Mol. Cells 2014; 37(4): 330-336
Published online April 8, 2014
https://doi.org/10.14348/molcells.2014.0003
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: lym@knu.ac.kr
Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount
Keywords endothelial-mesenchymal transformation, heart valve formation, thymosin beta4, zebrafish
Thymosin beta 4 (TB4) is a water-soluble 43-amino acid poly-peptide that does not possess signal sequences for secretion or nuclear localization signaling (NLS). TB4 has numerous functions, the most prominent of which involves the sequestration of G-actin monomers and the actin-cytoskeletal organization necessary for cell motility, organogenesis, and other cellular events (Huff et al., 2001). TB4 promotes skin and corneal wound healing by enhancing the endothelial cell migration, proliferation, differentiation, and the survival of a variety of cells (Malinda et al., 1997). It has been reported that TB4 can activate the survival kinase Akt and play a potent role in the protection of cardiac muscle from cell death after ischemic damage, as occurs in the setting of myocardial infarction (MI) (Bock-Marquette et al., 2004). TB4 can also promote angiogenesis in ischemic areas (Bock-Marquette et al., 2009; Smart et al., 2006), contribute to heart wound healing (Malinda et al., 1997; 1999) and promote cancer metastasis (Sribenja et al., 2013) and inflammatory responses (Huff et al., 2001; Sosne et al., 2002). Furthermore, TB4 has been reported to prime epicardium-derived progenitors to differentiate into cardiomyocytes (Smart et al., 2011) or coronary vessels (Bock-Marquette et al., 2009). Thus, it appears that TB4 has pleiotropic effects in various cellular responses, which included the promotion of cardiac repair (Bock-Marquette et al., 2004; Goldstein et al., 2012; Thatcher et al., 2012), cancer growth, and metastasis (Ji et al., 2013; Sribenja et al., 2013). TB4 has been reported to be present at elevated levels in the developing blood vessels and heart endocardial cushions of early mouse embryos post-implantation (Gomez-Marquez, 1996), which suggests it plays a key role in the formation of the cardiovascular system. TB4 is upregulated by tenascin-C signaling, which is likely involved in valve calcification (Li et al., 2002). However, the precise role played and the mechanism of TB4 in valve formation has not been investigated.
The developmental processes involved in heart formation have widely studied in experimental animal models, and it is known that cardiac valve formation is initially started by myocardial inductive signals in the non-chamber region. The human heart tube is composed of an outer layer of myocardium and an inner lining of endocardial cells, separated by an extensive extracellular matrix (ECM), which is referred to as cardiac jelly. After rightward looping of the heart, the cardiac jelly overlying the future atrioventricular canal (AVC) and outflow tract (OT) expands into cardiac cushions (Derynck and Zhang, 2003). Endothelial mesenchymal transformation (EMT) is an essential event characterized by delamination and invasion of cardiac jelly by endocardial cells in cardiac cushions (Markwald et al., 1977). Therefore, we tried to determine whether TB4 is involved in EMT during cardiac valve formation. In this study, we used a zebrafish model because this model offers several distinct advantages as a genetic and developmental model system for studies of cardiovascular defects. Furthermore, this model allows investigation of genes required for cardiovascular development because the zebrafish has a transparent heart during the early stage of heart development (Bakkers, 2011). During zebrafish heart development, heart looping starts at 36 h post-fertilization (hpf) and functional valves are almost completely formed by 48 hpf, although valve development is completed at around 55 hpf (Stainier et al., 2002). In this study, we found that the only member of the zTB family expressed in lateral plate mesoderm (LMP) during the early stage and in the heart region in the late stage was zTB4. Furthermore, knock-down of zTB4 with antisense morpholino oligomers induced linear heart tube formation and prevented AVC formation and the expressions of AVC marker genes. However, zTB4 gene rescued heart defects and the AVC marker gene loss observed in zTB4 morphants. Knock-down of zTB4 also attenuated EMT marker gene expressions in zebrafish AVC and in sheep endothelial cells induced by TGFβ1. Taken together, we suggest that TB4 plays an essential role in the regulation of EMT in zebrafish AVC and facilitates heart valve development.
