Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Sep 15.
Published in final edited form as: Dev Biol. 2013 Jul 11;381(2):389–400. doi: 10.1016/j.ydbio.2013.06.025

Small heat shock proteins Hspb7 and Hspb12 regulate early steps of cardiac morphogenesis

Gabriel E Rosenfeld 1, Emily J Mercer 1, Christopher E Mason 2,3, Todd Evans 1,*
PMCID: PMC3777613  NIHMSID: NIHMS506598  PMID: 23850773

Abstract

Cardiac morphogenesis is a complex multi-stage process, and the molecular basis for controlling distinct steps remains poorly understood. Because gata4 encodes a key transcriptional regulator of morphogenesis, we profiled transcript changes in cardiomyocytes when Gata4 protein is depleted from developing zebrafish embryos. We discovered that gata4 regulates expression of two small heat shock genes, hspb7 and hspb12, both of which are expressed in the embryonic heart. We show that depletion of Hspb7 or Hspb12 disrupts normal cardiac morphogenesis, at least in part due to defects in ventricular size and shape. We confirmed that gata4 interacts genetically with the hspb7/12 pathway, but surprisingly, we found that hspb7 also has an earlier, gata4-independent function. Depletion perturbs Kupffer’s vesicle (KV) morphology leading to a failure in establishing the left-right axis of asymmetry. Targeted depletion of Hspb7 in the yolk syncytial layer is sufficient to disrupt KV morphology and also causes an even earlier block to heart tube formation and a bifid phenotype. Recently, several genome-wide association studies found that HSPB7 SNPs are highly associated with idiopathic cardiomyopathies and heart failure. Therefore, GATA4 and HSPB7 may act alone or together to regulate morphogenesis with relevance to congenital and acquired human heart disease.

Introduction

Cardiomyopathies contribute significantly to morbidity and mortality, representing the most common congenital defects in newborns. Underlying most congenital cardiomyopathies are believed to be defects in heart morphogenesis (Gittenberger-de Groot et al., 2005). The GATA4 gene encodes a key transcriptional regulator that coordinates complex cardiac morphogenetic programs. Congenital cardiomyopathies caused by mutations in GATA4 are associated with a spectrum of common morphogenetic abnormalities including septal, outflow tract, and valvular defects (Rajagopal et al., 2007). Yet it remains unclear how mutations in GATA4 cause defects in heart development.

The heat shock protein family comprises a set of structurally similar chaperones regulating protein folding in response to environmental stress to maintain normal protein homeostasis. The classical heat shock response is activated by elevated temperatures and induces transcriptional upregulation of heat shock proteins by heat shock factor 1 (HSF1; (Westerheide et al., 2009). During embryonic development, environmental stressors can lead to abnormal aggregation of protein intermediates or cause protein mis-function (Balch et al., 2008). Moreover, embryogenesis itself generates stress as germ layers are organized and subsequently organs are constructed through morphogenetic rearrangements including gastrulation, convergent extension, epithelial-mesenchymal transitions, and tissue deformation (Davidson, 2011). Members of the heat shock family with molecular weights between 15–43 kDa are termed the small heat shock proteins (heat shock proteins-beta; HSPBs). HSPBs are expressed during embryonic development and may function as chaperones to ensure protein homeostasis. In contrast to HSF1-mediated responses to stress, the regulation of HSPBs for mediating organogenesis during embryonic development is less well investigated.

Characterized by a C-terminal alpha-crystallin domain, several diverse biological roles for HSPBs have been described, including remodeling of the cytoskeleton, regulation of apoptosis, and as a structural protein in the eye lens (Franck et al., 2004). Hspb1-9 are evolutionarily conserved from mammals to teleosts. Hspb10 is restricted to mammals while only lower vertebrate genomes encode Hspb11, Hspb12, and Hspb15 (Marvin et al., 2008). Mutations in HSPB proteins can cause several degenerative diseases including Charcot-Marie Tooth Disease (Evgrafov et al., 2004), Distal Hereditary Motor Neuropathy (dHMN, (Irobi et al., 2004), and cataract formation (Berry et al., 2001; Litt et al., 1998). Intriguingly, members of this family display tissue-specific expression patterns, and not all are responsive to heat shock, suggesting specific developmental functions independent of traditional environmental stressors (Marvin et al., 2008).

Cardiogenesis occurs through a series of extensive tissue morphogenetic orchestrations, from generation of a primordial heart tube, through cardiac looping, chamber formation, valve formation, and septation. One of the earliest steps of cardiac morphogenesis is dependent on the establishment of left-right asymmetry. Left-right asymmetry in zebrafish initiates from Kupffer’s vesicle (KV), analogous to the mammalian node, which generates a flow of signaling molecules toward the embryonic left side (Essner et al., 2005). Asymmetric gene expression is initiated by the feed-forward activation of spaw expression on the embryonic left side (Long et al., 2003). Downstream expression of the nodal antagonists lft1 and lft2 results in propagation of left-right asymmetry to the cardiac mesoderm (Long et al., 2003; Meno et al., 1999). Previous work has implicated gata4 functioning downstream of WNT/beta-catenin signaling to regulate the competence of cardiac mesoderm to express left-sided genes (Lin and Xu, 2009).

We showed previously that gata4 regulates cardiac morphogenesis and that embryos depleted of gata4 develop with a primordial heart tube that fails to loop or grow (Holtzinger and Evans, 2005). Here we investigated at the whole genome level the developmental program regulated by gata4 in myocardium prior to the looping stage of heart morphogenesis. When gata4 was depleted, we identified among the most down-regulated transcripts those encoding the small heat shock proteins Hspb7 and Hspb12. We analyzed loss-of-function phenotypes for these two HSPBs during zebrafish embryogenesis and discovered a previously unknown role for them in controlling cardiac morphogenesis. Surprisingly, we also discovered earlier roles for Hspb7 in the yolk syncytial layer (YSL) controlling precursor migration and left-right asymmetry, functions that are independent of gata4.

Materials and Methods

Zebrafish husbandry

All zebrafish strains were maintained as described (Westerfield, 1993) and staged according to standard developmental time points (Kimmel et al., 1995). The myl7:gfp strain was originally obtained from H.J. Tsai (Taiwan). The myl:actn3b-gfp and myl7-mKate-Caax lines were kindly provided by Deborah Yelon (UCSD, CA). The myl7:dsRed-nls; myl7:gfp line was generously provided by Nathalia Glickman Holtzman (Queens College, NY). The sox17:gfp strain was obtained from the Zebrafish International Resource Center (Eugene, Oregon).

