BACKGROUND
one of the signatures of life is the ability of organisms to maintain internal concentrations of ions independent of their environment  <cit> .
the homeostatic control of zinc is a notable example of this capability; most vertebrates contain approximately  <dig>  mmol zinc kg- <dig>  which is about  <dig>  times higher than that of typical uncontaminated waters  <cit> .
zinc is an essential micronutrient for all organisms.
there are estimated  <dig>  zinc binding proteins in the human genome of which over  <dig> are enzymes of all classes  <cit> .
however, the majority of zinc-proteins use zinc for structural purposes, generating folds characteristic of zinc- and ring-finger domains .
in addition, there is an increasing appreciation for the involvement of cytosolic zn2+ transients in cell signalling processes through its interactions with specific enzymes  <cit> .
therefore, concentration of free zn2+ in the cytoplasm has to be maintained in the picomolar range to avoid triggering signalling events  <cit> .
acquiring appropriate zinc to maintain this balance is a particular challenge for aquatic organisms, such as freshwater fish, which may encounter water zinc concentrations ranging five orders of magnitude,  <dig>  -  <dig>  μg l- <dig>  <cit> .
it is evident from physiological studies that zinc regulation in fish is indeed highly dynamic and efficient  <cit> .
in vertebrate cells zinc is tightly controlled by uptake, efflux, and compartmentalisation to maintain adequate levels and prevent toxic overaccumulation.
several zinc uptake transporters have been characterised and they are mainly from the zip family  of metal transporters  <cit> .
although exceptions exist, expression of zip transporters or recruitment of these to the plasma membrane is typically increased in zinc-limited conditions, while zinc excess generally results in down-regulation of zip expression and abundance at the plasma membrane.
the efflux transporters, called the znt family , are members of the cation diffusion facilitator  super-family of metal ion transporters.
at least nine znt proteins have been found in mammalian cells  <cit> .
znt <dig> was the first zinc transporter to be identified in mammals  <cit>  and fish  <cit>  and was shown to be responsible for extrusion of zinc from the cell.
with the exception of a splice variant of znt <dig>  which is localised to the plasma membrane and can mediate bi-directional movement of zinc  <cit> , all other characterised znt proteins regulate zinc transport into different intracellular membrane compartments  <cit> .
the zebrafish genome carries a set of zinc transporters that map very closely to the human repertoire and only in a single instance  does the zebrafish have a zinc transporter paralog not present in mammals  <cit> .
as in mammals expression of several zebrafish zinc transporters is regulated by zinc status  <cit> .
another important system that controls the concentration of labile zn2+ in cells is the metallothioneins   <cit> .
mts are a family of low molecular weight and high metal content proteins ubiquitously present in animals, plants, fungi, and cyanobacteria.
they control the concentration of labile zn2+ by binding or releasing zinc when necessary  <cit> .
they are induced by toxic metals such as cadmium and silver, or essential trace elements such as zinc and copper  <cit> .
there are four principal isoforms of mt genes in mammals, among which mt-i and mt-ii function in non-neural tissues to detoxify heavy metals, regulate zinc and copper homeostasis, and limit oxidative damage.
the mts found in different fish species are homologous to the mammalian mt and are functionally equivalent to the mt-i and mt-ii isoforms  <cit> .
zebrafish has at least two in-species paralogs of mt, designated mt <dig> and mt <dig>  <cit> .
zinc-driven transcription of mt genes is controlled by metal-regulatory transcription factor  <dig>   <cit> .
upon association of zinc to its zinc-finger domain in the cytosol, mtf <dig> moves to the nucleus where it binds to metal-response elements  in target genes.
in addition to mt, znt <dig> is also induced by mtf <dig> and we recently showed that zebrafish mtf <dig> is a repressor of zip <dig> gene expression  <cit> .
there are several other genes regulated by mtf <dig>  but it is not obvious how or if these are involved in zinc homeostasis  <cit> .
interestingly, there is a remarkable overrepresentation of genes involved in organism development among predicted zebrafish mtf <dig> targets.
this fits well with observations in mouse that mtf <dig> is essential for embryonic organ development, whereas targeted deletion post-partum is not lethal  <cit> .
although mtf <dig> is an important and specific zinc signalling factor there is increasing evidence for direct roles of zinc signalling at all levels of signal transduction, including modulatory effects on protein tyrosine phosphatases and ca2+-atpases  <cit> .
thus, the cellular regulation of zinc is complex and zinc itself is intimately involved in regulation of numerous cellular processes.
changes in cellular zinc levels evoke complex responses within an organism, making it difficult to explain effects observed at the organ and organism levels based on one or a few regulatory entities.
because of this an integrated functional genomics approach to study zinc homeostasis is appropriate.
there are several informative publications on global changes in gene expression of cultured cells following zinc supplementation  <cit> .
less is known about effects of zinc supplementation on gene expression profiles in vivo, with animal studies showing transcriptome responses at single time-points in oesophagus, liver and brain  <cit> .
to understand regulation of zinc uptake it would be useful to have information on time-dependent transcriptional responses at the site of uptake.
the fish gill is arguably a perfect organ for such studies as it is in direct contact with surrounding water, has well-defined zinc transporting cells, is responsible for a substantial part of the total zinc uptake, and is also the primary target organ for zinc toxicity  <cit> .
we have studied the 14-day time-course of changes in gene expression of zinc transporters in gills of zebrafish treated with zinc depletion or supplementation in water and diet  <cit> .
of the  <dig> zinc transporter genes analysed, four  showed consistent changes in mrna levels in the gills in response to changes in zinc supply.
therefore the aims of the present study were to determine the time-course of global gene expression changes in the gill linked to changes in zinc uptake associated with zinc supplementation, and to use this data to reverse-engineer the biological processes and regulatory networks involved in the acclimation process.
a  <dig> k oligonucleotide microarray was employed to quantify global transcript changes within zebrafish gill during a 14-day time course of acclimation to zinc supplemented diet and water.
the physiological response of the organism was assessed by measuring gill zn2+ influx and whole body nutrient composition .
the analyses revealed maximal alteration in gene expression at day  <dig>  followed by decline as the fish acclimated with a total of  <dig> genes whose expression was impacted by zinc supplementation independent of time.
many genes displayed cyclic expression, typical for homeostatic control mechanisms.
annotation enrichment analysis revealed overrepresentation of a number of functional categories including transcription factors with specific association evident with steroid hormone receptors.
additional pathways overrepresented link to developmental processes, which were dominated by genes involved in development of anatomical structure, nervous system, and cellular differentiation.
a significant bias in genes associated with the insulin signalling pathway was also observed, together with differential expression of molecules involved in apoptosis, cell division, and dna damage.
analysis of transcription factor binding sites in the regulatory region of genes exhibiting altered expression resolved initial regulation of genes responsive to a few key transcription factors  followed by hedgehog and bone morphogenic protein signalling, possibly leading to stem cell differentiation.
corresponding analysis of the functional pathways and regulatory network associated with zebrafish subjected to zinc depletion is presented in a parallel paper  <cit> .
