Identification and study of microRNAs regulated by Tbx5during cardiovascular development and differentiation in zebrafish (Danio rerio)

UNIVERSITY OF PISA

Ph.D. SCHOOL IN MOLECULAR AND BIOLOGICAL SCIENCES

Research Doctorate Course in Molecular Biotechnology

SSD BIO/06

Identification and study of microRNAs regulated by

Tbx5 during cardiovascular development and

differentiation in zebrafish (Danio rerio)

TUTOR:

CANDIDATE:

Dr. Letizia Pitto

Elena Chiavacci

INDEX

Abstract

Chapter I: Introduction

1.1 Tbx5

1.1.1 The T-box transcription factor family 8 1.1.2 The T-box transcription factor Tbx5 11

1.2 microRNAs

1.2.1 Genomic organization of miRNAs 14

1.2.2 miRNAs biogenesis and action 14

1.2.3 miRNAs in cardiac development and diseases 17

1.3 Danio rerio (Zebrafish) as HOS model system

1.3.1 Zebrafish embryogenesis 18

1.3.2 Zebrafish as model system in cardiovascular research 23

1.3.3 Zebrafish heart development 24

1.3.4 Zebrafish tbx5a and tbx5b 28

1.3.5 The study of miRNAs in zebrafish heart development:

gain-of-function, loss-of-function, Tol2 transgenensis 29

Chapter 2: Materials and Methods

2.1 Zebrafish husbandry

2.2 Zebrafish genomic DNA extraction

2.3 Vectors construction for transgenesis

2.5 CreER

_{induction treatment}

2.6 miRNome

2.7 Morpholino and miRNA - mimic treatments

2.8 Embryos injection and phenotyping

2.9 Imaging

2.10 In situ mRNA hybridization

2.11 In situ miRNA hybridization

2.12 In silico mRNA-target analysis

2.13 S1Pr1 vectors construction

2.14 Luciferase assay

Chapter 3: Results and Discussion

3.1 miR-218a

3.1.1 Identification of miR-218a 41

3.1.2 Tg(cmlc2:creERt2_{) 41}

3.1.3 Tg(ubb: switch, miR-218a) 42

3.1.4 Tg(cmlc2:creERt2_{; ubb: switch miR-218a) 47}

3.2 miR-19a

3.2.1 miRNome 49

3.2.2 In vivo screening 51

3.2.3 miR-19a gain of function (LOF), loss of function (GOF) 56 3.2.4 miR-19a silencing worsen the Tbx5a silencing 62 3.2.5 miR-19a rescues the HOS-like phenotype induced by Tbx5a

downregulation 64

3.2.6 miR-17-92 cluster is downregulated in Tbx5a silenced embryos 68 3.2.7 miRNAs of 17-92 cluster are not able to rescue Tbx5a silencing 70

3.2.8 miR-19a rescues the proepicardium 74 3.2.9 The Sphingosine-1-phosphate receptor 1 (s1pr1) is a miR-19a

putative target 76

3.2.10 Dysregulation of S1Pr1 impacts the cardiovascular

Development 78

3.2.11 miR-19a directly target the s1pr1 3' UTR 80 3.2.12 S1Pr1 downregulation rescues the HOS phenotype 83

Chapter 4: Conclusions

4.1 Conclusions

Bibliography

ABSTRACT

Cardiac malformations are the most prevalent birth defects and the main cause of infant mortality, moreover the heart diseases remain the primary cause of mortality also in the adult life. The T-box gene Tbx5 is a key transcription factor of vertebrate heart development. Tbx5 function in heart is gene dosage sensitive, as either haploinsufficiency or gene duplication give rise to Holt−Oram syndrome (HOS), an highly penetrant autosomal dominant disease characterized by congenital malformations of heart and upper limb. Nonetheless, molecular mechanisms accounting for gene dosage sensitiveness are not known. Several genes regulated by mouse Tbx5 are transcription factors (TFs), or are involved in transcriptional regulation, thus suggesting that HOS might be in part the results of the “regulation of regulators”. MicroRNAs (miRNAs) are small 22 nucleotide RNA molecules that are emerging as important regulators of gene expression. Indeed, recent work has begun to elucidate the key role of miRNAs in cardiac development and disease. There is increasing evidence that TFs and miRNAs can work cooperatively regulating each other. Up to date over 500 miRNAs have been cloned and sequenced, and for many, their targets are unknown. Thanks to recent advances in miRNAs sequencing by Next Generation Sequencing (NGS) platforms, the characterization and quantification of miRNAs at massive scale is now a new powerful instrument for miRNA profiling to be exploited for investigate the complexity of HOS miRNA-mRNA interaction network. However the power of NGS technology requires animal models which allow a massive-scale screening, such as Danio rerio (zebrafish). By coupling the high-throughput DNA sequencing with the advantages offered by zebrafish as model system we investigated the role of Tbx5 related miRNAs during heart development. Among the identified miRNAs we demonstrated that miR-218a have a crucial role in mediating the effects of Tbx5 dosage on heart development, so we focus our attention in the construction of two specific transgenic lines for the temporal and tissue specific regulation of miR-218a. We also identified a second Tbx5 regulated miRNA: miR-19a, establishing a complete novel role for miR-19a as heart and proepicardial (PE) development regulator. Moreover our data shows that miR-19a is capable to partially rescue the HOS-like phenotype by exerting its action through its target S1Pr1. As result we propose a model where mir-19a, acting as functional effector of Tbx5, controls different aspects of heart morphogenesis also by repressing S1Pr1.

1.1 Tbx5

1.1.1 The T-box transcription factor family

The T-box family, defined by a common DNA binding domain known as the T-box, is evolutionarily ancient, probably arising in the common ancestor of metazoan organisms (Fig.1a). The T-box DNA binding is essential for T-box protein function: the T-box domain of Brachyury, the founder member of the T-box family, recognizes in vitro a 20-bp almost perfect palindromic consensus site [T(G/C)ACACCT/ AGGTGTGAAATT, halfsite underlined] with high affinity (Stirnimann et al., 2010). Depending on context, T-box proteins may homodimerize, heterodimerize, or cooperatively bind other transcription factors. Moreover individual T-box proteins can exhibit either activation or repression activities, and single gene may have both functions depending on promoter context (Naiche et al., 2005) cells where the T-box genes are coexpressed one T-box protein can competing off another at a particular promoter making it difficult to predict which is relevant to a particular target gene. Extensive studies have demonstrated that all T-box proteins tested are capable of binding the T-half site as monomers (Naiche et al., 2005), although some have different optimal target sequences (Ghosh et al., 2001) (Lingbeek et al., 2002). Comparisons between T-box proteins have shown preference for different synthetic combinations of T-half sites in varying orientations, numbers, and spacing (Conlon et al., 2001) (Sinha et al., 2000), which may help create promoter specificity for target genes. T-box gene expression is widespread during embryonic development and has been noted almost in all stages, from the oocyte to the adult. In the early embryo, T-box genes are required for both evolutionarily ancient processes, such as gastrulation and comparatively recent developmental processes such as uterine implantation and umbilicus formation (Bruce et al., 2003), (Yamada et al., 2000). During the embryonal development, many T-box genes are found to be key morphogens for the correct heart development, such as Tbx1, Tbx2, Tbx3, Tbx5, Tbx18, and Tbx20 (Naiche et al., 2005). Despite overlapping expression patterns, experimental studies reveal that each gene has unique developmental functions. This aspect is explained by the identification of cardiac transcriptional binding partners with differential affinities for individual T-box factors. The resultant different combined regulatory proteic complexes influence multiple, related and coexpressed genes; as result, during the heart organogenesis, a lot of unique downstream target gene expression patterns were generated. T-box genes are highly conserved in development from hydra to humans (Papaioannou, 2001); many T-box genes have important functions during

mesoderm formation and patterning in the vertebrate gastrula: T and Eomesodermin in the mouse; Xbra, Xeomesodermin, and XvegT in Xenopus; no tail, spadetail, eomesodermin, and tbx6 in zebrafish (Naiche et al., 2005). These genes are critical for mesoderm formation at various axial levels and regulate mesodermal key factor signaling and cell migration. However, T-box gene functions during early embryonal development are recursive and overlapping, creating a complex web of developmental roles and signaling interactions (Showell et al., 2004). A different scenery is for the limb development, where the impact of T-box genes is still crucial, but is due to two T-box genes only, as Tbx4 and Tbx5 conferred hindlimb and forelimb identities, possibly in cooperation with Pitx1 (Naiche et al., 2005). In recent years, both spontaneous and induced mutations in T-box genes have demonstrated that these genes are important regulators of a wide range of tissues and organs during development, as well as major contributors to several human syndromes (Fig.1b). All of the known autosomal human T-box gene syndromes, with the exception of the recessive isolated ACTH deficiency, occur in heterozygous individuals, whereas homozygotes have not been identified, presumably due to embryonic lethality (Naiche et al., 2005). Mouse studies indicate that the full spectrum of tissues affected in homozygous mutants is not necessarily affected by haploinsufficiency. Anyway, in comparing human and mouse phenotypes, there is a high degree of similarity in tissues affected and in the nature of the mutant defects. The dose sensitivity may vary considerably, leading to examples of genes that have much more severe or extensive heterozygous phenotypes in humans than that observed in mice (Naiche et al., 2005)

a) b)