Morpholino design and embryo injection

Morpholinos (MOs) were obtained from Genetools, LLC (Philomath, OR). The MOs targeting the 5′UTR of gata4 (G4MO) or the splice acceptor site of exon 3 of gata4 (G4MO1) were previously validated for specificity and were used as described (Holtzinger and Evans, 2005). All injections used 2 nl of MO dissolved in water. A standard control MO (5′-CCTCTTACCTCAGTTACATTTATA) was used at 0.25 mM. Two MOs were designed to target either the hspb7 sequence at the splice acceptor site of intron 1 (5′-CCTGTTCTGTCTGATGAAAAACATA, named 7MO) or at the start codon (5′-AGAAGAATTGCTCGCGCTCATTAGT, named 7MO1). We also generated two MOs that target hspb12 at the splice donor site of intron 2 (5′-GCGCTCCTCGCTTACTCTTTATGTG, called 12MO) or the start codon (5′-CATATAGAGAGCCGCAGCAGTGCAT-3′), called 12MO1, respectively. For single injection experiments, a concentration of 0.25 mM and 0.2 mM was used for hspb7 and hspb12 splice site MOs, respectively. 7MO1 and 12MO1 generated cardiac defects at 0.5 mM and above. For injection of both 7MO and 12MO, each was used at 0.2 mM. For dual knockdown experiments with 7MO1 at 0.7 mM, 7MO was used at 0.15 mM. For dual knockdown experiments with G4MO1 at a sub-threshold concentration of 0.25mM, 7M0 or 12MO were used at a concentration of 0.15mM.

Fluorescence activated cell sorting (FACS)

The gata4 morphant embryos or controls were dechorinated at 24 hpf and batches of ~200 embryos placed into 1.5 ml tubes. Embryos were dissociated by manual agitation with a pellet pestle (Fisher) and trypsinized with pre-heated TrypLE (Life Technologies) at 32C for 15 min on a rotator. Trypsinized samples were pipetted through a 35 μm cell strainer into a 5 ml tube, trypsin was inhibited by addition of 4 ml FACS buffer (L-15 medium supplemented with 1% heat inactivated FCS, 0.8 mM CaCl2, 50 U/ml penicillin, and 0.05 mg/ml streptomycin) followed by addition of FCS to 7.5% final concentration. Cells were pelleted at 300 RCF for 5 min and then washed with FACS buffer. Dissociated embryonic cells were resuspended at 7.5x106 cells/ml in FACS buffer. FACS was performed on a Vantage cell sorter (BD) into Trizol LS (Life Technologies), and stored at −80C until RNA isolation.

RNA Isolation and sequencing

RNA was isolated according to the Trizol protocol except that after adding ethanol, the solution was transferred to an RNeasy minElute column (Qiagen). On-column DNase digestion, subsequent washing, and RNA elution was performed according to the manufacturer’s protocol. Samples were prepared for RNA sequencing using either the mRNA-Seq or TruSeq Kit according to the manufacturer’s recommendations (Illumina) and sequenced using the Illumina GIIx platform.

Quantitative RT-PCR

The cDNA was prepared with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) according to manufacturer’s recommendations. The qPCR was performed on a LightCycler 480 II (Roche) using the LightCycler 480 Sybr Green I Master mix (Roche). Primers used are listed in Supplemental Table ST1. Primers used for 18S RNA were described (Sedletcaia and Evans, 2011). Relative gene expression was determined as described (Holtzinger et al., 2010).

Whole-mount in situ hybridization

Analysis was performed as described (Holtzinger and Evans, 2005) with the exception that overnight hybridization was performed at 68C. Briefly, embryos were treated with 0.003% phenylthiourea (PTU) to inhibit pigment formation. Embryos were fixed in 4% paraformaldehyde (PFA) and those older than 24 hpf were treated with 10 μg/ml proteinase K. Hybridization was in 50% formamide buffer with digoxigenin-labeled RNA anti-sense probes unless otherwise stated. Hybridization was in 65% formamide buffer for hspb7 and hspb12 probes before 25 somites. The probes for lft1 and lft2 were as described (Yen et al., 2006). Spaw probe was as described (Long et al., 2003). For the hspb7 probe, a full-length clone was purchased (Open Biosystems), verified by sequencing, and the XhoI insert subcloned into pCS2+. To generate the anti-sense RNA probe, the pCS2-hspb7FL plasmid was linearized with SnaB1 and RNA transcribed with SP6 polymerase. The sense RNA control for hspb7 was generated in the same way using a separate pCS2+ clone with the cDNA in reverse orientation (pCS2-hspb7FL-reverse). For the hspb12 probe, zebrafish cDNA was generated from 24 hpf embryos and used for a PCR reaction according to manufacturer’s guidelines (Phusion hot start flex, NEB). The forward (5′-CCGTTAGGTCCTTCAGTGGA) and reverse (5′-CTGCTGGAAGCACAGACTTG) primers generated a 391 base pair product that was cloned into pCRII Topo-blunt plasmid and confirmed by sequencing. For anti-sense hspb12 probe, the pCRII-hspb12 plasmid was linearized with EcoRV and RNA transcribed with SP6 polymerase. For sense hspb12 probe, the same plasmid was linearized with BamHI and transcribed with T7 polymerase.

Immunohistochemistry

Embryos were fixed with 4% PFA and embryonic hearts were dissected with fine forceps and a micro-dissecting knife (Fine Science Tools). Hearts were permeabilized and processed as described (Yang and Xu, 2012). For staining of anti-acetylated alpha-tubulin, whole-mount immunohistochemistry of embryos was performed. Embryos were fixed in 4% PFA, washed (1x PBS supplemented with 10 μg/ml BSA and 1% v/v DMSO) and permeabilized with PBT (the same buffer with 1% Tritonx-100) overnight at 4C. Blocking was performed at RT in blocking solution (wash buffer with 5% goat serum). Primary and secondary antibody incubations were performed overnight in blocking solution at 4C and all wash steps between incubations were performed in wash buffer at RT. S46 supernatant (Developmental Studies Hybridoma Bank) was used at 1:15 dilution. Polyclonal rabbit anti-dsRed antibody (Clontech) was used at 1:1000 dilution. Anti-TagRFP antibody (Evrogen) was used at 1:400 dilution. Anti-acetylated alpha-tubulin antibody (Invitrogen) was used at 1:200 dilution. Anti-GFP antibody (Invitrogen) was used at 1:1000 dilution.

