BACKGROUND
hypoxic injury complicates up to 60 % of preterm births and leads to a broad range of neurological birth defects including epilepsy, autism, adhd, and mental retardation  <cit> .
despite the significant clinical impact, the specific effects of hypoxia on the developing central nervous system  are poorly characterized, and the molecular mechanisms largely unknown.
imaging studies show altered patterns of connectivity in brain magnetic resonance imaging  of children born prematurely  <cit> .
the timing of preterm birth, from  <dig> up to 37 weeks gestation, encompasses a key cns developmental window characterized by widespread axon pathfinding and synaptogenesis, and thus makes the brain particularly vulnerable to disruptions of connectivity.
major pathways established during this at-risk period in premature infants include the extension of the corticospinal tracts, the development of the corpus callosum, and the formation of connections in the cortical hemispheres .
development of connectivity in the cns is a critical step in neural development and appears to be disrupted in disorders including autism and intellectual disability .
the development of connectivity more precisely involves the processes of axon guidance and pathfinding, synapse development and stabilization; and in the mature cns, circuit properties and functional networks  <cit> .
experimental studies have demonstrated that developmental hypoxia can disrupt axon pathfinding  <cit>  and alter synapse gene expression  <cit> .
hif1α, a basic helix-loop-helix transcription factor whose protein expression is stabilized in hypoxia, controls many of the downstream responses to hypoxia including in the cns  <cit> .
in addition, hif1α is necessary for normal cns development  <cit> , although its precise role is unclear.
a small number of target genes involved in cns connectivity and dysregulated by hypoxia have been identified and experimentally validated; including adenosine a <dig> receptor, brn3b, ephrinb <dig>  netrin- <dig>  and vab- <dig> .
however, a global understanding of the changes in the genetic regulatory landscape of the developing cns connectome caused by hypoxia is lacking.
developmentally, it is not known if there are critical hypoxia exposure times.
functionally, it is unclear if certain types of connectivity genes are more affected by hypoxia, and what classes or types of genes are at risk.
to address these questions in a comprehensive, reproducible, and experimentally accessible fashion we performed our experiments in the small vertebrate zebrafish .
advantages of zebrafish are that it combines the relevancy of vertebrate cns structures and genes, with rapidity and efficiency for testing molecular mechanisms.
its low cost and ability to generate large numbers of animals in a range of experimental conditions provide significant statistical power.
in addition, fundamental developmental, neurobiological, and genomic mechanisms are conserved with mammals .
our objective was to identify the gene expression changes occurring with hypoxia in the developing cns, and to determine if there were patterns of gene expression changes that would inform understanding of the effects of hypoxia.
our focus was on characterizing the effects of hypoxia on connectivity genes.
we compared gene expression profiles across development during the first three days of zebrafish embryogenesis, when the majority of cns development occurs, including neurogenesis, axon pathfinding, and synapse formation.
analysis was limited to those genes involved in cns connectivity development.
we found that the majority of cns connectivity genes were unaffected by hypoxia.
however, for a small subset of genes hypoxia significantly affected their expression profiles, and caused changes in both timing and levels of expression that could have significant effects on connectivity development.
RESULTS
studying the effects of hypoxia on cns connectivity development
to study the effects of hypoxia on development of the cns, and in particular the effects of chronic hypoxia on the formation of connectivity in the cns, we assayed for changes in gene expression profiles.
we collected total rna from zebrafish at five different embryonic developmental stages ,  <dig> hpf,  <dig> hpf,  <dig> hpf,  <dig> hpf): the five stages reflect key epochs in early zebrafish development, and encompass the majority of cns connectivity development, from neurogenesis through synapse formation .
for the majority of our subsequent analyses, we restricted our analysis to  <dig> genes with known or likely roles in the development of cns connectivity .fig.
<dig> schematic illustrations of experiments.
a diagram of zebrafish developmental stages  and timing of rna sample collection.
following collection, rnaseq, and alignment to the zebrafish reference genome, subsequent analysis was based on  <dig> genes with roles in cns connectivity development.
b illustration of timing of hypoxia exposure and rna sample collection; and comparison to key events in cns connectivity development.
percent oxygen for hypoxia shown in red boxes
identification and cluster analysis of differentially expressed genes
after read mapping, gene expression was calculated using deseq <dig>  <cit>  and the abundances of genes were expressed as fpkm .
principal component analysis  using the entire transcriptome of  <dig>  genes and performed with both the normoxia and hypoxia data sets showed some separation of samples in earlier developmental stages, but tighter clustering at older ages .
this trend remained when the normoxic and hypoxic samples were analyzed separately .
the pca results show that the rnaseq read variance for the experimental replicates at any given developmental stage or experimental condition  were more similar to each other than to samples from different ages or conditions.
thus, the pca shows that the different conditions  were the contributors for the variance.fig.
<dig> principal component analysis of gene expression.
principal component analysis of entire data set  shows closer clustering at later time stages , but more spread at earlier time points.
separate analysis of normoxic-only or hypoxic-only data sets displays similar findings
expression analysis across development
gene expression analysis across development during normoxia showed that the  <dig> genes fell into ten k-means clusters .
differentially expressed genes, compared to their average expression across development, were defined as genes with a false discovery rate  less than  <dig>  and a more than a 2-fold expression change .fig.