Zebrafish strains were maintained as previously described (Westerfield, 1993). Animal experiments were performed according to the guidelines for the care and use of laboratory animals issued by the institutional ethical animal care committee of Kyungpook National University (Korea). The AB strain was used as a wild-type control. We maintained transgenic lines expressing enhanced green fluorescent protein (EGFP) under the control of a cmlc2 promoter, Tg(cmlc2:EGFP) (Huang et al., 2003), and flk-1 promoter, Tg(flk-1:EGFP)s843 (Jin et al., 2005). Embryos were dechorionated with pronase at each developmental stage, and treated with 0.003% 1-phenyl-2-thio-urea (PTU) (Sigma) to inhibit pigment formation.
zTB4 morpholino was synthesized by Gene-Tool, LLC (USA); its sequence was 5′-AGGTCTGAAAAAGGAAATGACAGGA-3′. MO was resuspended in diethylpyrocarbonate (DEPC)-treated water and then injected into one-or two-cell stage embryos collected by mating transgenic zebrafish using a micro-injector (M3301R, WPI, USA).
Digoxigenin-labeled antisense RNA probes specific for
Whole mount zebrafish embryos were immunostained with a mouse monoclonal antibody against Zn5 (1:500; Hybridoma Bank, USA) and Alexa Fluor 594-conjugated anti-mouse IgG antibody (1:1000, Molecular Probes, USA) as a secondary antibody. Images of the immunostained hearts were obtained with a Zeiss LSM5 Pascal confocal microscope.
Sheep aortic valve endothelial cells (AVECs) were kindly provided Dr. Joyce Bischoff’s Lab in Children’s Hospital, Boston (Harvard Medical School) (Paranya et al., 2001) and cultured on gelatin-coated tissue culture plates at 37°C in a humidified atmosphere incubator containing 5% CO2 in EBM (Hyclone, USA) supplemented with 20% fetal bovine serum (FBS, Hyc-lone) and antibiotics. Ovine AVECs were used at passages 7?10.
A pool of 4 distinct siRNAs directed against TB4 was synthesized by Qiagen Genosolution siRNA (USA). Subconfluent sheep valve endothelial cells (passage 6?10) were transfected with TB4 siRNA using a microporator (Invitrogen, USA).
Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA synthesis from total RNA was performed using the first strand cDNA synthesis kit (Promega, USA). cDNA was used as a template for PCR-reactions, which were conducted using specific primers (Table 1).
Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Whatman, England). After blocking membranes with 5% non-fat milk in Tris buffered saline (TBS) containing 0.1% Tween 20 for 1 h at RT, membranes were incubated with the appropriate primary antibody at 4°C overnight, and then with horseradish peroxidase-conjugated mouse or rabbit IgG antibody at RT for 1 h. Blots were developed using West Pico chemiluminescent substrate (PIERCE, USA).
Ovine aortic endothelial cells on coverslips were fixed with 10% formaldehyde solution for 10 min at RT. Cells were then permeabilized with 0.5% TritonX-100 in PBS for 10 min, and blocked with 1% BSA in PBS. After blocking, anti-CD31 and anti-α-smooth muscle actin (α-SMA) antibodies in PBS containing 1% BSA were added at 4°C overnight. The cells were then washed with PBS-T and incubated with anti-mouse FITC-conjugated and anti-goat Texas red-conjugated antibodies for 1 h. The cells on coverslips were then washed with PBS-T and mounted with DAPI mounting solution onto slides. Fluorescent images were obtained using a Zeiss fluorescence microscope (LSM5, Pascal, Germany).
To start a comparative analysis on sequence homologies of thymosin family members in zebrafish and man, we used the Genebee database and compared human TB4 with zebrafish thymosin family amino acid sequences. Phylogenetic analysis showed that human TB4 (hTB4; NM021109) protein clustered with zebrafish TB1 (zTB1; DQ989583), zebrafish zTB4 (zTB4; DQ119892), and zebrafish TB10 (zTB10; BC115156) (Fig. 1A). Multiple sequence alignment (MSA) analysis revealed high similarity between zTB4 and hTB4. More specifically, the percentage similarity between zTB4 and hZTB4 was 81%, and the amino acid sequences of G-actin sequestering sites in zTB family and hTB4 were identical (Figs. 1B and 1C). MSA revealed 81?100% identity at the protein level between zTB4 and its orthologs in man (NM021109) and in mouse (NM021278) (Fig. 1C).
