BACKGROUND
although the production of farmed atlantic halibut  has increased over the last ten years it still faces significant bottlenecks.
gametes are obtained by hand-stripping and oocytes are frequently of variable quality because of non-optimal timing of gamete collection and stress to broodstock fish .
compared to other farmed marine teleosts, atlantic halibut embryos are poorly developed at hatching with an associated long yolk-sac resorption time and a requirement for extended feeding on live prey before they can be fed with commercial diets  <cit> .
the long live feeding period makes the larvae vulnerable to bacterial and viral diseases and increases their mortality  <cit> .
gene expression studies of the atlantic halibut immune system have so far focused on larvae and juveniles .
however, both innate and adaptive immune system-relevant factors are maternally transferred into teleost oocytes and present during embryonic development, before hatching  <cit> .
maternally transmitted immune factors have not yet been characterized in atlantic halibut.
the relatively slow growth of atlantic halibut juveniles and its sex-dependent growth dimorphism are the main obstacles to commercially viable atlantic halibut production during the on-growing phase  <cit> .
the muscle growth characteristics of juvenile and adult stages of teleosts are moderated by environmental conditions at the embryonic stages, particularly temperature  <cit> .
embryonic gene expression patterns of single myogenic regulatory factors, such as myogenic differentiation  <dig> , myogenic differentiation  <dig> , and myogenin , as well as structural muscle proteins such as myosin heavy chain , myosin light chain 2a , and myosin light chain 2b , have been described in atlantic halibut  <cit> .
all-female halibut populations are preferred in commercial farming because of its sex-dependent growth dimorphism.
all-female atlantic halibut production has been established in canada and norway  <cit> .
however, to ensure controlled reproduction, the underlying molecular mechanisms of gamete development in atlantic halibut need to be better understood.
for example, the present knowledge of genes controlling proliferation and migration of primordial germ cells , precursors of gametes, is restricted in atlantic halibut to embryonic expression of askopos  and tudor domain-containing protein  <dig>   <cit> .
early embryonic development in teleosts is driven until the start of zygotic transcription by maternally supplied mrnas that are incorporated into the oocyte during oogenesis  <cit> .
maternal mrnas are critical to embryonic development since they implement basic biosynthetic processes, direct first mitotic divisions, and specify initial cell fate and embryonic patterning  <cit> .
hence, maternal mrnas are potential molecular markers for oocyte quality.
in aquaculture, early estimation of oocyte quality can avoid costly and unnecessary incubation of low-quality material and improve production and predictability  <cit> .
several maternal mrnas have been identified as molecular markers for oocyte quality in farmed mammals .
in commercially farmed teleosts, only prohibitin  <dig>  in rainbow trout  has been found to be related to oocyte quality  <cit> .
previously, we have idenfied three uncharacterized maternal transcripts, correlating significantly with atlantic halibut embryo quality  <cit> .
in addition, we found indications that the transition from maternal to zygotic transcripts  in atlantic halibut takes place between the blastula stage and germ ring stage  <cit> .
a microarray created by douglas et al.
<cit>  has been used to study gene expression in five developmental stages  of atlantic halibut, including larvae introduced to microencapsulated diet and juveniles fed fish meal replacement diets  <cit> .
since the production of the first atlantic halibut microarray  <cit> , the number of atlantic halibut ests available from ncbi genbank  has increased by 40%, including  <dig> maternal new ests  <cit> .
in the present study, we produced a new 10 k custom oligonucleotide array to profile embryonic expression of transcripts involved in immune system, germline, and muscle development, and to identify maternal transcripts.
we also examined differentially expressed maternal transcripts in 8-cell embryos from batches with either high  or low hatching success  in order to identify potential molecular markers of embryo quality.
RESULTS
microarray transcript ontology
of the  <dig> probes designed and printed onto the microarray,  <dig>  represented transcripts with putative identification based on sequence similarity by blastx searches .
go annotations were obtained for  <dig>  probes.
most probes  were classified as representing transcripts involved in cellular , metabolic , and regulatory processes .
their molecular function was dominated by binding , catalytic activities , and cellular compartments were cell  and organelle .
a full list of go annotation for the three domains is presented in additional files  <dig>   <dig> and  <dig>  after signal processing, data normalization, and filtering,  <dig>  probes were kept for further analysis of transcript expression during early embryonic development and  <dig>  for analysis of differentially expressed transcripts between high and low quality embryos at 8cs.
transcript expression during embryonic development
among the four developmental stages,  <dig> transcripts were differentially expressed and  <dig>  were up-regulated only in one of the four developmental stages .
out of the  <dig> up-regulated transcripts,  <dig>  were strictly maternal transcripts, only expressed at 8cs, and  <dig> ,  <dig>   <dig>  transcripts were only expressed at gr, 10ss and ht, respectively .
no significantly enriched go terms were found in any of the four groups of transcripts.