Imaging analysis

Images of the sox17:gfp, myl7:dsRednls; myl7:gfp, and myl7:actn3b-gfp; myl7:mKate-caax strains were acquired on an LSM510 confocal microscope. Processed hearts were embedded in glycerol and positioned such that the atrium and ventricle lay in parallel to each other on the x-y plane of the microscope stage. Embryos stained for KV morphology were positioned in 1% low melting point agarose so that the dorsal side of the vesicle faced the camera during imaging. Whole-mount in situ hybridization and morphogenetic defects in the myl7:gfp background were imaged on a Nikon SMZ1500 dissecting microscope. Images were analyzed with ImageJ (NIH) as previously described (Lin et al., 2012) except confocal stacks were processed into single images using ImageJ.

Bead Injection and Movies

Wildtype embryos at the 6-somite stage were dechorinated and embedded in 1% low melting point agarose with the lumen visible dorsally. Embryos were injected with 0.5 micron FluoSpheres (Invitrogen F8812) before the 10-somite stage. 10-somite stage embryos were positioned so that the KV was imaged dorsally using an Axio Observer microscope fitted with an AxioCam mRm camera. 50 frame movies were taken at 10 frames per second and analysis of movies was performed with ImageJ and the module “Manual Tracking”.

Bioinformatics

Chip-Seq data for input control, BirA control, and Gata4 immuno-precipitation was obtained from GEO accession number GSE21529 (NCBI). The SRA files were converted to Fastq format using the SRA toolkit (NCBI). Fastq files were uploaded to the gobyweb application (http://gobyweb.apps.campagnelab.org/). Sequences were aligned to the mouse reference genome (mm9) using the BWA alignment algorithm. Alignment files were visualized with IGV 2.0 to determine the density of sequence reads along the genome (Broad Institute). Zebrafish cardiomyocyte RNA sample sequence reads were analyzed with gobyweb and aligned to the zebrafish reference genome (Zv9.61) with the BWA algorithm. Differential expression analysis was performed with the DESeq package. The downregulated gene list was submitted to Database for Annotation, Visualization and Integrated Discovery (NCBI) to determine gene ontologies. RNA-seq files are available from GEO (accession number GSE44233).

Transcription activation-like effector nuclease (TALEN) Generation and Injection

TALENs were generated as described (Cermak et al., 2011) to target the following sequence in the first exon of the hspb7 locus: 5′-acctctgcaaaccca-3′. TAL1 repeat variable diresidue (RVD) sequence is N terminus-HD-HD-NG-HD-HD-NG-HD-NI-NG-HD-NG-NG-HD-NI-NG-HD-HD-NG-HD-NG-C terminus while TAL2 RVD sequence is N terminus-HD-HD-NG-HD-NN-NN-HD-NG-HD-NG-NG-HD-NG-HD-HD-NI-NG-NN-NG-NI-C terminus. 100 pg of synthesized RNA was injected per embryo. Genomic DNA was isolated from uninjected embryos or siblings injected with both TALEN RNAs using Trizol reagent (Invitrogen). PCR reactions were performed on the genomic DNA according to manufacturer’s recommendations (Phusion hot start flex, NEB) and PCR products purified (Qiaquick PCR Purification Kit). 1 μg of purified PCR product was digested with HpyCH4V (NEB) and run on a gel along with undigested control.

Statistics

All statistical analyses were performed with Excel 2011 (Microsoft). Unless otherwise indicated, Student’s two-tailed t-test was performed to determine statistical significance between standard control and experimental groups.

Results

The hspb7 and hspb12 genes are regulated by gata4 during early zebrafish heart development

Zebrafish embryos depleted of gata4 or mouse embryos mutant for Gata4 in the heart develop similar cardiomyopathies following formation of the primitive heart tube (Holtzinger and Evans, 2005; Watt et al., 2004). While some target genes related to growth control have been identified in the mouse heart (Rojas et al., 2008), it remains poorly understood why cardiogenesis fails in the absence of gata4. We used the zebrafish model to define at the whole genome level the changes in transcripts that occur in cardiomyocytes when gata4 is depleted during embryogenesis. For this purpose, cardiomyocytes were flow-sorted from either wildtype or gata4 morphant embryos derived from the myl7:gfp transgenic reporter strain. Three independent samples were prepared from hearts of embryos isolated at 24 hours post fertilization (hpf) and RNA-sequencing was carried out using the Illumina platform to define significant changes in transcript levels caused by gata4 depletion. Cardiomyocytes were enriched by FACS to 70–80% purity, and approximately 75% of the sequence reads were successfully aligned to the zebrafish genome (Supplemental Fig. S1). Genes down-regulated at least 2-fold were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Huang et al., 2009a, b) to identify those annotated specifically for expression in the heart (Table I). Genes encoding small heat shock proteins Hspb7 and Hspb12 were identified in this subset. Using independent sets of flow-sorted wildtype and morphant cardiomyocytes, RNA samples were analyzed by quantitative RT-PCR (qPCR) and this validated the RNA-seq data, showing that loss of gata4 causes a significant reduction in transcript levels for hspb7 and hspb12 (Supplemental Fig. S2). During embryogenesis transcripts for these genes could be readily detected by qPCR from the 11 somite (hspb7) or the 20 somite (hspb12) stage (Supplemental Fig. S3A–B). In situ hybridization was used to define expression patterns for both genes. The hspb7 transcripts could be detected throughout the early embryo by 5 somites, and remained ubiquitous throughout the 18–20 somites stages (Fig. 1A–E). During these stages, hspb12 levels were not reliably detected by in situ hybridization. However, by the 25 somite stage, transcripts for both genes are detected in the developing heart, corresponding to the cardiac cone stage (Fig. 1F). Both genes are expressed in the heart tube at 24 hpf and 48 hpf (Fig. 1G–H), consistent with previous observations (Marvin et al., 2008). By 48 hpf, both hspb7 and hspb12 are expressed in the ventricular and atrial myocardium, coincident with the cardiac marker myl7 (Supplementary Fig. S3).

Fig. 1.

Fig. 1

Open in a new tab

Expression of hspb7 and hspb12 during zebrafish embryogenesis. A–E) in situ hybridization expression data for hspb7 at 5–6 somites, 8–10 somites, 12–14 somites, 16–18 somites, and 18–20 somites, as indictated. Upper panels (7-sense) are controls at each time points for comparison to anti-sense (hspb7), demonstrating ubiquitous staining pattern between 5–20 somites. F–H) In situ hybridization expression data for hspb7 and hspb12 at 25-somites, 24 hours post fertilization, and 48 hours post fertilization, as indicated. Insets in right panels of F show dorsal views of boxed regions in the lateral views of the left panels. Arrows indicate areas of expression including the cardiac cone (F), heart tube and somites (G), and heart (H).