Expression of thymosin family in early zebrafish embryos was analyzed with zTB family anti-sense probes by whole mount
Because zTB4 was found to be expressed in heart, we sought to determine its function during embryonic heart development. Accordingly, we designed splicing inhibitory morpholinos that target the splice donor site of exon E2 to knockdown zTB4 (Fig. 2A). As shown in Fig. 2B, zTB4 morpholino was effective at disrupting the splicing of zTB4 pre-mRNA, and resulted in remarkable reductions in mature mRNA levels. Based on the severity of phenotypic changes observed at 48 hpf, we categorized the morphants into three types (Supplementary Fig. 2). Type I embryos with heart edema were used in further experiments. We confirmed that the first obvious phenotype was pericardial edema at 48 hpf, indicating a cardiac defect (Fig. 2C). zTB4 morphants had a linearized morphology (Fig. 2C, b, d) in contrast to control embryos, which showed a s-shaped looping heart morphology at this time in the valve region (Fig. 2C, a, c). These observations indicate that zTB4 might regulate looping of the heart during development.
It has been reported that heart valve development is tightly regulated by signaling between endocardial and underlying myocardial cell layers in the atrio-ventricular canal (AVC) (Johnson et al., 2003). Glycosaminoglycan hyaluronic acid (HA) exists as a hydrated gel that expands the extracellular space, regulates ligand availability, and interacts with numerous ECM components including proteoglycan (versican), a major constituent of cardiac jelly in the AVC (Armstrong and Bischoff, 2004). To investigate the underlying basis of chamber maturation and normal cardiac tube looping, we examined the expression of the cardiac specific marker,
To confirm the role of zTB4 during heart valve formation, we examined the differentiation of endocardial AVC cells in zTB4 morphants. Anti-Zn5 antibody recognizes Dm-grasp, a cell-surface adhesion molecule of the immunoglobulin superfamily in zebrafish AVC cushions (Beis et al., 2005), and enables the visualization of myocardial and endocardial cells undergoing EMT in AVCs. In the AVC region of control
To confirm the role played by TB4 in the EMT of valve endothelial cells, we treated sheep aortic valve endothelial cells with TGF-β1 (1 ng/ml) for 8 days, because TGF-β1 is known to induce EMT in these cells (Paranya et al., 2001). TGF-β1-treated cells demonstrated slower growth kinetics and exhibited a spindle-shaped morphology instead of the cobble-stone morphology of non-treated control cells (Fig. 4B). Upregulation of TB4 mRNA expression was observed in TGF-β1-treated cells, while knockdown of TB4 by small interfering (si) RNA clearly abolished TGF-β1-induced TB4 gene expression (Fig. 4C). Furthermore, TB4 mRNA expression was upregulated in TGF-β1-treated cells, and knockdown of TB4 by small interfering (si) RNA clearly abolished TGF-β1-induced TB4 gene expression (Fig. 4C). In addition, TGF-β1 induced α-smooth muscle actin (SMA) expression but decreased CD31 expression at the mRNA and protein levels. However, knockdown of TB4 recovered CD31 but decreased α-SMA in the presence of TGF-β1, suggesting that TB4 is necessary for TGF-β1-induced EMT (Fig. 4C). Furthermore, immunofluorescent assays confirmed these findings (Fig. 4D). Taken together, our results demonstrate that TB4 is involved in the EMT required for cardiac valve cushion formation in zebrafish embryos.
Here, we identified for the first time that TB4 is a regulator of heart valve formation in zebrafish embryos. TB4 has multiple functions in cellular responses during embryonic organ development and pathogenic states, but the role of TB4 in heart valvular development has not been well identified. Valvular developmental mechanisms should be identified because adult heart valve diseases, such as, cardiac valve fibrosis can be treated by redirecting or amplifying disease-related EMT. In the present study, zTB4 was expressed in developing LPM and in the heart valve region. zTB4 morphants showed a linear heart tube and reduced expressions of valve marker genes, whereas recovery of the zTB4 gene in morphants restored abnormal phenotypes and gene expressions. The EMT marker protein, Dm-grasp was stained with Zn5 antibody to observe distinct cellular differentiation patterns in valve regions. However, Zn5 staining disappeared in AVC linear heart tubes in zTB4 morphants, indicating that zTB4 is essential for the EMT required for cardiac valve formation.