Fig. 1: a) Schematic phylogenetic tree of the vertebrates T-box gene family; b) Comparison of the effects of mutations in human and mouse T-box genes showing the prevalence of dosage sensitivity of phenotypes (Naiche et al., 2005).

1.1.2 The T-box transcription factor Tbx5

Tbx5 is a transcription factor (TF) member of the T-box family, and one of the earliest genes to be expressed in the cardiac field during embryonic development (Bruneau et al., 1999). The consensus sequence of human TBX5 is composed by 8-bp-long DNA half-site (A/G)GGTGT (C/G/T)(A/G) (Ghosh et al., 2001). Recently, thanks to X-Ray cristallography, the structure of this TF also has been resolved (Fig. 2), confirming that mutations in the T-box domain cause the TBX5 loss of capacity to bind its consensus sequence (Stirnimann et al., 2010). In addition to the DNA binding domain, TBX5 possesses a poorly conserved N-terminal extension (residues 1–50), while the C-terminal region following the T-box domain harbours a transactivation domain including a nuclear localization signal (Stirnimann et al., 2010).

Fig. 2) Structural features of human TBX5-DNA complex (bases sequence of DNA is showed below the structure). TBX5 is depicted in yellow, parts interacting with DNA are in red, and the DNA duplex is in blue/grey. The unstructured loop is depicted as yellow broken line. β-Strands involved in the β-barrel are labeled with capital letters (A–G), and β strands from the β- pleated sheets are in lowercase letters (a–g) (Stirnimann et al., 2010).

Tbx5 associates directly with other conserved cardiac transcription factors, including Nkx2.5 and Gata4 (Bruneau et al., 2001), (Hiroi et al., 2001), (Garg et al., 2003), as well as with the transcriptional coactivators Tip60, a histone acetyltransferase (Barron et al., 2005), and Baf60c, a component of the Swi/Snf-like BAF chromatin remodeling complex (Lickert et al., 2004). In mouse Tbx5 is expressed early in the cardiac progenitors and then in a graded fashion along the heart tube, with the highest levels in the sinoatrial region, the graded pattern is established by retinoic acid signaling, which is essential for the formation of the sinoatrial junction (Abu-Issa and Kirby, 2007). Mutation of mammalian Tbx5 produces dominant phenotypes affecting cardiac septation as well as electrophysiological defects including atrioventricular conduction block (NewburyEcob et al., 1996) (Bruneau

et al., 2001). In humans, mutations of TBX5 leads to Holt-Oram syndrome (HOS), (OMIM

#142900). HOS is the most common heart-limb syndrome, affecting 1:100.000 live births (Stirnimann et al., 2010), besides cardiac and forelimb morphological defects HOS is also characterized by progressive cardiac conduction disease. Actually more than 60 mutations including nonsense, frameshift, splice site, deletion, and missense mutations in the TBX5 gene have been identified as HOS related, most of them mapping in the T-box domain (Stirnimann et al., 2010). Mammalian models of HOS indicate an essential role for Tbx5 in cardiac morphogenesis, as for transgenic mice Tbx5 knock-out recapitulate almost all the symptomatology of HOS (Bruneau et al., 1999) (Bruneau et al., 2001). In Tbx5-null mouse embryos, the left ventricular and sinoatrial regions are severely hypoplastic, and numerous chamber markers, including Nppa, are not expressed (Bruneau et al., 2001). Although Tbx5 is a transcription factor able to modulate hundred of genes (Mori et al., 2006), only few direct interaction are known, the best described remain the cardiac-specific natriuretic peptide precursor type A (ANF or Nppa) and the connexin (cx40) (Stirnimann et al., 2010). However, even so the Tbx5 mechanisms of action are not fully elucidated yet, it is known that this TF exert a fundamental role in the differentiation of cardiomyocyte progenitors (Garry and Olson, 2006), together with an anti-proliferative effect (Hatcher et al., 2001), this is consistent with the Tbx5 role as heart chamber identity regulator (Stennard and Harvey, 2005) (Fig. 3).

Fig. 3) Mammalian of model T-box transcription factor regulation of cardiac chamber specification. Chamber myocardium is showed in red, non-chamber myocardium is showed in grey, the inflow and outflow tracts are shown in green (Stennard and Harvey, 2005).

1.2 microRNAs

1.2.1 Genomic organization of miRNAs

Over the last decade, animal studies and advances in human genetics have highlighted the need for precise regulation of key molecular pathways during embryonic development. This is particularly true for the cardiovascular system, where haploinsufficiency of essential genes, such as Tbx5, often causes human cardiac malformations. The dosage of cardiogenic pathways can be controlled at numerous levels. Recently, post-transcriptional regulation by small non-coding RNAs, such as microRNAs (miRNAs), has emerged as a central regulator of many cardiogenic processes. miRNAs act as ‘‘rheostats’’ and ‘‘switches’’ to modulate multiple facets of cardiac development, function, and disease (Liu and Olson, 2010). miRNAs are a large class of evolutionarily conserved, small, non-coding RNAs, typically 20 to 26 nucleotides in length, that primarily function post-transcriptionally by interacting with the 3' untranslated region (UTR) of specific target mRNAs in a sequence-specific manner. There are estimated to be as many as 1000 miRNAs encoded by the human genome, about half of which have been cloned and confirmed (Liu and Olson, 2010). Based on their locations in the genome, these regulatory miRNAs can be classified as intergenic, intronic, and exonic miRNAs. Intergenic miRNAs are derived from their own transcriptional units in the intergenic regions of the genome, while intronic and exonic miRNAs are located within the introns and exons of host genes (protein-coding or non-protein coding genes), respectively, and are usually cotranscribed and coexpressed with their host genes (Bartel, 2004). A subset of miRNAs are also encoded within introns, but are transcribed in the opposite orientation of the host genes and have their own cis-regulatory elements (Bartel, 2004).