<dig> genome-wide temporal profiles of connectivity development mrna expression.
a developmental profiles of connectivity genes , displayed as a heat-map profile of groups of genes showing similar expression pattern profiles across development.
b k-means cluster heat-map display of connectivity genes across development.
c k clusters shown graphically as lines; relative log <dig> fold expression on y-axis; developmental time-points on x-axis.
d go pathway analysis of k clusters .
e-h, gene expression profiles comparing hypoxia to normoxia expression profiles of connectivity genes.
e developmental profiles of connectivity genes , displayed as a heat-map profile.
f k cluster heat-map display of connectivity genes.
g k clusters shown graphically as lines; relative log <dig> fold expression on y-axis.
h go pathway analysis of k clusters
expression analysis comparing hypoxia and normoxia and across development
for hypoxia, the relative expression of the genes was compared to their normoxic levels .
to adjust for changes in levels due to changes associated with development, we normalized expression for each gene to its mean level across development in each experimental condition , and then compared hypoxic to normoxic levels at each developmental time-point .
we found that the  <dig> genes grouped into fourteen k-means clusters.
the largest cluster , cluster  <dig>  had minimal changes across development or with hypoxic conditions.
several other clusters showed a developmental increase in gene expression but with little or no effect from hypoxia .
in total  <dig> genes  were unaffected by hypoxia.
the remaining clusters demonstrated differential responses of gene expression to hypoxia during development.
expression analysis showing three representative genes from clusters  <dig>   <dig>  and  <dig> displayed as lines demonstrates how different genes had differential responses to hypoxia across development .
interestingly, clusters  <dig> and  <dig>  which displayed the most dynamic responses to hypoxia, contained a total of only nine genes.
however, although transcription factors constituted a majority of the two clusters , there were not other apparent shared biological features of the clusters, including a pathway commons network analysis showing no direct protein-protein interactions between the members of clusters  <dig> or  <dig>   <cit> .
go pathway analysis was most descriptive for cluster  <dig>  the largest cluster; while clusters  <dig>   <dig>  and  <dig>  which showed increases in expression during development but no effect from hypoxia, contained a higher proportion of receptor/ligand genes .
thus, there did not appear to be a certain type or category of genetic response to hypoxia, as least as tested with pathway commons network analysis or go pathway analysis.fig.
<dig> normalized k cluster analysis demonstrates altered timing of expression caused by hypoxia.
a k cluster groups shown as heat maps, in which gene expression is averaged across development, and deviations both from the developmental average, and hypoxia versus normoxia, are shown.
values in parentheses indicate the range of log <dig> fold change of genes in the cluster.
b expression analysis of three representative gene expression changes from hypoxia across development shown as lines; lighter shade indicates expression in hypoxia.
x-axis developmental age, y-axis log2fpkm.
c genes in clusters  <dig> and 11; transcription factors are disproportionately represented.
d qrt-pcr of genes from cluster 5; error bars standard deviation; two-tailed t test; * p <  <dig> ; ** p <  <dig> .
e pathway commons network analysis, displayed with pcviz, shows no shared paths between the genes in cluster  <dig> or  <dig>  f kegg analysis of k clusters.
go categories are only loosely organized into clusters, indicating that the effects of hypoxia on gene expression are not based on gene type/category
we also tested whether analysis of the entire original transcriptome  would result in an alternative pattern of clustering of expression changes.
k-means analysis of the entire transcriptome resulted in eighteen clusters.
genes that clustered together in the connectivity gene analysis were often split into different clusters when the entire transcriptome was analyzed .
the k cluster analysis for the entire transcriptome often provided decreased resolution compared to when only connectivity genes were analyzed.
for example, some genes from clusters  <dig> and  <dig> in the pan-transcriptome analysis, when analyzed in the connectivity gene set could be observed to in fact have differential expression profiles at  <dig> and  <dig> hpf .
however, it is reassuring that for most genes their cluster assignments and grouping remained constant in both the whole transcriptome and connectivity-only analyses .
of note, analysis of the entire transcriptome included the hypoxia-inducible factor  genes.
hif1ab, hif1an, and hif <dig> were in cluster 1; hif <dig> and hif1al <dig> were in cluster 13; and hif1aa was in cluster  <dig>  thus, we think that the use of the entire transcriptome for k means clustering does not provide additional insight and actually obscures some of the findings from analysis of the  <dig> connectivity genes.fig.
