Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT^−/−) cardiac myocytes, the thick and thin filaments and α-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT^−/− cardiac myocytes showed regular Ca²⁺-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca²⁺ handling systems develop independently of the contractile system. In contrast, heterozygous cTnT^+/− mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.     

Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2

Various hypotheses have been proposed to explain the molecule processes of sarcomere assembly, partially due to the lack of systematic genetic studies of sarcomeric genes in an in vivo model. Towards the goal of developing zebrafish as a vertebrate model for this purpose, we characterized myofibrillogenesis in a developing zebrafish heart and went on to examine the functions of cardiac troponin T (tnnt2). We found that sarcomere assembly in zebrafish heart was initiated from a non-striated actin filament network at the perimembrane region, whereas sarcomeric myosin is independently assembled into thick filaments of variable length before integrating into the thin filament network. Compared to Z-discs that are initially aligned to form shorter periodic dots and expanded longitudinally at a later time, M-lines assemble later and have a constant length. Depletion of full-length tnnt2 disrupted the striation of thin filaments and Z-bodies, which sequentially affects the striation of thick filaments and M-lines. Conversely, truncation of a C-terminal troponin complex-binding domain did not affect the striation of these sarcomere sub-structures, but resulted in reduced cardiomyocyte size. In summary, our data indicates that zebrafish are a valuable in vivo model for studying both myofibrillogenesis and sarcomere-based cardiac diseases.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

In Situ Time-Resolved FRET Reveals Effects of Sarcomere Length on Cardiac Thin-Filament Activation

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca²⁺ and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca²⁺-induced opening of N-cTnC and is presumed to also destabilize cTnI–actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca²⁺-sensitizing N-cTnC–cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca²⁺-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)_AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca²⁺ and 1.80 and 2.2-μm sarcomere length. Mg²⁺-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca²⁺] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca²⁺ sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.     

Intracellular Ca dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastrucural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca²⁺] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca²⁺ overload. The [Ca²⁺] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.     

Functional characterization of human myosin-binding protein C3 variants associated with hypertrophic cardiomyopathy reveals exon-specific cardiac phenotypes in zebrafish model

Myosin-binding protein C 3 (MYBPC3) variants are the most common cause of hypertrophic cardiomyopathy (HCM). HCM is a complex cardiac disorder due to its significant genetic and clinical heterogeneity. MYBPC3 variants genotype–phenotype associations remain poorly understood. We investigated the impact of two novel human MYBPC3 splice-site variants: V1: c.654+2_654+4dupTGG targeting exon 5 using morpholino MOe5i5; and V2: c.772+1G>A targeting exon 6 using MOe6i6; located within C1 domain of cMyBP-C protein, known to be critical in regulating sarcomere structure and contractility. Zebrafish MOe5i5 and MOe6i6 morphants recapitulated typical characteristics of human HCM with cardiac phenotypes of varying severity, including reduced cardiomyocyte count, thickened ventricular myocardial wall, a drastic reduction in heart rate, stroke volume, and cardiac output. Analysis of all cardiac morphological and functional parameters demonstrated that V2 cardiac phenotype was more severe than V1. Coinjection with synthetic human MYBPC3 messenger RNA (mRNA) partially rescued disparate cardiac phenotypes in each zebrafish morphant. While human MYBPC3 mRNA partially restored the decreased heart rate in V1 morphants and displayed increased percentages of ejection fraction, fractional shortening, and area change, it failed to revert the V1 ventricular myocardial thickness. These results suggest a possible V1 impact on cardiac contractility. In contrast, attempts to rescue V2 morphants only restored the ventricular myocardial wall hypertrophy phenotype but had no significant effect on impaired heart rate, suggesting a potential V2 impact on the cardiac structure. Our study provides evidence of an association between MYBPC3 exon-specific cardiac phenotypes in the zebrafish model providing important insights into how these genetic variants contribute to HCM disease.     

Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes.

Troponin I is the putative molecular switch for Ca(2+)-activated contraction within the myofilament of striated muscles. To gain insight into functional troponin I domain(s) in the context of the intact myofilament, adenovirus-mediated gene transfer was used to replace endogenous cardiac troponin I within the myofilaments of adult cardiac myocytes with the slow skeletal isoform or a chimera of the slow skeletal and cardiac isoforms. Efficient expression and myofilament incorporation were observed in myocytes with each exogenous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture. Contractile function studies in single, permeabilized myocytes expressing exogenous troponin I provided support for the presence of a Ca(2+)-sensitive regulatory domain in the carboxyl terminus of troponin I and a second, newly defined Ca(2+)-sensitive domain residing in the amino terminus of troponin I. Additional experiments demonstrated that the isoform-specific, acidic pH-induced contractile dysfunction in myocytes appears to lie in the carboxyl terminus of troponin I. Functional results obtained from adult cardiac myocytes expressing the chimera or isoforms of troponin I now define multiple troponin I regulatory domains operating in the intact myofilament and provide new insight into the Ca(2+)-sensitive properties of troponin I during contraction.     

