during vertebrate development , heart is formed by a highly stereotypic and organized process , and the expression of genes in these processes need be tightly regulated in a spatiotemporal manner ( barnett et al . , 2012 ; wang , 2012 ) .
recent reports suggest that epigenetic regulation modulates genetic program during differentiation and development of various organs including heart and somitic lineages ( wamstad et al . , 2012 ) .
for instance , modifications of histone substantially alter the accessibility and structure of chromatin , therefore modifying transcriptional activity of specific gene clusters .
histones are known to undergo specific modification such as methylation and acetylation : while methylation and demethylation of histone are coordinated by histone methyltransferases ( hmts ) and demethylase ( hdms ) , acetylation of histone is modulated by acetyltransferases ( hat ) and deacetylases ( hdac ) ( bhaumik et al . , 2007 ; helin and dhanak , 2013 ) . due to their significance in regulating genetic program during development and diseases ,
recent efforts were directed to identify specific factors that mediate this process ( arrowsmith et al .
, 2012 ; cayuso mas et al . , 2011 ; kim et al . , 2012a ; 2012b ) .
in case of methylation , histone h3 lysine 4 residue ( h3k4 ) is sequentially modified through mono- , di- , and tri - methylation status , each of which step is mediated by specific enzymes
. mlls ( mll15 ) , setdb1 , and setdb2 have broad roles for h3k4 methylation , and can methylate all type of h3k4 , such as non- , mono- , di - methylated h3k4 ( black et al . , 2012 ; sims and reinberg , 2004 ) .
in contrast , smyd3 methylate mono- and di - methylated h3k4 , and setd7 can only catalase the non - methylated h3k4 to transit to mono - methylated h3k4 ( hamamoto et al . , 2004 ; wang et al . , 2001 )
set and mynd domain - containing proteins ( smyds ) , involving smyd15 , are high conserved protein family across plant , fungi , and animal as well as some protozoa , and have two functional protein domains , set and mynd domains ( del rizzo and trievel , 2011 ; dillon et al . , 2005 ) .
especially , set domain is important for histone lysine methylation activity and mynd domain can mediate the protein - protein interaction and bind to dna motifs .
smyd3 was originally reported as histone lysine methyltransferase ( hmt ) , which methlylates the mono-(h3k4me1 ) and di - methylated lysine 4 residue ( h3k4me2 ) to generate h3k4me3 , of histone h3 , but smyd3 also can bind to 5-ccctcc-3 motif on promoter region of nkx2.8 gene to induce its expression in cancer cells ( hamamoto et al . , 2004 ) .
recent researches showed the global decrease of histone h4 lysine 5 ( h4k5 ) methylation in smyd3 knock - down condition and structural preference for histone h4 lysine 20 ( h4k20 ) of smyd3 , suggesting the substrate diversity of smyd3 ( foreman et al .
it is known that smyd3 is highly expressed in a various cancers , including liver , prostate , rectal , and breast carcinomas , and stimulates the oncogenic activities , such as cell proliferation , adhesion and migration , of tumor cells ( frank et al . , 2006 ; hamamoto et al . , 2006 ; silva et al . ,
in addition , smyd3 is essential for myogenic differentiation through myod regulation ( proserpio et al . ,
actually , the deficient zebrafish embryos displayed the developmental cardiac and somite abnormalities , illuminating its functions in cardiac and somite muscle formation ( fujii et al .
, 2011 ) . set domain containing protein 7 ( setd7 ) , also termed as set7/9 , is another type of histone lysine methyltransferase ( hmt ) and only have set domain for methyltransferase activity , but not mynd domain ( wang et al . , 2001 ) .
it was initially discovered as a specific methyltransferase for only non - methylated histone h3 lysine 4 ( h3k4 ) that converted to mono - methylated histone h3 lysine 4 ( h3k4me1 ) ( wang et al . , 2001 ) .
subsequently , recent accumulating evidence suggests that non - histone proteins , such as yap , dnmt1 , e2f1 , and stat3 , are also the substrates for setd7 ( estve et al . , 2009 ; oudhoff et al . ,
2013 ; pradhan et al . , 2009 ; yang et al . , 2010 ) .
