BACKGROUND
comparative embryology of various animal groups has suggested the idea of a conserved stage in embryogenesis.
in  <dig>  karl von baer was the first to describe revolutionary laws for animal development  <cit> , inferring that the later the stage of embryogenesis of related organisms, the less they resemble each other  <cit> .
this "progressive divergence model" was challenged by seidel, sander, and elinson  <cit> , who noted that earlier stages of development  have relatively divergent morphological patterns among species, and so do not always fit the rule.
today, the revised version of the laws, called the "hourglass model"  <cit>  or the "egg-timer model"  <cit> , is widely accepted  <cit> , and the bottleneck stage, representing maximum resemblance among various vertebrates, is commonly referred to as the "phylotypic stage"  <cit> .
however, much controversy remains about when the phylotypic stage appears; ballard first proposed the pharyngula stage  <cit>  as the phylotypic stage, whereas wolpert proposed the early somite segmentation stage  <cit> , and slack, the tailbud stage  <cit> .
moreover, a few detailed morphological studies have thrown doubt on the existence of a phylotypic stage  <cit> .
these problems are due mainly to the difficulty in evaluating conserved or constrained developmental stages by morphological resemblance.
in particular, heterochrony, or variations in the relative rate or timing of developmental events , makes it extremely difficult to conclude which embryonic stage is the vertebrate phylotypic stage  <cit> .
the problem of heterochrony prevents us from concluding whether each vertebrate really has a phylotypic stage, and whether any morphological features are associated with the stage.
recent progress in developmental biology has revealed marked similarities of the molecular mechanisms of morphogenesis in bilaterians, leading to the "zootype" hypothesis  <cit> .
the zootype is defined by a common pattern of hox gene expression among bilaterians, and is considered to be displayed most clearly at the phylotypic stage.
we note, however, many conserved genes other than hox are also involved in animal development, so comprehensive evaluation of these genes should be included for a more accurate definition of zootype.
further, the fact that they are expressed differently during development among bilaterians prevents us from concluding which stage is zootype-related constrained.
recently, using mouse and human genome information, hazkani-covo et al.
evaluated the protein distances of each gene expressed during mouse embryogenesis to show the existence of the vertebrate phylotypic stage  <cit> .
although they could not specify the timing of the phylotypic stage, they raised the possibility of a genome-based approach to examining the phylotypic stage hypothesis.
here, we sought to develop a novel method to evaluate the evolutionary ancestral nature of each embryonic stage comprehensively and quantitatively, without the use of comparative morphology but instead by evaluating the expression of conserved genes.
by using this method, we tackled two questions.
first, we asked whether the mouse embryo really passes through a highly constrained stage that is in accord with the vertebrate phylotypic stage hypothesis.
second, we asked whether the hourglass model could be expanded to cover the embryogenesis of vertebrates as members of the bilaterians, in other words, whether vertebrates pass through stages conserved among bilaterians.
RESULTS
estimating the ancestral nature of mouse embryogenesis
on the basis that the vertebrate phylotypic stage should express the highest ratio of genes that conceivably existed in ancestral vertebrates, or vertebrate genes , we established the following index:
vertebrate ancestor index at developmental stage k = vk/nk
where vk: number of non-redundant vertebrate genes expressed at stage k; nk: number of non-redundant total genes expressed at stage k.
similarly, analyses with bilaterian, chordate, tetrapod, and amniote genes would also reveal the nature of each embryonic stage in a taxonomic context .
to analyze the ancestor indexes of mouse embryonic stages, we collected mouse gene sets from the ensembl database  <cit>  and gene expression profiles from the expressed sequence tags database   <cit> , and considered a gene to be "expressed" if the corresponding unigene  <cit>  id was present in any of the categorized est libraries.