RESULTS
to identify genes regulated under zinc supplementation we utilised a convenient model system, zebrafish gills, combined with microarray gene expression analysis.
zinc was introduced to a group of fish in continuous flow of reconstituted water with a final zinc concentration of  <dig>  μm in the water and  <dig>  mmol kg- <dig> in the diet, compared to a control group with  <dig>  μm and  <dig>  mmol kg- <dig> of zinc in the water and diet, respectively.
the experiment was carried out for  <dig> days with sampling points at  <dig> hours,  <dig>   <dig>   <dig>  and  <dig> days.
physiological characterisation of zinc treatment
the zinc supplementation regime caused no effects on survival, body weight, lipid or protein content at the end of the two-week experiment .
at the termination of the experiment a spectrum of whole body elements was analysed, including zn, cu, ca, fe, na, and k, in both zinc supplemented and control fish.
two metal ions exhibited significant  alterations in body load: zinc, which increased by 50% over the control, and copper, which decreased by 34% .
there was also an 18% decrease in whole body iron, but this was not statistically significant .
it was of interest to examine any alteration in the flux of zinc across the fish gill to evaluate whether the organism is moderating zinc uptake rates in response to the elevated zinc concentrations within the diet and water.
since an important pathway to control zinc homeostasis is flux across the gill surface we evaluated zinc uptake across the gill in eight fish at each time-point using a unidirectional zinc influx assay .
compared to the control group the zinc influx of the zinc supplemented group gradually increased, reached a peak at day  <dig>  and decreased thereafter to equal that observed within the control group after  <dig> days of exposure.
this increase in zinc uptake was contrary to the expected flux compensation, which was predicted to reduce zinc influx in response to supplementation.
the interpretation of this observation is therefore unclear.
it is possible that the up-regulation of zinc chelator  and basolateral exporter  resulted in an increased rate of transcellular zinc movement, while keeping intracellular labile zn2+ at a manageable concentration.
real-time pcr validation
since the present study was part of a larger study involving the effect of zinc depletion, a comprehensive joint qpcr validation of the microarray method was carried out, and the result is presented in the parallel article  <cit> .
the validation involved qpcr analysis of mrna abundance for  <dig> genes using nine biological replicates  per group and per time point, including the five gill samples used for microarrays.
the directionality of responses to zinc supplementation was in full agreement between microarrays and qpcr.
general effect of zinc status on differential gene expression in gills
five fish were randomly withdrawn at each of five time points  from the zinc supplemented and control groups , and their gills removed for gene expression analysis.
one control sample from day  <dig> was removed because of failure to pass quality control.
thus, a total number of  <dig> microarrays were hybridised and analysed with n =  <dig> or  <dig>  of approximately  <dig>  zebrafish reporters  <dig>  provided a significant  signal in at least three independent arrays.
this indicates that at least  <dig>  ±  <dig> % of all analysed genes were expressed in gill tissue independent of zinc treatment.
gene expression in gills of fish maintained in zinc supplemented feed and water was compared to branchial gene expression in fish under normal zinc supply at each time point.
genes having a fold-change greater than  <dig>  and an adjusted p-value less than  <dig> , controlling for a 10% false discovery rate  with the benjamini-hochberg method, were regarded as differentially regulated by zinc depletion compared to control group.
a total of  <dig> genes showed differential expression at one or more time points .
there was a remarkable variation in gene expression over time and of the  <dig> k reporters interrogated, only those for mt <dig> were consistently regulated in one direction  throughout the experiment.
expression of many transcripts appeared to oscillate, showing an up-regulation at one time point and down-regulation at another.
the number of genes with up- or down-regulation across five time points is shown in figure  <dig>  it can be clearly seen that zinc supplementation induced maximal differential gene regulation at day  <dig>  whilst the number of transcripts displaying significantly altered expression at  <dig> hours or  <dig> days were four-fold less.
this latter observation indicates that cellular compensation mechanisms, such as reduced apical influx, increased basolateral efflux , and cytosolic chelating capacity.
i.e.
elevation of mt, might have been well established by  <dig> days of exposure.
in the zinc supplementation condition many more genes were up-regulated than down-regulated at days  <dig> and 4; however, the trend was reversed at day  <dig>
functional classification of 'known' genes
in order to investigate the global impact zinc supplementation had on biological processes and molecular functions we performed annotation enrichment analysis to determine whether genes associated with specific gene ontology terms were significantly overrepresented within our lists of differentially regulated genes.
initial analysis interrogating the database with the unigene identifiers, assigned to the zebrafish genes targeted by our reporters, yielded less than 20%  recognised by the david  functional annotation tool  <cit>  .
the consequence of this low direct association with functional annotation was that ontological analysis of the gene lists from individual time points, where substantially smaller gene lists were involved, had low power.
however, combined consideration of all genes regulated across all time points revealed significant enrichment of ontology terms associated with transcriptional responses such as 'regulation of gene expression', and indicated a strong association of zinc and hormonal control through overrepresentation of 'steroid hormone receptor activity' and 'ligand-dependent nuclear receptor activity' , the latter category containing the retinoic acid receptor beta.
*mf is the abbreviation for molecular function, bp for biological process and cc for cellular component.
** categories enriched in the data-set at p <  <dig>  and containing three or more genes are listed.
to compensate for the low number of directly attributed ontological terms the zebrafish genes represented on our array were mapped across to their closest human orthologs.
this processes yielded human orthologs for 70%  of the differentially expressed zebrafish genes, of which  <dig> matched to annotated david gene objects.
using these orthologs we analysed the representation bias of gene ontology categories for all differentially expressed genes combined, as well as those regulated at specific time points .
the significantly enriched  gene ontology  terms associated with biological process and molecular function among all genes combined were grouped into semantically similar categories and displayed together with the representation bias at each time points .
the result confirmed all of the biased gene-function associations found using zebrafish unigene identifiers , and also detected further enriched annotations.
annotation enrichment analysis with human orthologs reinforced the link between zinc and ontologies relating to 'gene regulation' and 'nuclear receptors', with  <dig> genes denoted 'transcription factor activity'  and  <dig> genes as 'sequence specific dna binding'  .
intriguingly, these terms were only significantly overrepresented at days  <dig> and  <dig>  suggesting the existence of two phases of the transcriptional response to zinc supplementation.
the gata family of transcription factors  were highly represented, with gata <dig>   <dig>  and  <dig> all differentially regulated in response to zinc .
in fact, there was a significant bias among regulated genes overall for the interpro  term 'zn-finger, gata-type' .
tfs with links to the immune system were notable, including those involved in interleukin regulation, zeb <dig> and nfil <dig>  differential expression was observed in other systems where zinc has previously been implicated, including pax <dig>  a central tf involved in development of the eye and nervous system.
however, the most compelling tf association was that with 'steroid hormone receptor activity' , which includes esr <dig>  and rxrb .
it is reassuring that there was also a coincidental bias for genes with the ontological description 'response to steroid hormone stimulus'  , suggesting a cascade of events that initiated the alteration of tf expression.
athe closest human homologues with sequence similarity >45% are listed
barrows pointing up represent up-regulation and those pointing down represent down-regulation.
it is not surprising that some differentially expressed transcriptional regulators, such as dlx <dig>  hox2a, smad <dig>  and gli <dig>  are closely associated with development.
in general there was a strong overrepresentation of genes linked to the 'developmental process' .
the 'development' cluster is dominated by genes involved in development of anatomical structure , nervous system , and cellular differentiation .
genes associated with development started being overrepresented at day  <dig> and were maximally represented at day  <dig>  while no bias for development terms was apparent on day  <dig>  it is evident that zinc is playing a critical role at the level of 'cellular development' , and may contribute to cellular differentiation through zinc regulated genes associated with 'cell fate commitment'  .
the role of zinc as a global regulator was underlined through the myriad of terms associated with closely linked terms of 'positive regulation of biological process'  and 'positive regulation of cellular process' .
again the differential regulation of genes associated with immune system was notable, including genes encoding complement proteins c8g, c <dig> and c <dig> and the interferon regulatory factor irf <dig> .
the influence of zinc on the expression of these and other non-genomic regulators demonstrates the involvement of zinc on complex cell signalling systems.
given the prevalence of global regulators affected together with those associated with functions in cellular development, it is unsurprising that the underlying cellular metabolism was impacted, with over 50% of the differentially regulated genes being associated with 'primary metabolic processes' .
among more interesting groups of proteins were those categorised as involved in the 'lipid biosynthetic process'  since disruption of lipid metabolism is often associated with low level metal toxicity  <cit>  and these changes can be associated with those regulated by the insulin axis.
it has recently been shown that zinc can substitute for insulin in cell culture with the two compounds eliciting similar growth and metabolic characteristics  <cit> .
indeed, four regulated genes, including the insulin receptor itself , mapped onto the kegg 'insulin signalling pathway.'