EMT is a process that generates valve mesenchyme and this process is followed by endocardial cell proliferation, which elongate valve leaflets (Armstrong and Bischoff, 2004). TGFβ is a growth factor that induces EMT (Paranya et al., 2001), but this process is inhibited by VEGF in isolated valve endothelial cells (Yang et al., 2008). VEGF signaling has been importantly implicated during valve formation, and VEGFR2 acts to direct morphogenesis in AVC cushions (Lee et al., 2006), whereas VEGFR1 signaling does in the outflow tract (Stankunas et al., 2010). Furthermore, it has been shown that TB4 mediates the transcriptions of a number of genes, including VEGF and matrix metalloproteinases (MMPs), and therefore, increases endothelial cell migration and angiogenesis (Dube et al., 2012). TB4 also induces angiogenesis
TB4 has been suggested to have therapeutic potential for cardiac repair and regeneration because of its pleiotropic effects on various cellular responses, such as, wound healing, angiogenesis, and cardiomyocyte survival (Dube et al., 2012). In addition to its role in the regeneration of cardiomyocytes after myocardiac infarction, it might also be useful for cardiac valve regeneration in congenital heart disease or adult heart valve fibrosis by increasing EMT. In conclusion, our results demonstrate that TB4 participates in valve formation by promoting EMT in cardiac cushions. However, the detailed molecular and cellular mechanisms involved in the regulation of EMT in valve endocardial cells by TB4 remain to be determined.
. PCR primer sequences
Gene name | Primers sequences |
---|---|
TB4 | F : 5′-CTCCTTCTCCTGACCAGCTT-3′, |
α-SMA | F : 5′-TCTTTGAAGCCAACGACCTG-3′, |
CD31 | F : 5′-CCT GACATCTCCACCAATG-3′, |
F : 5′-GATCCACATCTGCTGGAA-3′, |
Mol. Cells 2014; 37(4): 330-336
Published online April 30, 2014 https://doi.org/10.14348/molcells.2014.0003
Copyright © The Korean Society for Molecular and Cellular Biology.
Sun-Hye Shin1,2, Sangkyu Lee1, Jong-Sup Bae1, Jun-Goo Jee1, Hee-Jae Cha3, and You Mie Lee1,2,*
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu 702-701, Korea, 2School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea, 3Department of Parasitology and Genetics, Kosin University College of Medicine, Busan 602-703, Korea
Correspondence to:*Correspondence: lym@knu.ac.kr
Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount
Keywords: endothelial-mesenchymal transformation, heart valve formation, thymosin beta4, zebrafish
Thymosin beta 4 (TB4) is a water-soluble 43-amino acid poly-peptide that does not possess signal sequences for secretion or nuclear localization signaling (NLS). TB4 has numerous functions, the most prominent of which involves the sequestration of G-actin monomers and the actin-cytoskeletal organization necessary for cell motility, organogenesis, and other cellular events (Huff et al., 2001). TB4 promotes skin and corneal wound healing by enhancing the endothelial cell migration, proliferation, differentiation, and the survival of a variety of cells (Malinda et al., 1997). It has been reported that TB4 can activate the survival kinase Akt and play a potent role in the protection of cardiac muscle from cell death after ischemic damage, as occurs in the setting of myocardial infarction (MI) (Bock-Marquette et al., 2004). TB4 can also promote angiogenesis in ischemic areas (Bock-Marquette et al., 2009; Smart et al., 2006), contribute to heart wound healing (Malinda et al., 1997; 1999) and promote cancer metastasis (Sribenja et al., 2013) and inflammatory responses (Huff et al., 2001; Sosne et al., 2002). Furthermore, TB4 has been reported to prime epicardium-derived progenitors to differentiate into cardiomyocytes (Smart et al., 2011) or coronary vessels (Bock-Marquette et al., 2009). Thus, it appears that TB4 has pleiotropic effects in various cellular responses, which included the promotion of cardiac repair (Bock-Marquette et al., 2004; Goldstein et al., 2012; Thatcher et al., 2012), cancer growth, and metastasis (Ji et al., 2013; Sribenja et al., 2013). TB4 has been reported to be present at elevated levels in the developing blood vessels and heart endocardial cushions of early mouse embryos post-implantation (Gomez-Marquez, 1996), which suggests it plays a key role in the formation of the cardiovascular system. TB4 is upregulated by tenascin-C signaling, which is likely involved in valve calcification (Li et al., 2002). However, the precise role played and the mechanism of TB4 in valve formation has not been investigated.