est libraries categorized to each stage are listed in table  <dig>  additionally, to make this analysis tolerant to the fluctuation in timing of embryonic stages, we took advantage of moving group analysis  to calculate the ancestor index of two sequential stages:
vertebrate ancestor index at grouped stage k = vk, k+1/nk, k+1
where vk, k+1: number of non-redundant vertebrate genes expressed at stage k or k+1; nk, k+1: number of non-redundant total genes expressed at stage k or k+ <dig>
with these methods, we calculated the bilaterian, chordate, vertebrate, tetrapod, and amniote ancestor indexes .
what surprised us initially was the appearance of an early peak around grouped stages  <dig> to  <dig> from the analyses of bilaterian and chordate genes .
broadly evaluated analysis also supported this tendency; both the early  and middle embryonic periods  have significantly higher ancestor indexes than the late period .
the prominently higher ancestor indexes shown in figure 2a,b indicate the existence of novel, well-conserved stages conceivably carrying characteristics of the bilaterians at around the cleavage to blastula stages in mouse embryogenesis.
vertebrate phylotypic stage in mouse embryogenesis
we next calculated ancestor indexes to search for a putative phylotypic stage in mouse embryogenesis.
in contrast to the bilaterian and chordate ancestor indexes, vertebrate, tetrapod, and amniote ancestor indexes did not show prominent peaks , covering the presumptive periods of the vertebrate phylotypic stage proposed by comparative morphological studies .
importantly, the vertebrate ancestor index of the middle embryonic period was significantly higher than those of both the early and late embryonic periods , as also seen in the tetrapod and amniote ancestor indexes .
all of these curves  were significantly different from those obtained and shown in figure 2a,b .
if we were to see the body plan and morphological patterning as readouts of genetic programs for development, then the ancestor indexes calculated for developmental genes would refine our understanding of the conservation of the stages between  <dig> and  <dig>  on this assumption, we focused on mouse developmental genes defined in the gene ontology database  to derive ancestor indexes of individual stages.
the highest ancestor index occurred at grouped stage  <dig> , and was significantly higher than values of both the early and late embryonic periods .
similar peaks in vertebrate, tetrapod, and amniote ancestor indexes indicate that e <dig> – <dig>  has been the stage most conserved throughout the evolution of vertebrates.
additionally, the peak of the amniote ancestor index at grouped stage  <dig>  indicates that these stages express the smallest ratio of newest developmental genes.
in light of the recent report that newer genes tend to evolve faster than older genes  <cit> , the e <dig> – <dig>  period is the most genetically unmodifiable or contains the strongest developmental constraints in mouse embryogenesis.
thus, our analyses with developmental genes reveal that mouse embryogenesis indeed has a highly conserved stage characteristic of ancestral vertebrates, conceivably at around e <dig>  to e <dig>  , which is in accord with the concepts of a vertebrate phylotypic stage  <cit> .
notably, many of the mice with mutations in vertebrate developmental genes expressed at the putative vertebrate phylotypic stage have fatal or systemic phenotypes: embryonic or perinatal lethal phenotypes , and abnormal phenotypes of the nervous , craniofacial , cardiovascular , respiratory , or skeletal  system .
thus, many of these gene products could be critical factors for defining fundamental morphological features of embryos.
this information in turn implies that mutations of developmental genes expressed at the putative phylotypic stage are not easily accommodated, which also supports the idea that this stage tends to be evolutionarily conserved.
existence of bilaterian-related stage
bilaterian and chordate ancestor indexes calculated with developmental genes again showed patterns significantly different from those of vertebrate genes , with their highest values at grouped stages  <dig> and  <dig>  similar to the results obtained with genome-level analysis, both of these peaks were earlier than the putative vertebrate phylotypic stage, spanning from the period of cleavage to the onset of gastrulation .
these results suggest that this early embryonic period carries the basic body plan of bilaterians , making it the "bilateriotypic" stage.
gene expression information of ciona intestinalis embryogenesis further allowed us to verify the existence of the putative bilaterian-related stage in this organism at the early embryonic stage .
the highest value of bilaterian ancestor indexes of genes expressed during c. intestinalis embryogenesis occurred at the cleavage stage, and was significantly higher than the ancestor index of the later embryonic stage .
however, we could not obtain evidence at the level of "developmental genes" owing to the scarcity of gene ontologies associated with c. intestinalis genes.