1.2.2 miRNAs biogenesis and action

Most animal miRNAs share common biogenesis and effector pathways, they are transcribed by RNA polymerase II as precursor molecules called pri-miRNAs, which can encode single or multiple miRNAs (Liu and Olson, 2010). Pri-miRNAs fold into hairpin structures containing imperfectly base-paired stems and are processed by the endonuclease Drosha and its cofactor DiGeorge syndrome critical region 8 (DGCR8) into 60–100 nt hairpins known as pre-miRNAs (Liu and Olson, 2010). The pre-miRNAs are exported from the nucleus to the cytoplasm by exportin 5, where they are cleaved by the

endonuclease Dicer to yield imperfect miRNA-miRNA duplexes (Yi et al., 2003). A miRNA strand generally indicated as guide is selected to become a mature miRNA, while the other miRNA strand, called passenger, is degraded; occasionally, both strands give rise to functional miRNAs (Liu and Olson, 2010). The mature miRNA is incorporated into the RNA-induced silencing complex (RISC), which recognizes specific targets and induces post-transcriptional gene silencing (Schwarz et al., 2007) (Fig.4). Several mechanisms have been proposed for this kind of regulation, typically a miRNA binds to the 3' UTRs of its mRNA target with imprecise complementarity, except for the so called “seed region” situated at the 5' end of the miRNA, which presents perfect matches (Cordes and Srivastava, 2009). As result we could have an inhibition of translational initiation, a marking of target mRNAs for degradation by deadenylation, or a sequesteration of the target into cytoplasmic P bodies (Filipowicz et al., 2008) (Fig. 4). miRNAs have also been detected in circulating plasma microvesicles, called exosomes, indicating that miRNAs can be secreted and may mediate intercellular communication (Liu and Olson, 2010). Recently, pseudogenes also have been implicated in regulating miRNA activity. Pseudogene transcripts containing conserved miRNA binding sites, referred to as competing endogenous RNAs (ceRNAs), act as decoys or sponges by sequestering miRNAs and preventing them from binding to their mRNA targets (Salmena et al., 2011). Among the hundreds of miRNAs identified thus far, only a limited number have been assigned target mRNAs. Several algorithmic databases have been designed for miRNA target prediction that rely, for the most part, on the following criteria: 1) conservation across species; 2) complementarity of the 5' miRNA seed match to the 3' UTR; 3) G:U wobbles in the seed; 4) the thermodynamic context of target mRNA binding sites; and 5) multiple miRNA binding sites in 3' UTR (Cordes and Srivastava, 2009). These computational programs are continuously updated to integrate new knowledge from validated miRNA/mRNA interactions.

fig. 4) schematic representation of miRNAs biogenesis and mechanism of action (Liu and Olson, 2010).

1.2.3 miRNAs in cardiac development and diseases

Forward genetic studies performed by acting on Dicer complex highlight the importance of miRNAs in the heart organogenesis and development. In mouse, cardiac deletion of Dicer using Cre- recombinase under control of cardiac regulatory DNA sequences, results in lethality at different developmental stages depending on the temporal expression pattern of the Cre transgene (Zhao et al., 2007). Postnatal deletion of Dicer in cardiomyocytes using Cre recombinase under control of the a-myosin heavy chain (MHC) promoter causes dysregulation of cardiac contractile proteins and sarcomere disarray, resulting in dilated cardiomyopathy, heart failure, and lethality (Chen et al., 2008). Similarly, deletion of Dicer in the adult heart using a tamoxifen- inducible Cre recombinase causes heart failure and death (da Costa Martins et al., 2008). While these studies demonstrate the importance of miRNAs in heart development and function, it is unclear whether the severe phenotypes resulting from Dicer deletion reflect requisite roles of specific miRNAs or, more likely, the collective functions of numerous miRNAs in multiple essential processes. The first report implicating miRNAs in heart disease was published in 2006 (van Rooij et al., 2006), in which signature patterns of miRNA expression were shown to correlate with heart failure and cardiac hypertrophy in mice and humans. Since then, numerous follow-up studies have described changes in miRNA expression in diseased human hearts and vascular tissues, as for a lot of gain- and loss- of function studies have uncovered prominent roles for miRNAs in cardiovascular disorders including myocardial infarction, cardiac hypertrophy, heart failure, angiogenesis, vascular stenosis and fibrosis (van Rooij and Olson, 2012).

1.3 Danio rerio (Zebrafish) as HOS model system

1.3.1 Zebrafish embryogenesis

The zebrafish (Danio rerio) system has emerged in the past years as an ideal vertebrate model organism to study a wide variety of biological processes, including the genetic analysis of developmental pathways, and it is currently used as a model for human disease and clinical research. Zebrafish is a small, freshwater teleost native of Ganges River in Southeast India. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200–300 eggs per week, external fertilization, that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, with the hatching occurring around 48-72hpf (Kimmel et al., 1995). The transparency of embryos is a key feature, allowing the direct observation of developing internal organs and tissues in vivo (Santoro, 2011). Other valuable advantages are the small size and low cost. Usually for the identification of zebrafish stages during the first 72 hours of embryogenesis we refer to the staging series described by kimmel et al., consisting in: zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching (Kimmel et al., 1995). This classification is necessay because different embryos, even together within a single clutch, develop at slightly different rates depending on incubation temperature. The optimal temperature for zebrafish raising and maintenance is 28.5°C (Westerfield, 2007); temperatures lower than this one can set back the development, while higher temperatures can accelerate it. The standard developmental time is designated by “h” letter and is defined as normalized hours after fertilization (hpf) at 28.5°C. The development starts with the egg fertilization, while the first cleavage occurs 40 minutes later. After the first cleavage the cells, called blastomeres, divide every 15 minutes in a meroblastic way, so they remain interconnected by cytoplasmatic bridges. At 128-cell stage embryo enters in the midblastula transition, the yolk syncytial layer forms and epiboly begins. During the epiboly phase we have a lot of morphogenetic cell movements including involution, convergence and extension, together with the specification of the primary germ layers and the embryonic axis. Around 5,25hpf the gastrula period ends (for stages and times of development refer to fig. 5) and we have the tail bud, notochord rudiment, and neural keel. At 10,2hpf we have the formation of the first somite and the segmentation starts. Around 24hpf the heart tube is formed and the beating starts, together with the blood circulation. At 42hpf the pectoral fin bud origins, while the hatching occurs at 48hpf and ends at 72hpf. Morphogenesis of many of the organs rudiments is now rather

complete. By day 3 post fertilization (dpf) animal is no more an embryo and starts to be called “larva” (Kimmel et al., 1995).

Fig. 5) Camera lucida of embryos at various stages of development, from zygote to larva (Kimmel et al., 1995).

1.3.2 Zebrafish as model system in cardiovascular research

The heart is the first organ that becomes functional in the vertebrate embryo, approximately 24 h after its induction at HH stage 4 in the chick and E7.25 in mouse, the pre-cardiac mesoderm forms a primitive tubular heart, which starts beating at HH stage 10 in the chick, E8 in mouse, and at about 3 weeks of gestation in man (Sissman, 1970). The heart is an asymmetric structure, which receives its polarity from the three body axes, anterior–posterior (A-P), dorsal–ventral (D-V), and left–right (L-R). Several cell populations that have their origin outside the heart field migrate into the heart and provide additional cell types and play a major role in sculpturing the embryonic heart. Heart morphogenesis is a complex process and therefore it is not surprising that many null mutants in mice display some form of an embryonic cardiovascular phenotype. Currently, about 150 induced mutations in mice have aberrant cardiac development (http://research.bmn.com/mkmd). The complexity of genetic control of heart development can also be deduced from the large number of zebrafish mutants that harbour mutations in genes that are essential for cardiac development (Yelon, 2001).The attraction of zebrafish for heart development studies relies in some unique characteristics. By 24 h after fertilization the zebrafish embryo has already developed a functional cardiovascular system composed by: beating heart, aorta, cardinal vein and blood (Stainier, 2001). Moreover embryos without cardiac contraction or blood thus develop normally until their size outstrips diffusive oxygenation (Stainier et al., 1996). This characteristic allows to follow the consequences of genetic manipulation on cardiovascular development for longer than it would be possible in mammals, in which abnormal heart or vascular development is fatal very early in development. Moreover the optical transparency of embryos offer the possibility of live in vivo imaging of the cellular and physiologic processes during cardiac morphogenesis in a unique manner.