Loss of hspb7 or hspb12 disrupts normal heart tube looping

We designed splice-blocking MOs to target specifically hspb7 or hspb12 and showed that these successfully deplete, with specificity, endogenous RNA when injected into fertilized eggs (Supplementary Fig. S4). Both splice-blocking MOs generate cardiac phenotypes when injected at or above a concentration of 0.2 mM, correlating with significant knockdown of target transcripts. Knockdown of hspb7 or hspb12 results with high penetrance in the development of dysmorphic or non-looping heart tubes (Fig. 2A,B). The results suggest that cardiac small heat shock proteins function downstream of gata4 mediating cardiac morphogenesis. To test this hypothesis, sub-threshold concentrations of MOs were injected targeting hspb7 or hspb12 that generate on their own a low percentage of looping defects. These were injected with or without a sub-threshold concentration of MO targeting gata4 that also on its own does not cause looping defects. Combined knockdown of Gata4 with Hspb7 or Hspb12 resulted in a much larger number of embryos with defective looping, demonstrating genetic interactions (Fig. 2C). Injection of hspb7 RNA was not sufficient to rescue the looping defect in the gata4 morphant embryos (data not shown) suggesting that hspb7 is only one of multiple downstream components mediating looping morphogenesis.

Fig. 2.

Fig. 2

Open in a new tab

Cardiac small heat shock proteins are required for morphogenesis of the heart tube and they interact genetically with gata4. A) Representative cardiac phenotypes observed at 3 dpf of myl7:gfp embryos. Non-looping is defined by the failure of the ventricle to align perpendicular to the atrium. B) Quantification of the cardiac phenotypes in wildtype embryos (wt, n = 54), embryos injected with standard control MO (control, n = 46), hspb7 MO (hspb7, n = 67), or hspb12 MO (hspb12, n = 89). C) Quantification of the cardiac phenotypes in wildtype embryos (wt, n = 20) or embryos injected with sub-threshold concentrations of MO specific for gata4 (n = 38), hspb7 (n = 36), or hspb12 (n = 29), or with the same concentrations combined (gata4 + hspb7, n = 29) or (gata4 + hspb12, n = 49).

Loss of hspb7 or hspb12 disrupts ventricular cardiomyocyte development

The ventricles appear smaller and dysmorphic in the HSPB morphants (Fig. 2A), which could be caused by a reduction in the numbers of cells. Experiments were performed to evaluate morphants derived from the myl7:gfp; myl7:dsRed-nls reporter line, which facilitates quantification of cardiomyocytes. To compare chambers, the embryos were also stained with S46 antibody, which labels specifically atrial cardiomyocytes. Ventricular cardiomyocytes were decreased in a statistically significant manner in both hspb12 morphants and double hspb7/12 morphants (Fig. 3). Interestingly, the number of atrial cardiomyocytes was not affected, and cardiomyocyte numbers were not significantly depleted in hspb7 morphants, suggesting that hspb12 modulates ventricular cell number.

Fig. 3.

Fig. 3

Open in a new tab

Hspb12 is required for normal numbers of ventricular cardiomyocytes. A) Representative cardiac phenotypes observed at 48 hpf development in embryos derived from the myl7:gfp; myl7:dsRed-nls reporter strain stained with S46 antibody to differentiate the atrium (blue) from the ventricle (green). B) Quantification of cardiomyocytes in wildtype embryos (wt, n=7) or embryos injected with standard control MO (control, n=7), hspb7 MO (hspb7, n=7), hspb12 MO (hspb12, n=8), or both hspb7 and hspb12 MOs (hspb7 + 12, n=6). * p < 0.05 by Student’s t-test in comparison to standard control group. The bars represent 10 micron.

Although cell number was not altered, the ventricles of hspb7 morphants appear dysmorphic; therefore we investigated the possibility that size, shape, or sub-cellular structure of ventricular cardiomyocytes are disturbed. Ventricular sarcomere formation and cardiomyocyte cell size was investigated in morphants derived from the myl7:actn3b-gfp; myl7:mKate-caax transgenic reporter strain that labels both the sarcomeric Z-lines and cardiomyocyte cell boundaries (Lin et al., 2012). By 48 hpf, sarcomere formation had proceeded normally in the hspb7 morphants (Supplementary Fig. S5). On the other hand, this analysis showed that embryos injected with MOs targeting hspb7 or both HSPBs showed statistically significant defects in ventricular cardiomyocyte cell size, defined by measuring cardiomyocyte area (Fig. 4). Transmission electron microscopy was used to more closely evaluate sarcomere structures in hspb7 morphants. The results suggest that overall myofiber formation and sarcomere structure is at least grossly normal (Supplemental Fig. S6), indicating that cardiac defects in the hspb7 morphants is not caused by a major breakdown in muscle cell structure.

Fig. 4.

Fig. 4

Open in a new tab

Hspb7 regulates ventricular cardiomyocyte size. A) Shown are representative phenotypes demonstrating the ventricular cell size observed in the indicated experimental conditions at 48 hpf in embryos derived from the myl7:actn3b; myl7:mKate-Caax reporter strain. The bars represent 10 micron. B) Wildtype embryos (wt, n=3) or embryos injected with the standard control MO (control, n=4), hspb7 MO (hspb7, n=5), hspb12 MO (hspb12, n=3), or hspb7+12 MOs (hspb7+12, n=4) were evaluated with ImageJ to measure average cell area. At least 7 cells were analyzed for each embryo to determine the average. The asterisk indicates p < 0.05 by Student’s t-test in comparison to the standard control group.