CONCLUSIONS
we have developed a comprehensive quantitative method to evaluate evolutionarily constrained stages during the development of individual embryos.
our results show that mouse embryogenesis indeed passes through a highly constrained stage, as the hourglass model implies, at the mid-embryonic period .
they also show that the stage may be the most developmentally conserved stage since the evolution of ancestral vertebrates, making it a candidate for the phylotypic stage.
notably, it exhibits the major characteristics of the phylotypic stage proposed by morphological studies: pharyngula stage  <cit> , early somite segmentation stage  <cit> , and tailbud stage  <cit> .
at the pharyngula stage, animals should contain a series of paired branchial grooves, a notochord, a post-anal tail, and a dorsal hollow nerve cord.
in mice, maxillary components of the 1st branchial arch become prominent at about e <dig> .
the 3rd branchial arch is still absent when mandibular components appear at around e <dig> .
the first somite pairs appear at e <dig> , and  <dig> to  <dig> somite pairs are found at e <dig> .
the tailbud stage is e <dig> – <dig> , later than the pharyngula and early somite segmentation stages.
thus, the period of e <dig> – <dig>  corresponds to the onset of the pharyngula stage and the early somite segmentation stage in mice.
however, the exact characteristics of the phylotypic stage cannot be concluded from our results, as it should be common to all vertebrate embryos.
our method will be a powerful tool for comparison among diverse vertebrates.
in contrast to morphological studies that evaluate developmental constraints qualitatively by morphological resemblance among embryos, our genome-based method evaluates developmental constraints quantitatively in a single species.
this latter point is especially important to the testing of the phylotypic stage hypothesis: our method allows us to define constrained or conserved stages in each organism, and thus to compare data among various vertebrate embryos.
this approach would clarify the common developmental events and gene sets that characterize the phylotypic stage .
in addition, our results suggest the existence of a bilateriotypic stage within the cleavage to gastrulation period in mouse embryogenesis, and the possible conservation of this stage in c. intestinalis.
these results demonstrate that although the early developmental period  is relatively modifiable on an evolutionary time-scale compared with the phylotypic stage, the cleavage-to-gastrulation period still retains highly ancestral characteristics.
on the basis of this perspective, we propose that mouse embryogenesis possesses two distinct, nested hourglasses, the vertebrate and bilaterian ones.
these hourglasses may represent two major developmental constraints from distinct evolutional pasts – ancestors of vertebrates and bilaterians – in mouse embryogenesis.
this extended version of the developmental hourglass model once again highlights the questioned, hierarchical nature of embryogenesis proposed by von baer, albeit partly: "general features common to a large group of animals appear earlier in the embryo than do specialized features."
similar and further analyses in various animals will be required to verify this model, to explain how the bilateriotypic period defined by our analysis is related to the "zootype" hypothesis proposed by slack  <cit> , and to clarify certain aspects of the cleavage period shared among bilaterians  <cit> .
comprehensive gene expression data of various animals  and further development of gene ontology  databases would make this kind of analysis more effective and definitive.
defining conserved stages and extracting genes by our method would be a powerful tool to answer what kind of developmental events and gene sets are evolutionarily constrained.
the information in turn would help explain how the conserved stages limit the possible variations of animal body plan in the context of animal evolution.