1.3.3 Zebrafish heart development

The development of the heart is a dynamic and complex process starting with the specification of myocardial, endocardial, and vascular/endothelial precursors. It culminate in the morphogenesis of these differentiated cells/tissues into a functional cardiac pumping organ. The heart organogenensis begins with the specification at 5hpf of the two populations of myocardial and endocardial progenitors, situated in the lateral marginal zone on either side of the embryo. The commitment of the two populations is restricted by retinoic acid signalling (Keegan et al., 2005) (Fig. 6 A). More specifically, the ventricular pool appears to reside more dorsally and closer to the margin than the atrial pool. In contrast to the myocardial progenitor cells, endocardial ventricular and atrial progenitors are distributed throughout the marginal zone without apparent organization (Keegan et al., 2004). By 6-9 somites, cardiac progenitors are observed within the posterior half of the anterior lateral plate mesoderm (ALPM) and are marked by the transcription factor genes

gata4 and hand2 (Schoenebeck et al., 2007). As they differentiate into ventricular cardiomyocytes (ventricular myosin heavy chain/vmhc expressing cells), medially at 12 somites and atrial cardiomyocytes (atrial myosin heavy chain/amhc/myh6 expressing cells), laterally at 15 somites (Berdougo et al., 2003), these cardiac precursors begin to express both the cardiac transcription factor gene nkx2.5 and the cardiac regulatory myosin

light chain 2 gene (cmlc2 also referred as myl7), (Yelon, 2001) (Fig. 6 B, C). This cardiac field within the ALPM is restricted anteriorly by cranial vascular and hematopoietic precursors (Schoenebeck et al., 2007) and posteriorly by pectoral fin precursors (Waxman et al., 2008). After forming in the ALPM, the bilateral myocardial progenitors migrate towards the embryonic midline to assemble into a heart tube (Fig. 6 D, E). This event appears to be regulated by not only cell autonomous but also cell non-autonomous mechanisms from the surrounding tissues including the overlying anterior endoderm, underlying YSL, and the endocardium. However, the endodermal signals that regulate myocardial progenitor cell migration remain unclear (Bakkers, 2011). Once the bilateral heart fields have fused at the mid-line, the cardiac tissue forms a disc, in which the future ventricle cells are located at the circumference, the endocardial cells at the centre, and the future atrial cells at the periphery (Bakkers, 2011) (Fig. 6 D). Finally, the endocardial cells, the inner lining of the myocardial heart tube, are required for the formation of the heart tube. These progenitor cells, which are more anteriorly located in the ALPM, migrate medially and posteriorly to line the interior of the developing heart disc (Schoenebeck et al., 2007) (Fig. 6 D). Once the linear heart tube has formed we start to observe

cardiomyocyte contractions. Initial contractions are irregular and unco-ordinated, but show a co-ordinated pattern once the heart tube has completely formed; contractions are initiated at the venous pole (Bakkers, 2011). Owing to slow conduction of the impulse throughout the heart tube, these contractions are initially peristaltic but, as the heart develops, the rate of contractions increases, changing from a peristaltic contraction into a sequential contraction of the atrium and ventricle (Bakkers, 2011). As the heart continues to develop and undergo morphologic changes, additional cardiomyocytes are required to create the final adult cardiac structures. Recently, taking advantage of several zebrafish transgenic fluorescent reporter lines, such as Tg(cmlc2:EGFP), Tg(cmlc2:nuc-dsRed) and Tg(cmlc2:kaede), developmental-timing studies revealed the presence of a further late-differentiating atrial cardiomyocyte population at the venous pole from 16 to 26 somites. This finding suggests the presence of a secondary heart field (SHF) in zebrafish (Zhou et al., 2011) (Fig. 6 C, F). At 24hpf, the heart tube begins to extend and turn such that the ventricle becomes anterior and right-sided, whereas the atrium becomes more posterior and left-sided (Fig. 6 E). These initial morphogenetic events lead to the looping of the heart, followed by the ballooning of the cardiac chambers as well as the development of the atrioventricular canal (Bakkers et al., 2009) (Fig. 6 F). After the linear heart tube forms by 30 hpf, a second wave of new ventricular cardiomyocytes is added to the arterial pole. During the addition of new cardiomyocytes the pre-existing cardiomyocytes within the developing zebrafish heart appears to slowly divide (de Pater et al., 2009). Around 37hpf the atrioventricular canal (AV) also starts to form at the border between the atrium and the ventricle (Tu and Chi, 2012) (Fig 6 G).

Fig. 6) Stages of zebrafish cardiac development. Blastula: white field; yolk cells: yellow field; V: ventral-, D: dorsal- side of the embryo. (Modified from: Bakkers et al., 2009, Tu and Chi, 2012).

During linear heart tube and looping stages, the heart wall is composed of only two cellular layers, the myocardium and endocardium. However during these last few years a third lay-er of tissue came out to the genlay-eral attention as a fundamental one for the comprehension

of heart development and pathogenesis: the heart surrounding layer epicardium. The epi-cardium develops from an extracardiac population of cells called the proepiepi-cardium (PE). The proepicardium can be distinguished morphologically at 48 hpf as a group of spherical cells located in close proximity of the ventral wall of the looped heart (Fig. 6 G). At 72 hpf, it has increased in size and starts to spreads over the myocardial surface of the looped heart (Serluca, 2008) (Liu and Stainier, 2010).Molecularly, the PE is characterized by the onset of expression of wt1a, tbx18 and tcf21 rising from 40-48 hpf (Bakkers, 2011) (Peralta et al., 2013). Specification of the PE requires a Bmp ligand, which is not derived from the liv-er bud, and tbx5a expression in the latliv-eral plate mesodliv-erm during the early somite stages (Liu and Stainier, 2010). Last year the mechanisms guiding the PE specification and morphogenesis have been elucidated; Peralta et al. showed that the hydrodinamic force due to the pericardiac fluid flow is essential for epicardium formation (Peralta et al., 2013). As final morphogenetic event, for the chambers to balloon and expand, cardiomyocytes in each chamber differentially undergo towards cell shape changes. As result, by 48-56 hpf, the ventricular concave inner curvature (IC) cardiomyocytes nearest the non-chambered myocardium (i.e. atrioventricular canal, inflow/outflow tract) remain more rounded, whereas the ventricular convex outer curvature (OC) cardiomyocytes become more elong-ated. Once that cardiac looping, chamber ballooning, and AV canal formation have oc-curred, zebrafish ventricular cardiomyocytes invaginate into the ventricular lumen at 60 hpf to initiate cardiac trabeculae formation indicating the maturation toward the adult heart (Peshkovsky et al., 2011).

1.3.4 Zebrafish tbx5a and tbx5b

Tbx5 is one of the earliest genes to be expressed in the cardiac field during embryonic development. In zebrafish there are two Tbx5 paralogue genes, called Tbx5a and Tbx5b (Albalat et al., 2010), as result of the teleosts whole genome duplication event take place ~270 mya (Parrie et al., 2013). The tbx5a gene is expressed in the embryonic heart, dorsal optic cup, and pectoral fins (Begemann and Ingham, 2000). The tbx5a/heartstrings (hst) mutant displays a loss-of-function phenotype that recapitulates aspects of HOS in both the heart and forelimb (Garrity et al., 2002). Despite overtly normal heart tube formation and slight bradycardia, the hearts of homozygous hts mutant embryos later fail to loop and progressively deteriorate, leading to heart failure and lethality (Garrity et al., 2002). Although in various species Tbx5 function impacts the numbers of cardiomyocytes, in zebrafish this aspect was not observed, neither for tbx5a or tbx5b (Parrie et al., 2013). The overlapping expression of both zebrafish tbx5 paralogs in the embryonic heart (Albalat et al., 2010) raised the possibility that these genes might share certain functions during cardiogenesis. However more recently Parrie et al., well demonstrated that, even the zebrafish T-box transcription factor tbx5b is essential for cardiac development and pectoral fin morphogenesis, the two tbx5a and tbx5b paralogs have distinct, non-redundant functions for heart and pectoral fin development (Parrie et al., 2013).