<dig> normalized k cluster analysis of all genes compared to connectivity genes only shows improved resolution of expression differences.
a analysis of all genes , k cluster groups shown as heat maps, in which gene expression is averaged across development, and deviations both from the developmental average, and hypoxia versus normoxia, are shown.
values in parentheses indicate log <dig> range fold change of genes in the cluster.
b examples of differential assignment of genes to different clusters .
green lines show different groups of genes assigned to different clusters in the two analyses for cluster  <dig>  red lines show differential assignment for cluster  <dig>  c table representation of differences in gene assignment to k clusters, comparing all genes  to connectivity genes only
in vivo testing, qrt-pcr validation, and biological significance
to perform testing of the rnaseq results and to validate rnaseq results for individual genes, we performed experiments in the embryonic zebrafish and compared hypoxic to normoxic animals.
we analyzed expression for three genes from three different clusters by both in situ hybridization and quantitative rt-pcr .
ryk, a receptor-tyrosine kinase from cluster  <dig>  has decreased expression at  <dig> hpf in hypoxia , but otherwise is relatively constant across development and in normoxia versus hypoxia.
nrxn1a, a synaptic transmembrane protein in cluster  <dig>  demonstrates increased expression levels at later developmental stages.
however, hypoxia diminishes expression at  <dig> hpf and  <dig> hpf .
fezf <dig>  a transcription factor important in neuronal fate specification in cluster  <dig>  had higher levels of expression in early development.
interestingly hypoxia caused a significant increase in expression levels at  <dig> hpf .
thus, for these genes the qrt-pcr results match the in situ results.
however, there are a few minor discordances compared to the rnaseq results ; for example for ryk at  <dig> hpf, nrxn1a at  <dig> hpf, and fezf <dig> at  <dig> hpf, the rnaseq change in hypoxia is discordant with the in situ and qrt-pcr results, emphasizing the importance of follow-up experimental validation.fig.
<dig> in situ validation of rnaseq results, and schematic of hypoxia-associated dysynchrony.
a examples of gene expression changes across development, and hypoxia compared to normoxia.
clusters refer to k analysis, fig.
<dig>  whole-mount in situ images for ryk, nrxn1a, and fezf2; lateral views, dorsal to top, rostral to left.
scale bar 50 μm.
ryk expression is decreased in hypoxia at  <dig> hpf, but then is otherwise relatively invariant across development and in hypoxia compared to normoxia.
nrxn1a and fezf <dig> also demonstrate dynamic changes in expression at different developmental stages, and in hypoxia/normoxia.
b qrt-pcr results for ryk, nrxn1a, and fezf2; normalized to elfa with relative value set to “1” for  <dig> hpf normoxia.
error bars, standard deviation; two-way t test; ** p <  <dig> .
c schematic of effects of hypoxia on disrupting normal connectivity gene expression interactions.
relative expression at  <dig> hpf is shown for axon pathfinding and at 72hpf for synaptogenesis.
normal/normoxic expression level is set at “1”; fold-change of gene expression following hypoxia is shown by the red bars.
two examples each of ligand/receptor gene pairs are shown for axon guidance; and two pairs of pre-/post-synaptic genes for synaptogenesis
the temporal changes demonstrated that hypoxia could affect gene regulation and action by changes in overall expression levels.
we then considered whether hypoxia could cause a dysynchrony by altering relative expression of genes known to interact, for example receptor-ligand pairs.
a change in relative expression of one or both genes that interact could potentially have additive effects on disrupting signaling.
we examined four pairs of genes with potential interactions and roles in connectivity: plxna <dig> and sema3ab; dcc and unc5b; dlg <dig> and syn1; and nrxn1a and nlgn <dig> .
at  <dig> hpf plxna <dig> and sema3ab were both up-regulated by hypoxia; these genes are a cell-surface receptor/ligand pair  <cit> , and increased expression of both genes would disrupt normal axon guidance.
dcc and unc5b at 24hpf are also up-regulated by hypoxia; both genes are receptors for netrin- <dig> and are necessary for normal midline axon guidance  <cit> .
examination of the effects of hypoxia at  <dig> hpf showed minimal effects on nrxn1a and nlgn <dig>  which have roles in synapse adhesion  <cit> .
dlg <dig>  a post-synaptic scaffolding protein, and syn <dig>  a pre-synaptic vesicle-associated protein, show contrasting up- and down-regulation by hypoxia at  <dig> hpf, which could interfere with normal synaptic function.
to test whether the connectivity genes fit into known protein interaction networks we analyzed genes with the most significant changes from hypoxia, while controlling for their expression changes across development .
the differential expression test was applied to each gene individually, and a p value was determined for each individual gene.
then an adjusted p value was calculated from all the individual p values with adjustment for multiple testing.
<dig> genes had adjusted p values <  <dig> , but only a minority of this group  had direct protein-protein interactions with each other   <cit> , and kegg term analysis had sparse representation in a few groups , and go term analysis revealed no statistically significant pathway memberships.
<dig> genes had unadjusted p values <  <dig>  , and showed multiple interactions in many kegg pathways .
an enlarged figure  shows gene names more clearly for fig.
7a, c.fig.
<dig> protein-protein interactions network.
a string analysis of most significant  genes interactions, n = 57; color key for interaction type is shown to the right .
b kegg analysis of the most common pathways for pathways with  <dig> or more genes.
c string analysis with relaxed criteria , n =  <dig> .
d kegg analysis of pathways using relaxed criteria for pathways with  <dig> or more genes.
e heat-map profile of gene expression changes across development and experimental conditions.
red boxes show clusters in which all the groups had the same age but had both hypoxic and normoxic samples.
yellow boxes show clusters in which both the age and the experimental condition varied.
at younger ages hypoxia is noted to have a larger effect by causing differential clustering based on the presence of hypoxia/normoxia  rather than based solely on age
when we compared overall gene expression changes with development and with experimental condition, we found that at later ages genes clustered primarily by developmental time, and less by hypoxia versus normoxia .
in contrast, at earlier ages the relative effects of hypoxia were more significant, and expression changes clustered in a mixture of time and hypoxia effects.
with this analysis the dysynchrony of expression is more notable- for example, that the gene expression profile of animals at  <dig> hpf of normoxia is more similar to that of hypoxic animals at  <dig> hpf.