 comparative embryology of various animal groups has suggested the idea of a conserved stage in embryogenesis. in karl von baer was the first to describe revolutionary laws for animal development, inferring that the later the stage of embryogenesis of related organisms, the less they resemble each other. this "progressive divergence model" was challenged by seidel, sander, and elinson, who noted that earlier stages of development have relatively divergent morphological patterns among species, and so do not always fit the rule. today, the revised version of the laws, called the "hourglass model" or the "egg-timer model", is widely accepted, and the bottleneck stage, representing maximum resemblance among various vertebrates, is commonly referred to as the "phylotypic stage". however, much controversy remains about when the phylotypic stage appears; ballard first proposed the pharyngula stage as the phylotypic stage, whereas wolpert proposed the early somite segmentation stage, and slack, the tailbud stage. moreover, a few detailed morphological studies have thrown doubt on the existence of a phylotypic stage. these problems are due mainly to the difficulty in evaluating conserved or constrained developmental stages by morphological resemblance. in particular, heterochrony, or variations in the relative rate or timing of developmental events, makes it extremely difficult to conclude which embryonic stage is the vertebrate phylotypic stage. the problem of heterochrony prevents us from concluding whether each vertebrate really has a phylotypic stage, and whether any morphological features are associated with the stage. recent progress in developmental biology has revealed marked similarities of the molecular mechanisms of morphogenesis in bilaterians, leading to the "zootype" hypothesis. the zootype is defined by a common pattern of hox gene expression among bilaterians, and is considered to be displayed most clearly at the phylotypic stage. we note, however, many conserved genes other than hox are also involved in animal development, so comprehensive evaluation of these genes should be included for a more accurate definition of zootype. further, the fact that they are expressed differently during development among bilaterians prevents us from concluding which stage is zootype-related constrained. recently, using mouse and human genome information, hazkani-covo et al. evaluated the protein distances of each gene expressed during mouse embryogenesis to show the existence of the vertebrate phylotypic stage. although they could not specify the timing of the phylotypic stage, they raised the possibility of a genome-based approach to examining the phylotypic stage hypothesis. here, we sought to develop a novel method to evaluate the evolutionary ancestral nature of each embryonic stage comprehensively and quantitatively, without the use of comparative morphology but instead by evaluating the expression of conserved genes. by using this method, we tackled two questions. first, we asked whether the mouse embryo really passes through a highly constrained stage that is in accord with the vertebrate phylotypic stage hypothesis. second, we asked whether the hourglass model could be expanded to cover the embryogenesis of vertebrates as members of the bilaterians, in other words, whether vertebrates pass through stages conserved among bilaterians.  estimating the ancestral nature of mouse embryogenesis on the basis that the vertebrate phylotypic stage should express the highest ratio of genes that conceivably existed in ancestral vertebrates, or vertebrate genes, we established the following index: vertebrate ancestor index at developmental stage k = vk/nk where vk: number of non-redundant vertebrate genes expressed at stage k; nk: number of non-redundant total genes expressed at stage k. similarly, analyses with bilaterian, chordate, tetrapod, and amniote genes would also reveal the nature of each embryonic stage in a taxonomic context. to analyze the ancestor indexes of mouse embryonic stages, we collected mouse gene sets from the ensembl database and gene expression profiles from the expressed sequence tags database, and considered a gene to be "expressed" if the corresponding unigene id was present in any of the categorized est libraries. est libraries categorized to each stage are listed in table additionally, to make this analysis tolerant to the fluctuation in timing of embryonic stages, we took advantage of moving group analysis to calculate the ancestor index of two sequential stages: vertebrate ancestor index at grouped stage k = vk, k+1/nk, k+1 where vk, k+1: number of non-redundant vertebrate genes expressed at stage k or k+1; nk, k+1: number of non-redundant total genes expressed at stage k or k+ with these methods, we calculated the bilaterian, chordate, vertebrate, tetrapod, and amniote ancestor indexes. what surprised us initially was the appearance of an early peak around grouped stages to from the analyses of bilaterian and chordate genes. broadly evaluated analysis also supported this tendency; both the early and middle embryonic periods have significantly higher ancestor indexes than the late period. the prominently higher ancestor indexes shown in figure 2a,b indicate the existence of novel, well-conserved stages conceivably