1.3.5 The study of miRNAs in zebrafish heart development: gain-of-function, loss-of-function, Tol2 transgenensis

miRNAs are highly conserved among species, so Zebrafish have proven to be a valuable model system to investigate miRNA function and characterize miRNA/mRNA target interactions. Actually many cases of miRNAs involved in zebrafish heart development have been reported, as for miR-138 (Morton et al., 2008) and miR-21(Banjo et al., 2013). The zebrafish system can be easily used to perform gain-of-function (GOF) and loss-of-function (LOF) studies of miRNA loss-of-functions, as synchronous populations of several hundred fertilized zebrafish eggs are easily available for microinjection of reporter constructs, RNA molecules or chemically modified nucleic acids (Santoro, 2011). For LOF, antisense morpholino phosphorodiamidate oligonucleotides (MOs) have been extensively used from years to knock down gene functions in zebrafish embryos and larvae by inhibition of miRNA processing, (Nasevicius and Ekker, 2000). On the other hand, for GOF studies the most successful strategy is to promote single miRNA expression by injection of double stranded miRNA-mimics (Zeng et al., 2009) (Chiavacci et al., 2012). More recently, a sophisticated genetic techniques based on the CreERT2_{/loxP system has} been described (Hans et al., 2009) (Hans et al., 2011) (Mosimann et al., 2011) and could be combined to the Tol2kit (Kwan et al., 2007) to allow temporal and tissue-specific miRNA expression in the zebrafish model system. The Tol2kit is a system exploiting the Tol2 transposase elements (Kawakami, 2007) for easily generating expression constructs and stable transgenics zebrafish lines. For both spatial and temporal control of the desired element expression, the system has to be based on the cross-mating of two different transgenic lines: one is called 'driver line', it harbour an inducible recombinase under a tissue-specific promoter, so it can 'drive' the expression of a gene/miRNA of interest in a tissue-specific manner. The second one is the so called 'responder line', it harbour the gene/miRNA of interest (or a sponge system in case of a miRNA downregulation) downstream of a stop cassette flanked by LoxP sites, all together guided by a strong ubiquitous promoter. In normal condition the presence of the stop cassette block the expression of all the downstream elements but, when the site-specific inducible recombinase is present, the loxP sites recombine and the stop cassette is excided, event that allows the temporal control of the miRNA of interest. Actually the most used site-specific inducible recombinase is the CreERt2_{, (Hans et al., 2009) which promotes strand exchanges} between two 34-bp loxP target sites without any additional cofactors. Each loxP site consists of two 13-bp repeats flanking an 8-bp asymmetric spacer sequence that confers

directionality, head-to-head orientation causes inversion of the DNA between the two sites, whereas head-to-tail orientation results in the excision of the intervening DNA sequence, an irreversible reaction due to the loss of the excised product. Altogether the GOF, LOF, and transgenesis techniques propose the zebrafish system as an optimal model to study miRNAs function during the development.

2.1 Zebrafish husbandry

All zebrafish lines used in this study were raised and maintained under standard laboratory conditions (Westerfield, 2007). Transgenic reporter line used in this study is Tg(cmlc2:EGFP), kindly provided by Prof. Santoro.

2.2 Zebrafish genomic DNA extraction

To avoid the problems due to genetic variation (Coe et al., 2009), genomic DNA extraction was performed starting from an homogenate of different zebrafish adult strains comprehensive of AB wild-type, Tg(cmlc2:EGFP), and Tg(kdrl:EGFP), using the DNeasy Blood & Tissue Kit (QIAGEN) according to manufacturer protocol.

2.3 Vectors construction for transgenesis

For generation of the Tol2(ubb:LoxP-EGFP-LoxP, DsRED, miR-218a) vector, shortened Tol2(ubb: switch miR-218a), the ubb:LoxP-EGFP-LoxP construct was generated by gateway recombination technology in PdestTol2PA vector after removal of the cmlc2:EGFP transgenesis marker cassette (Kwan et al., 2007) by BglII digestion. The pri-miR218a was PCR amplified from genomic DNA with primers Fw 5' AAG CCA TGC TGA CAT ACC AG 3' and Re 5' ACG CGT GAA CTG TAT GTG TAT 3', using the proof reading Herculase Taq polimerase (agilent). PCR profile as follow: 35 cycles [Denaturation 94°C 30”, Annealing 52°C 30”, Elongation 72°C 40”]. Insert was cloned in MluI restriction site; the correct sequence and orientation was confirmed by sequencing. The final vector map is showed in Fig. 7a. The generation of the Tol2(cmlc2:CreERt2_{) vector}

was performed thanks to multisite gateway technology as described by Kwan et al. (Kwan et al., 2007), by combining the 3' entry clone P3E-PolyA, the entry middle clone CreERt2 (obtained from Dr. Cellerino Lab), and the the 5' entry clone 228-cmlc2 obtained as follow: zebrafish cmlc2 promoter was amplified from pdestTol2-PA vector with primers Fw 5' AA AGC TTA AAT CAG TTG TG 3' and Re T GGT CAC TGT CTG CTT TGC TG 3'. PCR bottom-up profile was: 5 cycles [Denaturation 94°C 30”, Annealing 55°C 30”, Elongation 72°C 60”] + 25 cycles [Denaturation 94°C 30”, Annealing 60°C 30”, Elongation 72°C 60”]. The obtained fragment was blunt cloned in 228-p5E-MCS vector and the resulting 228-cmlc2 vector used as 5' entry clone. The multisite gateway reaction was performed with Gateway® LR Clonase® II Enzyme mix (Invitrogen™) following the manufacturer

instructions, using the pdestTol2-PA2 as destination vector. The resulting construct was the Tol2(cmlc2:CreERt2_{) vector, showed in fig. 7b.}

Fig. 7 a) map of Tol2(ubb: switch miR-218a) vector Fig. 7 b) map of Tol2(cmlc2:CreERt2_{) vector}

2.4 Transgenic zebrafish lines generation

Zebrafish transgenic lines were generated using Tol2-mediated transgenesis: 25 ng/ul of each transgenesis vector (Kwan et al., 2007) were combined with 15ng/nl Tol2 mRNA, and injected into one-cell-stage eggs derived from AB wild-type crosses. For the Tg(ubb: switch miR-218a) line individual Tg(ubb: switch miR-218a) F0 were screened for EGFP mosaicism by flurescence microscopy at 24hpf, and 6dpf. We achieved almost 100% efficiency of mosaicism, reflecting the extremely goodness efficiency of Tol2 mediated transgenesis. Individual F0 founders were raised until adulthood and their progeny were screened for the EGFP transgenesis marker outcrossed to AB, and their F1 progeny. For the Tg(cmlc2:CreERt2_{) generation F0 founders were raised until adulthood and outcrossed with} wild-type AB, their progeny was raised and juvenile fishes (2moths aged) genotyped as described in ZIRC, Zebrafish International Resource Centre http://zebrafish.org/zirc/documents/protocols/pdf/Genotyping/D_Sample_Preparation.pdf, modified as follow: after proteinase K step, a phenol/chloroform DNA extraction was performed. Genomic DNA purified was resuspended in 20ul of nuclease free sterile water and diluted 1:100 as to be used for PCR. Primers used for PCR screening were Cre Fw 5' TGG AAA ATG CTT CTG TCC GTT 3', and Cre Re 5' GCT AAG TGC CTT CTC TAC ACC 3'. PCR touch-down genotyping profile was: 10 cycles [Denaturation 94°C 30”, Annealing 61-> 56 °C 30”, Elongation 72°C 1',50”] + 25 cycles [Denaturation 94°C 30”, Annealing 56 °C 30”, Elongation 72°C 1',50”].

2.5 CreER

_{induction treatment}

4-Hydroxytamoxifen (4-OHT) was dissolved in ethanol (EtOH) at a final stock concentration of 10 mM and kept in single-use aliquots in the dark at –20°C. To induce Cre activity in CreERt2_{-expressing embryos, 5-10 stage-matched embryos per single well of a} 24-well culture plate were washed with filtered husbandry water, and replaced with filtered husbandry water freshly mixed with 5 μM 4-OHT. The treated embryos were immediately put into a closed and dark 28.5°C incubator to allow for efficient induction and remained in 4-OHT-containing husbandry waters over night (O.N.). The embryos were subsequently washed twice in husbandry water to removed all traces of 4-OHT, then they were placed in a 24-well culture plate containing fresh husbandry waters and grown until 6 dpf.

For high-throughput DNA sequencing total RNA was extracted from zebrafish embryos coinjected with 1,5ng of MO-Tbx5a plus 1,5ng MO-Tbx5b and collected at 48 hpf. Total RNA was extracted with miRNeasy® Mini kit (QIAGEN), according to manufacturer protocol. Embryos injected with 3ng of MO-Ct were treated in parallel. miRNomes were performed with Illumina 454 pyrosequencing system, according to the laboratory protocol described in Baumgart et al. 2012 (Baumgart et al., 2012). Differential expression analysis was performed as follow: miRNA read count, identified by miRExpress, were analysed using Bioconductor's package DESeq after a library size normalization step.