immune defence
seven transcripts encoding complement system proteins were identified in atlantic halibut embryos .
mannose binding lectin  was highly expressed at 8cs, and to a lower degree at gr and ht.
complement factor b  was highly expressed at gr and complement factor c <dig>  at 10ss.
complement factor c <dig> , h  and i  were highly expressed at 10ss and ht.
complement factor d  was only expressed at ht.
two types of pattern recognition receptors  were expressed: toll-like receptors , and c-type lectin receptors .
toll-like receptor  <dig>  was highly expressed at 8cs and gr.
c-type lectin domain family  <dig> member c  was expressed at 10ss and ht while c-type lectin domain family  <dig> member e  was only expressed at ht.
among transcripts encoding cytokines or related proteins, interferon regulatory factor  <dig>  was expressed at 8cs and gr, interferon-induced protein  <dig>  at 8cs and interferon regulatory factor  <dig>  at gr and 10ss.
interleukin enhancer binding factor  <dig> , interleukin  <dig> , and interferon-induced protein  <dig>  were only expressed at gr.
interleukin  <dig>  was expressed from gr to ht.
suppressor of cytokine signaling  <dig>  was highly expressed at gr.figure  <dig>
clustering of transcript groups.
a: immune system, b: germ cells, c: muscle.
transcripts were clustered using the un-weighted pair-group method  using arithmetic averages with normal euclidian distance as distance measurements.
developmental stages were: embryos at 8-cell stage ; germ ring stage , 10-somite stage , and hatched embryos .
data were standardized against the first stage 8cs.
colour bar indicates relative expression in relation to 8cs.
high intensity expression is represented in red colours while blue colours represent lower expression intensity .
among the major histocompatibility complex  receptor sub-chain transcripts, mhc class 1a chain  and mhc class 2b chain  were highly present at gr while mhc class 2a chain  transcript level peaked at 10ss.
transcripts of the t-cell receptor a chain  and the t-cell surface glycoprotein cd <dig> e chain  were found at 8cs and gr while t-cell receptor d chain  was expressed from gr to ht.
three transcripts coding t-cell co-signaling regulators were identified: lymphocyte antigen  <dig> , expressed at 8cs and gr, tumor necrosis factor superfamily member  <dig> , expressed from gr to ht, and programmed cell death  <dig> ligand  <dig>  highly expressed at 8cs.
cathepsin s  was expressed at 8cs and gr.
primordial germ cells
all selected transcripts involved in primordial germ cell proliferation and migration were found at both 8cs and gr, except for tudor domain containing  <dig>  being only expressed at 8cs .
transcript level of tudor domain containing  <dig> , tudor domain containing  <dig> , tudor domain containing  <dig> , nanos homolog  <dig> , dead-end homolog  <dig> , piwi-like  <dig>  and piwi-like  <dig>  was highest at 8cs, while the level of vasa  and askopos  peaked at gr.
muscle development
myosin light chain  <dig>  and  <dig> , myosin heavy chain , fast myotomal troponin t embryonic isoform , fast myotomal muscle troponin t <dig>  and t <dig>  and creatine kinase mitochondrial  <dig>  were expressed at 8cs, and increasingly at 10ss and ht .
capping protein actin filament muscle z-line a  was highly expressed at 10ss and ht, and capping protein actin filament muscle z-line b  was predominantly expressed at 10ss.
tropomyosin  <dig> , myosin light chain  <dig> , and myosin binding protein c cardiac  were highly expressed at ht.
parvalbumin  was highly expressed at gr and tropomyosin  <dig>  at gr, 10ss, and ht.
differential transcript expression in high and low quality eggs
twenty transcripts were differentially expressed between high and low quality 8cs embryos .
gene set enrichment analysis resulted in no significant differences in functional terms.
the highest differences in transcript levels were found for irf <dig>  and mhc2a .
nine transcripts  had significant blast hits representing genes involved in immune response, metabolism, rna transcription, protein degradation, cell signalling, and cytoskeleton.
conserved domain searches in the remaining  <dig> differentially expressed transcripts without significant blast hits resulted in identification of hh_ <dig> as belonging to the chaperonin-like superfamily of proteins.
two differentially expressed transcripts, eef1a2bp and pd-l <dig> showed strictly maternal expression and were expressed only before gr .figure  <dig>
clustering of differentially expressed transcripts in high  and low  quality oocytes.
transcripts were clustered using the un-weighted pair-group method  using arithmetic averages with normal euclidian distance as distance measurement.
high intensity expression is represented in red colours while blue colours represent lower expression intensity .
differentially expressed transcripts in high and low quality atlantic halibut 8cs embryos
p
p
for each transcript, fold-change difference  in low quality oocytes estimated by microarray  and qpcr  is given.
transcripts are grouped according to their function.
validation of expression patterns by quantitative pcr .