CONCLUSIONS
we combined a high-resolution transcriptional time course analysis, developmental comparisons of expression, and effects of hypoxia, to characterize and validate the role of hypoxia on connectivity gene expression in the cns.
we had several major findings.
first, we found that the majority of cns connectivity genes are not significantly affected by hypoxia during development.
second, for the minority of connectivity genes which are affected by hypoxia do not represent a single type or category of gene or protein.
third, connectivity genes affected by hypoxia display a dysynchrony.
that is, their relative expression level is aberrantly increased or decreased at an inappropriate time of development.
hypoxia also altered expression levels at defined developmental times of interacting gene pairs, including axon guidance ligand/receptor pairs and synaptic proteins, that would be predicted to disrupt their normal combinatorial functioning.
while following hypoxia embryos showed a slowing in their developmental progression, the gene response to hypoxia was not simply a delay in the timing of their normal expression, and in some cases the hypoxia in fact caused an “acceleration” with increased levels of expression.
previous work from our group and others has shown that hypoxic injury can disrupt the normal development of axon and synaptic connectivity .
the work presented here extends those findings by demonstrating that hypoxia acts at least in part by a causing a dysynchronous response of genes including of interacting receptor/ligand pairs.
for example, the receptor/ligand pair plxna <dig> and sema3ab are both up-regulated by hypoxia, which could lead to elevated gtpase activity and increase repulsive axon guidance  <cit> .
or, as we found, hypoxia causing a decrease in pre-synaptic syn <dig> coupled with a post-synaptic increase in dlg <dig>  could alter synaptic stabilization and function  <cit> .
while the changes in gene expression reveal some of the functional logic of the hypoxic response in our analysis we are not able to differentiate direct or indirect effects.
genes could be directly regulated by hypoxia-responsive transcription factors; or could be downstream targets in a multi-step cascade.
our findings provide an excellent starting point for deciphering the regulation of genes by hypoxia.
in addition, future experiments can address why certain genes are particularly vulnerable to hypoxia, which has important implications for understanding the effects of hypoxia on cns connectivity development.
in our analysis we focused our attention on a subset of  <dig> genes with known roles in connectivity development, in particular on genes with roles in axon pathfinding and synapse development and stabilization.
since the genes regulating connectivity development are relatively well-established  <cit> , our limitation of analysis to this subset provided an enhanced ability to detect trends and patterns that would otherwise have been obscured by including the entire transcriptome.
future studies could also examine whether certain neuron groups are more susceptible to the effects of hypoxia.
while we used rna from the head and brain, changes in expression from other non-neural tissues could have affected our results.
subsequent studies could use genetically-defined methods to limit analysis to neural tissue only, for example with fluorescence-activated cell sorting  analysis or translating ribosomal affinity purification   <cit> .
the transcription factor hif1α is a master regulator of the cellular response to hypoxia  <cit>  along with a collection of other hif genes and transcription factors  <cit> .
hif1α is necessary for normal brain development  <cit> , but in the developing brain hypoxia or ectopic expression of hif1α interrupts normal axon guidance  <cit> .
this adverse effect of hypoxia on connectivity has also been observed to affect synapse development in a rodent model  <cit> .
when we analyzed the changes in expression of the different hif isoforms in response to hypoxia we noted relatively minor changes.
this is similar to a previous report in zebrafish  <cit> , and is likely due to the regulation of hif by hypoxia predominantly occurring at the post-transcriptional  stage  <cit> .
chronic hypoxia and injury to the developing brain in premature infants can lead to adverse neurocognitive and neurodevelopmental outcomes  <cit> .
premature infants can experience extended bouts of hypoxia  <cit> , and mri studies have demonstrated altered connectivity in ex-premature infants  <cit> .
our findings suggest that certain key genetic pathways may be affected in premature infants by hypoxia.
in summary, our data suggests two central findings concerning the effects of hypoxia on cns connectivity development.
first, that the major effects of hypoxia are due to a dysynchrony of gene expression; and second, that hypoxia disproportionately affects a subset of connectivity genes.
these results should lead to further investigations on why certain genes are prone to the effects of hypoxia, and what effects those gene responses have on the developing cns.