2.7 Morpholino and miRNA - mimic treatments

Morpholino antisense oligonucleotide against the Tbx5a translational start site was: MO-Tbx5a 5'-GAA AGG TGT CTT CAC TGT CCG CCA T-3' (Garrity et al., 2002), while Tbx5b morpholino antisense oligonucleotide against was MO-Tbx5b 5'-GGA TTC GCC ATA TTC CCG TCT GAG T-3' (Chiavacci et al., 2012). Morpholino antisense against the translational stat site of S1Pr1 was 5'-AGT TGT CTG GCG ATT AGG TCA TCC AT-3'

(Mendelson et al., 2013). The sequence of the control MO was 5’-CCT CTT ACC TCA

GTT ACA ATT TAT A-3’. A morpholino against mature miR-19a was designed to silence miR-19a, the sequence was 5'-CAT CAG TTT TGC ATA GAT TTG CAC A-3'. All morpholinos were supplied by Gene Tools LLC. Mature miR-19a mimic for gain-of-function (GOF) and morpholino coinjections was synthesized by GenePharma (Shanghai, China), the mir-19a sequence was 5'-UGU GCA AAU CUA UGC AAA ACU GAU U-3'; as control a non-coding miRNA (miR-Ct) was used with the sequence 5'-CUC UAG GUU AAA CUC CUG GUU-3' (Li et al., 2011).

2.8 Embryos injection and phenotyping

Zebrafish morpholinos and miRNA-mimic were injected into the yolk of 1-2 cell stage embryos. The cap-mRNAs were injected into the cell at 1 cell stage. All the injections were performed with a constant injection volume, ~ 1 nl, using a microinjector (Tritech Research, Los Angeles CA USA). For each experiment were injected at least 220 embryos in 2/3/4 different independent batches of 120 embryos each. Mortality was assayed at 24hpf on the total injected embryos. The phenotypical counts were performed at 72hpf as follow: first, because of the heterozigosity of Tg(cmlc2:EGFP) strain, the few EGFP

negative embryos were discarded in order to avoid brood biases, then the embryos were distributed in 96-well culture plates (one single embryo for a single well), subsequently embryos were screened one by one at optical or fluorescence microscopy, depending on needs. The number of embryos for each phenotypical category of two independent injections were then added and totals converted on percentages referred to alive EGFP positive embryos at 72hpf.

2.9 Imaging

Optical phase contrast imaging was performed with Leica M80. For fluorescence imaging embryos were embedded in soft agar low-melting 1,2% and screened with Nikon Eclipse E600 microscope. For inverted fluorescence imaging embryos were single-dispensed in 96-well cell plate and counted with Leica DM-IL inverted microscope. Images were acquired with CoolSnap-CF camera, thanks to NIS-Elements software 2.0 ver. For confocal imaging embryos were embedded in low-melting soft agar 1,0 % in WillCo-dish® glass bottom plates, and acquired with Leica DM-IRE2 confocal microscope, stacks were processed with ImageJ 1.48o and mounted thanks to Gimp 2.6.

2.10 In situ mRNA hybridization

Whole mount in situ hybridization was performed as previously described (McMahon et al., 2009) with some modifications: the anti-DIG antibody-alkaline phosphatase Fab fragment (Roche) was diluted 1:5.000 in MABlock buffer (2% Roche blocking reagent in 100 mM Maleic acid and 150 mM NaCl) and incubation was performed at + 4 °C. After incubation with the anti-DIG antiserum, embryos were washed in PBT (PBS plus tween 20 0,1%), and then incubated in the alkaline Tris buffer solution containing 2 mM Tetramisole (Sigma). The final staining step was performed in BM Purple AP-Substrate precipitating buffer (Roche) according to the manufacturer’s recommendations. After dye development embryos were incubated for 10 minutes in pre-bleaching buffer (SSC 0,5X and 6% Formamide). After discarding the pre-bleaching buffer, bleaching buffer (pre-bleaching buffer containing 33% H2O2) was added to the embryos which were subsequently exposed to a bright source of light for 15 minutes, while shaking. Afterwards embryos were transferred back to 87% glycerol in PBS incubated at room temperature under slow agitation for 4 h and then stored at –20° C. wt1a probe was obtained from Englert lab (Bollig et al., 2009); tbx5a probe was obtained from Wilson lab, tbx18 probe was obtained

from Begemann lab. Probe for primary transcript of miR-17-92 cluster was generated in Cellerino lab as follow: the entire cluster was amplified from zebrafish genomic DNA with primers Fw 5'-ACT GCA GTG GAG GCA CTT CTA G-3' and Re 5' – TAT TGC ACT TGT CCC GGC CTG GUT AAT ACG ACT CAC TAT AGG G -3', harbouring the T7 promoter. Then 200ng of PCR product were transcribed with T7 RNApol (thermoscientific) and DIG-RNA Labeling Mix (Roche), according to manufacturer instruction. Hybridized probes were 100ng for 15-20 embryos batch in case of wt1a, tbx5a, tbx18, and 800ng in case of miR-17-92 cluster probe. Hybridization temperature was 68°C for all the probes used.

2.11 In situ miRNA hybridization

Whole mount in situ hybridization for miRNA were performed as previously described by Lagendijk et al. (Lagendijk et al., 2012) with some modifications: for the fixation step the

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC) was used. As

miR-19a probe a double-DIG miRCURY LNA Detection probe, with the following structure: 5'/DigN/ TCA GTT TGC ATA GAT TTG CAC A /DigN/3', was used. Hybridization temperature was 37°C

2.12 In silico mRNA-target analysis

For the prediction of the mRNA-target we used miRWalk prediction software package (Dweep et al., 2011), with the predicted validated target module updated on 15th March 2013. The TargetScanFish release used was the 6.2 http://www.targetscan.org/fish_62/ .

2.13 S1Pr1 vectors construction

3'UTR S1Pr1 luciferase repoter vector (pGLU S1Pr1) was constructed as follow: the fragment of 3'UTR containing the putative miR-19a binding site was cloned from zebrafish cDNA obtained with QuantiTect Reversion Transcription kit (QIAGEN), with primers Fw 5' GGT ACC TCT TCT TCT TAA AGG 3' and Re 5' GAA CAG GGA CAA AAC TGG CTC 3'. The PCR fragment was cloned with SalI and KpnI in pGLU 3'UTR reporter vector (Poliseno et al., 2010). The pCS2-s1pr1-CDS vector containing the complete coding sequence (CDS) of S1Pr1 was obtained by cloning the entire CDS with primers Fw 5' GGA TCC AAA CCA TGG ATG ACC 3' and Re 5' GTT CTA GAA TAG TCC CTT TAA

3' in the pGEM-T Easy Vector (Promega). Then the CDS was sub-cloned with BamHI and SalI/XhoI in pCS2-EGFP plasmid (kindly provided by Cremisi lab). The 3'UTR of S1Pr1 was successively insert in the pCS2-s1pr1-CDS as follow: the S1Pr1 3'UTR was excised from p-GLU S1Pr1 and XbaI cloned downstream the S1Pr1 CDS. The vector resulting was the s1pr1-full. Mature capped mRNA was obtained from NotI linearised pCS2-s1pr1-full with mMESSAGE mMACHINE Sp6 Kit (Ambion), according to manufacturer instructions.

2.14 Luciferase assay

293T cells were seeded at a density of 1,5x105 _{cells per well in 12 well dishes in} Dulbecco’s modified Eagle Medium (Gibco DMEM) with 10% FBS, 2μg/ml L-glutamine and 50μg/ml streptomycin. Cells were grown for 24 hours at 37°C in a humidified atmosphere containing 5% CO2. Twenty-four hours later, cells were transfected. Polyfect (QIAGEN) was used as transfectant, according to the manufacturer’s recommendations. In each transfection 100ng/μl of p-GLU S1Pr1 plasmid was cotrasfected with different concentrations of miR-19a associated with different concentration of miR-Ct, in the presence of 100ng/μl of renilla expressing plasmid as internal standard. After 24 hours at 37°C cells were washed with PBS for two times. Luciferase assay was performed using Dual luciferase Reporter assay System. Firefly luciferase activity was normalized to renilla activity for each transfected dishes.