relative gene expression  in atlantic halibut embryonic developmental stages estimated by microarray  and qpcr .
the embryonic developmental stages  were: 8-cell stage , germ ring , 10-somite stage , and hatched larvae .
qpcr data were normalized with luc.
data were standardized against the 8cs stage.
a: irf <dig>  b:  <dig>  c: hh_ <dig>  d: eef1a2bp, e: cyp2n, f: hh_ <dig>  g: hh_ <dig>  h: hh_ <dig>  i: hh_ <dig>  j: rnf <dig>  k: hh_contig <dig>  l: psmb <dig>  m: hh_ <dig>  n: hh_ <dig>  o: pd-1 l, p: ms4a8a, q: hh_contig <dig>  and r: mhc1a.
microarray validation
a significant correlation was found between qpcr and microarray results .
significant differences in transcript levels between high and low quality 8cs embryos by microarray were confirmed by qpcr for all  <dig> differentially expressed transcripts .
expression patterns of the differentially expressed transcripts during embryonic development  followed the same general trend when estimated by qpcr, with a few exceptions.
microarray relative expression data revealed strictly maternal expression  of two transcripts  while according to qpcr data, four transcripts were strictly maternal .
membrane-spanning 4-domains subfamily a member 8a  was expressed only zygotically  according to microarray results, but showed maternal-zygotic expression  according to qpcr expression results .
hh_contig <dig> expression was detected at 8cs and ht by microarray and addition in gr by qpcr .
mhc class 1a chain  expression was apparent in gr and ht by microarray, but only in gr by qpcr .
for  <dig> out of  <dig> tested transcripts, qpcr expression levels were up to  <dig>  times higher in one or more developmental stages, compared to the microarray results .
discussion
in hatched atlantic halibut embryos, the anterior part of the head kidney is present, but no haematopoietic tissue or cells can be observed.
liver, thymus, and spleen, important for the development of the adaptive immune system, are not present yet  <cit> .
therefore, the innate immune system is the first line of immune protection.
the complement system is a major non-cellular component of the innate immune system  <cit> .
the presence of maternally transferred mbl transcripts, an activator of the lectin pathway , indicates that it is the first complement defense mechanism active in atlantic halibut embryos .
between gr and ht, expression of transcripts encoding central complement components  and transcripts encoding alternative pathway  regulatory proteins  increases.
as previously found in zebrafish and rainbow trout, the ap seems to be functional as immune defense in atlantic halibut embryos, before the adaptive system is developed  <cit> .
despite the lack of igm-bearing cells and organs of the adaptive immune system in atlantic halibut embryos, transcripts of mhc receptors , t-cell receptors , and t-cell co-signaling regulators  were present.
these maternally transferred transcripts of the adaptive immune system add to the immune protection of the developing embryo and larvae.
a similar transfer of maternal transcripts involved in the innate and adaptive immune system have been identified in in half-smooth tongue sole  and rainbow trout embryos  <cit> .
transcripts of three tdrd genes, tdrd <dig>  tdrd <dig> and tdrd <dig>  showed similar expression pattern as piwil <dig> and piwil <dig>  reflecting the close interaction of the corresponding protein products .
tdrd <dig>  tdrd <dig>  and tdrd <dig>  together with tdrd <dig>  tdrd <dig>  tdrd <dig> and tdrd <dig> form piwi-tdrd complexes that are essential in retrotransposon silencing, chromatoid body assembly and spermiogenesis  <cit> .
tdrd <dig> maternal expression has previously been identified in atlantic halibut embryos  <cit> .
among tdrd transcripts, tdrd <dig> was the only transcript expressed mainly at 8cs.
tdrd <dig> preferably binds to asymmetric dimethyl arginine marks  in somatic cells acting as a transcriptional co-activator  <cit> .
expression profiles of nanos <dig> and dnd <dig> clustered together with piwil and tdrd transcripts with the exception of increased expression at h. dnd <dig> and nanos <dig> code germ plasm and pgcs specific rna-binding proteins involved in differentiation and survival of pgcs  <cit> .
through binding to 3’utrs of target mrnas, dnd <dig> counteracts mirna-mediated posttranscriptional repression in pgcs  <cit> .
expression of dnd <dig> and nanos <dig> in atlantic halibut embryos was similar to expression patterns found in teleost embryos previously  <cit> .
expression of kop and vasa peaked at gr in atlantic halibut, diminishing during later embryonic development and at ht.
a similar expression pattern of kop, coding a pgc-specific p-loop protein of unknown function, has previously been identified in zebrafish and atlantic halibut  <cit> .
vasa, an atp-dependent rna helicase, is mostly known as a pgc marker, but has recently also been identified as a regulator of cell cycle progression in somatic cells  <cit> .
most muscle related transcripts identified during atlantic halibut embryonic development represented isoforms of transcripts coding structural muscle proteins members of myosin , troponin , tropomyosin  and parvalbumin .