The developmental processes involved in heart formation have widely studied in experimental animal models, and it is known that cardiac valve formation is initially started by myocardial inductive signals in the non-chamber region. The human heart tube is composed of an outer layer of myocardium and an inner lining of endocardial cells, separated by an extensive extracellular matrix (ECM), which is referred to as cardiac jelly. After rightward looping of the heart, the cardiac jelly overlying the future atrioventricular canal (AVC) and outflow tract (OT) expands into cardiac cushions (Derynck and Zhang, 2003). Endothelial mesenchymal transformation (EMT) is an essential event characterized by delamination and invasion of cardiac jelly by endocardial cells in cardiac cushions (Markwald et al., 1977). Therefore, we tried to determine whether TB4 is involved in EMT during cardiac valve formation. In this study, we used a zebrafish model because this model offers several distinct advantages as a genetic and developmental model system for studies of cardiovascular defects. Furthermore, this model allows investigation of genes required for cardiovascular development because the zebrafish has a transparent heart during the early stage of heart development (Bakkers, 2011). During zebrafish heart development, heart looping starts at 36 h post-fertilization (hpf) and functional valves are almost completely formed by 48 hpf, although valve development is completed at around 55 hpf (Stainier et al., 2002). In this study, we found that the only member of the zTB family expressed in lateral plate mesoderm (LMP) during the early stage and in the heart region in the late stage was zTB4. Furthermore, knock-down of zTB4 with antisense morpholino oligomers induced linear heart tube formation and prevented AVC formation and the expressions of AVC marker genes. However, zTB4 gene rescued heart defects and the AVC marker gene loss observed in zTB4 morphants. Knock-down of zTB4 also attenuated EMT marker gene expressions in zebrafish AVC and in sheep endothelial cells induced by TGFβ1. Taken together, we suggest that TB4 plays an essential role in the regulation of EMT in zebrafish AVC and facilitates heart valve development.
Zebrafish strains were maintained as previously described (Westerfield, 1993). Animal experiments were performed according to the guidelines for the care and use of laboratory animals issued by the institutional ethical animal care committee of Kyungpook National University (Korea). The AB strain was used as a wild-type control. We maintained transgenic lines expressing enhanced green fluorescent protein (EGFP) under the control of a cmlc2 promoter, Tg(cmlc2:EGFP) (Huang et al., 2003), and flk-1 promoter, Tg(flk-1:EGFP)s843 (Jin et al., 2005). Embryos were dechorionated with pronase at each developmental stage, and treated with 0.003% 1-phenyl-2-thio-urea (PTU) (Sigma) to inhibit pigment formation.
zTB4 morpholino was synthesized by Gene-Tool, LLC (USA); its sequence was 5′-AGGTCTGAAAAAGGAAATGACAGGA-3′. MO was resuspended in diethylpyrocarbonate (DEPC)-treated water and then injected into one-or two-cell stage embryos collected by mating transgenic zebrafish using a micro-injector (M3301R, WPI, USA).
Digoxigenin-labeled antisense RNA probes specific for
Whole mount zebrafish embryos were immunostained with a mouse monoclonal antibody against Zn5 (1:500; Hybridoma Bank, USA) and Alexa Fluor 594-conjugated anti-mouse IgG antibody (1:1000, Molecular Probes, USA) as a secondary antibody. Images of the immunostained hearts were obtained with a Zeiss LSM5 Pascal confocal microscope.
Sheep aortic valve endothelial cells (AVECs) were kindly provided Dr. Joyce Bischoff’s Lab in Children’s Hospital, Boston (Harvard Medical School) (Paranya et al., 2001) and cultured on gelatin-coated tissue culture plates at 37°C in a humidified atmosphere incubator containing 5% CO2 in EBM (Hyclone, USA) supplemented with 20% fetal bovine serum (FBS, Hyc-lone) and antibiotics. Ovine AVECs were used at passages 7?10.
