BACKGROUND
darwin  <cit>  proposed that evolution by natural selection is a gradual process that results in continuous phenotypic variation among species.
however, there are many examples where discontinuous phenotypes are observed among related species and thus appear to evolve rapidly.
that evolution could suddenly "leap forward" led to extensions of darwin's theory to account for the rapid origin of novel phenotypes.
one very old idea is that novel and dramatically different phenotypes originate via saltational evolution from mutations of genes that regulate key developmental or physiological processes during ontogeny.
in particular, goldschmidt  <cit>  proposed that mutations occasionally yield individuals within populations that deviate radically from the norm and referred to such individuals as "hopeful monsters".
if the novel phenotypes of hopeful monsters arise under the right environmental circumstances, they may become fixed, and the population will found a new species.
while this idea was discounted during the modern synthesis  <cit> , aspects of the hopeful monster hypothesis have been substantiated in recent years.
for example, it is clear that dramatic changes in phenotype can occur from few mutations of key developmental genes and phenotypic differences among species often map to relatively few genetic factors  <cit> .
these findings are motivating renewed interest in the study of hopeful monsters and the perspectives they can provide about the evolution of development  <cit> .
in contrast to mutants that are created in the lab, hopeful monsters have been shaped by natural selection and are therefore more likely to reveal mechanisms of adaptive evolution.
at least three lines of evidence led goldschmidt  <cit>  to cite the mexican axolotl  as one of the original hopeful monsters.
first, the axolotl follows a different ontogeny from other closely related tiger salamanders.
whereas some tiger salamanders undergo an obligatory metamorphosis during ontogeny that allows for a transition from an aquatic habitat to a more terrestrial habitat, the axolotl has a non-metamorphic life cycle that is often referred to as paedomorphic  <cit> .
this extreme example of discontinuous phenotypic variation supports a model of evolution by heterochrony: larval morphological traits of ancestral metamorphic forms are observed in the adult stages of derived paedomorphic forms.
in the minds of early evolutionary biologists, these patterns were so clearly supportive of heterochrony that the mexican axolotl became the exemplar of evolution by neoteny  <cit> .
the second reason goldschmidt cited the axolotl was physiological - huxley  <cit>  had shown that a single molecule - thyroid hormone  - was capable of rescuing metamorphosis in the axolotl.
thus, the axolotl seemed to be an example of evolution waiting around for the right macromutation to happen - simply block a single physiological step in th regulation and a novel form is originated.
the third reason goldschmidt cited the axolotl was ecological.
previous researchers had noted that the axolotl was endemic to the high quality, permanent aquatic habitats of xochimilco, which is near present day mexico city  <cit> .
the evolution of paedomorphosis seemingly allowed the axolotl to exploit an empty niche in an environment that was devoid of predators.
since goldschmidt, the axolotl has remained a quintessential hopeful monster  <cit> .
speculation that the paedomorphic condition of the axolotl could have a simple mechanistic basis was supported when a quantitative trait locus  was identified for the segregation of metamorphic and paedomorphic phenotypes in interspecific crosses  <cit> .
previous physiological studies had also established that axolotls do not produce a sufficient titer of thyroid hormone during larval development to initiate anatomical metamorphosis .
the evolution of axolotl hypothyroidism is thought to be associated with a mechanism that affects the development and/or function of neuroendocrine axes that regulate the release of thyroid hormone from the thyroid glands  <cit> .
conceivably, this mechanism could function during early stages of development or it could function later in the larval period when metamorphosis is initiated.
regardless, whether paedomorphic and metamorphic larvae show similar or different patterns of neurological development and function has not been previously investigated.
in this study, microarray analysis was used to investigate transcription within whole brains  of the paedomorphic axolotl and a closely related metamorphic species .
the primary objective was to identify patterns of gene expression during early ontogeny that could provide new mechanistic insights about paedomorphic and metamorphic modes of development.
transcripts were sampled from both species at four chronologically matched times post hatching to obtain temporal profiles of gene expression during the early larval period and during early stages of morphological metamorphosis in a. t. tigrinum.
hundreds of genes showed different or unique patterns of expression between the species, many of which were initiated very early in the larval period and prior to the onset of morphological metamorphosis.
the results suggest considerable potential for transcriptional divergence between closely related vertebrate species and highlight the tiger salamander/axolotl model system for examining mechanisms in the developing brain that determine adult phenotypic outcomes.
RESULTS
larval growth and metamorphosis
under normal growth conditions, larvae of metamorphic and paedomorphic species of ambystoma increase in size but only larvae of metamorphic species show changes in morphology  that are indicative of anatomical metamorphosis.
in this experiment, tiger larvae were larger than axolotls at  <dig> dph and exhibited higher growth rates early in the larval period .
as development proceeded, tiger salamander growth rates decelerated while axolotl growth rates remained constant.
none of the tiger larvae showed changes in morphology suggestive of initiation of metamorphosis at the three earliest time points .
however at  <dig> dph, 23%  of tiger larvae showed bulging eyes and subtle changes in head morphology.
by  <dig> dph, all tiger larvae were undergoing anatomical metamorphosis and 20%  of these had rudiments of gills that were less than  <dig> mm in length.
these larvae, and all larvae that were examined at  <dig> dph , were considered metamorphs.
thus, some tiger salamanders initiated anatomical metamorphosis between  <dig> and  <dig> dph and most larvae completed metamorphosis between  <dig> and  <dig> dph.