myosin molecules consist of six subunits, two heavy chains  of ~200 kda and four light chains  of ~20 kda.
myhc expression has previously been identified at the 17-somite stage and onwards in atlantic halibut  <cit> .
in the same study, two isoforms of mylc <dig> were identified to be stage-specific .
in zebrafish, myl <dig> and myl <dig> are expressed at the 10-somite stage, followed by myl <dig> expression at the 12-somite stage.
here we have identified isoforms of myl <dig> and myl <dig> with expression at 8cs, 10ss, and ht, while myl <dig> was mainly expressed at ht.
one embryonic/larval troponin isoform  and two adult isoforms have previously been identified  in atlantic halibut larvae during metamorphosis  <cit> .
in the present study, ftnt <dig>  ftnt <dig> and eftnt showed similar expression patterns, indicating that they represented embryonic/larval isoforms.
in atlantic halibut embryos, tpm <dig> expression started after somite formation  and was highly expressed at ht.
isoforms of tpm <dig> have been previously found to be essential for embryonic development in mice, but the underlying mechanism is unknown  <cit> .
both tpm <dig>  and mybpc <dig> are known to be specifically expressed in cardiac muscle in zebrafish  <cit> .
while tpm <dig> expression was detected from gr to ht, mybpc <dig> expression was restricted to ht.
in the present study, pval expression peaked earlier, at gr, in atlantic halibut embryos compared to its expression in zebrafish, where it was first detected in 15-somite stage embryos  <cit> .
as a ca2+-binding protein, pval is usually present in high concentrations in fast muscle cells, to a lower extend in specific neurons of the central and peripheral nervous system, and in cells of endocrine glands  <cit> .
the two maternal transcripts, irf <dig> and mhc2a, were identified as potential markers for quality in atlantic halibut due to their high level of expression differences in high and low quality 8cs embryos .
the three uncharacterized transcripts  had the same potential, but their functions have to be further characterized.
irf <dig> is the primary regulator of type i interferon  production and its absence impairs antiviral innate immunity  <cit> .
the significantly lower levels of irf <dig> transcripts in low quality 8cs embryos may result in reduced immune response capacity resulting in further poor embryonic and larval development.
most of the transcripts showing elevated expression in low quality embryos are involved in adaptive immune response and protein degradation .
their elevated concentrations could be the result of a maternally transferred immune response during the oogenesis triggered by unknown inflammation, infection or immune-activated stress.
polyadenylation of maternal mrnas during oocyte maturation usually protects mrnas from degradation and activates their translation  <cit> .
in contrast, regulatory rna or protein-mediated deadenylation triggers mrna degradation and translational repression to allow normal embryonic development after the maternal-zygotic transition   <cit> .
in xenopus tropicalis, oocyte post-ovulatory aging  induced a general decrease in maternal transcripts and a female-specific shortening of maternal mrnas by deadenylation in oocytes that developed into embryos experiencing high malformation and mortality rates  <cit> .
in contrast, poa induced both a decrease and increase in specific maternal transcripts in rainbow trout oocytes  <cit> .
in the present study, timing of hand-stripping atlantic halibut females was synchronised to their individual ovulation rhythms to avoid poa.
whether low maternal transcript levels are the result of a lack or sub-optimal polyadenylation during atlantic halibut oocyte maturation, leading to poor transcript translation and/or degradation in low quality early embryos, requires further investigation.
due to the experimental design, it was not possible to estimate whether the transcript level differences in high and low quality 8cs embryos were based on individual female differences and/or inheritability.
however, no major differences in transcript levels were observed within the three oocyte batches tested .
to test the potential of irf <dig> and mhc2a as markers for atlantic halibut oocyte quality, future studies should nevertheless be performed across a higher number of batches from females of known genetic background.
both abundance of differentially expressed transcripts in high and low quality oocytes  and their expression patterns during embryonic development  were successfully confirmed by qpcr.
the qpcr and microarray analysis have inherent technical and data normalization challenges that can lead to variability in results.
following strict quality assessment procedures in both techniques,  and data filtering after normalization  resulted in high correlation between qpcr and microarray data   <cit> .
due to the requirement of designing qpcr primers across exon/introns boundaries, microarray probes and qpcr primer locations varied.
this could result in the observed variation between qpcr and microarray data.
compared to the previously created atlantic halibut microarray by douglas et al.
<cit> , the new microarray contains a higher number of transcripts corresponding to genes expressed during the embryonic development.
this microarray was successfully used to screen maternal transcript expression, and to identify differential transcripts expression in low and high quality 8cs embryos.
it has proven to be suitable for future analysis of atlantic halibut embryonic transcript expression which is likely to advance our understanding of important developmental processes in teleosts.