TnI Structural Interface with the N-Terminal Lobe of TnC as a Determinant of Cardiac Contractility

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca²⁺ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca²⁺ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca²⁺ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca²⁺ transient. Free-energy perturbation calculations of cTnC-Ca²⁺ binding indicate no difference in cTnC-Ca²⁺ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells

Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus.     

Differential requirement for BMP signaling in atrial and ventricular lineages establishes cardiac chamber proportionality

The function of an organ relies upon the proper relative proportions of its individual operational components. For example, effective embryonic circulation requires the appropriate relative sizes of each of the distinct pumps created by the atrial and ventricular cardiac chambers. Although the differences between atrial and ventricular cardiomyocytes are well established, little is known about the mechanisms regulating production of proportional numbers of each cell type. We find that mutation of the zebrafish type I BMP receptor gene alk8 causes reduction of atrial size without affecting the ventricle. Loss of atrial tissue is evident in the lateral mesoderm prior to heart tube formation and results from the inhibition of BMP signaling during cardiac progenitor specification stages. Comparison of the effects of decreased and increased BMP signaling further demonstrates that atrial cardiomyocyte production correlates with levels of BMP signaling while ventricular cardiomyocyte production is less susceptible to manipulation of BMP signaling. Additionally, mosaic analysis provides evidence for a cell-autonomous requirement for BMP signaling during cardiomyocyte formation and chamber fate assignment. Together, our studies uncover a new role for BMP signaling in the regulation of chamber size, supporting a model in which differential reception of cardiac inductive signals establishes chamber proportion.     

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca²⁺ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca²⁺ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca²⁺ current (I_{Ca, T}) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of I_{Ca, T} and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of I_{Ca, T} mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in I_{Ca, T} and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.     

Suppression of Rap1 Impairs Cardiac Myofibrils and Conduction System in Zebrafish

Numerous studies have revealed that Rap1 (Ras-proximate-1 or Ras-related protein 1), a small GTPase protein, plays a crucial role in mediating cAMP signaling in isolated cardiac tissues and cell lines. However, the involvement of Rap1 in the cardiac development in vivo is largely unknown. By injecting anti-sense morpholino oligonucleotides to knock down Rap1a and Rap1b in zebrafish embryos, and in combination with time-lapsed imaging, in situ hybridization, immunohistochemistry and transmission electron microscope techniques, we seek to understand the role of Rap1 in cardiac development and functions. At an optimized low dose of mixed rap1a and rap1b morpholino oligonucleotides, the heart developed essentially normally until cardiac contraction occurred. Morphant hearts showed the myocardium defect phenotypes, most likely due to disrupted myofibril assembly and alignment. In vivo heart electrocardiography revealed prolonged P-R interval and QRS duration, consistent with an adherens junction defect and reduced Connexons in cardiac myocytes of morphants. We conclude that a proper level of Rap1 is crucial for heart morphogenesis and function, and suggest that Rap1 and/or their downstream factor genes are potential candidates for genetic screening for human heart diseases.     

Sarcomere thin filament regulatory isoforms. Evidence of a dominant effect of slow skeletal troponin I on cardiac contraction

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

miR-731 modulates the zebrafish heart morphogenesis via targeting Calcineurin/Nfatc3a pathway

BackgroundZebrafish miR-731 is orthologous of human miR-425, which has been demonstrated to have cardio-protective roles by a variety of mechanisms. The miR-731 morphants show pericardium enlargement, and many DEGs (differentially expressed genes) are enriched in ‘Cardiac muscle contraction’ and ‘Calcium signaling pathway’, implying that miR-731 plays a potential role in heart function and development. However，the in vivo physiological role of miR-731 in the heart needs to be fully defined.MethodsZebrafish miR-731 morphants were generated by morpholino knockdown, and miR-731 knockout zebrafish was generated by CRISRP/Cas9. We observed cardiac morphogenesis based on whole-mount in situ hybridization. Furthermore, RNA-seq and qRT-PCR were used to elucidate the molecular mechanism and analyze the gene expression. Double luciferase verification and Western blot were used to verify the target gene.ResultsThe depletion of miR-731 in zebrafish embryos caused the deficiency of cardiac development and function, which was associated with reduced heart rate, ventricular enlargement and heart looping disorder. In addition, mechanistic study demonstrated that Calcineurin/Nfatc3a signaling involved in miR-731 depletion induced abnormal cardiac function and developmental defects.ConclusionMiR-731 regulates cardiac function and morphogenesis through Calcineurin/Nfatc3a signaling.General significanceOur studies highlight the potential importance of miR-731 in cardiac development.     

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s,Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.