differentially expressed genes identified independently from axolotls and tiger salamanders
a custom affymetrix genechip  was used to estimate mrna abundances at four chronologically matched times  to obtain temporal profiles of gene expression for tiger salamander and axolotl larvae.
statistical and fold-change criteria were then used to identify genes that were differentially expressed within each of the species.
thus, these analyses took the conservative approach of independently identifying genes from each species that were differentially expressed as a function of time and comparing the list of differentially expressed genes  from tiger salamander with the list of degs identified from axolotl.
this approach is conservative because it reduces the risk of identifying false positive expression differences between species that are caused by heterologous hybridization  <cit> .
more than twice as many degs were identified from tiger salamander than axolotl larvae  .
there was considerable overlap between the two species as more than half  of the genes identified from axolotls were also identified from tiger salamander larvae.
the remaining degs changed uniquely in only one species.
thus, many shared and unique degs were identified, with more unique degs identified from tiger salamander larvae.
of the  <dig> degs identified from both species, 60% exhibited the same regression profile and 99% exhibited the same generalized direction of differential expression .
of the  <dig> degs uniquely identified from axolotls , 55% showed increasing mrna abundances  during the larval period while 45% showed decreasing abundances  .
the opposite pattern was observed for the  <dig> unique tiger salamander degs with 55% showing decreasing mrna abundances, 43% showing increasing abundances, and 1% showing a pattern of transient increase .
only a small percentage  of shared and unique degs exhibited ≥  <dig>  fold difference in mrna abundance between the earliest time points .
however, between the latest time points , ~32% of the shared degs, 64% of unique axolotl degs, and 61% of unique tiger salamander degs showed a ≥  <dig>  fold difference.
thus, abundances of uniquely expressed transcripts tended to increase throughout larval development, showing the largest fold differences at later larval stages.
the degs identified independently from both species that showed significant sequence identity to a human refseq protein were assumed to be salamander-human orthologs and were annotated with biological process information from the gene ontology  database.
many of the same go terms were represented among the three deg lists - the degs that were expressed in common between the species  and the separate lists of degs that were identified uniquely from each species .
the commonly expressed degs were statistically associated with  <dig> terms, and many of these genes are predicted to function in cell cycle processes .
nearly all of the degs that annotated to cell cycle terms exhibited decreasing mrna abundances during larval development in both species, as did six of seven annotated transcription factors.
for example, two biomarkers of neural development and differentiation  were both categorized as ld in axolotl and tiger salamander.
conversely, the majority of degs associated with system development were up regulated in both species.
these results suggest that some aspects of neural development and function are similarly regulated between axolotl and tiger salamander larvae.
this also seems to be true for some but not all hemoglobin loci, and genes that function in stress and immunological pathways.
relatively few uniquely expressed axolotl genes were identified overall  and thus only  <dig> broad go terms were identified as statistically enriched : regulation of cellular process , regulation of biological process , and biological regulation .
the unique axolotl genes are predicted to function in some but not all of the biological processes observed for the shared deg list .
for example, six genes that function in apoptosis  were uniquely identified for axolotls.
degs known to be associated with vertebrate brain development were identified, including genes that code for extracellular matrix constituents and cell adhesion .
also, several biomarkers of mammalian brain pathologies were uniquely up regulated in axolotls .
these and other uniquely expressed genes may be associated with the axolotl's paedomorphic mode of development.
the larger list of unique degs from tiger larvae yielded more biological process annotations and were statistically associated with  <dig> go terms .
as was observed for the shared gene list, the cell cycle go term was significantly enriched and these genes showed decreasing mrna abundances during larval development.
however, several go terms were identified that were not represented in the shared or unique axolotl list, including biological processes associated with chromatin organization and biogenesis.
for example, lmx1b showed a pattern of decreasing transcript abundance, as did several other genes that function in chromatin organization, modification, and gene silencing .
it is possible that some of these unique degs are associated with the maturation of brain regions that orchestrate metamorphic events.
for example, several genes that function to regulate the secretion of hypothalamic, pituitary, and interrenal hormones were uniquely expressed in tiger salamander larvae, including nr3c <dig>  prl, and sstr <dig>  in addition to these genes, pomc and crhr <dig> exhibited higher expression levels in tiger salamander larvae .
thus, the microarray analysis identified expression differences between axolotl and tiger larvae that may correlate with hpi axis regulation and function.
direct comparison of transcription between axolotl and tiger salamander
to complement the statistical analyses described above, a subset of affymetrix probesets were identified that could be used to reliably compare transcript abundance estimates directly between the species.
othologous genes from axolotl and tiger salamander were aligned to identify  <dig> affymetrix probe-sets with zero mismatches between the species.
ten of these probesets were identical to other probesets.
of the remaining non-redundant probesets, 31%  registered statistically distinct expression profiles between the species.
this analysis identified some of the unique, temporally regulated degs described above, and additionally, genes that exhibited flat regression profiles that differed significantly in elevation between the species.
indeed, 84%  of the genes identified from this analysis showed higher expression levels throughout the larval period in tiger salamanders .
this list is statistically enriched with genes that function in oxygen/gas transport , lipid transport , and heterocycle metabolic processes .
with respect to oxygen transport, several hemoglobin genes  exhibited >  <dig> fold differences between the species.