CONCLUSIONS
using a new atlantic halibut 10 k custom oligonucleotide array, we have demonstrated maternal transfer of innate and adaptive immune system transcripts into atlantic halibut embryos and profiled their expression in early developmental stages.
we identified several transcripts, including irf <dig> and mhc2a, as potential molecular markers for embryo quality.
microarray validation did prove the usefulness of the tool for further transcript quantification in atlantic halibut.
both the established information and microarray provide useful resources to improve commercial production of atlantic halibut.

 although the production of farmed atlantic halibut has increased over the last ten years it still faces significant bottlenecks. gametes are obtained by hand-stripping and oocytes are frequently of variable quality because of non-optimal timing of gamete collection and stress to broodstock fish. compared to other farmed marine teleosts, atlantic halibut embryos are poorly developed at hatching with an associated long yolk-sac resorption time and a requirement for extended feeding on live prey before they can be fed with commercial diets. the long live feeding period makes the larvae vulnerable to bacterial and viral diseases and increases their mortality. gene expression studies of the atlantic halibut immune system have so far focused on larvae and juveniles. however, both innate and adaptive immune system-relevant factors are maternally transferred into teleost oocytes and present during embryonic development, before hatching. maternally transmitted immune factors have not yet been characterized in atlantic halibut. the relatively slow growth of atlantic halibut juveniles and its sex-dependent growth dimorphism are the main obstacles to commercially viable atlantic halibut production during the on-growing phase. the muscle growth characteristics of juvenile and adult stages of teleosts are moderated by environmental conditions at the embryonic stages, particularly temperature. embryonic gene expression patterns of single myogenic regulatory factors, such as myogenic differentiation, myogenic differentiation, and myogenin, as well as structural muscle proteins such as myosin heavy chain, myosin light chain 2a, and myosin light chain 2b, have been described in atlantic halibut. all-female halibut populations are preferred in commercial farming because of its sex-dependent growth dimorphism. all-female atlantic halibut production has been established in canada and norway. however, to ensure controlled reproduction, the underlying molecular mechanisms of gamete development in atlantic halibut need to be better understood. for example, the present knowledge of genes controlling proliferation and migration of primordial germ cells, precursors of gametes, is restricted in atlantic halibut to embryonic expression of askopos and tudor domain-containing protein. early embryonic development in teleosts is driven until the start of zygotic transcription by maternally supplied mrnas that are incorporated into the oocyte during oogenesis. maternal mrnas are critical to embryonic development since they implement basic biosynthetic processes, direct first mitotic divisions, and specify initial cell fate and embryonic patterning. hence, maternal mrnas are potential molecular markers for oocyte quality. in aquaculture, early estimation of oocyte quality can avoid costly and unnecessary incubation of low-quality material and improve production and predictability. several maternal mrnas have been identified as molecular markers for oocyte quality in farmed mammals. in commercially farmed teleosts, only prohibitin in rainbow trout has been found to be related to oocyte quality. previously, we have idenfied three uncharacterized maternal transcripts, correlating significantly with atlantic halibut embryo quality. in addition, we found indications that the transition from maternal to zygotic transcripts in atlantic halibut takes place between the blastula stage and germ ring stage. a microarray created by douglas et al. has been used to study gene expression in five developmental stages of atlantic halibut, including larvae introduced to microencapsulated diet and juveniles fed fish meal replacement diets. since the production of the first atlantic halibut microarray, the number of atlantic halibut ests available from ncbi genbank has increased by 40%, including maternal new ests. in the present study, we produced a new 10 k custom oligonucleotide array to profile embryonic expression of transcripts involved in immune system, germline, and muscle development, and to identify maternal transcripts. we also examined differentially expressed maternal transcripts in 8-cell embryos from batches with either high or low hatching success in order to identify potential molecular markers of embryo quality.  microarray transcript ontology of the probes designed and printed onto the microarray, represented transcripts with putative identification based on sequence similarity by blastx searches. go annotations were obtained for probes. most probes were classified as representing transcripts involved in cellular, metabolic, and regulatory processes. their molecular function was dominated by binding, catalytic activities, and cellular compartments were cell and organelle. a full list of go annotation for the three domains is presented in additional files and after signal processing, data normalization, and filtering, probes were kept for further analysis of transcript expression during early embryonic development and for analysis of differentially expressed transcripts between high and low quality embryos at 8cs. transcript expression during embryonic development among the four developmental stages, transcripts were differentially expressed and were up-regulated only in one of the four developmental stages. out of the up-regulated transcripts, were strictly maternal transcripts, only expressed at 8cs, and, transcripts were only expressed at gr, 10ss and ht, respectively. no significantly enriched go terms were found in any of the four