for striated muscle cells are first expressed in early chordates, confirming findings in earlier studies. the alpha-skeletal actins of vertebrates group together, except for the alpha-skeletal actin of zebrafish, which clusters with the actin of the urochordate ciona savignyi. in earlier studies it was already shown that the real alpha-skeletal actins evolved in early amphibians or stem reptiles, explaining why the zebrafish actin is not found on the same branch as the other vertebrates. interestingly, the sequence of the urochordate c. savignyi is closer related to the vertebrate alpha-skeletal actins than the sequence of the branchiostoma b. belcheri. a possible explanation might be that the used branchiostoma sequence is not the expected alpha-skeletal actin, although it was identified as the most homologues gene when searched with alpha-skeletal actin sequences of different species. sequence comparison and evolution of genomic architectures the alignment of the amino acid sequence shows extremely high conservation of the protein in a broad range of species. most of the differences are located in the n-terminus of the protein, as has already been shown in a previous study. the shading shows the mode of conservation, the darker blocks being more conserved. in detail black background, conserved in 100% of sequences, dark grey background conserved in more than 70% of sequences, light grey background conserved in more than 60% of sequences and white background below 60% of sequences. we have identified here the intron-exon boundaries of the alpha-skeletal actin genes in several vertebrates, chordates, nematodes, insects, plants and yeast. we compare the genomic organization of these alpha-skeletal actin genes and derive an evolutionary model for the development of their gene architecture. the yeast gene consists of one exon, which is also the case for the studied insect actin genes. in plants and nematodes the gene consists of four exons, but only the first exon shares the same boundaries in these two groups. surprisingly, the intron between the second and third exon in plants, is located on the same site as the intron in the ciona species. the other exon boundaries differ from plants and nematodes. the last exon in ciona is split in two separate exons in the vertebrates. the zebrafish gene shows another split of the second exon in three separate pieces. the gene architectures of the other vertebrate alpha-skeletal actin genes are even more conserved. the genes in human, mouse, chicken, snake and frog have the same intron-exon boundaries, differing only in the length of the untranslated regions. in summary, we can show here that in plants, like arabidopsis thaliana,oryza sativa and populus trichocarpa, exons were present. in the course of evolution some species like the studied insect species and also yeast have lost all introns within their alpha-skeletal actin gene. in the nematodes,caenorhabditis elegans and caenorhabditis briggsae, the second intron was lost and exon and fused. however this intron as seen in our evolutionary model in figure has been conserved from plants to higher chordates. these results show that the genomic organization of the alpha-skeletal actin differs significantly between species. molecular modeling we modelled here the dimensional protein structures of different actins and show the actins for which structural differences were found. the molecular models of all vertebrate species, s. purpuratus, d. melanogaster and c. elegans do not show a structural difference when compared to each other. additionally, we have found no visible difference between the models of the six different human actins. for each of the organisms that were used here, we list the expression domains found in this study in the upper row. newly in this study discovered expression domains are shown in bold. expressions domains that have been described in previous studies are listed in the second row. however, we found important structural differences between the other species investigated. the major changes between the other muscle actins in secondary structures are pointed out by arrows. these sites are also mapped on the alignment of the primary protein sequences. the b. belcheri model shows an additional coil in the alpha helix in subdomain. this is also the case for yeast, which also shows a difference in the structure on the right in subdomain. in c. savignyi the beta sheets in the middle of the molecule in subdomain are a little shorter. in the a. thaliana model the secondary structures in subdomain are shorter, whereas the alpha helix in subdomain is longer. the structure in subdomain and the beta sheet in subdomain are also longer. besides, the a. thaliana protein seems to miss the short structure in the middle of the molecule in subdomain, which is present in all the other models. this structure might as well be fused with the beta sheet in subdomain, explaining why this structure is significantly longer in a. thaliana. not all differences in the protein model can easily be recognized in the amino acid sequence. the corresponding regions in the alignment are highlighted by colored arrows. the ultimate configuration of the protein depends on several regions and their allosteric interactions, sometimes a change in the amino acid sequence can have an effect on a distant part of the protein, where the respective amino acid is actually not located. this explains why some regions, which show differences in the model, do not show clear differences in the alignment. at the same time, the alignment shows less conserved regions, which are not detectable as such in the 3d model. the structural differences we describe here can point to important functional differences or affect specific molecular interactions of actins. it is important to note, that the structural models presented here, are based on predictions and that the subtle changes in structure we observe here, are not likely to have an influence on actins major structural functions e.g. filament assembly or myosin binding. further studies are necessary to delineate these structure/function relationships and to identify novel functions of actin which could be related to them. here, molecular approaches or bioinformatic analysis of potential interactors for example, could give better insights on the significance of our findings. expression in mouse in the early stage mouse embryo expression seems to be present in several brain areas. also the heart, branchial arches, lens, hind limb bud, forelimb bud, liver and somites are clearly stained. the strongest staining in the limb bud is located at the most distal part, the distal progress zone and the apical ectodermal ridge. the mouse at stage e shows staining in the heart, branchial arches, lens, hind limb bud, forelimb bud, liver and somites. in addition, the otic vesicle and distinct parts of the brain show staining. detailed pictures of the atrium are shown for the early stage embryo and the late stage embryo. expression in chicken in the early stage of the chicken embryo the hind limb bud and forelimb bud are strongly stained. the heart, branchial arches, otic vesicle, nasal pituitary, lens, and somites are also stained. in addition, parts of the brain show expression. at the older stage of the chicken-embryo there is clear staining in the heart, branchial arches, otic vesicle, liver, forelimb bud and hind limb bud. details of the atrium are shown for the embryos of both stages. expression in snake the youngest snake embryo shows strong expression in the proliferative cell