the complete list of genes that showed higher abundances in tiger salamanders annotate to many different biological processes, including mrna biosynthesis , protein translation , post-translational modification of proteins , neural function and development , stress and immune response , and hypothalamic-pituitary function .
the relatively few genes  that showed consistently higher transcript abundances in axolotl larvae also annotate to a number of different biological processes, including dna metabolism , cell cycle regulation , collagen metabolism , neural development , and cellular metabolism .
it is unlikely that the interspecific expression differences described above are artifacts of heterologous hybridization because > 80% of the probe-sets were designed from the species  that showed consistently lower transcriptional abundances across genes.
these results further demonstrate that genes are differentially expressed between the developing brains of axolotl and tiger salamander larvae, and that mrna abundance is on average higher for tiger salamander orthologs.
further investigation of gene expression using qpcr
some of the degs that were identified by microarray analysis suggest the hpi axis is differentially regulated between tiger salamander and axolotl larvae.
for example, microarray analysis estimated higher transcript abundances for nr3c <dig> in tiger salamanders, while nr3c <dig> did not differ between the species.
in adult mammals and presumably amphibians, glucocorticoids feedback to regulate brain development and function by binding to nr3c <dig> and nr3c <dig>  which like thyroid hormone receptors, act as transcription factors.
to verify transcript abundance estimates for these genes, and extend the analysis to two additional hpi axis genes , quantitative real-time reverse transcription pcr  was used to examine mrna expression across a broader range of time points .
both nr3c <dig> and nr3c <dig> showed a pattern of increasing transcript abundance with maximal levels attained at  <dig> dph for nr3c <dig> and  <dig> dph for nr3c2; after these times, levels decreased.
whereas nr3c <dig> showed the same pattern of expression in both species, nr3c <dig> abundance was two fold higher in tiger salamander larvae at  <dig> dph.
a pattern of increasing transcript abundance was also observed across the larval period for pomc while crhr <dig> mrna levels remained relatively flat.
for both of these genes, mrna levels were significantly higher in tiger salamander larvae.
these qpcr results corroborate the earlier microarray results and show that hpi axis genes are more highly expressed in tiger salamanders during larval development.
the microarray analysis also showed dramatic changes in hemoglobin mrna abundances both within and between species.
presumably, these gene expression patterns reflect transcription within erythrocytes that were isolated from vasculature within the brain.
the ambystoma genome appears to encode multiple paralogs for almost all hemoglobin loci and these are expressed differently in some cases .
for example, paralogs of hba in tiger salamander showed both increasing  and decreasing  patterns of mrna abundance, while all axolotl hba paralogs showed flat or increasing patterns .
qpcr yielded a flatter profile for axolotl hba than was suggested by the microarray analysis, however qpcr replicated the dramatic decrease in mrna abundance for tiger salamander hba, as well as the expression patterns of three additional hemoglobin loci for both species .
overall, the patterns suggest that axolotls and tiger salamanders similarly up regulate some of the same hemoglobin loci  during the larval period, although levels were consistently higher in tiger salamander larvae.
the only locus that was similarly down regulated was hbz.
interestingly, axolotls maintain higher embryonic-type hemoglobin  mrna levels than tigers throughout the larval period, a pattern that is consistent with a paedomorphic mode of development.
discussion
this study used a functional genomics approach to detail larval brain transcription between the paedomorphic mexican axolotl and metamorphic tiger salamander.
the results show that larvae of these species have different transcriptional programs that are distinguishable in two important respects.
first, although shared expression patterns were observed between the species, most of the genes that were identified as differentially expressed during the larval period showed species-specific patterns of expression.
gene expression was more similar between the species at earlier time points, with pronounced differences observed at  <dig> and  <dig> dph, which coincided with the onset of anatomical metamorphosis in a subset of the tiger salamander larvae.
second, the abundance of mrnas tended to be higher for genes that were up regulated during tiger salamander development, relative to those that were up regulated during axolotl development.
approximately 31% of genes that could be reliably and directly compared between the species were differentially expressed and 84% of these showed higher mrna abundances in tiger salamander larvae.
below, we discuss these primary results and explore their relationships to transcriptional programming that may correlate with metamorphic and paedomorphic modes of development.
similar expression patterns were observed for 99% of the genes that were commonly differentially expressed in axolotl and tiger salamander larvae as a function of time.
this suggests that some biological processes are regulated similarly between axolotl and tiger salamander larvae during development.
for example, it is possible that some of the genes that function in the specification and proliferation of neuronal cell types are similarly expressed during development in both metamorphic and paedomorphic salamanders.
genes associated with vertebrate brain development such as sox <dig>  <cit> , msx <dig>  <cit> , and npy  <cit>  significantly increased in abundance in both species.
also clu, a gene expressed at low levels in the central nervous systems of embryonic mice before increasing during postnatal life  <cit> , was similarly up regulated during the axolotl and tiger salamander larval periods.
many aspects of brain development and function are highly conserved among vertebrates.
if these functions depend upon conserved patterns of gene transcription, then similarities are expected whether a salamander follows a metamorphic or paedomorphic mode of development.
although many degs were expressed similarly between the species, approximately four times as many were uniquely differentially expressed in tiger salamander larvae.
it is possible that many of these gene expression differences represent transcriptional responses in tiger larvae that are necessary for metamorphosis.
three lines of evidence support this idea.
more genes were uniquely expressed in tiger larvae and the majority of these showed larger fold changes later in the larval period, when larvae were undergoing anatomical metamorphosis.