groups of transcripts. immune defence seven transcripts encoding complement system proteins were identified in atlantic halibut embryos. mannose binding lectin was highly expressed at 8cs, and to a lower degree at gr and ht. complement factor b was highly expressed at gr and complement factor c at 10ss. complement factor c, h and i were highly expressed at 10ss and ht. complement factor d was only expressed at ht. two types of pattern recognition receptors were expressed: toll-like receptors, and c-type lectin receptors. toll-like receptor was highly expressed at 8cs and gr. c-type lectin domain family member c was expressed at 10ss and ht while c-type lectin domain family member e was only expressed at ht. among transcripts encoding cytokines or related proteins, interferon regulatory factor was expressed at 8cs and gr, interferon-induced protein at 8cs and interferon regulatory factor at gr and 10ss. interleukin enhancer binding factor, interleukin, and interferon-induced protein were only expressed at gr. interleukin was expressed from gr to ht. suppressor of cytokine signaling was highly expressed at gr.figure clustering of transcript groups. a: immune system, b: germ cells, c: muscle. transcripts were clustered using the un-weighted pair-group method using arithmetic averages with normal euclidian distance as distance measurements. developmental stages were: embryos at 8-cell stage; germ ring stage, 10-somite stage, and hatched embryos. data were standardized against the first stage 8cs. colour bar indicates relative expression in relation to 8cs. high intensity expression is represented in red colours while blue colours represent lower expression intensity. among the major histocompatibility complex receptor sub-chain transcripts, mhc class 1a chain and mhc class 2b chain were highly present at gr while mhc class 2a chain transcript level peaked at 10ss. transcripts of the t-cell receptor a chain and the t-cell surface glycoprotein cd e chain were found at 8cs and gr while t-cell receptor d chain was expressed from gr to ht. three transcripts coding t-cell co-signaling regulators were identified: lymphocyte antigen, expressed at 8cs and gr, tumor necrosis factor superfamily member, expressed from gr to ht, and programmed cell death ligand highly expressed at 8cs. cathepsin s was expressed at 8cs and gr. primordial germ cells all selected transcripts involved in primordial germ cell proliferation and migration were found at both 8cs and gr, except for tudor domain containing being only expressed at 8cs. transcript level of tudor domain containing, tudor domain containing, tudor domain containing, nanos homolog, dead-end homolog, piwi-like and piwi-like was highest at 8cs, while the level of vasa and askopos peaked at gr. muscle development myosin light chain and, myosin heavy chain, fast myotomal troponin t embryonic isoform, fast myotomal muscle troponin t and t and creatine kinase mitochondrial were expressed at 8cs, and increasingly at 10ss and ht. capping protein actin filament muscle z-line a was highly expressed at 10ss and ht, and capping protein actin filament muscle z-line b was predominantly expressed at 10ss. tropomyosin, myosin light chain, and myosin binding protein c cardiac were highly expressed at ht. parvalbumin was highly expressed at gr and tropomyosin at gr, 10ss, and ht. differential transcript expression in high and low quality eggs twenty transcripts were differentially expressed between high and low quality 8cs embryos. gene set enrichment analysis resulted in no significant differences in functional terms. the highest differences in transcript levels were found for irf and mhc2a. nine transcripts had significant blast hits representing genes involved in immune response, metabolism, rna transcription, protein degradation, cell signalling, and cytoskeleton. conserved domain searches in the remaining differentially expressed transcripts without significant blast hits resulted in identification of hh_ as belonging to the chaperonin-like superfamily of proteins. two differentially expressed transcripts, eef1a2bp and pd-l showed strictly maternal expression and were expressed only before gr.figure clustering of differentially expressed transcripts in high and low quality oocytes. transcripts were clustered using the un-weighted pair-group method using arithmetic averages with normal euclidian distance as distance measurement. high intensity expression is represented in red colours while blue colours represent lower expression intensity. differentially expressed transcripts in high and low quality atlantic halibut 8cs embryos p p for each transcript, fold-change difference in low quality oocytes estimated by microarray and qpcr is given. transcripts are grouped according to their function. validation of expression patterns by quantitative pcr. relative gene expression in atlantic halibut embryonic developmental stages estimated by microarray and qpcr. the embryonic developmental stages were: 8-cell stage, germ ring, 10-somite stage, and hatched larvae. qpcr data were normalized with luc. data were standardized against the 8cs stage. a: irf b: c: hh_ d: eef1a2bp, e: cyp2n, f: hh_ g: hh_ h: hh_ i: hh_ j: rnf k: hh_contig l: psmb m: hh_ n: hh_ o: pd-1 l, p: ms4a8a, q: hh_contig and r: mhc1a. microarray validation a significant correlation was found between qpcr and microarray results. significant differences in transcript levels between high and low quality 8cs embryos by microarray were confirmed by qpcr for all differentially expressed transcripts. expression patterns of the differentially expressed transcripts during embryonic development followed the same general trend when estimated by qpcr, with a few exceptions. microarray relative expression data revealed strictly maternal expression of two transcripts while according to qpcr data, four transcripts were strictly maternal. membrane-spanning 4-domains subfamily a member 8a was expressed only zygotically according to microarray results, but showed maternal-zygotic expression according to qpcr expression results. hh_contig expression