the mouse knock out allele of setd7 is a viable and did not show any developmental and gross defect ( campaner et al .
knock - down of setd7 showed the defects in skeletal muscle formation and myofibril structures ( tao et al . , 2011 ) .
here , we examined the function of smyd3 and setd7 in heart development in zebrafish . collectively , our results demonstrate that smyd3 and setd7 function synergistically for skeletal muscle development and non - redundantly for heart morphogenesis .
zebrafish ab strain as wild - type control for all experiments , expect the experiments using tg(cmlc2:egfp ) line .
zebrafish strains were maintained as previously described ( westerfield , 1993 ) . to visualize the heart morphology in live embryos , tg(cmlc2:egfp )
control , smyd3 , and setd7 mos were designed by author and ordered to genetools company .
mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations .
the sequences of mos used are control mo , 5-cctcttacctcagttacaatttata-3 ; smyd3 mo , 5-aagagctcttacctgacactgaacg-3 ; and setd7 mo , 5-agcttctcgtaccttccaccacttc-3. embryos injected mos were observed or collected at desired developmental stages for further experiment . to make digoxigenin - labelled antisense ribo - probe , zebrafish smyd1a ( nm_205540.1 ) , smyd2 ( nm_001013550.1 ) , smyd3
( nm_001037400.1 ) and setd7 ( nm_001002456.1 ) full orfs were cloned into pgem - t vector ( promega ) after amplification of 24 hpf cdna .
following primers were used for pcr reaction : smyd1a forward , 5-agcatgaccgtggagaagac-3 ; smyd1a reverse , 5-tgttcatgctttgatctgcac-3 ; smyd2 forward , 5-agca gcaggtcaatagcgatg-3 ; smyd2 reverse , 5-ttcctcccattatgtctgctc-3 ; smyd3 forward , 5-atggacggtcactg cagatga-3 ; smyd3 reverse , 5-ctagagcgtgttgagctc agc-3 ; setd7 forward ,
5-aacatggacagcgatgatgac-3 , and setd7 reverse , 5-caggtgttcagaggagtc-3. the reaction producing ribo - probes were carried out according to manufacture protocol ( roche ) .
whole mount in situ hybridization were routinely performed as previously described ( thisse and thisse , 2008 ) . as brief ,
to experiment , fixed embryos washed and digested with proteinase k solution as desired concentration and times .
hybridization reaction was carried out at 65c , and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate ( roche ) .
staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation . to generate the synthetic smyd3 and setd7 mrnas , we first cloned the full open reading frames ( orf ) of smyd3 and setd7 into pcs2 + expression vector , which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription .
these plasmids were cut by noti digestion to linearize , and capping mrna were produced using m7g(5)ppp(5)g ( roche , germany ) and sp6 rna polymerase ( roche , germany ) .
synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution .
injection was achieved at 12 cell stages as optimal concentrations ( about 100200 pg / embryo ) .
zebrafish ab strain as wild - type control for all experiments , expect the experiments using tg(cmlc2:egfp ) line .
zebrafish strains were maintained as previously described ( westerfield , 1993 ) . to visualize the heart morphology in live embryos , tg(cmlc2:egfp )
control , smyd3 , and setd7 mos were designed by author and ordered to genetools company .
mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations .
the sequences of mos used are control mo , 5-cctcttacctcagttacaatttata-3 ; smyd3 mo , 5-aagagctcttacctgacactgaacg-3 ; and setd7 mo , 5-agcttctcgtaccttccaccacttc-3. embryos injected mos were observed or collected at desired developmental stages for further experiment .
to make digoxigenin - labelled antisense ribo - probe , zebrafish smyd1a ( nm_205540.1 ) , smyd2 ( nm_001013550.1 ) , smyd3 ( nm_001037400.1 ) and setd7 ( nm_001002456.1 ) full orfs were cloned into pgem - t vector ( promega ) after amplification of 24 hpf cdna .