hundreds of genes were differentially expressed between axolotls and tiger salamanders throughout larval development, including the earliest time point sampled .
genes and gene functions that are likely associated with later metamorphic regulation were identified as degs.
for example, several genes that encode chromatin structure and modifying proteins were uniquely identified from tiger larvae or were differentially expressed between the species .
also, several genes that may function in post-transcriptional modification of chromatin proteins were identified as differentially expressed between the species .
it is well established that metamorphosis in amphibians and insects requires programming events that activate new transcriptional programs  <cit> .
indeed, knocking out smt <dig>  in drosophila, a gene associated with chromatin remodeling by sumoylation, is known to extend the pupal stage and inhibit metamorphosis  <cit> .
in addition to chromatin-associated genes, the expression of several genes involved in cellular metabolic processes  and hormone pathways  increased or were expressed at higher levels  during tiger salamander development.
while it is not unexpected to discover gene expression differences between closely related species, the results are intriguing because transcriptional output was generally higher in tiger salamanders, for the majority of loci that showed differential expression.
moreover, some of the genes that were differentially expressed were identified early in the larval period, well before the onset of morphological metamorphosis.
this suggests that metamorphic and paedomorphic modes of development are distinct in a transcriptional sense at very early stages of ontogeny, perhaps tracing back to embryogenesis  <cit> .
paedomorphosis is a heterochonic term that is classically defined as a change in the timing of development that leads to the retention of ancestral, juvenile characteristics in adults of evolutionarily derived lineages  <cit> .
the simplest model to explain such a pattern is a change that delays the overall rate of development.
patterns of gene expression were discovered that support the idea of developmental delay: axolotls maintained relatively constant hbe and hba transcript abundances, suggesting the maintenance of an embryonic hemoglobin expression profile throughout larval development.
in comparison, transcripts for these genes declined precipitously midway during the larval period in tiger salamanders, perhaps coinciding with the initiation of early, metamorphic changes.
however, developmental delay cannot explain all of the axolotl-tiger salamander expression differences.
as was noted above, axolotls shared some expression patterns with tiger salamander larvae, which presumably present aspects of the ancestral metamorphic pattern.
in addition, axolotls showed unique expression patterns not observed in tiger salamander larvae.
axolotls uniquely up regulated genes that are associated with vertebrate brain development and mammalian brain pathologies, including ctss and aging  <cit> , ogn and pituitary cancer  <cit> , and cd <dig> and alzheimers  <cit> .
also, an expression difference was identified that supports the idea of a depressed hpt axis in axolotls  <cit> .
tef, a bzip transcription factor implicated in the activation of mammalian tshb  <cit> , showed significantly lower abundances across all time points in axolotl larvae.
finally, consider the transcription of nr3c <dig> and nr3c <dig> during development.
the glucocorticoid receptor  showed a dynamic temporal expression profile that was statistically indistinguishable in both species.
conversely, the mineralocorticoid receptor  was expressed at significantly lower levels in axolotl larvae.
thus, gene expression patterns in larval axolotls appear to be mosaic: some patterns are shared with tiger salamanders and some patterns are novel.
some of the novel patterns may be neutral in effect and fixed by genetic drift.
however, in the case of transcription factors that may influence hypothalamic-pituitary development and activity  or hemoglobins that allow for physiological adaptation to changing oxygen needs, the axolotl provides a model to study expression changes that have likely been selected to better suit large and reproductively competent "larval forms" for a totally aquatic life history.
in considering how discontinuous phenotypes evolve via darwinian means, gould  <cit>  proposed an answer in the case of the axolotl:
"the problem of reconciling evident discontinuity in macroevolution with darwinism is largely solved by the observation that small changes early in embryology accumulate through growth to yield profound differences among adults.... delay the onset of metamorphosis and the axolotl of lake xochimilco reproduces as a tadpole with gills and never transforms into a salamander."
although once a controversial idea, it is accepted now that evolution can act on early stages of development to yield novel phenotypes  <cit> .
there is a lengthy temporal disconnect between embryogenesis and the time a tiger salamander larva first shows morphological changes indicative of metamorphosis .
during this time, there is ample time for genetic and environmental factors to affect brain development in ways that alter hypothalamic-pituitary activity late in the larval period.
the results show that many genes in the brains of axolotl larvae are transcribed at lower levels than they are in tiger salamander larvae.
these include genes that function in the regulation of hypothalamic-pituitary activities that orchestrate anatomical metamorphosis.
this suggests the following hypothesis: an axolotl's failure to undergo metamorphosis late in the larval period traces to mechanisms that act early in development to broadly program transcription.
this hypothesis can be tested by over-expressing tiger salamander genes in axolotl embryos that function to program gene expression in the brain during early development.
other hypotheses can be tested in axolotls to investigate mechanisms that direct brain development in a predictable manner, towards a hopeful monster outcome.
CONCLUSIONS
this study shows that axolotl and tiger salamander larvae present different brain transcriptional programs and these programs diverge early in development.
these early transcriptional differences include genes whose functions associate with a number of biological processes, including cell cycle, apoptosis, chromatin structure and remodeling, cellular metabolism, transcription, post-translational modification, neural development, and regulation of the hpi and hpt axes.
studies of other metamorphic and paedomorphic species of salamander are needed to disentangle species-specific gene expression responses from those that distinguish metamorphic and paedomorphic modes of development.