 hypoxic injury complicates up to 60 % of preterm births and leads to a broad range of neurological birth defects including epilepsy, autism, adhd, and mental retardation. despite the significant clinical impact, the specific effects of hypoxia on the developing central nervous system are poorly characterized, and the molecular mechanisms largely unknown. imaging studies show altered patterns of connectivity in brain magnetic resonance imaging of children born prematurely. the timing of preterm birth, from up to 37 weeks gestation, encompasses a key cns developmental window characterized by widespread axon pathfinding and synaptogenesis, and thus makes the brain particularly vulnerable to disruptions of connectivity. major pathways established during this at-risk period in premature infants include the extension of the corticospinal tracts, the development of the corpus callosum, and the formation of connections in the cortical hemispheres. development of connectivity in the cns is a critical step in neural development and appears to be disrupted in disorders including autism and intellectual disability. the development of connectivity more precisely involves the processes of axon guidance and pathfinding, synapse development and stabilization; and in the mature cns, circuit properties and functional networks. experimental studies have demonstrated that developmental hypoxia can disrupt axon pathfinding and alter synapse gene expression. hif1α, a basic helix-loop-helix transcription factor whose protein expression is stabilized in hypoxia, controls many of the downstream responses to hypoxia including in the cns. in addition, hif1α is necessary for normal cns development, although its precise role is unclear. a small number of target genes involved in cns connectivity and dysregulated by hypoxia have been identified and experimentally validated; including adenosine a receptor, brn3b, ephrinb netrin- and vab-. however, a global understanding of the changes in the genetic regulatory landscape of the developing cns connectome caused by hypoxia is lacking. developmentally, it is not known if there are critical hypoxia exposure times. functionally, it is unclear if certain types of connectivity genes are more affected by hypoxia, and what classes or types of genes are at risk. to address these questions in a comprehensive, reproducible, and experimentally accessible fashion we performed our experiments in the small vertebrate zebrafish. advantages of zebrafish are that it combines the relevancy of vertebrate cns structures and genes, with rapidity and efficiency for testing molecular mechanisms. its low cost and ability to generate large numbers of animals in a range of experimental conditions provide significant statistical power. in addition, fundamental developmental, neurobiological, and genomic mechanisms are conserved with mammals. our objective was to identify the gene expression changes occurring with hypoxia in the developing cns, and to determine if there were patterns of gene expression changes that would inform understanding of the effects of hypoxia. our focus was on characterizing the effects of hypoxia on connectivity genes. we compared gene expression profiles across development during the first three days of zebrafish embryogenesis, when the majority of cns development occurs, including neurogenesis, axon pathfinding, and synapse formation. analysis was limited to those genes involved in cns connectivity development. we found that the majority of cns connectivity genes were unaffected by hypoxia. however, for a small subset of genes hypoxia significantly affected their expression profiles, and caused changes in both timing and levels of expression that could have significant effects on connectivity development.  studying the effects of hypoxia on cns connectivity development to study the effects of hypoxia on development of the cns, and in particular the effects of chronic hypoxia on the formation of connectivity in the cns, we assayed for changes in gene expression profiles. we collected total rna from zebrafish at five different embryonic developmental stages, hpf, hpf, hpf, hpf): the five stages reflect key epochs in early zebrafish development, and encompass the majority of cns connectivity development, from neurogenesis through synapse formation. for the majority of our subsequent analyses, we restricted our analysis to genes with known or likely roles in the development of cns connectivity.fig. schematic illustrations of experiments. a diagram of zebrafish developmental stages and timing of rna sample collection. following collection, rnaseq, and alignment to the zebrafish reference genome, subsequent analysis was based on genes with roles in cns connectivity development. b illustration of timing of hypoxia exposure and rna sample collection; and comparison to key events in cns connectivity development. percent oxygen for hypoxia shown in red boxes identification and cluster analysis of differentially expressed genes after read mapping, gene expression was calculated using deseq and the abundances of genes were expressed as fpkm. principal component analysis using the entire transcriptome of genes and performed with both the normoxia and hypoxia data sets showed some separation of samples in earlier developmental stages, but tighter clustering at older ages. this trend remained when the normoxic and hypoxic samples were analyzed separately. the pca results show that the rnaseq read variance for the experimental replicates at any given developmental stage or experimental condition were more similar to each other than to samples from different ages or conditions. thus, the pca shows that the different conditions were the contributors for the variance.fig. principal component analysis of gene expression. principal component analysis of entire data set shows closer clustering at later time stages, but more spread at earlier time points. separate analysis of normoxic-only or hypoxic-only data sets displays similar findings expression analysis across development gene expression analysis across development during normoxia showed that the genes fell into ten k-means clusters. differentially expressed genes, compared to their average expression across development, were defined as genes with a false discovery rate less than and a more than a 2-fold expression change.fig. genome-wide temporal profiles of connectivity development mrna expression. a developmental profiles of connectivity genes, displayed as a heat-map profile of groups of genes showing similar expression pattern profiles across development. b k-means cluster heat-map display of connectivity genes across development. c k clusters shown graphically as lines; relative log fold expression