A pool of 4 distinct siRNAs directed against TB4 was synthesized by Qiagen Genosolution siRNA (USA). Subconfluent sheep valve endothelial cells (passage 6?10) were transfected with TB4 siRNA using a microporator (Invitrogen, USA).
Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA synthesis from total RNA was performed using the first strand cDNA synthesis kit (Promega, USA). cDNA was used as a template for PCR-reactions, which were conducted using specific primers (Table 1).
Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Whatman, England). After blocking membranes with 5% non-fat milk in Tris buffered saline (TBS) containing 0.1% Tween 20 for 1 h at RT, membranes were incubated with the appropriate primary antibody at 4°C overnight, and then with horseradish peroxidase-conjugated mouse or rabbit IgG antibody at RT for 1 h. Blots were developed using West Pico chemiluminescent substrate (PIERCE, USA).
Ovine aortic endothelial cells on coverslips were fixed with 10% formaldehyde solution for 10 min at RT. Cells were then permeabilized with 0.5% TritonX-100 in PBS for 10 min, and blocked with 1% BSA in PBS. After blocking, anti-CD31 and anti-α-smooth muscle actin (α-SMA) antibodies in PBS containing 1% BSA were added at 4°C overnight. The cells were then washed with PBS-T and incubated with anti-mouse FITC-conjugated and anti-goat Texas red-conjugated antibodies for 1 h. The cells on coverslips were then washed with PBS-T and mounted with DAPI mounting solution onto slides. Fluorescent images were obtained using a Zeiss fluorescence microscope (LSM5, Pascal, Germany).
To start a comparative analysis on sequence homologies of thymosin family members in zebrafish and man, we used the Genebee database and compared human TB4 with zebrafish thymosin family amino acid sequences. Phylogenetic analysis showed that human TB4 (hTB4; NM021109) protein clustered with zebrafish TB1 (zTB1; DQ989583), zebrafish zTB4 (zTB4; DQ119892), and zebrafish TB10 (zTB10; BC115156) (Fig. 1A). Multiple sequence alignment (MSA) analysis revealed high similarity between zTB4 and hTB4. More specifically, the percentage similarity between zTB4 and hZTB4 was 81%, and the amino acid sequences of G-actin sequestering sites in zTB family and hTB4 were identical (Figs. 1B and 1C). MSA revealed 81?100% identity at the protein level between zTB4 and its orthologs in man (NM021109) and in mouse (NM021278) (Fig. 1C).
Expression of thymosin family in early zebrafish embryos was analyzed with zTB family anti-sense probes by whole mount
Because zTB4 was found to be expressed in heart, we sought to determine its function during embryonic heart development. Accordingly, we designed splicing inhibitory morpholinos that target the splice donor site of exon E2 to knockdown zTB4 (Fig. 2A). As shown in Fig. 2B, zTB4 morpholino was effective at disrupting the splicing of zTB4 pre-mRNA, and resulted in remarkable reductions in mature mRNA levels. Based on the severity of phenotypic changes observed at 48 hpf, we categorized the morphants into three types (Supplementary Fig. 2). Type I embryos with heart edema were used in further experiments. We confirmed that the first obvious phenotype was pericardial edema at 48 hpf, indicating a cardiac defect (Fig. 2C). zTB4 morphants had a linearized morphology (Fig. 2C, b, d) in contrast to control embryos, which showed a s-shaped looping heart morphology at this time in the valve region (Fig. 2C, a, c). These observations indicate that zTB4 might regulate looping of the heart during development.