Hspb7 has an earlier, Gata4-independent role in cardiac morphogenesis

The primitive heart tube normally jogs to the left as an initial morphogenetic movement prior to overt looping. We noticed that over 30% of hspb7 morphants displayed aberrant heart tube jogging (Fig. 5). This jogging defect was rarely seen in control-injected, gata4 or hspb12 morphants (Fig. 5 and data not shown). To confirm that this directional jogging defect was due specifically to targeting of hspb7, additional controls were performed. First, embryos were injected with sub-threshold concentrations of either a splice-blocking or a translation-blocking morpholino, such that all embryos jogged hearts normally to the left. When the same concentrations of MOs were co-injected, over 30% of the embryos failed to jog normally to the left, consistent with an hspb7-specific defect (Supplemental Fig. S7). Second, as an independent test of hspb7 function, TALENS were designed (Cermak et al., 2011) to target the first exon of the hspb7 locus. Injection of RNA encoding a matched pair of hspb7-specific TALENs results in a significant percentage of embryos with developing heart tubes that failed to loop normally towards the left (Supplemental Fig. S8A–B). PCR-amplified fragments from genomic DNA of uninjected control or TALEN-injected siblings were subjected to restriction analysis with the enzyme HpyCH4V, which specifically digests the non-mutated TALEN target site. Consistent with successful targeting, a notable fraction of the PCR-amplified genomic DNA from TALEN-injected embryos was resistant to HpyCH4V digestion (Supplemental Fig. S8C).

Fig. 5.

Fig. 5

Open in a new tab

Hspb7 is required for normal jogging of the heart tube. A) Shown are representative phenotypes of morphant embryos derived from myl7:gfp reporter fish displaying normal (left) or abnormal (center or right) heart tube jogging at 30 hpf. B) Quantification of the phenotypes from several independent experiments for uninjected wildtype embryos (wt, n=110), standard control MO injected (control, n=100), hspb7 morphants (hspb7, n=75), or hspb12 morphants (hspb12, n=64).

Heart jogging defects in hspb7 morphants result from failure to establish a normal left-right axis

Proper heart tube jogging to the left depends upon appropriate establishment of overall left-right asymmetry during early stages of embryogenesis. One of the first steps in the pathway is left-side initiation of spaw expression, followed by downstream activation in the cardiac mesoderm of lft1 and lft2. In embryos injected with MOs targeting hspb7, all three markers were aberrantly expressed on the right side (or both sides) in a significant number of morphants (Fig. 6). Left-right asymmetry was not affected in control MO-injected or the hspb12 morphants (Fig. 6). Embryos depleted of Gata4 also correctly restrict spaw expression appropriately to the left side, consistent with a gata4-independent function of hspb7 (Supplemental Fig. S9).

Fig. 6.

Fig. 6

Open in a new tab

Hspb7 is required for establishment of left-right asymmetry. A–C) Shown are representative in situ hybridization expression patterns of the left-right asymmetry markers lft1 (A), lft2 (B), or spaw (C). Panels in A and B show, left to right, normal (leftward), bilateral, or right-sided expression in embryos at the 22-somite stage. Panels in C are likewise, but the embryos are at the 18 somite stage, and some hspb7 morphants show a partial pattern of bilateral expression (far right panel). All views are dorsal, with anterior to the top. D–F shows quantification of these phenotypes, and in each case n is at least 18 embryos.

Normally, left-side laterality markers are amplified specifically on the left side due to the function of Kupffer’s Vesicle (KV), which generates an asymmetric flow of signaling molecules to the left side of the embryo (Essner et al., 2005). The disrupted expression of spaw in hspb7 morphants suggests that Hspb7 is involved at an early stage in the generation of this left-right asymmetry, possibly at the level of the KV. The KV is generated from dorsal forerunner cells (DFCs), a type of initially non-involuting surface epithelial cells that migrate at the dorsal blastomere margin during epiboly stages (Oteiza et al., 2008). Unlike most embryonic cells, the DFCs retain cytoplasmic bridges with the yolk cell. Therefore, it is possible to deregulate gene expression specifically in these KV progenitors by targeting injections into the yolk syncytial layer (YSL) at the 1000-cell (1K) stage (Wang et al., 2013). At this point the YSL remains a syncytium that is non-contiguous with blastomeres of the embryo proper.

We tested whether Hspb7 is required in the KV or YSL by targeting hspb7 MOs specifically in the YSL at the 1K stage, and examining morphants for cardiac morphology. Embryos were co-injected with fluorescent dextran beads in order to confirm proper targeting to the YSL. Knockdown of Hspb7 in the YSL resulted in an early arrest in cardiac development, as most of the embryos developed with a non-looping partially fused heart tube, or cardia bifida (Fig. 7). Injection with lower doses of MO caused laterality defects. As expected, these phenotypes were not found with similar injections using the hspb12 MO, since this gene is not expressed at such early stages. The phenotype is relieved in a significant number of embryos by co-injection with hspb7 RNA into the YSL. These data support the hypothesis that the laterality defects arise from specific loss of Hspb7 function in the KV and/or YSL.

Fig. 7.

Fig. 7

Open in a new tab

Hspb7 is required in the YSL for proper cardiac tube formation. A) Representative phenotypes observed at 24 hpf in myl7:gfp embryos. B) Quantification of phenotypes in wildtype embryos (wt, n = 37) or embryos injected into the YSL with standard control MO (control, n = 29), hspb7 RNA (n = 30), hspb7 MO (n = 53), hspb7 MO + RNA (n = 74), or hspb12 MO (n = 21). *p<0.05 comparing the embryos co-injected with RNA compared to those injected only with MO, according to Chi-squared test.

YSL-dependent function of hspb7 is required for normal KV development and left-right asymmetry

Since the DFCs, precursors to the KV, are associated early on with the YSL, we tested if KV development was affected by knockdown of Hspb7 in the YSL. Hspb7-specific MOs were injected into the YSL of embryos derived from the sox17:gfp reporter strain that marks the DFCs (Oteíza et al., 2008). Embryos were also stained with anti-acetylated alpha tubulin antibody to mark the cilia. Overall KV morphology was found to be abnormal in morphants, with overall reduced width, and in addition decreased cilia length (Fig. 8). As a control, similar injections with the hspb12-specific MO did not disrupt KV development. To test whether these morphological defects are functionally relevant, cilia-mediated KV flow was quantified. For this purpose, 0.5 micron fluorescent beads were injected into the KV of YSL-targeted morphants for hspb7 or (as control) hspb12. Clear defects in KV flow were evident in the hspb7 morphants. Average bead velocity per embryo was decreased significantly (Fig. 9), confirming that Hspb7 regulates KV morphology and function; defects in this function therefore lead to failure in establishing normal laterality.

Fig. 8.