layer. furthermore, various brain regions and cephalic musculature are stained. expression in the heart, branchial arches, nasal pituitary and somites is visible as well. within the heart the strongest staining is located in the primary atrial septum. in the older snake embryo the proliferative cell layer, heart, brain regions, nasal pituitary and somites show clear expression. detailed pictures of the heart are shown for the two embryos. expression in salamander the youngest salamander embryo only shows staining in the somites. in the older embryo there is also expression in the heart. pigmentation is visible throughout the embryo. this is clearly visible in the detailed picture of the head and heart region. expression in zebrafish as early as the 4cell stage, there is staining in the blastomeres. in the k stage the embryo shows expression in the germinal plate. at the shield stage and the tail bud stage the staining focuses at the area where the somites will develop. at prim- there is clear expression at the somites, at the lens and in the proliferative cell layer. there is also some expression in the head region. a detail from the head additionally shows staining in the hindbrain, cerebellum, epiphysis and telencephalon. at the prim- stage the somites are strongly stained. the lens and proliferative cell layer are a bit weaker stained. staining is also visible in the midbrain, hindbrain and heart. at the next stage, long-pec there is high expression in the somites, heart, hindbrain, lens, proliferative cell layer, branchial arches, cephalic musculature, midbrain, hindbrain and the midcerebral vein. the last stage, dpf shows a strong staining, especially in the branchial arches, jaw musculature, lens, midbrain, liver and somites. comparison of expression patterns in this study all used embryos displayed clear staining in the somites. except for the early stage salamander embryo, all embryos showed also expression in the heart. table gives an overview of expression domains of alpha-skeletal actin in several vertebrate species found in this study. in addition we summarize in table the findings of other studies on alpha-skeletal actin gene expression. in general, staining is detected in regions which are bound to give rise to skeletal muscle. this is illustrated by the strong staining in the somites and limb buds. we also show here staining in a number of muscles in the head and neck, which also has been described by other studies in rodents and zebrafish. in addition we show that the expression is specifically strong in the branchial arches, which will give rise to a number of facial muscles and muscles that are associated with the larynx and pharynx. all current studies in mouse or chicken have detected expression in the heart as well but expression we show here in the zebrafish heart has not been described before. here, we also discover alpha-skeletal actin expression in the liver of all species except the salamander. furthermore, staining in the eye, otic vesicle, nasal pituitary and brain regions has not previously been described. we can not entirely rule out that there was no crossreactivity of the probe with other isoactins. however, hybridization conditions were very stringent and the fact that most other actins are expressed ubiquitously and that we do not see ubiquitous staining, argues against this. still, crossreactivity with cardiac actin is not ruled out by this argument but here other studies also have shown that alpha-skeletal actin is weakly expressed in the vertebrate heart. discussion in this study, we have compared the gene expression patterns of alpha-skeletal actin in five different vertebrate species and describe several novel expression domains for this functional and structural important gene. because of the high conservation of the actin multigene family, a single probe could be used for the in situ hybridization. this proved to be a useful method when comparing expression patterns of the alpha-skeletal actin gene in these vertebrate species. an important question remains still, how the expression patterns of alpha-skeletal actin and the other actin isoforms are regulated. here, results from experiments on differentiated, cultured cells have often differed from in vivo studies. factors such as innervation, growth factors and mitogens, oncogenes, cell density, hormones, mechanical and biological stress and cell-cell interactions may all contribute to the regulation of this multigene family. promoter regions of different actins have been extensively analyzed. so far it is clear that interaction of multiple cis-acting and trans-acting regulatory elements are involved. however, the sequences and factors that interact with these regions and confer to positive and/or negative transcriptional regulation are poorly understood. additionally, a variety of post-transcriptional modifications contribute to the regulation of actin protein expression. future studies should shed more light on these regulatory mechanisms and actin functions. here, a comparative evolutionary analysis of regulatory elements should help in unraveling some of the signaling pathways and transcriptional networks responsible.  the phylogenetic analysis, the alignment, the comparison of the genomic architecture and the 3d protein models we show here reveal extreme high conservation of alpha-skeletal actins. the high homology between higher vertebrate actins and muscle-like actins in plant and yeast is striking when looked at the evolutionary distance. furthermore, alpha-skeletal actin expression patterns show strong conservation in evolution and are very similar in all tested vertebrate species. interestingly, we find many novel expression domains of alpha-skeletal actin including several in non-muscular tissues and organs. despite all observed similarities, we show here also important structural and other differences between alpha-skeletal actins of different species. our evolutionary model illustrates that the genomic organization of the alpha-skeletal actin differs significantly between species. insects and yeast for example have lost all introns within their alpha-skeletallike actin genes. 
actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. we present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. we further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species. our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. in higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. all members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins. nonetheless, we find here important differences in 3d structures, in gene architectures and identify novel expression domains for this structural and functional important gene. in addition, we identify many novel non-muscular expression domains including several in the central nervous system.
 actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. in higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. all members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins.  we present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. we further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species. our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. in addition, we identify many novel non-muscular expression domains including several in the central nervous system.  our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. nonetheless, we find here important differences in 3d structures, in gene architectures and identify novel expression domains for this structural and functional important gene. 