the most overrepresented kegg pathway was otherwise that for 'pyrimidine metabolism' .
the interface of zinc with the 'cell cycle process'  was illustrated through the zinc based regulation of molecules involved in apoptosis , cell division , dna damage , and those like chek <dig> implicated in all of these processes.
the response involving cell cycle was maximally significant at day  <dig> but remained significant at day  <dig> , correlating with the transition from increased zinc influx across the gills to a return to steady state flux levels .
the only gene ontology term associated with direct metal binding which passed our significance criteria was 'iron binding' , which included several heme containing proteins, and in particular a selection of cytochrome p <dig> s .
analysis of semantic enrichment from other database such as swissprot pir keywords and interpro did provide highly significant enrichment of terms such as 'metal-binding' , 'metalloprotein' , 'zinc finger, nhr/gata-type' , 'iron' , and 'cytochrome p450' , whilst weaker associations with terms included 'calcium-binding ef-hand'  and 'calcium' .
the cellular exporter for ferrous iron, slc40a <dig> , was up-regulated  <dig> -fold by zinc supplementation at the  <dig> day time point, likely in compensation of compromised iron uptake.
only one zinc transporter, slc39a <dig> , was detected to respond to zinc supplementation; it was down-regulated by  <dig> -fold at days  <dig> and  <dig>  followed by a gradual return to the control level.
the biological process term 'detection of abiotic stimulus' was significantly overrepresented when genes whose expression had been significantly impacted by zinc independent of time were considered , but did not seem to gravitate towards any particular time-point .
an interesting facet of this group with possible significance is that four of the five genes in this cluster, gnat <dig>  gnat <dig>  rgr, and pdc, have well-described functions in the outer retina, one of the most zinc-rich tissues of the vertebrate body  <cit> .
analysis of transcription factor binding sites  in the 5' region of regulated genes
it was considered that many of the observed changes in gene expression were not caused by zinc itself, but rather by a transcriptional cascade involving a limited number of key regulators.
a large proportion of the genes that were regulated correspond to genes involved in regulation of transcription  and it is reasonable to assume that changes in expression of tfs would have resulted in further downstream effects on transcription of other genes.
to investigate the cascade effects that might result from changes in expression of these specific tfs, we extracted the upstream dna sequence of all regulated genes and searched for the occurrences of their cognate binding motifs  as defined by the transfac database  <cit> .
of the tfbs for the transcriptionally regulated tfs mapped to entries in the transfac  database , ppara, nr6a1a, stat <dig>  and gata <dig> showed statistically significant difference in frequency of occurrence among regulated genes relative to that of non-regulated control genes .
interestingly, the number of genes with binding sites for ppara was markedly underrepresented at the  <dig> hour time-point, compared with the stably expressed gene cohort as well as the genes regulated at all subsequent time-points.
although the expression of ppara was only defined as increased  at the day  <dig> sampling, it did show a  <dig> -fold higher expression in zinc supplemented fish compared with the control at the  <dig> hour time-point.
there was an overrepresentation of tfbs for stat <dig> and gata <dig> among genes regulated on days  <dig> to  <dig> of the experiment, although the genes for these tfs were significantly up-regulated on only day  <dig> .
however, gata factors have the same consensus binding site and gata <dig> showed increased expression on day  <dig>
although mtf <dig> mrna expression did not change under zinc supplementation in this experiment, mtf <dig> is a well-recognised zinc sensor  <cit> .
hence, binding motifs for mtf <dig>  were also included in the analysis.
the frequency of mres among regulated genes was statistically higher than that in non-regulated genes at the  <dig> hour and day  <dig> sampling points.
among the genes containing mres and displaying altered expression in response to zinc were mt <dig>  znt <dig>  and zip <dig>  all known to be regulated by mtf <dig> in zebrafish  <cit> .
pathway analysis of the transcriptional response to zinc supplementation
the molecular interactions between zinc and proteins encoded by regulated transcripts were explored and visualised using the pathwayarchitecht® software.
a continuous 'direct interaction network' was generated consisting of  <dig> nodes and  <dig> interactions .
an interactive version of this network is presented  where nodes and edges can be individually interrogated.
a remarkable number of the proteins corresponding to regulated genes interact with zinc, copper, iron, and calcium, homeostasis of which are all affected by zinc supplementation.
by far the highest number of interactions with a protein was found for connections with jun, which was indicated to be activated by zinc and connected to  <dig> nodes.
incidentally, the network also implicated jun to be a transcriptional regulator of thrp <dig>  the only transcription factor up-regulated at  <dig> hours into the zinc supplementation regime.
discussion
the present study describes for the first time the cascade of transcriptional responses to zinc supplementation in an animal zinc uptake epithelium.
there was a remarkable variation in gene expression over time, with the number of up-regulated genes dominating at days  <dig> and  <dig>  and down-regulation being predominant at  <dig> hours and  <dig> days.
there was a steady increase in the number of differentially expressed genes up to day  <dig>  and after  <dig> days of treatment few genes remained regulated.
a similar pattern was observed in gills of zebrafish treated with zinc-depleted water and feed  <cit> .
the conclusion from this global analysis may be that the transcriptional adjustments in the gill required for acclimation to a change in zinc availability culminates within a week and acclimation is established by two weeks.
this is in line with studies on rainbow trout  showing physiological acclimation to zinc supplementation within a two-week period  <cit> .
two previous studies on cultured human cells have investigated the effect of time on transcriptomic responses to altered zinc regimes  <cit> .
although these studies considered much shorter time-scales  than in the present study, they do confirm that there are large differences in gene expression with time.
taken together these new findings have quite profound implications on how microarray experiments concentrating on a single time-point should be interpreted, and emphasise the need for time-course studies.
although there was little overlap between genes regulated at adjacent time-points, they tended to belong to the same, or similar ontologies.
in addition, several of the enriched functional categories sorting under 'development', 'metabolism', and 'gene expression' were overrepresented, not only among genes regulated by zinc supplementation, but also during zinc depletion  <cit> .
these functional categories represented a catalogue of the diverse roles played by zinc in biology, and tended to focus on embryonic development and regulation of cellular processes, both at genomic and non-genomic levels.
this is fully in keeping with the view of zinc as a signalling substance with many essential functions, particularly during organism development, and the present findings fill gaps in the networks of such interactions  <cit> .
the go term 'developmental process' was one of the most significantly enriched annotations  among genes regulated in gills of zinc supplemented zebrafish .
this indicates a particular importance of zinc dependent gene expression during development.
mtf <dig> is believed to be the principal mediator of direct transcriptional responses to zinc  <cit> .
in mouse mtf <dig> is essential for embryonic development, but not post-parturition, where it is of primary importance to metal tolerance  <cit> .
accordingly, we previously found that almost half  of a set of  <dig> candidate mtf <dig> targets in zebrafish  were annotated with the go term "developmental process", compared with 23% in the whole genome.
the degree of enrichment was much smaller in the present study , which would be expected as most of these genes were likely to not be directly regulated by mtf <dig>  analysis of the 5' flanking regions of regulated genes showed a statistically significant bias for the mtf <dig> cognate binding motif  at the  <dig> hour and  <dig> day time-points, but the functionality of these mres was not investigated.
developmental genes tended to have the highest bias towards day  <dig>  possibly indicating a physical remodelling of the gill as a part of the acclimation process to the new zinc level.
the fish gill has a phenomenal morphological plasticity and dramatic alterations in cell-types can occur to allow the fish to cope with changes in water chemistry  <cit> .
in support of this idea  <dig> genes that were regulated on day  <dig> carried the annotation "developmental process" , and  <dig> of these are involved in cell differentiation.
several of the developmental genes that were regulated by zinc supplementation, map onto the kegg hedgehog signalling pathway.
these include gli <dig> , bmp7b , zic2a , and ptc <dig> .
in vertebrates the hedgehog family of proteins has a variety of functions during development, but also regulates stem cells involved in maintenance and regeneration of post-embryonic tissues  <cit> .