was detected at 8cs and ht by microarray and addition in gr by qpcr. mhc class 1a chain expression was apparent in gr and ht by microarray, but only in gr by qpcr. for out of tested transcripts, qpcr expression levels were up to times higher in one or more developmental stages, compared to the microarray results. discussion in hatched atlantic halibut embryos, the anterior part of the head kidney is present, but no haematopoietic tissue or cells can be observed. liver, thymus, and spleen, important for the development of the adaptive immune system, are not present yet. therefore, the innate immune system is the first line of immune protection. the complement system is a major non-cellular component of the innate immune system. the presence of maternally transferred mbl transcripts, an activator of the lectin pathway, indicates that it is the first complement defense mechanism active in atlantic halibut embryos. between gr and ht, expression of transcripts encoding central complement components and transcripts encoding alternative pathway regulatory proteins increases. as previously found in zebrafish and rainbow trout, the ap seems to be functional as immune defense in atlantic halibut embryos, before the adaptive system is developed. despite the lack of igm-bearing cells and organs of the adaptive immune system in atlantic halibut embryos, transcripts of mhc receptors, t-cell receptors, and t-cell co-signaling regulators were present. these maternally transferred transcripts of the adaptive immune system add to the immune protection of the developing embryo and larvae. a similar transfer of maternal transcripts involved in the innate and adaptive immune system have been identified in in half-smooth tongue sole and rainbow trout embryos. transcripts of three tdrd genes, tdrd tdrd and tdrd showed similar expression pattern as piwil and piwil reflecting the close interaction of the corresponding protein products. tdrd tdrd and tdrd together with tdrd tdrd tdrd and tdrd form piwi-tdrd complexes that are essential in retrotransposon silencing, chromatoid body assembly and spermiogenesis. tdrd maternal expression has previously been identified in atlantic halibut embryos. among tdrd transcripts, tdrd was the only transcript expressed mainly at 8cs. tdrd preferably binds to asymmetric dimethyl arginine marks in somatic cells acting as a transcriptional co-activator. expression profiles of nanos and dnd clustered together with piwil and tdrd transcripts with the exception of increased expression at h. dnd and nanos code germ plasm and pgcs specific rna-binding proteins involved in differentiation and survival of pgcs. through binding to 3’utrs of target mrnas, dnd counteracts mirna-mediated posttranscriptional repression in pgcs. expression of dnd and nanos in atlantic halibut embryos was similar to expression patterns found in teleost embryos previously. expression of kop and vasa peaked at gr in atlantic halibut, diminishing during later embryonic development and at ht. a similar expression pattern of kop, coding a pgc-specific p-loop protein of unknown function, has previously been identified in zebrafish and atlantic halibut. vasa, an atp-dependent rna helicase, is mostly known as a pgc marker, but has recently also been identified as a regulator of cell cycle progression in somatic cells. most muscle related transcripts identified during atlantic halibut embryonic development represented isoforms of transcripts coding structural muscle proteins members of myosin, troponin, tropomyosin and parvalbumin. myosin molecules consist of six subunits, two heavy chains of ~200 kda and four light chains of ~20 kda. myhc expression has previously been identified at the 17-somite stage and onwards in atlantic halibut. in the same study, two isoforms of mylc were identified to be stage-specific. in zebrafish, myl and myl are expressed at the 10-somite stage, followed by myl expression at the 12-somite stage. here we have identified isoforms of myl and myl with expression at 8cs, 10ss, and ht, while myl was mainly expressed at ht. one embryonic/larval troponin isoform and two adult isoforms have previously been identified in atlantic halibut larvae during metamorphosis. in the present study, ftnt ftnt and eftnt showed similar expression patterns, indicating that they represented embryonic/larval isoforms. in atlantic halibut embryos, tpm expression started after somite formation and was highly expressed at ht. isoforms of tpm have been previously found to be essential for embryonic development in mice, but the underlying mechanism is unknown. both tpm and mybpc are known to be specifically expressed in cardiac muscle in zebrafish. while tpm expression was detected from gr to ht, mybpc expression was restricted to ht. in the present study, pval expression peaked earlier, at gr, in atlantic halibut embryos compared to its expression in zebrafish, where it was first detected in 15-somite stage embryos. as a ca2+-binding protein, pval is usually present in high concentrations in fast muscle cells, to a lower extend in specific neurons of the central and peripheral nervous system, and in cells of endocrine glands. the two maternal transcripts, irf and mhc2a, were identified as potential markers for quality in atlantic halibut due to their high level of expression differences in high and low quality 8cs embryos. the three uncharacterized transcripts had the same potential, but their functions have to be further characterized. irf is the primary regulator of type i interferon production and its absence impairs antiviral innate immunity. the significantly lower levels of irf transcripts in low quality 8cs embryos may result in reduced immune response capacity resulting in further poor embryonic and larval development. most of the transcripts showing elevated expression in low quality embryos are involved in adaptive immune response and protein degradation. their elevated concentrations could be the result of a maternally transferred immune response during the oogenesis triggered by unknown inflammation, infection or immune-activated