 darwin proposed that evolution by natural selection is a gradual process that results in continuous phenotypic variation among species. however, there are many examples where discontinuous phenotypes are observed among related species and thus appear to evolve rapidly. that evolution could suddenly "leap forward" led to extensions of darwin's theory to account for the rapid origin of novel phenotypes. one very old idea is that novel and dramatically different phenotypes originate via saltational evolution from mutations of genes that regulate key developmental or physiological processes during ontogeny. in particular, goldschmidt proposed that mutations occasionally yield individuals within populations that deviate radically from the norm and referred to such individuals as "hopeful monsters". if the novel phenotypes of hopeful monsters arise under the right environmental circumstances, they may become fixed, and the population will found a new species. while this idea was discounted during the modern synthesis, aspects of the hopeful monster hypothesis have been substantiated in recent years. for example, it is clear that dramatic changes in phenotype can occur from few mutations of key developmental genes and phenotypic differences among species often map to relatively few genetic factors. these findings are motivating renewed interest in the study of hopeful monsters and the perspectives they can provide about the evolution of development. in contrast to mutants that are created in the lab, hopeful monsters have been shaped by natural selection and are therefore more likely to reveal mechanisms of adaptive evolution. at least three lines of evidence led goldschmidt to cite the mexican axolotl as one of the original hopeful monsters. first, the axolotl follows a different ontogeny from other closely related tiger salamanders. whereas some tiger salamanders undergo an obligatory metamorphosis during ontogeny that allows for a transition from an aquatic habitat to a more terrestrial habitat, the axolotl has a non-metamorphic life cycle that is often referred to as paedomorphic. this extreme example of discontinuous phenotypic variation supports a model of evolution by heterochrony: larval morphological traits of ancestral metamorphic forms are observed in the adult stages of derived paedomorphic forms. in the minds of early evolutionary biologists, these patterns were so clearly supportive of heterochrony that the mexican axolotl became the exemplar of evolution by neoteny. the second reason goldschmidt cited the axolotl was physiological - huxley had shown that a single molecule - thyroid hormone - was capable of rescuing metamorphosis in the axolotl. thus, the axolotl seemed to be an example of evolution waiting around for the right macromutation to happen - simply block a single physiological step in th regulation and a novel form is originated. the third reason goldschmidt cited the axolotl was ecological. previous researchers had noted that the axolotl was endemic to the high quality, permanent aquatic habitats of xochimilco, which is near present day mexico city. the evolution of paedomorphosis seemingly allowed the axolotl to exploit an empty niche in an environment that was devoid of predators. since goldschmidt, the axolotl has remained a quintessential hopeful monster. speculation that the paedomorphic condition of the axolotl could have a simple mechanistic basis was supported when a quantitative trait locus was identified for the segregation of metamorphic and paedomorphic phenotypes in interspecific crosses. previous physiological studies had also established that axolotls do not produce a sufficient titer of thyroid hormone during larval development to initiate anatomical metamorphosis. the evolution of axolotl hypothyroidism is thought to be associated with a mechanism that affects the development and/or function of neuroendocrine axes that regulate the release of thyroid hormone from the thyroid glands. conceivably, this mechanism could function during early stages of development or it could function later in the larval period when metamorphosis is initiated. regardless, whether paedomorphic and metamorphic larvae show similar or different patterns of neurological development and function has not been previously investigated. in this study, microarray analysis was used to investigate transcription within whole brains of the paedomorphic axolotl and a closely related metamorphic species. the primary objective was to identify patterns of gene expression during early ontogeny that could provide new mechanistic insights about paedomorphic and metamorphic modes of development. transcripts were sampled from both species at four chronologically matched times post hatching to obtain temporal profiles of gene expression during the early larval period and during early stages of morphological metamorphosis in a. t. tigrinum. hundreds of genes showed different or unique patterns of expression between the species, many of which were initiated very early in the larval period and prior to the onset of morphological metamorphosis. the results suggest considerable potential for transcriptional divergence between closely related vertebrate species and highlight the tiger salamander/axolotl model system for examining mechanisms in the developing brain that determine adult phenotypic outcomes.  larval growth and metamorphosis under normal growth conditions, larvae of metamorphic and paedomorphic species of ambystoma increase in size but only larvae of metamorphic species show changes in morphology that are indicative of anatomical metamorphosis. in this experiment, tiger larvae were larger than axolotls at dph and exhibited higher growth rates early in the larval period. as development proceeded, tiger salamander growth rates decelerated while axolotl growth rates remained constant. none of the tiger larvae showed changes in morphology suggestive of initiation of metamorphosis at the three earliest time points. however at dph, 23% of tiger larvae showed bulging eyes and subtle changes in head morphology. by dph, all tiger larvae were undergoing anatomical metamorphosis and 20% of these had rudiments of gills that were less than mm in length. these larvae, and all larvae that were examined at dph, were considered metamorphs. thus, some tiger salamanders initiated anatomical metamorphosis between and dph and most larvae completed metamorphosis between and dph. differentially expressed genes identified independently from axolotls and tiger salamanders a custom affymetrix genechip was used to estimate mrna abundances at four chronologically matched times to obtain temporal profiles of gene expression for tiger salamander and axolotl larvae. statistical and fold-change criteria were then used to identify genes that were differentially expressed within each of the species. thus, these analyses took the conservative approach of independently identifying genes from each species that were differentially expressed as a function of time and comparing the list of differentially expressed genes from tiger salamander with the list of degs identified from axolotl. this approach is conservative because it reduces the risk of identifying false positive expression differences between species that are caused by heterologous hybridization. more