in zebrafish sonic hedgehog  binds to its receptor, ptc <dig>  located at the plasma membrane.
this relieves the repression of another plasma membrane protein, smo, which in turns activates the gli family of transcription factors.
zebrafish gli <dig> mainly functions as a transcriptional repressor suppressed by shh signalling.
therefore, down-regulation of gli <dig>  promotes shh signalling  <cit> .
bmp <dig> was one of the regulated downstream targets of the hedgehog pathway in the present study.
this may be of special interest because bmp <dig> is involved in the development of ion-transporting ionocytes in the zebrafish gill  <cit> , and these cells are responsible for zinc uptake in fish  <cit> .
two other downstream targets of shh signalling  <cit>  were also found to be regulated by zinc supplementation, pax <dig> , and dlx2a .
there was only a single zinc transporter, slc39a <dig> , which passed the data filters and was designated as significantly regulated.
in mammals zip <dig> is located in the golgi apparatus and is required for connective tissue development through its involvement in the bone morphogenic protein  and transforming growth factor beta  signalling pathways  <cit> .
like the hedgehog pathway, bmp-signalling contributes to several developmental processes, but are also important for stem cell fate and proliferation in post-embryonic tissues  <cit> .
in the present study we found that slc39a <dig> expression was down-regulated by zinc supplementation, and related to this we also found altered expression of bmp7b , bmp <dig> , and bmp <dig> .
downstream targets of bmp signalling include smad <dig> , gata <dig> , and matrix metalloproteases, mmp .
furthermore, dlx <dig> and dlx <dig>  which are part of the same pathway, were regulated in zebrafish gill by zinc depletion  <cit> .
dlx <dig> has been shown to induce expression of runx <dig> in chicken  <cit> , and runx <dig>  which is important for osteogenic differentiation, was recently found to be downregulated during zinc deficiency  <cit> , and abnormally accumulated in osteoblasts from slc39a <dig> null mice  <cit> .
these results provide compelling evidence for the involvement of slc39a <dig> and zinc signalling in the bmp/tgf-beta pathways, and indicate that this connection is conserved in the vertebrate lineage.
taken together, the present study suggests that homeostatic regulation of zinc uptake at the gill may involve reprogramming of epithelial stem cell fate and restructuring of the gill surface.
specifically, this appears to involve hedgehog and bmp signalling.
the present study is therefore delivering unique insight to the molecular mechanisms that might be involved in this process, although more focussed studies will be required to determine the exact signalling pathways and outcomes.
other zinc transporters did not appear in the gene lists of significantly regulated transcripts, either because their expression was below the detection limits of the array or they did not pass the stringent statistical criteria associated with fdr associated with parallel analysis of > <dig> k genes.
however, we have recently presented qpcr analysis of gills from the same fish showing that zinc supplementation results in down-regulation of the zinc importers slc30a <dig>  and slc39a <dig> , and up-regulation of the basolateral zinc exporter slc30a <dig>   <cit> .
we further showed that induction of znt <dig>  as well as repression of zip <dig>  was mediated by the intracellular zinc sensor mtf <dig>  <cit> .
however, mtf <dig> appeared not to be responsible for changes in znt <dig> expression, and an as yet unidentified transcription factor is thought to be responsible for zinc-mediated down-regulation of znt <dig> in humans  <cit> .
zinc supplementation caused no visible signs of toxicity to zebrafish.
however, the increased zinc content of feed and water did have negative effects on copper status, and caused a numerical, but non-significant , decrease in whole body iron content.
these are well-known effects of high dietary zinc supplementation  <cit> .
there was also a tendency for whole-body calcium content to be reduced and it has likewise been shown previously that zinc is a potent inhibitor of calcium uptake  <cit> .
although interactions between zinc, copper, iron, and calcium uptake may occur by competition for transporters, our network analysis of regulated genes  clearly show the complex interplay at different levels between zinc and homeostasis of these essential metal ions.
this interplay was recently highlighted in a microarray study on cultured cells, in which several copper and iron regulatory genes were regulated by zinc supplementation  <cit> .
in the present study we observed an up-regulation of slc40a <dig> , which mediates transport of iron from the enterocytes to the blood circulation.
similarly, kelleher et al.
<cit>  found that in the rat intestine negative effects of zinc supplementation on iron absorption were associated with increased ferroportin- <dig> expression, retention of iron in the intestinal tissue, and decreased hephaestin levels.
iron binding was also a gene ontology molecular function that was enriched among regulated genes in the present study .
this might be associated with the trend for zinc-induced decrease in whole body iron content, although other explanations are possible.
it is becoming increasingly apparent that labile intracellular zn2+ plays roles in non-genomic cell signalling  <cit> .
for example, an increase in labile zn2+ has been shown to activate the erk1/ <dig> pathway by inhibition of mapk phosphatases  <cit> .
an early increase in labile zn2+ in the gill cells as a result of the sudden increase in waterborne zinc at the beginning of the experiment may have led to mapk-mediated phosphorylation and activation of jun, which was one of the nodes with the most edges in our direct interaction network .
expression of jun was also increased on day  <dig> and this may have reinforced the influence of jun on early gene expression.
it has been shown previously that zinc induces jun expression in chicken bm <dig> cells and in human hpr- <dig> cells  <cit> .
agonists of ppara  have also been found to be inducers of jun expression  <cit> , and in the present study ppara was up-regulated on day  <dig>  whilst one of its heterodimer partners, rxrba, showed increased expression on day  <dig> .
ppara is a nuclear receptor which, along with other members of the ppar family, is involved in control of lipid metabolism, cell proliferation, cell differentiation, and immune and inflammatory responses.
in our study ppara expression in gills was up-regulated by zinc supplementation on day  <dig>  but there was already a non-significant  <dig> -fold increase in ppara mrna at  <dig> hours after the start of zinc supplementation.
it has been shown previously that the dna-binding activity of ppara is regulated by zinc  <cit>  and this may have been one of the very first ripples in the transcriptional cascade.
the increase in ppara expression in our experiment may result from the zinc induced increased ppara activity because its transcription can be autoregulated  <cit> .
interestingly, the tfbs for ppara was significantly underrepresented among genes regulated at the  <dig> hour sampling point, but was present in about 60% of all regulated genes thereafter.
our data show that at least  <dig> genes involved in lipid metabolism were regulated by zinc either directly or indirectly, and go terms, such as "cellular lipid metabolic process"  and lipid "biosynthetic process" , were overrepresented among regulated genes.
zinc supplementation resulted in up-regulation of apob, slc27a <dig>  stard <dig>  and hmgcs <dig>  apob  is the main apolipoprotein of chylomicrons and low density lipoproteins .
slc27a <dig> can convert free long-chain fatty acids into fatty acyl-coa esters and play a key role in lipid biosynthesis and fatty acid degradation.
stard <dig> is a sterol transporter involved in cholesterol homeostasis, which is regulated, at least in part, by sterol regulatory element -binding proteins  <cit> .
hmgcs <dig> catalyses the acetyl-coa to hmg-coa, which is further involved in ketone metabolism.
hmgcs <dig> was also down-regulated in zebrafish gill by zinc depletion of water and feed  <cit> .
the interaction between zinc supplementation and lipid metabolism is well established and has been suggested as a major negative side effect of dietary zinc supplementation, especially in an aging population  <cit> , since it results in an adverse affect on hdl cholesterol  <cit> .
in the present study we show that increased zinc accumulation is associated with profound changes in the expression of genes involved in lipid metabolism, helping to explain the molecular background to the known effect of zinc on lipid profiles.
our results echoed the findings in zinc-deficient rats, which showed profound changes in genes involved in hepatic lipid metabolisms, including ppar genes, fatty acid transporter genes, genes involved in acyl-coa metabolism, and several genes of the cytochrome p <dig> family  <cit> .
it is intriguing that activation of ppara has similar effects on lipid metabolism  <cit> , and therefore we may postulate this tf provides the mechanistic link between zinc and lipid metabolism.
it has been noted that zinc deficiency is associated with atherosclerosis and development of ischemic heart diseases, and zinc supplementation is used to help treat angina pectoris  <cit> .
ppars might play a major role in these processes, especially ppara  <cit> .
ppara inhibits inflammatory reactions, one of the key factors involved in atherosclerosis, by stopping nfκb signalling, positively regulating iκbα, inhibiting jun function, negatively regulating cox <dig>  etc  <cit> .