Fig. 8

Open in a new tab

Hspb7 is required in Kupffer’s vesicle for normal morphogenesis. A, B) Confocal stack and DIC images, respectively, of the KV in wildtype and morphant embryos derived from the sox17:gfp reporter strain, as indicated at the 8–10 somite stage. In this case, embryos were injected with MOs at the 1K stage, and selected for YSL-specific deposition using a reporter dye. Embryos in A were stained with anti-acetylated tubulin antibody (red). The bars represent 10 micron. C–E) Quantification of KV phenotypes noting average cilia number (C), cilia length (D) or KV width (E). In each sample n was at least 5 embryos. The asterisk indicates p<0.05 according to student’s T-test in comparison to the standard control group.

Fig. 9.

Fig. 9

Open in a new tab

Hspb7 is required for normal KV function. A) Traces of fluorescent bead movement in the KV from a representative 5 second video for wildtype or morphant embryos injected into the YSL at the 1K stage as indicated. Movies were acquired when the embryos reached the 10-somite stage. The bars represent 10 micron. B) Average bead velocity for each sample (n= at least 5). The asterisk indicates p < 0.05 by Student’s t-test in comparison to the standard control group. For each KV, the velocity of 5 beads was measured to determine the average.

Finally, we considered if early laterality defects are an indirect cause of the cardiac morphogenic defects we had described earlier. Hspb7 morphant embryos were separated at 30 hpf into cohorts demonstrating either normal (left-side) or abnormal jogging. A similar range of morphogenetic defects ensued, including dysmorphic and non-looped heart tubes, regardless of whether or not the early heart tube had jogged normally to the left (Supplemental Fig. S10). Thus, the later morphogenetic heart defects appear independent of the earlier role of Hspb7 in controlling laterality. Taken together these results support an early role for hspb7 in the YSL or KV establishing KV morphology, and thereby cardiac laterality. This early function is specific to hspb7 and independent of gata4 and hspb12. Subsequent functions during cardiac morphogenesis for looping and chamber formation are regulated by both hspb7 and hspb12 in a common pathway with gata4 (Fig. 10).

Fig. 10.

Fig. 10

Open in a new tab

Distinct functions for Hspb7 in YSL and cardiac morphogenesis. Hspb7 is expressed at 5-somites and is required in the YSL for YSL-dependent processes including KV morphogenesis and migration of the cardiac precursors to the midline. By 25-somites, both hspb7 and hspb12 are increasingly restricted to the myocardium and are under the regulation of gata4 during subsequent stages of cardiac morphogenesis.

Discussion

Hspb7 and Hspb12 are two small heat shock proteins each containing a conserved α-crystallin domain characteristic of this family. Little is known about the normal function of these proteins, which are presumed to carry out chaperone activity. Hspb7 has been shown to prevent the aggregation of polyQ proteins in a manner dependent upon macroautophagy. However, overexpression of Hspb7 does not activate autophagic pathways. Unlike other members of the HSPB family, Hspb7 is unable to facilitate the refolding of heat-denatured proteins, and is not dependent on the ATP-dependent Hsp70 machine for its activity (Vos et al., 2010).

Previous work characterized Hspb7 as most highly expressed in cardiac muscle, and it has been referred to as cardiac heat shock protein (Krief et al., 1999). In a cardiac cell culture system, overexpression of Hspb7 prevented tachypacing-induced F-actin stress fiber formation. While other HSPBs also were capable of preventing stress fibers, they appear to act differently, and the activity of Hspb7 was most potent. Hspb7 inhibits the initial polymerization of actin but has no effect on the rate of depolymerization (Ke et al., 2011). Hspb7 has been shown to interact with the cytoskeletal protein alpha-filamin (Krief et al., 1999) as well as other small heat shock proteins (Sun et al., 2004). Even less is known about the function of hspb12 in teleosts, although, as we confirmed, transcripts are also enriched in cardiac tissue (Marvin et al., 2008).

Our experiments are the first to test function for these genes during development, and revealed two previously unknown functions for Hspb7 and Hspb12. First, Hspb7 is essential for establishing the left-right axis of asymmetry, through controlling the morphology of the key signaling center, Kupffer’s vesicle. Hspb7 specifically regulates cilia length and KV size, which impacts KV function. The left-right asymmetry defects likely arise in hspb7 morphants because of defective KV function, although we cannot distinguish if this is due to Hspb7 in the KV or indirectly in the YSL. Hspb12, on the other hand, does not regulate KV development, and thus depletion of Hspb12 does not affect establishment of left-right asymmetry. Although Hspb7 is not present in the Ciliary Proteome Database (ciliaproteome.org), HSPB1 and numerous other HSPs are found in the photoreceptor sensory cilia complex (Liu et al., 2007), so it remains possible that Hspb7 has a direct role in KV ciliary morphogenesis. Although we discovered hspb7 as a downstream target of gata4, this early role is independent of gata4 since depletion of Gata4 during embryogenesis has no effect on jogging, nor for establishment of left-right asymmetry. Targeted loss of Hspb7 in the YSL often led to a cardia bifid phenotype, caused when cardiac progenitors fail to migrate properly to the midline, a process known to be regulated by the YSL (Langenbacher et al., 2012; Sakaguchi et al., 2006). This suggests a wider (and perhaps unrelated) function for hspb7 in the YSL beyond KV morphology. This bifid phenotype was rarely noted when MOs were injected into the embryos, presumably because this is hypermorphic with respect to the YSL function, which would be more fully targeted by injection directly into the YSL at the 1K stage. The range of phenotypes including bifid hearts is seen in embryos injected with RNA encoding hspb7-specific TALENs.

Second, we showed that Hspb12 is required to generate normal cardiomyocyte numbers in the ventricle, while Hspb7 is required for ventricular cardiomyocyte growth. These data suggest small heat shock proteins cooperate to ensure proper ventricular morphogenesis by impacting cell number and size. While functions for HSPBs in cardiac development were previously unrecognized, small heat shock proteins have been implicated already in cardiac biology and disease. An R120G mutation in alpha-B crystallin is associated with a heritable desminopathy, and forced expression in mice induces a cardiac hypertrophy with a similar desmin disorder (Wang et al., 2001). In addition, Hspb7 is involved in muscular aging (Doran et al., 2007) and the prevention of actin stress fiber formation in a tachycardia model system (Ke et al., 2011). Most interestingly, several recent studies identified HSPB7 in genome wide association analyses for advanced heart failure and idiopathic cardiomyopathies (Cappola et al., 2010; Stark et al., 2010; Villard et al., 2011). Our identification of hspb7 as a downstream target of gata4 is intriguing in this regard. Gata4 is known to be cardio-protective. For example, Gata4 overexpression is protective in models of anthracycline-induced cardiotoxicity, by inhibition of apoptosis (Aries et al., 2004; Kim et al., 2003; Kitta et al., 2003; Suzuki and Evans, 2004). This occurs at least in part through upregulation of Bcl-2, and through inhibition of doxorubicin-induced autophagy (Kobayashi et al., 2010). Based on published ChiP-seq data (He et al., 2011), Gata4 may regulate Hspb7 expression directly by binding to proximal promoter elements (Supplemental Fig. S11). Thus, it will be interesting to explore whether HSPBs function to mediate in part the cardio-protection afforded by Gata4. In conclusion, we found that gata4 and small heat shock proteins encoded by hspb7 and hspb12 comprise a pathway that controls cardiac morphogenesis. The function of hspb7 and hspb12 can explain at least part of the gata4-dependent activities controlling embryonic cardiogenesis, while hspb7 also functions earlier in the YSL, independent of gata4. Future studies will explore if HSPB function is relevant to autophagic cardio-protective mechanisms, which could reveal insight into heart failure disease mechanisms.