on y-axis; developmental time-points on x-axis. d go pathway analysis of k clusters. e-h, gene expression profiles comparing hypoxia to normoxia expression profiles of connectivity genes. e developmental profiles of connectivity genes, displayed as a heat-map profile. f k cluster heat-map display of connectivity genes. g k clusters shown graphically as lines; relative log fold expression on y-axis. h go pathway analysis of k clusters expression analysis comparing hypoxia and normoxia and across development for hypoxia, the relative expression of the genes was compared to their normoxic levels. to adjust for changes in levels due to changes associated with development, we normalized expression for each gene to its mean level across development in each experimental condition, and then compared hypoxic to normoxic levels at each developmental time-point. we found that the genes grouped into fourteen k-means clusters. the largest cluster, cluster had minimal changes across development or with hypoxic conditions. several other clusters showed a developmental increase in gene expression but with little or no effect from hypoxia. in total genes were unaffected by hypoxia. the remaining clusters demonstrated differential responses of gene expression to hypoxia during development. expression analysis showing three representative genes from clusters and displayed as lines demonstrates how different genes had differential responses to hypoxia across development. interestingly, clusters and which displayed the most dynamic responses to hypoxia, contained a total of only nine genes. however, although transcription factors constituted a majority of the two clusters, there were not other apparent shared biological features of the clusters, including a pathway commons network analysis showing no direct protein-protein interactions between the members of clusters or. go pathway analysis was most descriptive for cluster the largest cluster; while clusters and which showed increases in expression during development but no effect from hypoxia, contained a higher proportion of receptor/ligand genes. thus, there did not appear to be a certain type or category of genetic response to hypoxia, as least as tested with pathway commons network analysis or go pathway analysis.fig. normalized k cluster analysis demonstrates altered timing of expression caused by hypoxia. a k cluster groups shown as heat maps, in which gene expression is averaged across development, and deviations both from the developmental average, and hypoxia versus normoxia, are shown. values in parentheses indicate the range of log fold change of genes in the cluster. b expression analysis of three representative gene expression changes from hypoxia across development shown as lines; lighter shade indicates expression in hypoxia. x-axis developmental age, y-axis log2fpkm. c genes in clusters and 11; transcription factors are disproportionately represented. d qrt-pcr of genes from cluster 5; error bars standard deviation; two-tailed t test; * p < ; ** p < . e pathway commons network analysis, displayed with pcviz, shows no shared paths between the genes in cluster or f kegg analysis of k clusters. go categories are only loosely organized into clusters, indicating that the effects of hypoxia on gene expression are not based on gene type/category we also tested whether analysis of the entire original transcriptome would result in an alternative pattern of clustering of expression changes. k-means analysis of the entire transcriptome resulted in eighteen clusters. genes that clustered together in the connectivity gene analysis were often split into different clusters when the entire transcriptome was analyzed. the k cluster analysis for the entire transcriptome often provided decreased resolution compared to when only connectivity genes were analyzed. for example, some genes from clusters and in the pan-transcriptome analysis, when analyzed in the connectivity gene set could be observed to in fact have differential expression profiles at and hpf. however, it is reassuring that for most genes their cluster assignments and grouping remained constant in both the whole transcriptome and connectivity-only analyses. of note, analysis of the entire transcriptome included the hypoxia-inducible factor genes. hif1ab, hif1an, and hif were in cluster 1; hif and hif1al were in cluster 13; and hif1aa was in cluster thus, we think that the use of the entire transcriptome for k means clustering does not provide additional insight and actually obscures some of the findings from analysis of the connectivity genes.fig. normalized k cluster analysis of all genes compared to connectivity genes only shows improved resolution of expression differences. a analysis of all genes, k cluster groups shown as heat maps, in which gene expression is averaged across development, and deviations both from the developmental average, and hypoxia versus normoxia, are shown. values in parentheses indicate log range fold change of genes in the cluster. b examples of differential assignment of genes to different clusters. green lines show different groups of genes assigned to different clusters in the two analyses for cluster red lines show differential assignment for cluster c table representation of differences in gene assignment to k clusters, comparing all genes to connectivity genes only in vivo testing, qrt-pcr validation, and biological significance to perform testing of the rnaseq results and to validate rnaseq results for individual genes, we performed experiments in the embryonic zebrafish and compared hypoxic to normoxic animals. we analyzed expression for three genes from three different clusters by both in situ hybridization and quantitative rt-pcr. ryk, a receptor-tyrosine kinase from cluster has decreased expression at hpf in hypoxia, but otherwise is relatively constant across development and in normoxia versus hypoxia. nrxn1a, a synaptic transmembrane protein in cluster demonstrates increased expression levels at later developmental stages. however, hypoxia diminishes expression at hpf and hpf. fezf a transcription factor important in neuronal fate specification in cluster had higher levels of expression in early development. interestingly hypoxia caused a significant increase in expression levels at hpf. thus, for these genes the qrt-pcr results match the in situ results. however, there are a few minor discordances compared to the rnaseq results; for example for ryk at hpf, nrxn1a at hpf, and fezf at hpf, the rnaseq change in hypoxia is discordant with the in situ and qrt-pcr results, emphasizing the importance of follow-up experimental validation.fig. in situ validation of rnaseq results, and schematic of hypoxia-associated dysynchrony. a