CONCLUSIONS
in conclusion, the transcriptional response to zinc supplementation in the zebrafish gill epithelium goes through several phases that probably involve both genomic and non-genomic signalling.
our attempts to reverse-engineer the very complex time-dependent progression of regulated transcripts resulted in the conclusion that the early phase is characterised by regulation of genes that may be responsive to a few key transcription factors, such as mtf <dig>  jun, stat <dig>  ppara, and gata <dig>  these would have been responsible for the expression of other transcription factors as well as the initiation of compensatory measures, such as regulation of zinc transporters as shown previously  <cit> , and, interestingly, remodelling of the gill epithelium through stem-cell differentiation.
this reprogramming of stem cell fate likely involved hedgehog and bone morphogenic protein signalling.
the crescendo of the transcriptional cascade culminates after a week of zinc supplementation, which coincides with maximum repression of zinc importer  expression  <cit> .
the subsequent damping down of the transcriptional wave suggests that the acclimation process was concluded by the end of the two week experiment.

 one of the signatures of life is the ability of organisms to maintain internal concentrations of ions independent of their environment. the homeostatic control of zinc is a notable example of this capability; most vertebrates contain approximately mmol zinc kg- which is about times higher than that of typical uncontaminated waters. zinc is an essential micronutrient for all organisms. there are estimated zinc binding proteins in the human genome of which over are enzymes of all classes. however, the majority of zinc-proteins use zinc for structural purposes, generating folds characteristic of zinc- and ring-finger domains. in addition, there is an increasing appreciation for the involvement of cytosolic zn2+ transients in cell signalling processes through its interactions with specific enzymes. therefore, concentration of free zn2+ in the cytoplasm has to be maintained in the picomolar range to avoid triggering signalling events. acquiring appropriate zinc to maintain this balance is a particular challenge for aquatic organisms, such as freshwater fish, which may encounter water zinc concentrations ranging five orders of magnitude, - μg l-. it is evident from physiological studies that zinc regulation in fish is indeed highly dynamic and efficient. in vertebrate cells zinc is tightly controlled by uptake, efflux, and compartmentalisation to maintain adequate levels and prevent toxic overaccumulation. several zinc uptake transporters have been characterised and they are mainly from the zip family of metal transporters. although exceptions exist, expression of zip transporters or recruitment of these to the plasma membrane is typically increased in zinc-limited conditions, while zinc excess generally results in down-regulation of zip expression and abundance at the plasma membrane. the efflux transporters, called the znt family, are members of the cation diffusion facilitator super-family of metal ion transporters. at least nine znt proteins have been found in mammalian cells. znt was the first zinc transporter to be identified in mammals and fish and was shown to be responsible for extrusion of zinc from the cell. with the exception of a splice variant of znt which is localised to the plasma membrane and can mediate bi-directional movement of zinc, all other characterised znt proteins regulate zinc transport into different intracellular membrane compartments. the zebrafish genome carries a set of zinc transporters that map very closely to the human repertoire and only in a single instance does the zebrafish have a zinc transporter paralog not present in mammals. as in mammals expression of several zebrafish zinc transporters is regulated by zinc status. another important system that controls the concentration of labile zn2+ in cells is the metallothioneins. mts are a family of low molecular weight and high metal content proteins ubiquitously present in animals, plants, fungi, and cyanobacteria. they control the concentration of labile zn2+ by binding or releasing zinc when necessary. they are induced by toxic metals such as cadmium and silver, or essential trace elements such as zinc and copper. there are four principal isoforms of mt genes in mammals, among which mt-i and mt-ii function in non-neural tissues to detoxify heavy metals, regulate zinc and copper homeostasis, and limit oxidative damage. the mts found in different fish species are homologous to the mammalian mt and are functionally equivalent to the mt-i and mt-ii isoforms. zebrafish has at least two in-species paralogs of mt, designated mt and mt. zinc-driven transcription of mt genes is controlled by metal-regulatory transcription factor. upon association of zinc to its zinc-finger domain in the cytosol, mtf moves to the nucleus where it binds to metal-response elements in target genes. in addition to mt, znt is also induced by mtf and we recently showed that zebrafish mtf is a repressor of zip gene expression. there are several other genes regulated by mtf but it is not obvious how or if these are involved in zinc homeostasis. interestingly, there is a remarkable overrepresentation of genes involved in organism development among predicted zebrafish mtf targets. this fits well with observations in mouse that mtf is essential for embryonic organ development, whereas targeted deletion post-partum is not lethal. although mtf is an important and specific zinc signalling factor there is increasing evidence for direct roles of zinc signalling at all levels of signal transduction, including modulatory effects on protein tyrosine phosphatases and ca2+-atpases. thus, the cellular regulation of zinc is complex and zinc itself is intimately involved in regulation of numerous cellular processes. changes in cellular zinc levels evoke complex responses within an organism, making it difficult to explain effects observed at the organ and organism levels based on one or a few regulatory entities. because of this an integrated functional genomics approach to study zinc homeostasis is appropriate. there are several informative publications on global changes in gene expression of cultured cells following zinc supplementation. less is known about effects of zinc supplementation on gene expression profiles in vivo, with animal studies showing transcriptome responses at single time-points in oesophagus, liver and brain. to understand regulation of zinc uptake it would be useful to have information on time-dependent transcriptional responses at the site of uptake. the fish gill is arguably a perfect organ for such studies as it is in direct contact with surrounding water, has well-defined zinc transporting cells, is responsible for a substantial part of the total zinc uptake, and is also the primary target organ for zinc toxicity. we have studied the 14-day time-course of changes in gene expression of zinc transporters in gills of zebrafish treated with zinc depletion or supplementation in water and diet. of the zinc transporter genes analysed, four showed consistent changes in mrna levels in the gills in response to changes in zinc supply. therefore the aims of the present study were to determine the time-course of global gene expression changes in the gill linked to changes in zinc uptake associated with zinc supplementation, and to use this data to reverse-engineer the biological processes and regulatory networks involved in the acclimation process. a k oligonucleotide microarray was employed to quantify global transcript changes within zebrafish gill during a 14-day time course of acclimation to zinc supplemented diet and water. the physiological response of the organism was assessed by measuring gill zn2+ influx and whole body nutrient composition. the analyses revealed maximal alteration in gene expression at day followed by decline as the fish acclimated with a total of genes whose expression was impacted by zinc supplementation independent of time. many genes displayed cyclic expression, typical for homeostatic control mechanisms. annotation enrichment analysis revealed overrepresentation of a number of functional categories including transcription factors with specific association evident with steroid hormone receptors. additional pathways overrepresented link to developmental processes, which were dominated by genes involved in development of anatomical structure, nervous system, and cellular differentiation. a significant bias in genes associated with the insulin signalling pathway was also observed, together with differential expression of molecules involved in apoptosis, cell division, and dna damage. analysis of transcription factor binding sites in the regulatory region of genes exhibiting altered expression resolved initial regulation of genes responsive to a few key transcription factors followed by hedgehog and bone morphogenic protein signalling, possibly leading to stem cell differentiation. corresponding analysis of the functional pathways and regulatory network associated with zebrafish subjected to zinc depletion is presented in a parallel paper.  