than twice as many degs were identified from tiger salamander than axolotl larvae. there was considerable overlap between the two species as more than half of the genes identified from axolotls were also identified from tiger salamander larvae. the remaining degs changed uniquely in only one species. thus, many shared and unique degs were identified, with more unique degs identified from tiger salamander larvae. of the degs identified from both species, 60% exhibited the same regression profile and 99% exhibited the same generalized direction of differential expression. of the degs uniquely identified from axolotls, 55% showed increasing mrna abundances during the larval period while 45% showed decreasing abundances. the opposite pattern was observed for the unique tiger salamander degs with 55% showing decreasing mrna abundances, 43% showing increasing abundances, and 1% showing a pattern of transient increase. only a small percentage of shared and unique degs exhibited ≥ fold difference in mrna abundance between the earliest time points. however, between the latest time points, ~32% of the shared degs, 64% of unique axolotl degs, and 61% of unique tiger salamander degs showed a ≥ fold difference. thus, abundances of uniquely expressed transcripts tended to increase throughout larval development, showing the largest fold differences at later larval stages. the degs identified independently from both species that showed significant sequence identity to a human refseq protein were assumed to be salamander-human orthologs and were annotated with biological process information from the gene ontology database. many of the same go terms were represented among the three deg lists - the degs that were expressed in common between the species and the separate lists of degs that were identified uniquely from each species. the commonly expressed degs were statistically associated with terms, and many of these genes are predicted to function in cell cycle processes. nearly all of the degs that annotated to cell cycle terms exhibited decreasing mrna abundances during larval development in both species, as did six of seven annotated transcription factors. for example, two biomarkers of neural development and differentiation were both categorized as ld in axolotl and tiger salamander. conversely, the majority of degs associated with system development were up regulated in both species. these results suggest that some aspects of neural development and function are similarly regulated between axolotl and tiger salamander larvae. this also seems to be true for some but not all hemoglobin loci, and genes that function in stress and immunological pathways. relatively few uniquely expressed axolotl genes were identified overall and thus only broad go terms were identified as statistically enriched: regulation of cellular process, regulation of biological process, and biological regulation. the unique axolotl genes are predicted to function in some but not all of the biological processes observed for the shared deg list. for example, six genes that function in apoptosis were uniquely identified for axolotls. degs known to be associated with vertebrate brain development were identified, including genes that code for extracellular matrix constituents and cell adhesion. also, several biomarkers of mammalian brain pathologies were uniquely up regulated in axolotls. these and other uniquely expressed genes may be associated with the axolotl's paedomorphic mode of development. the larger list of unique degs from tiger larvae yielded more biological process annotations and were statistically associated with go terms. as was observed for the shared gene list, the cell cycle go term was significantly enriched and these genes showed decreasing mrna abundances during larval development. however, several go terms were identified that were not represented in the shared or unique axolotl list, including biological processes associated with chromatin organization and biogenesis. for example, lmx1b showed a pattern of decreasing transcript abundance, as did several other genes that function in chromatin organization, modification, and gene silencing. it is possible that some of these unique degs are associated with the maturation of brain regions that orchestrate metamorphic events. for example, several genes that function to regulate the secretion of hypothalamic, pituitary, and interrenal hormones were uniquely expressed in tiger salamander larvae, including nr3c prl, and sstr in addition to these genes, pomc and crhr exhibited higher expression levels in tiger salamander larvae. thus, the microarray analysis identified expression differences between axolotl and tiger larvae that may correlate with hpi axis regulation and function. direct comparison of transcription between axolotl and tiger salamander to complement the statistical analyses described above, a subset of affymetrix probesets were identified that could be used to reliably compare transcript abundance estimates directly between the species. othologous genes from axolotl and tiger salamander were aligned to identify affymetrix probe-sets with zero mismatches between the species. ten of these probesets were identical to other probesets. of the remaining non-redundant probesets, 31% registered statistically distinct expression profiles between the species. this analysis identified some of the unique, temporally regulated degs described above, and additionally, genes that exhibited flat regression profiles that differed significantly in elevation between the species. indeed, 84% of the genes identified from this analysis showed higher expression levels throughout the larval period in tiger salamanders. this list is statistically enriched with genes that function in oxygen/gas transport, lipid transport, and heterocycle metabolic processes. with respect to oxygen transport, several hemoglobin genes exhibited > fold differences between the species. the complete list of genes that showed higher abundances in tiger salamanders annotate to many different biological processes, including mrna biosynthesis, protein translation, post-translational modification of proteins, neural function and development, stress and immune response, and hypothalamic-pituitary function. the relatively few genes that showed consistently higher transcript abundances in axolotl larvae also annotate to a number of different biological processes, including dna metabolism, cell cycle regulation, collagen metabolism, neural development, and cellular metabolism. it is unlikely that the interspecific expression differences described above are artifacts of heterologous hybridization because > 80% of the probe-sets were designed from the species that showed consistently lower transcriptional abundances across genes. these results further demonstrate that genes are differentially expressed between the developing brains of axolotl and tiger salamander larvae, and that mrna abundance is on average higher for tiger salamander orthologs. further investigation of gene expression using qpcr some of the degs that were identified by microarray analysis suggest the hpi axis is differentially regulated between tiger salamander and axolotl larvae. for example, microarray analysis estimated higher transcript abundances for nr3c in tiger