carrying characteristics of the bilaterians at around the cleavage to blastula stages in mouse embryogenesis. vertebrate phylotypic stage in mouse embryogenesis we next calculated ancestor indexes to search for a putative phylotypic stage in mouse embryogenesis. in contrast to the bilaterian and chordate ancestor indexes, vertebrate, tetrapod, and amniote ancestor indexes did not show prominent peaks, covering the presumptive periods of the vertebrate phylotypic stage proposed by comparative morphological studies. importantly, the vertebrate ancestor index of the middle embryonic period was significantly higher than those of both the early and late embryonic periods, as also seen in the tetrapod and amniote ancestor indexes. all of these curves were significantly different from those obtained and shown in figure 2a,b. if we were to see the body plan and morphological patterning as readouts of genetic programs for development, then the ancestor indexes calculated for developmental genes would refine our understanding of the conservation of the stages between and on this assumption, we focused on mouse developmental genes defined in the gene ontology database to derive ancestor indexes of individual stages. the highest ancestor index occurred at grouped stage, and was significantly higher than values of both the early and late embryonic periods. similar peaks in vertebrate, tetrapod, and amniote ancestor indexes indicate that e – has been the stage most conserved throughout the evolution of vertebrates. additionally, the peak of the amniote ancestor index at grouped stage indicates that these stages express the smallest ratio of newest developmental genes. in light of the recent report that newer genes tend to evolve faster than older genes, the e – period is the most genetically unmodifiable or contains the strongest developmental constraints in mouse embryogenesis. thus, our analyses with developmental genes reveal that mouse embryogenesis indeed has a highly conserved stage characteristic of ancestral vertebrates, conceivably at around e to e, which is in accord with the concepts of a vertebrate phylotypic stage. notably, many of the mice with mutations in vertebrate developmental genes expressed at the putative vertebrate phylotypic stage have fatal or systemic phenotypes: embryonic or perinatal lethal phenotypes, and abnormal phenotypes of the nervous, craniofacial, cardiovascular, respiratory, or skeletal system. thus, many of these gene products could be critical factors for defining fundamental morphological features of embryos. this information in turn implies that mutations of developmental genes expressed at the putative phylotypic stage are not easily accommodated, which also supports the idea that this stage tends to be evolutionarily conserved. existence of bilaterian-related stage bilaterian and chordate ancestor indexes calculated with developmental genes again showed patterns significantly different from those of vertebrate genes, with their highest values at grouped stages and similar to the results obtained with genome-level analysis, both of these peaks were earlier than the putative vertebrate phylotypic stage, spanning from the period of cleavage to the onset of gastrulation. these results suggest that this early embryonic period carries the basic body plan of bilaterians, making it the "bilateriotypic" stage. gene expression information of ciona intestinalis embryogenesis further allowed us to verify the existence of the putative bilaterian-related stage in this organism at the early embryonic stage. the highest value of bilaterian ancestor indexes of genes expressed during c. intestinalis embryogenesis occurred at the cleavage stage, and was significantly higher than the ancestor index of the later embryonic stage. however, we could not obtain evidence at the level of "developmental genes" owing to the scarcity of gene ontologies associated with c. intestinalis genes.  we have developed a comprehensive quantitative method to evaluate evolutionarily constrained stages during the development of individual embryos. our results show that mouse embryogenesis indeed passes through a highly constrained stage, as the hourglass model implies, at the mid-embryonic period. they also show that the stage may be the most developmentally conserved stage since the evolution of ancestral vertebrates, making it a candidate for the phylotypic stage. notably, it exhibits the major characteristics of the phylotypic stage proposed by morphological studies: pharyngula stage, early somite segmentation stage, and tailbud stage. at the pharyngula stage, animals should contain a series of paired branchial grooves, a notochord, a post-anal tail, and a dorsal hollow nerve cord. in mice, maxillary components of the 1st branchial arch become prominent at about e. the 3rd branchial arch is still absent when mandibular components appear at around e. the first somite pairs appear at e, and to somite pairs are found at e. the tailbud stage is e –, later than the pharyngula and early somite segmentation stages. thus, the period of e – corresponds to the onset of the pharyngula stage and the early somite segmentation stage in mice. however, the exact characteristics of the phylotypic stage cannot be concluded from our results, as it should