to identify genes regulated under zinc supplementation we utilised a convenient model system, zebrafish gills, combined with microarray gene expression analysis. zinc was introduced to a group of fish in continuous flow of reconstituted water with a final zinc concentration of μm in the water and mmol kg- in the diet, compared to a control group with μm and mmol kg- of zinc in the water and diet, respectively. the experiment was carried out for days with sampling points at hours, and days. physiological characterisation of zinc treatment the zinc supplementation regime caused no effects on survival, body weight, lipid or protein content at the end of the two-week experiment. at the termination of the experiment a spectrum of whole body elements was analysed, including zn, cu, ca, fe, na, and k, in both zinc supplemented and control fish. two metal ions exhibited significant alterations in body load: zinc, which increased by 50% over the control, and copper, which decreased by 34%. there was also an 18% decrease in whole body iron, but this was not statistically significant. it was of interest to examine any alteration in the flux of zinc across the fish gill to evaluate whether the organism is moderating zinc uptake rates in response to the elevated zinc concentrations within the diet and water. since an important pathway to control zinc homeostasis is flux across the gill surface we evaluated zinc uptake across the gill in eight fish at each time-point using a unidirectional zinc influx assay. compared to the control group the zinc influx of the zinc supplemented group gradually increased, reached a peak at day and decreased thereafter to equal that observed within the control group after days of exposure. this increase in zinc uptake was contrary to the expected flux compensation, which was predicted to reduce zinc influx in response to supplementation. the interpretation of this observation is therefore unclear. it is possible that the up-regulation of zinc chelator and basolateral exporter resulted in an increased rate of transcellular zinc movement, while keeping intracellular labile zn2+ at a manageable concentration. real-time pcr validation since the present study was part of a larger study involving the effect of zinc depletion, a comprehensive joint qpcr validation of the microarray method was carried out, and the result is presented in the parallel article. the validation involved qpcr analysis of mrna abundance for genes using nine biological replicates per group and per time point, including the five gill samples used for microarrays. the directionality of responses to zinc supplementation was in full agreement between microarrays and qpcr. general effect of zinc status on differential gene expression in gills five fish were randomly withdrawn at each of five time points from the zinc supplemented and control groups, and their gills removed for gene expression analysis. one control sample from day was removed because of failure to pass quality control. thus, a total number of microarrays were hybridised and analysed with n = or of approximately zebrafish reporters provided a significant signal in at least three independent arrays. this indicates that at least ± % of all analysed genes were expressed in gill tissue independent of zinc treatment. gene expression in gills of fish maintained in zinc supplemented feed and water was compared to branchial gene expression in fish under normal zinc supply at each time point. genes having a fold-change greater than and an adjusted p-value less than, controlling for a 10% false discovery rate with the benjamini-hochberg method, were regarded as differentially regulated by zinc depletion compared to control group. a total of genes showed differential expression at one or more time points. there was a remarkable variation in gene expression over time and of the k reporters interrogated, only those for mt were consistently regulated in one direction throughout the experiment. expression of many transcripts appeared to oscillate, showing an up-regulation at one time point and down-regulation at another. the number of genes with up- or down-regulation across five time points is shown in figure it can be clearly seen that zinc supplementation induced maximal differential gene regulation at day whilst the number of transcripts displaying significantly altered expression at hours or days were four-fold less. this latter observation indicates that cellular compensation mechanisms, such as reduced apical influx, increased basolateral efflux, and cytosolic chelating capacity. i.e. elevation of mt, might have been well established by days of exposure. in the zinc supplementation condition many more genes were up-regulated than down-regulated at days and 4; however, the trend was reversed at day functional classification of'known' genes in order to investigate the global impact zinc supplementation had on biological processes and molecular functions we performed annotation enrichment analysis to determine whether genes associated with specific gene ontology terms were significantly overrepresented within our lists of differentially regulated genes. initial analysis interrogating the database with the unigene identifiers, assigned to the zebrafish genes targeted by our reporters, yielded less than 20% recognised by the david functional annotation tool. the consequence of this low direct association with functional annotation was that ontological analysis of the gene lists from individual time points, where substantially smaller gene lists were involved, had low power. however, combined consideration of all genes regulated across all time points revealed significant enrichment of ontology terms associated with transcriptional responses such as'regulation of gene expression', and indicated a strong association of zinc and hormonal control through overrepresentation of'steroid hormone receptor activity' and'ligand-dependent nuclear receptor activity', the latter category containing the retinoic acid receptor beta. *mf is the abbreviation for molecular function, bp for biological process and cc for cellular component. ** categories enriched in the data-set at p < and containing three or more genes are listed. to compensate for the low number of directly attributed ontological terms the zebrafish genes represented on our array were mapped across to their closest human orthologs. this processes yielded human orthologs for 70% of the differentially expressed zebrafish genes, of which matched to annotated david gene objects. using these orthologs we analysed the representation bias of gene ontology categories for all differentially expressed genes combined, as well as those regulated at specific time points. the significantly enriched gene ontology terms associated with biological process and molecular function among all genes combined were grouped into semantically similar categories and displayed together with the representation bias at each time points. the result confirmed all of the biased gene-function associations found using zebrafish unigene identifiers, and also detected further enriched annotations. annotation enrichment analysis with human orthologs reinforced the link between zinc and ontologies relating to'gene regulation' and'nuclear receptors', with genes denoted'transcription factor activity' and genes as'sequence specific dna binding'. intriguingly, these terms were only significantly overrepresented at days and suggesting the existence of two phases of the transcriptional response to zinc supplementation. the gata family of transcription factors were highly represented, with gata and all differentially regulated in response to zinc. in fact, there was a significant bias among regulated genes overall for the interpro term'zn-finger, gata-type'. tfs with links to the immune system were notable, including those involved in interleukin regulation, zeb and nfil differential expression was observed in other systems where zinc has previously been implicated, including pax a central tf involved in development of the eye and nervous system. however, the most compelling tf association was that with'steroid hormone receptor activity', which includes esr and rxrb. it is reassuring that there was also a coincidental bias for genes with the ontological description'response to steroid hormone stimulus', suggesting a cascade of events that initiated the alteration of tf expression. athe closest human homologues with sequence similarity >45% are listed barrows pointing up represent up-regulation and those pointing down represent down-regulation. it is not surprising that some differentially expressed transcriptional regulators, such as dlx hox2a, smad and gli are closely associated with development. in general there was a strong overrepresentation of genes linked to the'developmental process'. the'development' cluster is dominated by genes involved in development of anatomical structure, nervous system, and cellular differentiation. genes associated with development started being overrepresented at day and were maximally represented at day while no bias for development terms was apparent on day it is evident that zinc is playing a critical role at the level of'cellular development', and may contribute to cellular differentiation through zinc regulated genes associated with'cell fate commitment'. the role of zinc as a global regulator was underlined through the myriad of terms associated with closely linked terms of'positive regulation of biological process' and'positive regulation of cellular process'. again the differential regulation of genes associated with immune system was notable, including genes encoding complement proteins c8g, c and c and the interferon regulatory factor irf. the influence of zinc on the expression of these and other non-genomic regulators demonstrates the involvement of zinc on complex cell signalling systems. given the prevalence of global regulators affected together with those associated with functions in cellular development, it is unsurprising that the underlying cellular metabolism was impacted, with over 50% of the differentially regulated genes being associated with'primary metabolic processes'. among more interesting groups of proteins were those categorised as involved in the'lipid biosynthetic process' since disruption of lipid metabolism is often associated with low level metal toxicity and these changes can be associated with those regulated by the insulin axis. it has recently been shown that zinc can substitute for insulin in cell culture with the two compounds eliciting similar growth and metabolic characteristics. indeed, four regulated genes, including the insulin receptor itself, mapped onto the kegg'insulin signalling pathway.' the most overrepresented kegg pathway was otherwise that for'pyrimidine metabolism'. the interface of zinc with the'cell cycle process' was illustrated through the zinc based regulation of molecules involved in apoptosis, cell division, dna damage, and those like chek implicated in all of these processes. the response involving cell cycle was maximally significant at day but remained significant at day, correlating with the transition from increased zinc influx across the gills to a return to steady state flux levels. the only gene ontology term associated with direct metal binding which passed our significance criteria was'iron binding', which included several heme containing proteins, and in particular a selection of cytochrome p s. analysis of semantic enrichment from other database such as swissprot pir keywords and interpro did provide highly significant enrichment of terms such