salamanders, while nr3c did not differ between the species. in adult mammals and presumably amphibians, glucocorticoids feedback to regulate brain development and function by binding to nr3c and nr3c which like thyroid hormone receptors, act as transcription factors. to verify transcript abundance estimates for these genes, and extend the analysis to two additional hpi axis genes, quantitative real-time reverse transcription pcr was used to examine mrna expression across a broader range of time points. both nr3c and nr3c showed a pattern of increasing transcript abundance with maximal levels attained at dph for nr3c and dph for nr3c2; after these times, levels decreased. whereas nr3c showed the same pattern of expression in both species, nr3c abundance was two fold higher in tiger salamander larvae at dph. a pattern of increasing transcript abundance was also observed across the larval period for pomc while crhr mrna levels remained relatively flat. for both of these genes, mrna levels were significantly higher in tiger salamander larvae. these qpcr results corroborate the earlier microarray results and show that hpi axis genes are more highly expressed in tiger salamanders during larval development. the microarray analysis also showed dramatic changes in hemoglobin mrna abundances both within and between species. presumably, these gene expression patterns reflect transcription within erythrocytes that were isolated from vasculature within the brain. the ambystoma genome appears to encode multiple paralogs for almost all hemoglobin loci and these are expressed differently in some cases. for example, paralogs of hba in tiger salamander showed both increasing and decreasing patterns of mrna abundance, while all axolotl hba paralogs showed flat or increasing patterns. qpcr yielded a flatter profile for axolotl hba than was suggested by the microarray analysis, however qpcr replicated the dramatic decrease in mrna abundance for tiger salamander hba, as well as the expression patterns of three additional hemoglobin loci for both species. overall, the patterns suggest that axolotls and tiger salamanders similarly up regulate some of the same hemoglobin loci during the larval period, although levels were consistently higher in tiger salamander larvae. the only locus that was similarly down regulated was hbz. interestingly, axolotls maintain higher embryonic-type hemoglobin mrna levels than tigers throughout the larval period, a pattern that is consistent with a paedomorphic mode of development. discussion this study used a functional genomics approach to detail larval brain transcription between the paedomorphic mexican axolotl and metamorphic tiger salamander. the results show that larvae of these species have different transcriptional programs that are distinguishable in two important respects. first, although shared expression patterns were observed between the species, most of the genes that were identified as differentially expressed during the larval period showed species-specific patterns of expression. gene expression was more similar between the species at earlier time points, with pronounced differences observed at and dph, which coincided with the onset of anatomical metamorphosis in a subset of the tiger salamander larvae. second, the abundance of mrnas tended to be higher for genes that were up regulated during tiger salamander development, relative to those that were up regulated during axolotl development. approximately 31% of genes that could be reliably and directly compared between the species were differentially expressed and 84% of these showed higher mrna abundances in tiger salamander larvae. below, we discuss these primary results and explore their relationships to transcriptional programming that may correlate with metamorphic and paedomorphic modes of development. similar expression patterns were observed for 99% of the genes that were commonly differentially expressed in axolotl and tiger salamander larvae as a function of time. this suggests that some biological processes are regulated similarly between axolotl and tiger salamander larvae during development. for example, it is possible that some of the genes that function in the specification and proliferation of neuronal cell types are similarly expressed during development in both metamorphic and paedomorphic salamanders. genes associated with vertebrate brain development such as sox, msx, and npy significantly increased in abundance in both species. also clu, a gene expressed at low levels in the central nervous systems of embryonic mice before increasing during postnatal life, was similarly up regulated during the axolotl and tiger salamander larval periods. many aspects of brain development and function are highly conserved among vertebrates. if these functions depend upon conserved patterns of gene transcription, then similarities are expected whether a salamander follows a metamorphic or paedomorphic mode of development. although many degs were expressed similarly between the species, approximately four times as many were uniquely differentially expressed in tiger salamander larvae. it is possible that many of these gene expression differences represent transcriptional responses in tiger larvae that are necessary for metamorphosis. three lines of evidence support this idea. more genes were uniquely expressed in tiger larvae and the majority of these showed larger fold changes later in the larval period, when larvae were undergoing anatomical metamorphosis. hundreds of genes were differentially expressed between axolotls and tiger salamanders throughout larval development, including the earliest time point sampled. genes and gene functions that are likely associated with later metamorphic regulation were identified as degs. for example, several genes that encode chromatin structure and modifying proteins were uniquely identified from tiger larvae or were differentially expressed between the species. also, several genes that may function in post-transcriptional modification of chromatin proteins were identified as differentially expressed between the species. it is well established that metamorphosis in amphibians and insects requires programming events that activate new transcriptional programs. indeed, knocking out smt in drosophila, a gene associated with chromatin remodeling by sumoylation, is known to extend the pupal stage and inhibit metamorphosis. in addition to chromatin-associated genes, the expression of several genes involved in cellular metabolic processes and hormone pathways increased or were expressed at higher levels during tiger salamander development. while it is not unexpected to discover gene expression differences between closely related species, the results are intriguing because transcriptional output was generally higher in tiger salamanders, for the majority of loci that showed differential expression. moreover, some of the genes that were differentially expressed were identified early in the larval period, well before the onset of morphological metamorphosis. this suggests that metamorphic and paedomorphic modes of development are distinct in a transcriptional sense at very early stages of ontogeny, perhaps tracing back to embryogenesis. paedomorphosis is a heterochonic term that is classically defined as a change