following primers were used for pcr reaction : smyd1a forward , 5-agcatgaccgtggagaagac-3 ; smyd1a reverse , 5-tgttcatgctttgatctgcac-3 ; smyd2 forward , 5-agca gcaggtcaatagcgatg-3 ; smyd2 reverse , 5-ttcctcccattatgtctgctc-3 ; smyd3 forward , 5-atggacggtcactg cagatga-3 ; smyd3 reverse , 5-ctagagcgtgttgagctc agc-3 ; setd7 forward , 5-aacatggacagcgatgatgac-3 , and setd7 reverse , 5-caggtgttcagaggagtc-3. the reaction producing ribo - probes were carried out according to manufacture protocol ( roche ) .
whole mount in situ hybridization were routinely performed as previously described ( thisse and thisse , 2008 ) . as brief ,
zebrfish embryos were fixed by 4% paraformaldehyde ( pfa ) and stored in methanol . to experiment , fixed embryos washed and digested with proteinase k solution as desired concentration and times .
hybridization reaction was carried out at 65c , and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate ( roche ) .
staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation .
to generate the synthetic smyd3 and setd7 mrnas , we first cloned the full open reading frames ( orf ) of smyd3 and setd7 into pcs2 + expression vector , which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription .
these plasmids were cut by noti digestion to linearize , and capping mrna were produced using m7g(5)ppp(5)g ( roche , germany ) and sp6 rna polymerase ( roche , germany ) .
synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution .
injection was achieved at 12 cell stages as optimal concentrations ( about 100200 pg / embryo ) .
to delineate functions of hmts , such as smyd1a , smyd2 , smyd3 , and setd7 , during heart development , we have first analyzed the expression pattern of these genes by whole mount in situ hybridization analyses and conventional reverse - transcriptase ( rt ) pcr .
transcripts of all hmts examined appear to be maternally deposited , and widely detected at early stages . at later stages ,
the expression becomes gradually restricted to several organs , including somite , brain and eye ( figs . 1 and 2 ) .
while smyd1a and smyd2 showed higher expressions in developing somite region from 10 somite stage ( ss ) than smyd3 and setd7 , hinting their roles in somitogenesis ( figs . 1 and 2 ) ( ses et al . , 2013
) , the developmental expression regions of smyd3 and setd7 were highly expressed within the heart forming region at 36 h post - fertilization ( hpf ) and 48 hpf , respectively ( red arrow heads in fig .
2 ) . these gene expression profiling data suggest functions of zygotic smyd3 and setd7 in heart development . to further delineate the roles of hmts in heart development , we carried out knock - down experiments of smyd3 and setd7 .
first , to determine the functional requirement of smyd3 during zebrafish heart development , we knocked smyd3 down with a morpholino ( mo)-based gene targeting system . for this purpose ,
splicing blocking mo that binds to the junction of exon2 and intron2 of smyd3 pre - matured mrna was designed ( fig .
3a ) . to validate the mo efficacy , pcr primers were designed to detect normal and aberrant smyd3 transcripts in wild - type embryos and smyd3 morphants , and carried out conventional rt - pcr ( fig .
3b ) . in smyd3 morphant embryo , mature transcript that amplified by f+r2 primer was not presented , while aberrant transcript that produced by f+r1 primer was displayed ( fig .
in addition , smyd3 expression was drastically reduced in smyd3 morphants at 22 ss , suggesting non - sense mediated decay of aberrant transcript caused by mo injection ( fig .
3c ) . at optimal concentration ( approximately 3.0 ng / embryo ) , gross morphology of smyd3 morphants was comparable to control embryos ( fig .
3d and data not shown ) . however , pronounced heart edema ( red arrow ) was apparent in smyd3 morphants at 48 hpf , suggesting that smyd3 function may be essential for cardiac development ( fig .
mos targeting setd7 was designed in a way to target the junction of exon 1 and intron 1 of setd7 pre - mature mrna ( fig .
expression of setd7 was noticeably decreased by mo injection at 22 ss , suggest that setd7 mo is a functional to block the normal transcription of setd7 gene ( fig .