be common to all vertebrate embryos. our method will be a powerful tool for comparison among diverse vertebrates. in contrast to morphological studies that evaluate developmental constraints qualitatively by morphological resemblance among embryos, our genome-based method evaluates developmental constraints quantitatively in a single species. this latter point is especially important to the testing of the phylotypic stage hypothesis: our method allows us to define constrained or conserved stages in each organism, and thus to compare data among various vertebrate embryos. this approach would clarify the common developmental events and gene sets that characterize the phylotypic stage. in addition, our results suggest the existence of a bilateriotypic stage within the cleavage to gastrulation period in mouse embryogenesis, and the possible conservation of this stage in c. intestinalis. these results demonstrate that although the early developmental period is relatively modifiable on an evolutionary time-scale compared with the phylotypic stage, the cleavage-to-gastrulation period still retains highly ancestral characteristics. on the basis of this perspective, we propose that mouse embryogenesis possesses two distinct, nested hourglasses, the vertebrate and bilaterian ones. these hourglasses may represent two major developmental constraints from distinct evolutional pasts – ancestors of vertebrates and bilaterians – in mouse embryogenesis. this extended version of the developmental hourglass model once again highlights the questioned, hierarchical nature of embryogenesis proposed by von baer, albeit partly: "general features common to a large group of animals appear earlier in the embryo than do specialized features." similar and further analyses in various animals will be required to verify this model, to explain how the bilateriotypic period defined by our analysis is related to the "zootype" hypothesis proposed by slack, and to clarify certain aspects of the cleavage period shared among bilaterians. comprehensive gene expression data of various animals and further development of gene ontology databases would make this kind of analysis more effective and definitive. defining conserved stages and extracting genes by our method would be a powerful tool to answer what kind of developmental events and gene sets are evolutionarily constrained. the information in turn would help explain how the conserved stages limit the possible variations of animal body plan in the context of animal evolution. 
to overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag data, gene ontologies, and available genomes of various animals. our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. these results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von baer. this "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. however, much controversy remains about when the phylotypic stage appears, and whether it even exists. if strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage. the genes associated with the phylotypic stage identified by our method are essential in embryogenesis. identification of conserved stages and tissues by this method in various animals would be a powerful tool to examine the phylotypic stage hypothesis, and to understand which kinds of developmental events and gene sets are evolutionarily constrained and how they limit the possible variations of animal basic body plans. similar analyses revealed the existence of a bilaterian-related period, during which go-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. the numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day –, the time of appearance of the pharyngeal arch and somites. we established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. during this period, the mouse prominently expresses go-determined developmental genes shared among vertebrates.
 embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. this "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. however, much controversy remains about when the phylotypic stage appears, and whether it even exists. to overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag data, gene ontologies, and available genomes of various animals. if strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage.  we established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. the numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day –, the time of appearance of the pharyngeal arch and somites. during this period, the mouse prominently expresses go-determined developmental genes shared among vertebrates. similar analyses revealed the existence of a bilaterian-related period, during which go-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. the genes associated with the phylotypic stage identified by our method are essential in embryogenesis.  our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. these results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von baer. identification of conserved stages and tissues by this method in various animals would be a powerful tool to examine the phylotypic stage hypothesis, and to understand which kinds of developmental events and gene sets are evolutionarily constrained and how they limit the possible variations of animal basic body plans. 