in the timing of development that leads to the retention of ancestral, juvenile characteristics in adults of evolutionarily derived lineages. the simplest model to explain such a pattern is a change that delays the overall rate of development. patterns of gene expression were discovered that support the idea of developmental delay: axolotls maintained relatively constant hbe and hba transcript abundances, suggesting the maintenance of an embryonic hemoglobin expression profile throughout larval development. in comparison, transcripts for these genes declined precipitously midway during the larval period in tiger salamanders, perhaps coinciding with the initiation of early, metamorphic changes. however, developmental delay cannot explain all of the axolotl-tiger salamander expression differences. as was noted above, axolotls shared some expression patterns with tiger salamander larvae, which presumably present aspects of the ancestral metamorphic pattern. in addition, axolotls showed unique expression patterns not observed in tiger salamander larvae. axolotls uniquely up regulated genes that are associated with vertebrate brain development and mammalian brain pathologies, including ctss and aging, ogn and pituitary cancer, and cd and alzheimers. also, an expression difference was identified that supports the idea of a depressed hpt axis in axolotls. tef, a bzip transcription factor implicated in the activation of mammalian tshb, showed significantly lower abundances across all time points in axolotl larvae. finally, consider the transcription of nr3c and nr3c during development. the glucocorticoid receptor showed a dynamic temporal expression profile that was statistically indistinguishable in both species. conversely, the mineralocorticoid receptor was expressed at significantly lower levels in axolotl larvae. thus, gene expression patterns in larval axolotls appear to be mosaic: some patterns are shared with tiger salamanders and some patterns are novel. some of the novel patterns may be neutral in effect and fixed by genetic drift. however, in the case of transcription factors that may influence hypothalamic-pituitary development and activity or hemoglobins that allow for physiological adaptation to changing oxygen needs, the axolotl provides a model to study expression changes that have likely been selected to better suit large and reproductively competent "larval forms" for a totally aquatic life history. in considering how discontinuous phenotypes evolve via darwinian means, gould proposed an answer in the case of the axolotl: "the problem of reconciling evident discontinuity in macroevolution with darwinism is largely solved by the observation that small changes early in embryology accumulate through growth to yield profound differences among adults.... delay the onset of metamorphosis and the axolotl of lake xochimilco reproduces as a tadpole with gills and never transforms into a salamander." although once a controversial idea, it is accepted now that evolution can act on early stages of development to yield novel phenotypes. there is a lengthy temporal disconnect between embryogenesis and the time a tiger salamander larva first shows morphological changes indicative of metamorphosis. during this time, there is ample time for genetic and environmental factors to affect brain development in ways that alter hypothalamic-pituitary activity late in the larval period. the results show that many genes in the brains of axolotl larvae are transcribed at lower levels than they are in tiger salamander larvae. these include genes that function in the regulation of hypothalamic-pituitary activities that orchestrate anatomical metamorphosis. this suggests the following hypothesis: an axolotl's failure to undergo metamorphosis late in the larval period traces to mechanisms that act early in development to broadly program transcription. this hypothesis can be tested by over-expressing tiger salamander genes in axolotl embryos that function to program gene expression in the brain during early development. other hypotheses can be tested in axolotls to investigate mechanisms that direct brain development in a predictable manner, towards a hopeful monster outcome.  this study shows that axolotl and tiger salamander larvae present different brain transcriptional programs and these programs diverge early in development. these early transcriptional differences include genes whose functions associate with a number of biological processes, including cell cycle, apoptosis, chromatin structure and remodeling, cellular metabolism, transcription, post-translational modification, neural development, and regulation of the hpi and hpt axes. studies of other metamorphic and paedomorphic species of salamander are needed to disentangle species-specific gene expression responses from those that distinguish metamorphic and paedomorphic modes of development. 
transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. all but two of the shared degs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. this suggests that an axolotls failure to undergo anatomical metamorphosis late in the larval period is indirectly associated with a mechanism that acts earlier in development to broadly program transcription. the basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae that typically undergo a metamorphosis. the mexican axolotl is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral form. in particular, the axolotl showed a genome-wide reduction in mrna abundance across loci, including genes that regulate hypothalamic-pituitary activities. collectively, more differentially expressed genes were identified as unique to the axolotl and tiger salamander than were identified as shared. of these degs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. however, the axolotl also showed unique patterns of transcription early in development.
 the mexican axolotl is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral form. the basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae that typically undergo a metamorphosis.  transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. collectively, more differentially expressed genes were identified as unique to the axolotl and tiger salamander than were identified as shared. all but two of the shared degs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. a second, complementary analysis that directly compared the expression of genes between the species identified genes that differed as a function of species or the interaction between time and species. of these degs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times.  many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. however, the axolotl also showed unique patterns of transcription early in development. in particular, the axolotl showed a genome-wide reduction in mrna abundance across loci, including genes that regulate hypothalamic-pituitary activities. this suggests that an axolotls failure to undergo anatomical metamorphosis late in the larval period is indirectly associated with a mechanism that acts earlier in development to broadly program transcription. the axolotl hopeful monster provides a model to identify mechanisms of early brain development that proximally and ultimately affect the expression of adult phenotypes. 