4c ) . similar to smyd3 deficiency , the knock - down embryos of setd7 at optimal concentration ( 1.0 ng / embryo ) displayed cardiac edema ( red arrow ) without causing any obvious morphological defects ( fig .
collectively , our results illustrate that the proper activities of h3k4 methyltransferases , specifically smyd3 and setd7 , are important for zebrafish heart formation . during histone
h3 lysine 4 ( h3k4 ) methylation , setd7 can only methylate non - methylated h3k4 to produce mono - methylated h3k4 ( h3k4me1 ) , which then is further methylated by smyd3 to become h3k4me2 and h3k4me4 ( hamamoto et al . , 2004 ; wang et al . ,
individual roles of smyd3 and setd7 during cardiac and skeletal muscle development in zebrafish have been reported ( fujii et al . , 2011 ; tao et al . , 2011 ) .
given that smyd3 and setd7 successively methylate h3k4 , we can assume that these two enzymes function synergistically during zebrafish development . to test this possibility
, we examined whether co - injection of smyd3 and setd7 mos at a suboptimal concentration can give rise a synergistic effect for induction of developmental defects ( fig .
the smyd3 and setd7 mos , by themselves at optimal concentrations ( 3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7 ) , produced no discernible gross morphological defects except cardiac edema at 48 hpf ( figs . 3 and 4 ) . however , co - injection of these mos at optimal concentrations caused severe somitogenesis defects as indicated by the phenotype of shortened and curled body morphology ( fig .
, the severity of the abnormalities was sharpened by co - injection of increased concentrations of these mos ( fig .
as expected , these results suggest that smyd3 and setd7 have a synergistic function in zebrafish development .
next , we delineated the synergistic roles of smyd3 and setd7 in zebrafish heart development . for this purpose , we used the heart - specific transgenic fish tg(cmlc2:egfp ) line to allow easy visualization of the heart morphology in live embryos ( fig .
the injections of control , smyd3 , setd7 , and smyd3 + setd7 mos were carried out at the optimal concentrations ( 3.0 ng / embryo for smyd3 , 1.0 ng / embryo for setd7 ) .
morphants without body morphological defects were selected for further analyses of heart development . at 48 hpf ,
heart tubes in the smyd3 and setd7 morphants displayed defects in the looping process and remained linear in shape ( fig .
single knockdown of these genes caused a defect in heart morphogenesis , suggesting that each gene is indispensable for heart morphogenesis .
interestingly , double knock - down of smyd3 and setd7 presented a more severe defect than single knockdown of either of those genes ( fig .
however , the global expression level of clmc2 detected by whole mount in situ hybridization ( kim et al . , 2014 ) was not altered in these smyd3 , setd7 , and smyd3 + setd7 morphant embryos at 48 hpf ( data not shown ) , suggesting that specification and/or maintenance of cardiomyocytes were not compromised in those embryos .
collectively , our results show that smyd3 and setd7 likely function synergistically in zebrafish development . moreover , our data suggest that heart development is more sensitive than somitogenesis to the decrease of smyd3 and setd7 levels . to test whether the developing heart defects in the smyd3-and setd7-deficient embryos are caused by the expressional changes of early cardiac muscle genes
, we determined the early expressions of cmlc2 , vmhc , and amhc ( kim et al . , 2014 ) by whole mount in situ hybridization experiment .
the expression level of these genes was not altered by the knock - down of smyd3 and/or setd7 and the gathered shape of cardiac precursor cells in these morphant embryos was also comparable to wild - type embryos ( fig .
this finding suggests that smyd3 and setd7 are dispensable for proper expression of early cardiac muscle genes and differentiation of cardiac precursor cells . to investigate whether enforced expression of smyd3 and setd7 causes the cardiac defect in zebrafish , synthetic mrna encoding smyd3 or setd7
the co - injection of smyd3 and setd7 mrna as optimal dose ( each 100200 pg / embryo ) caused similar developmental defects as in embryos lack either smyd3 or setd7 , such as defective body axis formation .
moreover , these embryos showed pronounced cardiac edema , indicating the defects in heart formation ( red arrow in fig .