Supplementary Material

01

Table 1.

Gata4 regulates a specific developmental program in the cardiomyocytes. Downregulated genes from the RNAseq experiment at 24hpf were submitted to DAVID for gene ontology analysis. Those genes demonstrating a gene ontology association with the heart in ZFIN are shown along with the Log2 transformed relative expression of gata4 morphants as compared to uninjected controls.

Gene Name Ensembl Gene ID Log2 FC G4MO/WT
cardiac lim protein ENSDARG00000069975 −3.31
versican a ENSDARG00000078680 −2.29
calcium channel, voltage dependent, L type, alpha 1c ENSDARG00000008398 −2.22
cytochrome P450, family member 1, member A1 ENSDARG00000026039 −2.20
ryanodine receptor 2b ENSDARG00000003706 −2.19
ventricular myosin heavy chain like ENSDARG00000079782 −2.10
solute carrier family 8, member 1a ENSDARG00000013422 −2.09
heat shock protein, alpha crystallin related, b12 ENSDARG00000087665 −1.95
alpha smooth muscle actin ENSDARG00000045180 −1.85
ribonuclease T2 ENSDARG00000058413 −1.76
aquaporin 3a ENSDARG00000003808 −1.74
heart adaptor protein 1 ENSDARG00000060813 1.67
natriuretic peptide a ENSDARG00000052960 −1.66
nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 ENSDARG00000036168 −1.63
ADP ribosylhydrolase like 1 ENSDARG00000041589 −1.62
troponin c, type 1 ENSDARG00000011400 −1.61
heat shock protein family, alpha crystallin related, b7 ENSDARG00000011538 −1.58
myosin binding protein c, cardiac ENSDARG00000011615 −1.55
C type lectin domain family 19, member A ENSDARG00000037978 −1.51
PR domain containing 1, with ZNF domain ENSDARG00000002445 −1.32
Open in a new tab

Highlights.

  • The hspb7 and hspb12 genes are regulated by gata4 in embryonic cardiomyocytes.

  • Knockdown of Hspb7 or Hspb12 disrupts embryonic cardiac morphogenesis.

  • Hspb7 also has an earlier gata4-independent function in YSL morphogenesis.

  • Hspb7 regulates KV morphology and thereby embryonic laterality.