3.1. miR-218a

3.1.1 Identification of miR-218a

To identify miRNAs regulated by Tbx5 we developed a new bioinformatic tool to search for miRNAs within introns of Tbx5-controlled genes. As source of Tbx5 target genes, we crossed data from microarray analysis of a conditional mouse allelic series of Tbx5 (Bruneau et al., 2001) and data from mouse 1H cell line overexpressing Tbx5 (Plageman

and Yutzey, 2006). Four miRNAs were identified: 218-1, 678, mir-719 and

miR-335. We focused our attention on miR-218 for several reasons: miR-218 is conserved from human to zebrafish (www.mirbase.org); in mouse the miR-218-1 host gene, Slit2, is highly sensitive to Tbx5 mis-expression (Mori et al., 2006), and the secreted Slit ligands, together with their Robo receptors, contribute to the control of oriented cell tissue growth during chamber morphogenesis of the mammalian heart (Medioni et al., 2010). In zebrafish, as in mammals, there are two different sources of mature 218a, the 218a-1and miR-218a-2, embedded in slit3 and slit2 gene, respectively. In a previous work we showed in zebrafish by functional assays and rescue experiments that miR-218a is indirectly regulated by Tbx5a, as the silencing of miR-218a via specific morpholino is capable to attenuate the phenotype given by Tbx5a depletion (Chiavacci et al., 2012). As subsequent step we would like to better understand the role of miR-218a in zebrafish heart development, in doing so we decide to construct zebrafish transgenic lines capable to regulate the spatio and temporal expression of miR-218a. The system we choose for conditional transgenesis is based on the Tol2Kit, one of the most efficient system present at the moment for the introduction of exogenous nucleic acid chains in a naïve genome.

3.1.2 Tg(cmlc2:creERt2₎

For the temporal and spatial control of miR-218a we built a transgenic driver line harbouring the inducible recombinase CreERt2 _{upstream to the cardiac specific promoter,}

cmlc2 (Cardiac myosine light chain 2), .that allows the expression of the CreERt2 _{in the} cardiac district only. Moreover, since cmlc2 is one of the first cardiac genes to be expressed in zebrafish development and it is maintained during all the life period (see introduction, 1.3.3 section), the CreERt2 _{expression can be activated any time during the embryo} development. CreERt2 _{is probably the most widely used recombinase for site-specific} transgenesis, because of the best properties of ligand sensitivity and inducible

recombination efficiency (Hans et al., 2009). It is a chimeric protein composed by a recombinase fused to a mutated human ligand-binding domain of the estrogen receptor. When the estrogen is present it binds to its receptor and cleave it. As consequence the Cre protein is released in the cytoplasm and can reach the nucleus, where it can cut the loxP sites on the transgenic construct. This mechanism is based on the observation that the localization of proteins can be controlled by a ligand when fused to a ligand-binding domain of an extrogen, so it offersa temporal control of Cre mediated recombination, together with all the elements Cre-hanging (Hans et al., 2009). The CreERt2 _recombinase fusion protein is inducible by exposure to tamoxifen (TAM) or its active metabolite 4-Hydroxytamoxifen (4-OHT) (Feil et al., 1996) (Feil et al., 1997). To establish the Tg(cmlc2: CreERt2_{) line we assembled the Tol2(cmlc2:CreER}t2_{) reporter vector, and} injected it together with Tol2 transposase-encoding mRNA into one-cell-stage zebrafish embryos (Kikuta and Kawakami, 2009). Because of the lacking of a reporter gene, no visual screening was possible to select the transgenic fishes. Therefore we raises and crossbred the mosaic F0 progeny. We subsequently screened the F1 progeny by PCR genotyping assay, as described in material and methods section 2.4. As result of the screening PCR for CreERt2 _{allele we found 7 females and 1male as founders. To produce a} stable transgenic line we selected three independent F1 female founders to be mated with wild type males. Progeny will be screened by genotyping assay and CreERt2 _{positive fishes} will be raise to assure the line maintenance.

3.1.3 Tg(ubb: switch, miR-218a)

To conditionally express miR-218a we started by generating a responder transgenic line for 218a overexpression in the heart. The responder line we built is Tg(ubb: switch miR-218a), in this line we use the strong ubiquitous promoter ubiquitin B (ubb). In transient injections into zebrafish embryos it was been shown that ubb promoter is capable to drive strong and ubiquitous visible expression of an EGFP reporter gene within 4 hours post-injection (Mosimann et al., 2011), likely starting at the onset of zygotic transcription. Moreover, the stable Tg(ubb:EGFP) lines revealed strong expression in all analysed external and internal organs, including the retina, fin fold, and across all blood cell types from embryo to adulthood (Mosimann et al., 2011). After the ubb promoter we cloned the EGFP stop cassette flanked by loxP sites, followed by the dsRED as second reporter gene, with its 3'UTR harbouring a fragment of the slit2 intron embedding the pri-miR-218a. This particular construct with the two different reporter genes, loxP-reporter1-loxP-reporter2, is

called 'switch construct' (Mosimann et al., 2011), in fact when the recombinase is not active, only the first reporter (in our case EGFP) is expressed. When the recombinase activity is induced by TAM, the stop cassette is removed and the expression of the first reporter gene is lost, while the expression of the second reporter (in our case dsRED) is switched on. The switch from the first to the second reporter is indicative that the recombination event take place correctly and is followed by the expression of the 3'UTR, together with the miR-218a precursor. As result we have an upregulation of miR-218a in the tissue containing the induced CreERt2_{. We can conclude that the switch event marks} bona fide the tissue in which the overexpression of miR-218a is taking place. To establish the Tg(ubb: switch miR-218a) line we assembled the Tol2 (ubb: switch miR-218a) reporter vector and injected it together with Tol2 transposase-encoding mRNA into one-cell-stage zebrafish embryos (Kikuta and Kawakami, 2009). At 24hpf we check the injected embryos for EGFP and we could detect strong mosaic EGFP expression by fluorescence microscopy (Fig. 8a). To assess the correct functioning of the switch construct we inject the Tol2 (ubb: switch miR-218a) reporter vector together with Tol2 transposase-encoding mRNA and an high dose of CreERt2 _{encoding mRNA (800pg). At 24hpf embryos were treated O.N. with} the CreERt2 _{inductor 4-OHT. The active metabolite 4-OHT is of more difficult wieldy} compared to its precursor TAM, however 4-OHT shows a faster action and this feature is crucial in case of time-specific transgenesis. It has been reported that 4-OHT can be uptaken and can exert its action within 2-4 hours from the administration (Hans et al., 2009). As working concentration we choose 5μM because it was shown to be the highest concentration at which the recombination event takes place efficiently without toxicity effects (Mosimann et al., 2011). Because of the uniform distribution of the injected mRNA in the embryos, the excision of the EGFP stop cassette takes place in all the recombinated cells. Since the EGFP is a very stable protein capable to resist even 1-2 days in the cells once it was produced, the induction event leads to double labelled recombinated cells. As we can see, the injection of Tol2 (ubb: switch miR-218a) construct produces highly mosaic and strong EGFP expression in both the 4-OHT-treated and control-treated embryos (embryos treated with the ethanol solvent, EtOH-treated). On the contrary, CreERt2 activation in 4-OHT treated embryos, causes a strong dsRED expression in EGFP labelled cells, as observed at 72hpf (Fig. 8 b, c), whereas EtOH-treated embryos never produced detectable dsRED expression during the observation period (not shown).

Fig. 8 a) Mosaic expression of EGFP in 24 hpf embryos injected with Tol2 (ubb: switch miR-218a) vector. Switch test: embryos co-injected with Tol2 (ubb: switch miR-218a) and CreERt2 _{mRNA. At 24 hpf embryos were treated ON with 4-OHT; images of 72 hpf} embryo showing a strong mosaic double-labeled EGFP (b) and dsRED (c) cells. Scalebars: 100μm.