stress. polyadenylation of maternal mrnas during oocyte maturation usually protects mrnas from degradation and activates their translation. in contrast, regulatory rna or protein-mediated deadenylation triggers mrna degradation and translational repression to allow normal embryonic development after the maternal-zygotic transition. in xenopus tropicalis, oocyte post-ovulatory aging induced a general decrease in maternal transcripts and a female-specific shortening of maternal mrnas by deadenylation in oocytes that developed into embryos experiencing high malformation and mortality rates. in contrast, poa induced both a decrease and increase in specific maternal transcripts in rainbow trout oocytes. in the present study, timing of hand-stripping atlantic halibut females was synchronised to their individual ovulation rhythms to avoid poa. whether low maternal transcript levels are the result of a lack or sub-optimal polyadenylation during atlantic halibut oocyte maturation, leading to poor transcript translation and/or degradation in low quality early embryos, requires further investigation. due to the experimental design, it was not possible to estimate whether the transcript level differences in high and low quality 8cs embryos were based on individual female differences and/or inheritability. however, no major differences in transcript levels were observed within the three oocyte batches tested. to test the potential of irf and mhc2a as markers for atlantic halibut oocyte quality, future studies should nevertheless be performed across a higher number of batches from females of known genetic background. both abundance of differentially expressed transcripts in high and low quality oocytes and their expression patterns during embryonic development were successfully confirmed by qpcr. the qpcr and microarray analysis have inherent technical and data normalization challenges that can lead to variability in results. following strict quality assessment procedures in both techniques, and data filtering after normalization resulted in high correlation between qpcr and microarray data. due to the requirement of designing qpcr primers across exon/introns boundaries, microarray probes and qpcr primer locations varied. this could result in the observed variation between qpcr and microarray data. compared to the previously created atlantic halibut microarray by douglas et al., the new microarray contains a higher number of transcripts corresponding to genes expressed during the embryonic development. this microarray was successfully used to screen maternal transcript expression, and to identify differential transcripts expression in low and high quality 8cs embryos. it has proven to be suitable for future analysis of atlantic halibut embryonic transcript expression which is likely to advance our understanding of important developmental processes in teleosts.  using a new atlantic halibut 10 k custom oligonucleotide array, we have demonstrated maternal transfer of innate and adaptive immune system transcripts into atlantic halibut embryos and profiled their expression in early developmental stages. we identified several transcripts, including irf and mhc2a, as potential molecular markers for embryo quality. microarray validation did prove the usefulness of the tool for further transcript quantification in atlantic halibut. both the established information and microarray provide useful resources to improve commercial production of atlantic halibut. 
out of differentially expressed transcripts, were identified as maternal transcripts being specifically expressed at 8cs only. atlantic halibutegg qualityembryonic developmentmaternal transcriptsmicroarraytranscriptomeissue-copyright-statement© the author 2014 using a newly developed agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos, germ ring stage, 10-somite stage, and hatched embryos. we have demonstrated maternal transfer of innate and adaptive immune system transcripts into atlantic halibut embryos and their relation with future embryo developmental potential. we identified several transcripts as potential molecular markers of embryo quality. twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. commercial atlantic halibut farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. the developed microarray represents a useful resource for improving the commercial production of atlantic halibut. in addition, we identified differentially expressed transcripts in low and high quality eggs at 8cs to identify potential maternal markers for embryo quality.
 commercial atlantic halibut farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. maternally transferred components regulate early developmental processes; therefore, they have an effect on the future viability of the embryo. using a newly developed agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos, germ ring stage, 10-somite stage, and hatched embryos. in addition, we identified differentially expressed transcripts in low and high quality eggs at 8cs to identify potential maternal markers for embryo quality.  out of differentially expressed transcripts, were identified as maternal transcripts being specifically expressed at 8cs only. twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. several immune-related transcripts were identified as promising molecular markers of hatching success including interferon regulatory factor and mhc class 2a chain. differential expression was positively validated with quantitative real-time pcr.  we have demonstrated maternal transfer of innate and adaptive immune system transcripts into atlantic halibut embryos and their relation with future embryo developmental potential. we identified several transcripts as potential molecular markers of embryo quality. the developed microarray represents a useful resource for improving the commercial production of atlantic halibut. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords atlantic halibutegg qualityembryonic developmentmaternal transcriptsmicroarraytranscriptomeissue-copyright-statement© the author 2014 