7b ) . to better examine the defects caused by overexpression of these smyd3 and setd7
, we analyzed the expression of known cardiac markers , including cmlc2 , vmhc , and amhc . despite the obvious cardiac edema , the expression of these genes was unaltered in embryos overexpressing smyd3 and setd7 ( fig .
therefore , it appears that excessive h3k4 methyltransferase activity can lead to defects in heart and skeletal muscle development in zebrafish . taken together ,
our results indicate that methylation can provide essential regulatory input during organ development in zebrafish .
since similar developmental defects were observed in embryos with either excessive or reduced level of smdy3 and/or setd7 activity , it appears that appropriate level of histone methylation is critical to ensure the formation of cardiac and skeletal muscle in zebrafish .
heart morphogenesis in vertebrate development is a highly complicated event that achieved by correct cell specification , migration , proliferation , apoptosis , and transition as well as controlled beating of heart myocytes ( barnett et al . , 2012 ) .
in this process , proper gene expressions for structure of cardiac chamber at early developmental stages are critical for late heart forming event .
our data presented here indicates that proper level of histone methylation , which is mediated by a series of enzymes commonly known as hmts , including smyd3 , and setd7 appears to be essential for heart development . during zebrafish development
, the expression pattern of hmts indicates that transcripts encoding these proteins may be maternally deposited .
in addition , considering their ubiquitous expression , these proteins may provide essential functions in development by regulating histone methylation for all types of cells ( figs . 1 and 2 ) . however , knock - down of smyd3 and setd7 in zebrafish embryos did not cause any obvious defects in early development , such as in gastrulation movement , germ layer formation , cell proliferation , or cell death . therefore , these genes may redundantly function to regulate early vertebrate development ( figs . 1 and 2 ) . in later developmental stages ,
the expressions of all hmts tested in this paper were gradually restricted and often increased in specific organs , including somite , brain , eye primordial cells , fin primordial region , and heart .
for instance , smyd2 becomes highly expressed within eye primodium , while smyd3 and setd7 are enriched in developing heart , suggest that each hmts might have a non - redundant and organ - specific roles during late vertebrate developmental periods ( figs . 1 and 2 ) .
it was report that knocked - down smyd3 and setd7 by mo led to developmental defects in heart and/or skeletal muscle ( fujii et al .
consistent with previous reports , we also find that mo injections as high concentrations ( over 10 ng / embryo for smyd3 mo and 4.2 ng / embryo for setd7 mo ) cause the similar somite defects .
however , the optimal injection conditions ( concentrations under 3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7 mo ) morphant embryos showed only developmental heart abnormality without any somite defects , indicating that each organ may have different sensitivity to the fluctuation in smyd3 and setd7 level during organogenesis . interestingly , in skeletal muscle development , setd7 directly interacts with myod to induce myogenin and mef2 expression to generate differentiated myocytes ( tao et al . , 2011 ) . therefore , it is tempting to speculate that setd7 manipulation in zebrafish may lead to the alteration in expression of certain structural genes .
however , we did not find any discernible change in the expression of cmlc2 , vmhc , and amhc in our smyd3 and setd7 double - deficient or overexpressed embryos , suggesting the presence of distinct mechanisms underlying setd7-mediated differentiation of the cardiac muscle and skeletal muscle cells .
moreover , smyd3 and setd7 appear to be dispensable for the specification of cardiomyocytes , since the lack of smyd3 or setd7 did not abrogate the expression of cardiac markers ( figs . 6 and 7 ) . here
, we have shown the knock - down and overexpression phenotypes of smyd3 and setd7 in zebrafish development .
these findings extend our current knowledge of the roles of hmts and increase our understanding of the functional mechanism of hmts in heart development .
 while increasing evidence indicates the important function of histone methylation during development , how this process influences cardiac development in vertebrates has not been explored . here , we elucidate the functions of two histone h3 lysine 4 ( h3k4 ) methylation enzymes , smyd3 and setd7 , during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide ( mo)-based gene knockdown . 
 we find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock - down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers , including cmlc2 , vmhc , and amhc . 
 furthermore , double knock - down by coinjection of smyd3 and setd7 mos caused the synergistic defects in heart development . as similar to knock - down effect , overexpression of these genes also caused the heart morphogenesis defect in zebrafish . 
 these results indicate that histone modifying enzymes , smyd3 and setd7 , appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development . 
INTRODUCTION
METHODS AND MATERIALS
Zebrafish husbandry
Injection of anti-sense morpholino oligonucleotides (MOs)
Whole mount 
Over-expression of 
RESULTS
DISCUSSION