as'metal-binding','metalloprotein','zinc finger, nhr/gata-type','iron', and'cytochrome p450', whilst weaker associations with terms included'calcium-binding ef-hand' and'calcium'. the cellular exporter for ferrous iron, slc40a, was up-regulated -fold by zinc supplementation at the day time point, likely in compensation of compromised iron uptake. only one zinc transporter, slc39a, was detected to respond to zinc supplementation; it was down-regulated by -fold at days and followed by a gradual return to the control level. the biological process term'detection of abiotic stimulus' was significantly overrepresented when genes whose expression had been significantly impacted by zinc independent of time were considered, but did not seem to gravitate towards any particular time-point. an interesting facet of this group with possible significance is that four of the five genes in this cluster, gnat gnat rgr, and pdc, have well-described functions in the outer retina, one of the most zinc-rich tissues of the vertebrate body. analysis of transcription factor binding sites in the 5' region of regulated genes it was considered that many of the observed changes in gene expression were not caused by zinc itself, but rather by a transcriptional cascade involving a limited number of key regulators. a large proportion of the genes that were regulated correspond to genes involved in regulation of transcription and it is reasonable to assume that changes in expression of tfs would have resulted in further downstream effects on transcription of other genes. to investigate the cascade effects that might result from changes in expression of these specific tfs, we extracted the upstream dna sequence of all regulated genes and searched for the occurrences of their cognate binding motifs as defined by the transfac database. of the tfbs for the transcriptionally regulated tfs mapped to entries in the transfac database, ppara, nr6a1a, stat and gata showed statistically significant difference in frequency of occurrence among regulated genes relative to that of non-regulated control genes. interestingly, the number of genes with binding sites for ppara was markedly underrepresented at the hour time-point, compared with the stably expressed gene cohort as well as the genes regulated at all subsequent time-points. although the expression of ppara was only defined as increased at the day sampling, it did show a -fold higher expression in zinc supplemented fish compared with the control at the hour time-point. there was an overrepresentation of tfbs for stat and gata among genes regulated on days to of the experiment, although the genes for these tfs were significantly up-regulated on only day. however, gata factors have the same consensus binding site and gata showed increased expression on day although mtf mrna expression did not change under zinc supplementation in this experiment, mtf is a well-recognised zinc sensor. hence, binding motifs for mtf were also included in the analysis. the frequency of mres among regulated genes was statistically higher than that in non-regulated genes at the hour and day sampling points. among the genes containing mres and displaying altered expression in response to zinc were mt znt and zip all known to be regulated by mtf in zebrafish. pathway analysis of the transcriptional response to zinc supplementation the molecular interactions between zinc and proteins encoded by regulated transcripts were explored and visualised using the pathwayarchitecht® software. a continuous'direct interaction network' was generated consisting of nodes and interactions. an interactive version of this network is presented where nodes and edges can be individually interrogated. a remarkable number of the proteins corresponding to regulated genes interact with zinc, copper, iron, and calcium, homeostasis of which are all affected by zinc supplementation. by far the highest number of interactions with a protein was found for connections with jun, which was indicated to be activated by zinc and connected to nodes. incidentally, the network also implicated jun to be a transcriptional regulator of thrp the only transcription factor up-regulated at hours into the zinc supplementation regime. discussion the present study describes for the first time the cascade of transcriptional responses to zinc supplementation in an animal zinc uptake epithelium. there was a remarkable variation in gene expression over time, with the number of up-regulated genes dominating at days and and down-regulation being predominant at hours and days. there was a steady increase in the number of differentially expressed genes up to day and after days of treatment few genes remained regulated. a similar pattern was observed in gills of zebrafish treated with zinc-depleted water and feed. the conclusion from this global analysis may be that the transcriptional adjustments in the gill required for acclimation to a change in zinc availability culminates within a week and acclimation is established by two weeks. this is in line with studies on rainbow trout showing physiological acclimation to zinc supplementation within a two-week period. two previous studies on cultured human cells have investigated the effect of time on transcriptomic responses to altered zinc regimes. although these studies considered much shorter time-scales than in the present study, they do confirm that there are large differences in gene expression with time. taken together these new findings have quite profound implications on how microarray experiments concentrating on a single time-point should be interpreted, and emphasise the need for time-course studies. although there was little overlap between genes regulated at adjacent time-points, they tended to belong to the same, or similar ontologies. in addition, several of the enriched functional categories sorting under'development','metabolism', and'gene expression' were overrepresented, not only among genes regulated by zinc supplementation, but also during zinc depletion. these functional categories represented a catalogue of the diverse roles played by zinc in biology, and tended to focus on embryonic development and regulation of cellular processes, both at genomic and non-genomic levels. this is fully in keeping with the view of zinc as a signalling substance with many essential functions, particularly during organism development, and the present findings fill gaps in the networks of such interactions. the go term'developmental process' was one of the most significantly enriched annotations among genes regulated in gills of zinc supplemented zebrafish. this indicates a particular importance of zinc dependent gene expression during development. mtf is believed to be the principal mediator of direct transcriptional responses to zinc. in mouse mtf is essential for embryonic development, but not post-parturition, where it is of primary importance to metal tolerance. accordingly, we previously found that almost half of a set of candidate mtf targets in zebrafish were annotated with the go term "developmental process", compared with 23% in the whole genome. the degree of enrichment was much smaller in the present study, which would be expected as most of these genes were likely to not be directly regulated by mtf analysis of the 5' flanking regions of regulated genes showed a statistically significant bias for the mtf cognate binding motif at the hour and day time-points, but the functionality of these mres was not investigated. developmental genes tended to have the highest bias towards day possibly indicating a physical remodelling of the gill as a part of the acclimation process to the new zinc level. the fish gill has a phenomenal morphological plasticity and dramatic alterations in cell-types can occur to allow the fish to cope with changes in water chemistry. in support of this idea genes that were regulated on day carried the annotation "developmental process", and of these are involved in cell differentiation. several of the developmental genes that were regulated by zinc supplementation, map onto the kegg hedgehog signalling pathway. these include gli, bmp7b, zic2a, and ptc. in vertebrates the hedgehog family of proteins has a variety of functions during development, but also regulates stem cells involved in maintenance and regeneration of post-embryonic tissues. in zebrafish sonic hedgehog binds to its receptor, ptc located at the plasma membrane. this relieves the repression of another plasma membrane protein, smo, which in turns activates the gli family of transcription factors. zebrafish gli mainly functions as a transcriptional repressor suppressed by shh signalling. therefore, down-regulation of gli promotes shh signalling. bmp was one of the regulated downstream targets of the hedgehog pathway in the present study. this may be of special interest because bmp is involved in the development of ion-transporting ionocytes in the zebrafish gill, and these cells are responsible for zinc uptake in fish. two other downstream targets of shh signalling were also found to be regulated by zinc supplementation, pax, and dlx2a. there was only a single zinc transporter, slc39a, which passed the data filters and was designated as significantly regulated. in mammals zip is located in the golgi apparatus and is required for connective tissue development through its involvement in the bone morphogenic protein and transforming growth factor beta signalling pathways. like the hedgehog pathway, bmp-signalling contributes to several developmental processes, but are also important for stem cell fate and proliferation in post-embryonic tissues. in the present study we found that slc39a expression was down-regulated by zinc supplementation, and related to this we also found altered expression of bmp7b, bmp, and bmp. downstream targets of bmp signalling include smad, gata, and matrix metalloproteases, mmp. furthermore, dlx and dlx which are part of the same pathway, were regulated in zebrafish gill by zinc depletion. dlx has been shown to induce expression of runx in chicken, and runx which is important for osteogenic differentiation, was recently found to be downregulated during zinc deficiency, and abnormally accumulated in osteoblasts from slc39a null mice. these results provide compelling evidence for the involvement of slc39a and zinc signalling in the bmp/tgf-beta pathways, and indicate that this connection is conserved in the