This switch test proves the goodness of recombination and induction events of ubb: switch miR-218a construct. Next step was the establishment of a stable Tg(ubb: switch miR-218a) transgenic line. To accomplish this aim individual mosaic EGFP-positive F0 females were raised and crossed to wild-type males, their F1 progeny was screened at 24hpf (Fig. 9a) and 72hpf for ubiquitous EGFP expression (Fig. 9b). We selected three independent F1 male founders to be mated with wild type females. The F2 progeny showing EGFP expression will be further selected and rise for the line maintenance in the next years.

Fig. 9 a) F1 embryos Tg(ubb: switch miR-218a), at 24hpf (a) and 72hpf (b). Scalebar: 100μm.

3.1.4 Tg(cmlc2:creERt2_{; ubb: switch miR-218a)}

Once obtained both the F1 transgenic lines we proceeded to mate and induce the progeny with 4-OHT in order to assess the responsiveness of Tg(ubb: switch miR-218a) to the Tg(cmlc2: CreERt2_{). If the induction takes place correctly we should be able to obtain} homogeneous EGFP embryos with a dsRED labelled heart. Since we want to investigate the role of miR-218a during the heart development, we perform the induction at several stages in order to evaluate both the correct functioning of the construct and eventual efficiency gaps through embryo development. We get on by crossing Tg(ubb: switch miR-218a) F1 male founders to Tg(cmlc2: CreERt2_{) F1 female founders and the resulting double} transgenic embryos were induced with 5uM 4-OHT overnight in the dark. In parallel the same group of siblings, at same stages, was control treated with EtOH. The induction were performed at 24hpf and 36hpf stage. All the embryos were raised in the dark until 6dpf to allow the dsRED accumulation in the Cmlc2 positive cells. As result we observed that: 1) almost all the double transgenic inducted fishes showed some dsRED labelled cells in the heart region (Fig. 10 a', a'') compared to the control (fig10 b', b'') (Tab 1), proving the correct functioning of the Tg(cmlc2:creERt2_{; ubb: switch miR-218a) line. 2) The number of} embryos showing a correct induction after ON. 4-OHT exposition (marked by the dsRED expression in the heart) was not influenced by the embryo stage at which induction took place (Tab 1). 3) The efficiency of induction was not homogeneous and still reflected some inefficiency in the induction procedure that must be investigated in the future. This aspect is highlighted from several reports of different induction protocols which are reported in the literature, reports showing, sometimes, even discrepancies (Park and Leach, 2013).

Fig. 10) Green fluorescence field (a) and (b), red fluorescence field (a') and (b'), merge (a'') and (b'') of 6dpf double transgenic Tg(cmlc2:creERt2_{; ubb: switch miR-218a) larvae.} 4-OHT-treated larvae (a, a' and a'') showed some dsRED labelled cells in the cardiac region (white arrows), compared to the control EtOH-treated larvae (b, b' and b''). Scalebar: 100μm.

Tab. 1) Number of 6dpf Tg(cmlc2:creERt2_{; ubb: switch miR-218a) larvae showing cardiac} dsRED labelled cells after treatment at 24 hpf or at 36 hpf with 4-OHT. Larvae EtOH-treated never showed dsRED+ cells. Treatments were performed on 40 embryos (20 for 4-OHT and 20 for EtOH-treated) for each stage.

3.2 miR-19a

3.2.1 miRNome

Although the bioinformatic approach used for miR-218a identification worked well in the case of this specific miRNA, it is based on intronic miRNA searching, so it is useless in case of intergenic or exone embedded miRNAs. Moreover the data we used coming from microarray experiments, and it is well known that hybridization-based microarray technologies, although reliable and useful, offer a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes. Thanks to the introduction in these last few years of NGS (Next-Generation-Sequencing) technologies we were able to produce the miRNome of HOS-like zebrafish embryos and take an almost complete look on their miRNA profile. Since in zebrafish was recently isolated a second paralogue gene of tbx5a, called tbx5b (see section 1.3.4 and (Albalat et al., 2010)), we choose to silence both tbx5a and tbx5b paralogs to avoid the possible reduntant role of

tbx5b in heart development. Although a recent paper points out the predominancy of tbx5a

as the true heart development master-gene, is still true that tbx5b is required for heart and fins morphogenesis in heart and fin development (Parrie et al., 2013). The downregulation was accomplished by coinjection of two specific morpholinos against tbx5a and tbx5b translational start site, at 1,5ng each one (3ng total) as previously described (Chiavacci et al., 2012). The downregulated zebrafish embryos (from now called Tbx5a/b-morphants), were raised until 48hpf, then the Tbx5a/b-morphants showing an HOS-like phenotype, consisting in lack of pectoral fin, pericardial edema and heartstring (hts) (Garrity et al., 2002), were collected. Control embryos (Ct-morphants) were injected with 3ng of control morpholino (MO-Ct) and processed as for Tbx5a/b-morphants. Total RNA was extracted from Tbx5a/b-morphants and Ct-morphants, and processed in order to obtain the miRNome via high-throughput DNA sequencing. Raw-data obtained by NGS were further analysed in silico and a pool of miRNA candidates was selected. The main criteria for the in silico screening were: 1) miRNA abundance, evidenced by reads; 2) fold change variation compared to Ct-morphants. A third criteria was applied to 1) and 2): data already present in the literature of clues indicating a cardiac development involvement. We choose to investigate firstly downregulated miRNAs as putative direct targets of the transcription factor Tbx5. miRNAs selected from the screening are shown in tab 2.

Tab. 2) The five miRNA candidates selected from the in silico screening. The fold decrease indicate the downregulation rate of Tbxa/b-morphants compared to the Ct-morphants resulting from the miRNome analysis.

3.2.2 In vivo screening

As we obtained the first pool of miRNA candidates by the in silico screening, we proceeded with a second in vivo functional screening by rescue experiments. This approach consisted in the coinjection of 0,25ng amount of the candidate miRNA mimic together with 1,5ng of MO-Tbx5a in the Tg(cmlc2:EGFP) transgenic line. This particular transgenic line, expressing the EGFP under the control of the specific cardiac promoter cmlc2 (see 1.3.3 section), allows the direct visualization of the entire heart under fluorescence microscopy. Coinjected embryos were assayed for mortality at 24hpf, then they were phenotyped at 72hpf for pectoral fin recovery by optical microscopy, and for heart morphology recovery by fluorescence microscopy. Although there was a good correlation between the severity of cardiac and pectoral fin defects in Tbx5a-morphants and hts mutant, we decided to separate the two issues because of a possible candidate miRNA function in only one of these two compartments. Because of the wide range of pectoral fin defects generated by Tbx5a downregulation via morpholino (Garrity et al., 2002) (Ahn et al., 2002), in order to better discriminate the fin restoring rate we decided to group the pectoral fin phenotypes in two categories only: 1. presence of both wild-type pectoral fins (from now referred as WT-fins, fig. 11a), 2. affected pectoral fins, comprehensive of curtailed and/or up-turned two or one pectoral fins, and absence of one or both pectoral fins (from now referred as NO-fins fig. 11b).

Fig. 11) Pectoral fin phenotypes at 72 hpf: a) WT-fins; b) NO-fins. Arrows point the two pectoral fins, stars mark their absence. Scalebar: 200μm.

Similarly, we divided the cardiac phenotypes obtained as follow: 1. Wild-type shaped heart (WT fig.12 a, a'). 2. Heartstring-shaped heart (hts fig 12 b, b'), as previously described (Garrity et al., 2002) (Ahn et al., 2002). 3. Bifid heart (bif fig. 12 c, c'), in case of detection of two distinct cmlc2-positive cell mass. 4. Aspecific heart defects (asp fig. 12 d, d', e, e', f, f'), comprising all the observed heart defects besides heartstring and bifid heart (e.g. absent or incomplete looping , affection of one or both chambers shape, position or volume etc...).

Fig. 12) Cardiac phenotype categories at 72 hpf in Tg(cmlc2:EGFP) embryos injected with MO-Tbx5a (1,5ng) plus miRNA mimic candidate (0,25ng). a, a') WT: wild type heart; b, b') hts: heartstring; c, c') bif: bifid heart; d, d', e, e', f, f') asp: aspecific heart phenotypes. A: atrium; V: ventricle; OFT: out-flow tract (arrow). Scalebar: 100μm.