examples of gene expression changes across development, and hypoxia compared to normoxia. clusters refer to k analysis, fig.  whole-mount in situ images for ryk, nrxn1a, and fezf2; lateral views, dorsal to top, rostral to left. scale bar 50 μm. ryk expression is decreased in hypoxia at hpf, but then is otherwise relatively invariant across development and in hypoxia compared to normoxia. nrxn1a and fezf also demonstrate dynamic changes in expression at different developmental stages, and in hypoxia/normoxia. b qrt-pcr results for ryk, nrxn1a, and fezf2; normalized to elfa with relative value set to “1” for hpf normoxia. error bars, standard deviation; two-way t test; ** p < . c schematic of effects of hypoxia on disrupting normal connectivity gene expression interactions. relative expression at hpf is shown for axon pathfinding and at 72hpf for synaptogenesis. normal/normoxic expression level is set at “1”; fold-change of gene expression following hypoxia is shown by the red bars. two examples each of ligand/receptor gene pairs are shown for axon guidance; and two pairs of pre-/post-synaptic genes for synaptogenesis the temporal changes demonstrated that hypoxia could affect gene regulation and action by changes in overall expression levels. we then considered whether hypoxia could cause a dysynchrony by altering relative expression of genes known to interact, for example receptor-ligand pairs. a change in relative expression of one or both genes that interact could potentially have additive effects on disrupting signaling. we examined four pairs of genes with potential interactions and roles in connectivity: plxna and sema3ab; dcc and unc5b; dlg and syn1; and nrxn1a and nlgn. at hpf plxna and sema3ab were both up-regulated by hypoxia; these genes are a cell-surface receptor/ligand pair, and increased expression of both genes would disrupt normal axon guidance. dcc and unc5b at 24hpf are also up-regulated by hypoxia; both genes are receptors for netrin- and are necessary for normal midline axon guidance. examination of the effects of hypoxia at hpf showed minimal effects on nrxn1a and nlgn which have roles in synapse adhesion. dlg a post-synaptic scaffolding protein, and syn a pre-synaptic vesicle-associated protein, show contrasting up- and down-regulation by hypoxia at hpf, which could interfere with normal synaptic function. to test whether the connectivity genes fit into known protein interaction networks we analyzed genes with the most significant changes from hypoxia, while controlling for their expression changes across development. the differential expression test was applied to each gene individually, and a p value was determined for each individual gene. then an adjusted p value was calculated from all the individual p values with adjustment for multiple testing. genes had adjusted p values < , but only a minority of this group had direct protein-protein interactions with each other, and kegg term analysis had sparse representation in a few groups, and go term analysis revealed no statistically significant pathway memberships. genes had unadjusted p values < , and showed multiple interactions in many kegg pathways. an enlarged figure shows gene names more clearly for fig. 7a, c.fig. protein-protein interactions network. a string analysis of most significant genes interactions, n = 57; color key for interaction type is shown to the right. b kegg analysis of the most common pathways for pathways with or more genes. c string analysis with relaxed criteria, n = . d kegg analysis of pathways using relaxed criteria for pathways with or more genes. e heat-map profile of gene expression changes across development and experimental conditions. red boxes show clusters in which all the groups had the same age but had both hypoxic and normoxic samples. yellow boxes show clusters in which both the age and the experimental condition varied. at younger ages hypoxia is noted to have a larger effect by causing differential clustering based on the presence of hypoxia/normoxia rather than based solely on age when we compared overall gene expression changes with development and with experimental condition, we found that at later ages genes clustered primarily by developmental time, and less by hypoxia versus normoxia. in contrast, at earlier ages the relative effects of hypoxia were more significant, and expression changes clustered in a mixture of time and hypoxia effects. with this analysis the dysynchrony of expression is more notable- for example, that the gene expression profile of animals at hpf of normoxia is more similar to that of hypoxic animals at hpf.  we combined a high-resolution transcriptional time course analysis, developmental comparisons of expression, and effects of hypoxia, to characterize and validate the role of hypoxia on connectivity gene expression in the cns. we had several major findings. first, we found that the majority of cns connectivity genes are not significantly affected by hypoxia during development. second, for the minority of connectivity genes which are affected by hypoxia do not represent a single type or category of gene or protein. third, connectivity genes affected by hypoxia display a dysynchrony. that is, their relative expression level is aberrantly increased or decreased at an inappropriate time of development. hypoxia also altered expression levels at defined developmental times of interacting gene pairs, including axon guidance ligand/receptor pairs and synaptic proteins, that would be predicted to disrupt their normal combinatorial functioning. while following hypoxia embryos showed a slowing in their developmental progression, the gene response to hypoxia was not simply a delay in the timing of their normal expression, and in some cases the hypoxia in fact caused an “acceleration” with increased levels of expression. previous work from our group and others has shown that hypoxic injury can disrupt the normal development of axon and synaptic connectivity. the work presented here extends those findings by demonstrating that hypoxia acts at least in part by a causing a dysynchronous response of genes including of interacting receptor/ligand pairs. for example, the receptor/ligand pair plxna and sema3ab are both up-regulated by hypoxia, which could lead to elevated gtpase activity and increase repulsive axon guidance. or, as we found, hypoxia causing a decrease in pre-synaptic syn coupled with a post-synaptic increase in dlg could alter synaptic stabilization and function. while the changes in gene expression reveal some of the functional logic of the hypoxic response in our analysis we are not able to differentiate direct or indirect effects. genes could be directly regulated by hypoxia-responsive transcription