vertebrate lineage. taken together, the present study suggests that homeostatic regulation of zinc uptake at the gill may involve reprogramming of epithelial stem cell fate and restructuring of the gill surface. specifically, this appears to involve hedgehog and bmp signalling. the present study is therefore delivering unique insight to the molecular mechanisms that might be involved in this process, although more focussed studies will be required to determine the exact signalling pathways and outcomes. other zinc transporters did not appear in the gene lists of significantly regulated transcripts, either because their expression was below the detection limits of the array or they did not pass the stringent statistical criteria associated with fdr associated with parallel analysis of > k genes. however, we have recently presented qpcr analysis of gills from the same fish showing that zinc supplementation results in down-regulation of the zinc importers slc30a and slc39a, and up-regulation of the basolateral zinc exporter slc30a. we further showed that induction of znt as well as repression of zip was mediated by the intracellular zinc sensor mtf. however, mtf appeared not to be responsible for changes in znt expression, and an as yet unidentified transcription factor is thought to be responsible for zinc-mediated down-regulation of znt in humans. zinc supplementation caused no visible signs of toxicity to zebrafish. however, the increased zinc content of feed and water did have negative effects on copper status, and caused a numerical, but non-significant, decrease in whole body iron content. these are well-known effects of high dietary zinc supplementation. there was also a tendency for whole-body calcium content to be reduced and it has likewise been shown previously that zinc is a potent inhibitor of calcium uptake. although interactions between zinc, copper, iron, and calcium uptake may occur by competition for transporters, our network analysis of regulated genes clearly show the complex interplay at different levels between zinc and homeostasis of these essential metal ions. this interplay was recently highlighted in a microarray study on cultured cells, in which several copper and iron regulatory genes were regulated by zinc supplementation. in the present study we observed an up-regulation of slc40a, which mediates transport of iron from the enterocytes to the blood circulation. similarly, kelleher et al. found that in the rat intestine negative effects of zinc supplementation on iron absorption were associated with increased ferroportin- expression, retention of iron in the intestinal tissue, and decreased hephaestin levels. iron binding was also a gene ontology molecular function that was enriched among regulated genes in the present study. this might be associated with the trend for zinc-induced decrease in whole body iron content, although other explanations are possible. it is becoming increasingly apparent that labile intracellular zn2+ plays roles in non-genomic cell signalling. for example, an increase in labile zn2+ has been shown to activate the erk1/ pathway by inhibition of mapk phosphatases. an early increase in labile zn2+ in the gill cells as a result of the sudden increase in waterborne zinc at the beginning of the experiment may have led to mapk-mediated phosphorylation and activation of jun, which was one of the nodes with the most edges in our direct interaction network. expression of jun was also increased on day and this may have reinforced the influence of jun on early gene expression. it has been shown previously that zinc induces jun expression in chicken bm cells and in human hpr- cells. agonists of ppara have also been found to be inducers of jun expression, and in the present study ppara was up-regulated on day whilst one of its heterodimer partners, rxrba, showed increased expression on day. ppara is a nuclear receptor which, along with other members of the ppar family, is involved in control of lipid metabolism, cell proliferation, cell differentiation, and immune and inflammatory responses. in our study ppara expression in gills was up-regulated by zinc supplementation on day but there was already a non-significant -fold increase in ppara mrna at hours after the start of zinc supplementation. it has been shown previously that the dna-binding activity of ppara is regulated by zinc and this may have been one of the very first ripples in the transcriptional cascade. the increase in ppara expression in our experiment may result from the zinc induced increased ppara activity because its transcription can be autoregulated. interestingly, the tfbs for ppara was significantly underrepresented among genes regulated at the hour sampling point, but was present in about 60% of all regulated genes thereafter. our data show that at least genes involved in lipid metabolism were regulated by zinc either directly or indirectly, and go terms, such as "cellular lipid metabolic process" and lipid "biosynthetic process", were overrepresented among regulated genes. zinc supplementation resulted in up-regulation of apob, slc27a stard and hmgcs apob is the main apolipoprotein of chylomicrons and low density lipoproteins. slc27a can convert free long-chain fatty acids into fatty acyl-coa esters and play a key role in lipid biosynthesis and fatty acid degradation. stard is a sterol transporter involved in cholesterol homeostasis, which is regulated, at least in part, by sterol regulatory element -binding proteins. hmgcs catalyses the acetyl-coa to hmg-coa, which is further involved in ketone metabolism. hmgcs was also down-regulated in zebrafish gill by zinc depletion of water and feed. the interaction between zinc supplementation and lipid metabolism is well established and has been suggested as a major negative side effect of dietary zinc supplementation, especially in an aging population, since it results in an adverse affect on hdl cholesterol. in the present study we show that increased zinc accumulation is associated with profound changes in the expression of genes involved in lipid metabolism, helping to explain the molecular background to the known effect of zinc on lipid profiles. our results echoed the findings in zinc-deficient rats, which showed profound changes in genes involved in hepatic lipid metabolisms, including ppar genes, fatty acid transporter genes, genes involved in acyl-coa metabolism, and several genes of the cytochrome p family. it is intriguing that activation of ppara has similar effects on lipid metabolism, and therefore we may postulate this tf provides the mechanistic link between zinc and lipid metabolism. it has been noted that zinc deficiency is associated with atherosclerosis and development of ischemic heart diseases, and zinc supplementation is used to help treat angina pectoris. ppars might play a major role in these processes, especially ppara. ppara inhibits inflammatory reactions, one of the key factors involved in atherosclerosis, by stopping nfκb signalling, positively regulating iκbα, inhibiting jun function, negatively regulating cox etc.  in conclusion, the transcriptional response to zinc supplementation in the zebrafish gill epithelium goes through several phases that probably involve both genomic and non-genomic signalling. our attempts to reverse-engineer the very complex time-dependent progression of regulated transcripts resulted in the conclusion that the early phase is characterised by regulation of genes that may be responsive to a few key transcription factors, such as mtf jun, stat ppara, and gata these would have been responsible for the expression of other transcription factors as well as the initiation of compensatory measures, such as regulation of zinc transporters as shown previously, and, interestingly, remodelling of the gill epithelium through stem-cell differentiation. this reprogramming of stem cell fate likely involved hedgehog and bone morphogenic protein signalling. the crescendo of the transcriptional cascade culminates after a week of zinc supplementation, which coincides with maximum repression of zinc importer expression. the subsequent damping down of the transcriptional wave suggests that the acclimation process was concluded by the end of the two week experiment. 
dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. many genes displayed cyclic expression, typical for homeostatic control mechanisms. gill tissues were harvested at five time points and transcriptome changes analysed in quintuplicate using a k microarray with results anchored to gill zn2+ influx and whole body nutrient composition. the study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation. more specifically, early regulation of genes was linked to a few key transcription factors and was followed by hedgehog and bone morphogenic protein signalling. the results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". we exploited the zebrafish gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. in total genes were regulated by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. this provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status.
 dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. we exploited the zebrafish gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. this organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components.  fish were maintained with either zinc supplemented water and diet or water and diet containing zn2+ at μm and mg zinc kg- respectively. gill tissues were harvested at five time points and transcriptome changes analysed in quintuplicate using a k microarray with results anchored to gill zn2+ influx and whole body nutrient composition. the number of regulated genes increased up to day but declined as the fish acclimated. in total genes were regulated by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. many genes displayed cyclic expression, typical for homeostatic control mechanisms. annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". a suite of genes linked to "development" were also statistically overrepresented. more specifically, early regulation of genes was linked to a few key transcription factors and was followed by hedgehog and bone morphogenic protein signalling.  the results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. this provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status. the study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation. 