It has been reported that heart valve development is tightly regulated by signaling between endocardial and underlying myocardial cell layers in the atrio-ventricular canal (AVC) (Johnson et al., 2003). Glycosaminoglycan hyaluronic acid (HA) exists as a hydrated gel that expands the extracellular space, regulates ligand availability, and interacts with numerous ECM components including proteoglycan (versican), a major constituent of cardiac jelly in the AVC (Armstrong and Bischoff, 2004). To investigate the underlying basis of chamber maturation and normal cardiac tube looping, we examined the expression of the cardiac specific marker,
To confirm the role of zTB4 during heart valve formation, we examined the differentiation of endocardial AVC cells in zTB4 morphants. Anti-Zn5 antibody recognizes Dm-grasp, a cell-surface adhesion molecule of the immunoglobulin superfamily in zebrafish AVC cushions (Beis et al., 2005), and enables the visualization of myocardial and endocardial cells undergoing EMT in AVCs. In the AVC region of control
To confirm the role played by TB4 in the EMT of valve endothelial cells, we treated sheep aortic valve endothelial cells with TGF-β1 (1 ng/ml) for 8 days, because TGF-β1 is known to induce EMT in these cells (Paranya et al., 2001). TGF-β1-treated cells demonstrated slower growth kinetics and exhibited a spindle-shaped morphology instead of the cobble-stone morphology of non-treated control cells (Fig. 4B). Upregulation of TB4 mRNA expression was observed in TGF-β1-treated cells, while knockdown of TB4 by small interfering (si) RNA clearly abolished TGF-β1-induced TB4 gene expression (Fig. 4C). Furthermore, TB4 mRNA expression was upregulated in TGF-β1-treated cells, and knockdown of TB4 by small interfering (si) RNA clearly abolished TGF-β1-induced TB4 gene expression (Fig. 4C). In addition, TGF-β1 induced α-smooth muscle actin (SMA) expression but decreased CD31 expression at the mRNA and protein levels. However, knockdown of TB4 recovered CD31 but decreased α-SMA in the presence of TGF-β1, suggesting that TB4 is necessary for TGF-β1-induced EMT (Fig. 4C). Furthermore, immunofluorescent assays confirmed these findings (Fig. 4D). Taken together, our results demonstrate that TB4 is involved in the EMT required for cardiac valve cushion formation in zebrafish embryos.
Here, we identified for the first time that TB4 is a regulator of heart valve formation in zebrafish embryos. TB4 has multiple functions in cellular responses during embryonic organ development and pathogenic states, but the role of TB4 in heart valvular development has not been well identified. Valvular developmental mechanisms should be identified because adult heart valve diseases, such as, cardiac valve fibrosis can be treated by redirecting or amplifying disease-related EMT. In the present study, zTB4 was expressed in developing LPM and in the heart valve region. zTB4 morphants showed a linear heart tube and reduced expressions of valve marker genes, whereas recovery of the zTB4 gene in morphants restored abnormal phenotypes and gene expressions. The EMT marker protein, Dm-grasp was stained with Zn5 antibody to observe distinct cellular differentiation patterns in valve regions. However, Zn5 staining disappeared in AVC linear heart tubes in zTB4 morphants, indicating that zTB4 is essential for the EMT required for cardiac valve formation.
EMT is a process that generates valve mesenchyme and this process is followed by endocardial cell proliferation, which elongate valve leaflets (Armstrong and Bischoff, 2004). TGFβ is a growth factor that induces EMT (Paranya et al., 2001), but this process is inhibited by VEGF in isolated valve endothelial cells (Yang et al., 2008). VEGF signaling has been importantly implicated during valve formation, and VEGFR2 acts to direct morphogenesis in AVC cushions (Lee et al., 2006), whereas VEGFR1 signaling does in the outflow tract (Stankunas et al., 2010). Furthermore, it has been shown that TB4 mediates the transcriptions of a number of genes, including VEGF and matrix metalloproteinases (MMPs), and therefore, increases endothelial cell migration and angiogenesis (Dube et al., 2012). TB4 also induces angiogenesis
TB4 has been suggested to have therapeutic potential for cardiac repair and regeneration because of its pleiotropic effects on various cellular responses, such as, wound healing, angiogenesis, and cardiomyocyte survival (Dube et al., 2012). In addition to its role in the regeneration of cardiomyocytes after myocardiac infarction, it might also be useful for cardiac valve regeneration in congenital heart disease or adult heart valve fibrosis by increasing EMT. In conclusion, our results demonstrate that TB4 participates in valve formation by promoting EMT in cardiac cushions. However, the detailed molecular and cellular mechanisms involved in the regulation of EMT in valve endocardial cells by TB4 remain to be determined.
. PCR primer sequences.
Gene name | Primers sequences |
---|---|
TB4 | F : 5′-CTCCTTCTCCTGACCAGCTT-3′, |
α-SMA | F : 5′-TCTTTGAAGCCAACGACCTG-3′, |
CD31 | F : 5′-CCT GACATCTCCACCAATG-3′, |
F : 5′-GATCCACATCTGCTGGAA-3′, |
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