Acknowledgments

This work was supported by the NIH through R01HL11400 (TE) and T32HD060600 (GR). We thank Paul Zumbo for technical assistance with RNA sequencing and Fabien Compagne for advice and assistance using gobyweb. We appreciate the expert advice of Leona Cohen-Gould for the TEM analysis. Kelly McCartin, Ingrid Torregroza, and Ting-Chun Liu provided outstanding zebrafish husbandry. We are grateful to D. Yelon for providing the myl7:actn3b-gfp and myl7-mKate-Caax lines and N. Glickman-Holtzman for the myl7:dsRed-nls; myl7:gfp line. We also thank Joe Yost for helpful advice on measuring KV function.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci U S A. 2004;101:6975–6980. doi: 10.1073/pnas.0401833101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science. 2008;319:916–919. doi: 10.1126/science.1141448. [DOI] [PubMed] [Google Scholar]
  3. Berry V, Francis P, Reddy MA, Collyer D, Vithana E, MacKay I, Dawson G, Carey AH, Moore A, Bhattacharya SS, Quinlan RA. Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Am J Hum Genet. 2001;69:1141–1145. doi: 10.1086/324158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cappola TP, Li M, He J, Ky B, Gilmore J, Qu L, Keating B, Reilly M, Kim CE, Glessner J, Frackelton E, Hakonarson H, Syed F, Hindes A, Matkovich SJ, Cresci S, Dorn GW., II Common Variants in HSPB7 and FRMD4B Associated With Advanced Heart Failure. Circulation-Cardiovascular Genetics. 2010;3:147–U192. doi: 10.1161/CIRCGENETICS.109.898395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research. 2011;39:e82. doi: 10.1093/nar/gkr218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davidson LA. Embryo mechanics: balancing force production with elastic resistance during morphogenesis. Curr Top Dev Biol. 2011;95:215–241. doi: 10.1016/B978-0-12-385065-2.00007-4. [DOI] [PubMed] [Google Scholar]
  7. Doran P, Gannon J, O’Connell K, Ohlendieck K. Aging skeletal muscle shows a drastic increase in the small heat shock proteins alpha B-crystallin/HspB35 and cvHsp/HspB7. European Journal of Cell Biology. 2007;86:629–640. doi: 10.1016/j.ejcb.2007.07.003. [DOI] [PubMed] [Google Scholar]
  8. Essner JJ, Amack JD, Nyholm MK, Harris EB, Yost J. Kupffer’s vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development. 2005;132:1247–1260. doi: 10.1242/dev.01663. [DOI] [PubMed] [Google Scholar]
  9. Evgrafov OV, Mersiyanova I, Irobi J, Van Den Bosch L, Dierick I, Leung CL, Schagina O, Verpoorten N, Van Impe K, Fedotov V, Dadali E, Auer-Grumbach M, Windpassinger C, Wagner K, Mitrovic Z, Hilton-Jones D, Talbot K, Martin JJ, Vasserman N, Tverskaya S, Polyakov A, Liem RK, Gettemans J, Robberecht W, De Jonghe P, Timmerman V. Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet. 2004;36:602–606. doi: 10.1038/ng1354. [DOI] [PubMed] [Google Scholar]
  10. Franck E, Madsen O, van Rheede T, Ricard G, Huynen MA, de Jong WW. Evolutionary diversity of vertebrate small heat shock proteins. Journal of Molecular Evolution. 2004;59:792–805. doi: 10.1007/s00239-004-0013-z. [DOI] [PubMed] [Google Scholar]
  11. Gittenberger-de Groot AC, Bartelings MM, Deruiter MC, Poelmann RE. Basics of cardiac development for the understanding of congenital heart malformations. Pediatr Res. 2005;57:169–176. doi: 10.1203/01.PDR.0000148710.69159.61. [DOI] [PubMed] [Google Scholar]
  12. He A, Kong SW, Ma Q, Pu WT. Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart. Proc Natl Acad Sci U S A. 2011;108:5632–5637. doi: 10.1073/pnas.1016959108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holtzinger A, Evans T. Gata4 regulates the formation of multiple organs. Development. 2005;132:4005–4014. doi: 10.1242/dev.01978. [DOI] [PubMed] [Google Scholar]
  14. Holtzinger A, Rosenfeld GE, Evans T. Gata4 directs development of cardiac-inducing endoderm from ES cells. Dev Biol. 2010;337:63–73. doi: 10.1016/j.ydbio.2009.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang dW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009a;37:1–13. doi: 10.1093/nar/gkn923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang dW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009b;4:44–57. doi: 10.1038/nprot.2008.211. [DOI] [PubMed] [Google Scholar]
  17. Irobi J, Van Impe K, Seeman P, Jordanova A, Dierick I, Verpoorten N, Michalik A, De Vriendt E, Jacobs A, Van Gerwen V, Vennekens K, Mazanec R, Tournev I, Hilton-Jones D, Talbot K, Kremensky I, Van Den Bosch L, Robberecht W, Van Vandekerckhove J, Van Broeckhoven C, Gettemans J, De Jonghe P, Timmerman V. Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy. Nat Genet. 2004;36:597–601. doi: 10.1038/ng1328. [DOI] [PubMed] [Google Scholar]
  18. Ke L, Meijering RAM, Hoogstra-Berends F, Mackovicova K, Vos MJ, Van Gelder IC, Henning RH, Kampinga HH, Brundel BJJM. HSPB1, HSPB6, HSPB7 and HSPB8 Protect against RhoA GTPase-Induced Remodeling in Tachypaced Atrial Myocytes. Plos One. 2011:6. doi: 10.1371/journal.pone.0020395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim Y, Ma AG, Kitta K, Fitch SN, Ikeda T, Ihara Y, Simon AR, Evans T, Suzuki YJ. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol. 2003;63:368–377. doi: 10.1124/mol.63.2.368. [DOI] [PubMed] [Google Scholar]
  20. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995;203:253–310. doi: 10.1002/aja.1002030302. [DOI] [PubMed] [Google Scholar]
  21. Kitta K, Day RM, Kim Y, Torregroza I, Evans T, Suzuki YJ. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem. 2003;278:4705–4712. doi: 10.1074/jbc.M211616200. [DOI] [PubMed] [Google Scholar]
  22. Kobayashi S, Volden P, Timm D, Mao K, Xu X, Liang Q. Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death. J Biol Chem. 2010;285:793–804. doi: 10.1074/jbc.M109.070037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krief S, Faivre JF, Robert P, Le Douarin B, Brument-Larignon N, Lefrere I, Bouzyk MM, Anderson KM, Greller LD, Tobin FL, Souchet M, Bril A. Identification and characterization of cvHsp - A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues. J Biol Chem. 1999;274:36592–36600. doi: 10.1074/jbc.274.51.36592. [DOI] [PubMed] [Google Scholar]
  24. Langenbacher AD, Huang J, Chen Y, Chen JN. Sodium pump activity in the yolk syncytial layer regulates zebrafish heart tube morphogenesis. Developmental biology. 2012;362:263–270. doi: 10.1016/j.ydbio.2011.12.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin X, Xu X. Distinct functions of Wnt/beta-catenin signaling in KV development and cardiac asymmetry. Development. 2009;136:207–217. doi: 10.1242/dev.029561. [DOI] [PubMed] [Google Scholar]
  26. Lin YF, Swinburne I, Yelon D. Multiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart. Dev Biol. 2012;362:242–253. doi: 10.1016/j.ydbio.2011.12.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet. 1998;7:471–474. doi: 10.1093/hmg/7.3.471. [DOI] [PubMed] [Google Scholar]
  28. Liu Q, Tan G, Levenkova N, Li T, Pugh EN, Jr, Rux JJ, Speicher DW, Pierce EA. The proteome of the mouse photoreceptor sensory cilium complex. Mol Cell Proteomics. 2007;6:1299–1317. doi: 10.1074/mcp.M700054-MCP200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Long S, Ahmad N, Rebagliati M. The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry. Development. 2003;130:2303–2316. doi: 10.1242/dev.00436. [DOI] [PubMed] [Google Scholar]
  30. Marvin M, O’Rourke D, Kurihara T, Juliano CE, Harrison KL, Hutson LD. Developmental expression patterns of the zebrafish small heat shock proteins. Developmental Dynamics. 2008;237:454–463. doi: 10.1002/dvdy.21414. [DOI] [PubMed] [Google Scholar]
  31. Meno C, Gritsman K, Ohishi S, Ohfuji Y, Heckscher E, Mochida K, Shimono A, Kondoh H, Talbot WS, Robertson EJ, Schier AF, Hamada H. Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation. Mol Cell. 1999;4:287–298. doi: 10.1016/s1097-2765(00)80331-7. [DOI] [PubMed] [Google Scholar]
  32. Oteiza P, Koppen M, Concha ML, Heisenberg CP. Origin and shaping of the laterality organ in zebrafish. Development. 2008;135:2807–2813. doi: 10.1242/dev.022228. [DOI] [PubMed] [Google Scholar]
  33. Oteíza P, Köppen M, Concha ML, Heisenberg CP. Origin and shaping of the laterality organ in zebrafish. Development. 2008;135:2807–2813. doi: 10.1242/dev.022228. [DOI] [PubMed] [Google Scholar]
  34. Rajagopal SK, Ma Q, Obler D, Shen J, Manichaikul A, Tomita-Mitchell A, Boardman K, Briggs C, Garg V, Srivastava D, Goldmuntz E, Broman KW, Benson DW, Smoot LB, Pu WT. Spectrum of heart disease associated with murine and human GATA4 mutation. J Mol Cell Cardiol. 2007;43:677–685. doi: 10.1016/j.yjmcc.2007.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rojas A, Kong SW, Agarwal P, