BACKGROUND
much effort has been expended to define developmental stages: cellular and morphological hallmarks of critical points during embryogenesis.
stages, unlike developmental time alone, provide insights into cellular and molecular processes as simple as the eight-cell stage or as complex as the onset of circulation.
although numerous aspects of development have been discovered through studies of diverse species  <cit> , a comprehensive analysis of gene expression for each separate stage of vertebrate development is lacking.
among vertebrates, developmental processes are shared  <cit> ; thus insights from fish inform human studies  <cit> .
fundulus heteroclitus, similar to zebrafish and medaka , has external development and transparent eggs, which facilitate associations between morphological developmental changes and patterns of gene expression.
unlike many other developmental models, f. heteroclitus has a ~ <dig> day development that allows greater precision in defining specific stages than is possible in vertebrate species with short developmental times.
finally, this species has large population sizes, a well-described phylogeny, and locally adapted populations making it an exceptional model for environmental and evolutionary studies  <cit> .
to provide quantitative and statistical analyses of development, we used four biological replicates from all  <dig> developmental stages  of f. heteroclitus.
these biological replicates provide the data for statistical analyses of the expression of  <dig>  genes throughout embryogenesis and a better understanding of the differences among stages and developmental pathways.
although  <dig>  genes are not the full complement of genes expressed in vertebrates, they provide a statistically robust measure of differences between stages, which is important for experimental sciences that explore embryo responses to altered environments, chemical exposures and physiological differences.
RESULTS
expression of  <dig>  genes  differs significantly  among all stages.
our a priori query addressed the number of significant differences between adjacent stages: expression of  <dig>  genes  differs significantly between stages.
hierarchical clustering of these genes  groups stages into three main clusters: 1) stages 1-13: fertilization-early gastrula; 2) stages 14-25: blastoderm-heart formation; 3) stages 26-40: onset of circulation-late organogenesis and post-hatch.
these groups are in developmental order with a few exceptions .
thus, the patterns of gene expression recapitulate the series of developmental stages.
the number of genes that alter expression between adjacent stages differs widely, from  <dig> to  <dig> .
at a critical p-value of  <dig> , one expects approximately  <dig> differences due to type- <dig> errors.
fifteen pairs of adjacent stages have less than this false expectation.
three of these pairs of adjacent stages with few significant genes occur among gastrulation stages  and two are prior to the 4-cell stage .
not surprisingly, few differences in expression occur between fertilization and first cell division.
in contrast, the lack of changes in expression throughout formation of the germ layers during gastrulation is unexpected.
expression of  <dig>  and  <dig>  genes are significantly different between the 4-cell  and 8-cell  stages and the 8-cell and 16-cell  stages, respectively.
during zebrafish embryogenesis, these stages correspond to the timing of maternal gene degradation and onset of embryo gene activity  <cit> .
the large number of significantly differently expressed genes suggests similar timing for f. heteroclitus.
notice, a large number of genes show both significant increases and decreases from stage  <dig> to  <dig> .
however, between stages  <dig> and  <dig>  most significant differences  are increases in expression  suggesting that initiation of embryonic gene expression becomes more dominant at stage  <dig>
the greatest number of differentially expressed genes  occurs between pre-hatching  and hatching , which validates our incentive to distinguish these two stages in contrast to previous work  <cit> .
other notable differences occur between stages  <dig> and  <dig> , when most of the yolk is consumed by the free-swimming fundulus larvae  and between stages  <dig> and  <dig> , marked by the onset of circulation.
times to stage have large variances .
for example, stages  <dig> and  <dig> are on average one hour apart yet have a combined standard deviation greater than one hour.
variability in times to stage becomes more pronounced as later stages become longer .
for stages  <dig> and  <dig>  with  <dig> differences in expression, the mean times to stage differ by fourteen hours, which is nearly equal to the standard deviation for each stage.
thus, using time alone rather than developmental markers can lead to misinterpretations of gene expression changes.
fundulus heteroclitus development.
bolded p-values are significant.
time values are averages ± standard deviations from  <dig> families of  <dig> embryos each.
the wave of maximum gene expression throughout development provides insight into how the magnitude of expression relates to developmental processes .
four distinct quadrants, similar to the hierarchical clustering results, are formed: stages 1- <dig> , stages 14- <dig> , stages 26- <dig> , and stages 36- <dig> .
pre-mid gastrulation  has the most genes  with peak expression, followed by pre-hatching  and the 8-cell stage .
thus, although most genes have a significant increase in expression between  <dig> and  <dig> cell stages, the greatest number of genes reaches maximum expression during pre-mid gastrulation.
notably, the lack of many significant differences among gastrulation stages  corresponds to the large block of maximum expression.
these data suggest that both qualitative differences in expression  and quantitative differences  are important for defining developmental processes.
genes with peak expression during stage  <dig>  are significantly enriched for ribosomal genes , which show highly positively correlated expression patterns .
interestingly, these ribosomal genes are significantly negatively correlated with many of the other ribosomal genes expressed during development.
stage  <dig> also is significantly enriched for genes involved in oxidative phosphorylation, fatty acid metabolism, and translation as well as proteases .
taken together, these data suggest that peak gene expression during gastrulation enhances high-energy demands during cellular proliferation and protein synthesis and turnover.
among the  <dig>  genes significantly differently expressed across all stages, almost half  have a significant correlation coefficient :  <dig> % are significantly positively correlated and  <dig> % are significantly negatively correlated .
among pairs of genes with larger correlation coefficients, the ratio of significant positively to negatively correlated genes increases.
thus, when the absolute value of the correlation coefficients exceeds  <dig> , this ratio is  <dig>  , and when these correlation coefficients exceed  <dig> , it is  <dig>  .
the numerous correlated genes suggest concerted changes in gene expression throughout development.
moreover, the increase in the relative amount of positive associations with stronger correlations suggests common regulatory factors while the less significant negative correlations may reflect coordinate regulation, potentially using similar signalling pathways, but with a variety of different regulatory factors.
the last five stages  compared to the  <dig> embryonic developmental stages give insight into pre-adult versus developmental gene expression.
eight-hundred and eighty-nine genes  are significantly differently expressed during pre-hatching  versus post-hatching  ;  <dig> genes  have higher expression levels before hatching, while  <dig> genes  are up regulated after hatching .
several post-hatch up regulated genes have important functions in muscle tissue development and movement including parvalbumins alpha and beta , myosin regulatory light chain, skeletal muscle isoform , myosin light chain  <dig>  skeletal muscle gene , myosin binding protein c  and troponins i, t and c .
increased transcript levels of these genes suggest increases in movement and muscle activity of a free-swimming fundulus compared to restricted movement within a chorion microenvironment before hatching.
in addition, the creatine-kinase system is important for energy delivery in skeletal and cardiac muscle  <cit> , and the 3-fold up regulation of muscle type creatine-kinase post-hatch indicates increases in metabolic activity and atp consumption resulting from skeletal muscle activity caused by swimming.
low transcript levels pre-hatch, and higher levels post-hatch of both retinal-cone rhodopsin-sensitve cgmp  and photosystem i reaction center subunit ii-like gene  are associated with vision  <cit> .
since both genes are induced by light and fundulus embryos are exposed to higher light intensity after hatching than within the chorion  <cit> , the up regulation of these two genes suggest embryo photoreceptor system response to a brighter external environment.
finally, several up regulated genes post-hatch suggest changes in metabolic activity of the free-swimming fundulus.
these include nucleoside diphosphate kinase nbr-b , required for nucleoside triphosphate synthesis  and necessary for lipid and polysaccharide synthesis, protein elongation, signal transduction and microtubule polymerization  <cit> , trifunctional purine biosynthetic protein adenosine- <dig>  which plays a major role in purine biosynthesis  <cit>  and fatty acid-binding protein required for lipid transport and metabolism.
both liver and heart fatty acid binding protein genes are up regulated in fundulus post hatch , and the increased expression levels likely reflect changes taking place within the last two stages of fundulus development marked by the transition period between complete yolk consumption and increasing dependence on external food sources.
CONCLUSIONS
statistical analyses of nearly  <dig>  fundulus genes during all  <dig> stages of vertebrate embryogenesis highlight the temporal dynamics of developmental gene expression and stage clustering.
analyses of differences in gene expression between adjacent stages and onset of peak gene activity emphasize the importance of correctly identifying stages during embryogenesis.
these data show clear differences between the transitions among stages: 4- <dig> cell stages have many more significant changes than similar stages ; pre-post hatching involves the largest number of significant temporal changes in gene expression associated with transition from egg enclosed embryos to free swimming larvae.
these statistical analyses are possible because gene expression was quantified with biological replication among well-defined stages.

 much effort has been expended to define developmental stages: cellular and morphological hallmarks of critical points during embryogenesis. stages, unlike developmental time alone, provide insights into cellular and molecular processes as simple as the eight-cell stage or as complex as the onset of circulation. although numerous aspects of development have been discovered through studies of diverse species, a comprehensive analysis of gene expression for each separate stage of vertebrate development is lacking. among vertebrates, developmental processes are shared; thus insights from fish inform human studies. fundulus heteroclitus, similar to zebrafish and medaka, has external development and transparent eggs, which facilitate associations between morphological developmental changes and patterns of gene expression. unlike many other developmental models, f. heteroclitus has a ~ day development that allows greater precision in defining specific stages than is possible in vertebrate species with short developmental times. finally, this species has large population sizes, a well-described phylogeny, and locally adapted populations making it an exceptional model for environmental and evolutionary studies. to provide quantitative and statistical analyses of development, we used four biological replicates from all developmental stages of f. heteroclitus. these biological replicates provide the data for statistical analyses of the expression of genes throughout embryogenesis and a better understanding of the differences among stages and developmental pathways. although genes are not the full complement of genes expressed in vertebrates, they provide a statistically robust measure of differences between stages, which is important for experimental sciences that explore embryo responses to altered environments, chemical exposures and physiological differences.  expression of genes differs significantly among all stages. our a priori query addressed the number of significant differences between adjacent stages: expression of genes differs significantly between stages. hierarchical clustering of these genes groups stages into three main clusters: 1) stages 1-13: fertilization-early gastrula; 2) stages 14-25: blastoderm-heart formation; 3) stages 26-40: onset of circulation-late organogenesis and post-hatch. these groups are in developmental order with a few exceptions. thus, the patterns of gene expression recapitulate the series of developmental stages. the number of genes that alter expression between adjacent stages differs widely, from to. at a critical p-value of, one expects approximately differences due to type- errors. fifteen pairs of adjacent stages have less than this false expectation. three of these pairs of adjacent stages with few significant genes occur among gastrulation stages and two are prior to the 4-cell stage. not surprisingly, few differences in expression occur between fertilization and first cell division. in contrast, the lack of changes in expression throughout formation of the germ layers during gastrulation is unexpected. expression of and genes are significantly different between the 4-cell and 8-cell stages and the 8-cell and 16-cell stages, respectively. during zebrafish embryogenesis, these stages correspond to the timing of maternal gene degradation and onset of embryo gene activity. the large number of significantly differently expressed genes suggests similar timing for f. heteroclitus. notice, a large number of genes show both significant increases and decreases from stage to. however, between stages and most significant differences are increases in expression suggesting that initiation of embryonic gene expression becomes more dominant at stage the greatest number of differentially expressed genes occurs between pre-hatching and hatching, which validates our incentive to distinguish these two stages in contrast to previous work. other notable differences occur between stages and, when most of the yolk is consumed by the free-swimming fundulus larvae and between stages and, marked by the onset of circulation. times to stage have large variances. for example, stages and are on average one hour apart yet have a combined standard deviation greater than one hour. variability in times to stage becomes more pronounced as later stages become longer. for stages and with differences in expression, the mean times to stage differ by fourteen hours, which is nearly equal to the standard deviation for each stage. thus, using time alone rather than developmental markers can lead to misinterpretations of gene expression changes. fundulus heteroclitus development. bolded p-values are significant. time values are averages ± standard deviations from families of embryos each. the wave of maximum gene expression throughout development provides insight into how the magnitude of expression relates to developmental processes. four distinct quadrants, similar to the hierarchical clustering results, are formed: stages 1-, stages 14-, stages 26-, and stages 36-. pre-mid gastrulation has the most genes with peak expression, followed by pre-hatching and the 8-cell stage. thus, although most genes have a significant increase in expression between and cell stages, the greatest number of genes reaches maximum expression during pre-mid gastrulation. notably, the lack of many significant differences among gastrulation stages corresponds to the large block of maximum expression. these data suggest that both qualitative differences in expression and quantitative differences are important for defining developmental processes. genes with peak expression during stage are significantly enriched for ribosomal genes, which show highly positively correlated expression patterns. interestingly, these ribosomal genes are significantly negatively correlated with many of the other ribosomal genes expressed during development. stage also is significantly enriched for genes involved in oxidative phosphorylation, fatty acid metabolism, and translation as well as proteases. taken together, these data suggest that peak gene expression during gastrulation enhances high-energy demands during cellular proliferation and protein synthesis and turnover. among the genes significantly differently expressed across all stages, almost half have a significant correlation coefficient: % are significantly positively correlated and % are significantly negatively correlated. among pairs of genes with larger correlation coefficients, the ratio of significant positively to negatively correlated genes increases. thus, when the absolute value of the correlation coefficients exceeds, this ratio is, and when these correlation coefficients exceed, it is. the numerous correlated genes suggest concerted changes in gene expression throughout development. moreover, the increase in the relative amount of positive associations with stronger correlations suggests common regulatory factors while the less significant negative correlations may reflect coordinate regulation, potentially using similar signalling pathways, but with a variety of different regulatory factors. the last five stages compared to the embryonic developmental stages give insight into pre-adult versus developmental gene expression. eight-hundred and eighty-nine genes are significantly differently expressed during pre-hatching versus post-hatching; genes have higher expression levels before hatching, while genes are up regulated after hatching. several post-hatch up regulated genes have important functions in muscle tissue development and movement including parvalbumins alpha and beta, myosin regulatory light chain, skeletal muscle isoform, myosin light chain skeletal muscle gene, myosin binding protein c and troponins i, t and c. increased transcript levels of these genes suggest increases in movement and muscle activity of a free-swimming fundulus compared to restricted movement within a chorion microenvironment before hatching. in addition, the creatine-kinase system is important for energy delivery in skeletal and cardiac muscle, and the 3-fold up regulation of muscle type creatine-kinase post-hatch indicates increases in metabolic activity and atp consumption resulting from skeletal muscle activity caused by swimming. low transcript levels pre-hatch, and higher levels post-hatch of both retinal-cone rhodopsin-sensitve cgmp and photosystem i reaction center subunit ii-like gene are associated with vision. since both genes are induced by light and fundulus embryos are exposed to higher light intensity after hatching than within the chorion, the up regulation of these two genes suggest embryo photoreceptor system response to a brighter external environment. finally, several up regulated genes post-hatch suggest changes in metabolic activity of the free-swimming fundulus. these include nucleoside diphosphate kinase nbr-b, required for nucleoside triphosphate synthesis and necessary for lipid and polysaccharide synthesis, protein elongation, signal transduction and microtubule polymerization, trifunctional purine biosynthetic protein adenosine- which plays a major role in purine biosynthesis and fatty acid-binding protein required for lipid transport and metabolism. both liver and heart fatty acid binding protein genes are up regulated in fundulus post hatch, and the increased expression levels likely reflect changes taking place within the last two stages of fundulus development marked by the transition period between complete yolk consumption and increasing dependence on external food sources.  statistical analyses of nearly fundulus genes during all stages of vertebrate embryogenesis highlight the temporal dynamics of developmental gene expression and stage clustering. analyses of differences in gene expression between adjacent stages and onset of peak gene activity emphasize the importance of correctly identifying stages during embryogenesis. these data show clear differences between the transitions among stages: 4- cell stages have many more significant changes than similar stages; pre-post hatching involves the largest number of significant temporal changes in gene expression associated with transition from egg enclosed embryos to free swimming larvae. these statistical analyses are possible because gene expression was quantified with biological replication among well-defined stages. 
among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than five adjacent stages have disproportionately more significant changes in gene expression relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. this study presents statistical analyses of gene expression during all developmental stages in the teleost fundulus heteroclitus using four biological replicates per stage. yet, at stage the largest number of genes has peak expression. these data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development. the fewest differences among adjacent stages occur during gastrulation. unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. similar correlated patterns of expression occur among all significant genes. describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. yet gene expression patterns have not been described throughout vertebrate embryogenesis. patterns of gene expression for genes appear to be important as they recapitulate developmental timing.
 describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. yet gene expression patterns have not been described throughout vertebrate embryogenesis. this study presents statistical analyses of gene expression during all developmental stages in the teleost fundulus heteroclitus using four biological replicates per stage.  patterns of gene expression for genes appear to be important as they recapitulate developmental timing. among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than five adjacent stages have disproportionately more significant changes in gene expression relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. the fewest differences among adjacent stages occur during gastrulation. yet, at stage the largest number of genes has peak expression. this stage has an over representation of genes in oxidative respiration and protein expression. unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. similar correlated patterns of expression occur among all significant genes.  these data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development. 