factors; or could be downstream targets in a multi-step cascade. our findings provide an excellent starting point for deciphering the regulation of genes by hypoxia. in addition, future experiments can address why certain genes are particularly vulnerable to hypoxia, which has important implications for understanding the effects of hypoxia on cns connectivity development. in our analysis we focused our attention on a subset of genes with known roles in connectivity development, in particular on genes with roles in axon pathfinding and synapse development and stabilization. since the genes regulating connectivity development are relatively well-established, our limitation of analysis to this subset provided an enhanced ability to detect trends and patterns that would otherwise have been obscured by including the entire transcriptome. future studies could also examine whether certain neuron groups are more susceptible to the effects of hypoxia. while we used rna from the head and brain, changes in expression from other non-neural tissues could have affected our results. subsequent studies could use genetically-defined methods to limit analysis to neural tissue only, for example with fluorescence-activated cell sorting analysis or translating ribosomal affinity purification. the transcription factor hif1α is a master regulator of the cellular response to hypoxia along with a collection of other hif genes and transcription factors. hif1α is necessary for normal brain development, but in the developing brain hypoxia or ectopic expression of hif1α interrupts normal axon guidance. this adverse effect of hypoxia on connectivity has also been observed to affect synapse development in a rodent model. when we analyzed the changes in expression of the different hif isoforms in response to hypoxia we noted relatively minor changes. this is similar to a previous report in zebrafish, and is likely due to the regulation of hif by hypoxia predominantly occurring at the post-transcriptional stage. chronic hypoxia and injury to the developing brain in premature infants can lead to adverse neurocognitive and neurodevelopmental outcomes. premature infants can experience extended bouts of hypoxia, and mri studies have demonstrated altered connectivity in ex-premature infants. our findings suggest that certain key genetic pathways may be affected in premature infants by hypoxia. in summary, our data suggests two central findings concerning the effects of hypoxia on cns connectivity development. first, that the major effects of hypoxia are due to a dysynchrony of gene expression; and second, that hypoxia disproportionately affects a subset of connectivity genes. these results should lead to further investigations on why certain genes are prone to the effects of hypoxia, and what effects those gene responses have on the developing cns. 
despite the fundamental biological importance and clinical relevance of characterizing the effects of chronic hypoxia exposure on central nervous system development, the changes in gene expression from hypoxia are unknown. we perform transcriptional profiling at high temporal resolution to systematically determine and then experimentally validate the response of cns connectivity genes to hypoxia exposure. we characterized mrna changes during development, comparing the effects of chronic hypoxia exposure at different time-points. overall, our study identifies the response of cns connectivity genes to hypoxia exposure during development. we focused on changes in expression levels of a subset of genes selected for their roles in development of cns connectivity, including axon pathfinding and synapse formation. we found that the majority of cns connectivity genes were unaffected by hypoxia. the observed dysynchrony of gene expression could impair the development of normal cns connectivity maps. here, we use the small vertebrate zebrafish to study the effects of hypoxia on connectivity gene expression across development. however, for a small subset of genes hypoxia significantly affected their gene expression profiles. while for most genes hypoxia did not significantly affect expression, for a subset of genes hypoxia changed both levels and timing of expression. in particular, hypoxia appeared to affect both the timing and levels of expression, including altering expression of interacting gene pairs in a fashion that would potentially disrupt normal function.
 despite the fundamental biological importance and clinical relevance of characterizing the effects of chronic hypoxia exposure on central nervous system development, the changes in gene expression from hypoxia are unknown. it is not known if there are unifying principles, properties, or logic in the response of the developing cns to hypoxic exposure. here, we use the small vertebrate zebrafish to study the effects of hypoxia on connectivity gene expression across development. we perform transcriptional profiling at high temporal resolution to systematically determine and then experimentally validate the response of cns connectivity genes to hypoxia exposure.  we characterized mrna changes during development, comparing the effects of chronic hypoxia exposure at different time-points. we focused on changes in expression levels of a subset of genes selected for their roles in development of cns connectivity, including axon pathfinding and synapse formation. we found that the majority of cns connectivity genes were unaffected by hypoxia. however, for a small subset of genes hypoxia significantly affected their gene expression profiles. in particular, hypoxia appeared to affect both the timing and levels of expression, including altering expression of interacting gene pairs in a fashion that would potentially disrupt normal function.  overall, our study identifies the response of cns connectivity genes to hypoxia exposure during development. while for most genes hypoxia did not significantly affect expression, for a subset of genes hypoxia changed both levels and timing of expression. importantly, we identified that some genes with interacting proteins, for example receptor/ligand pairs, had dissimilar responses to hypoxia that would be expected to interfere with their function. the observed dysynchrony of gene expression could impair the development of normal cns connectivity maps. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hypoxiaconnectivitysynapseaxon pathfindingzebrafishhttp://dx.doi.org/ /100000025national institute of mental healthr mh107039http://dx.doi.org/ /100000002national institutes of healthdp mh100008http://dx.doi.org/ /100000054national cancer institutep ca042014milash brett http://dx.doi.org/ /100000912march of dimes foundationfy13-425issue-copyright-statement© the author 2016 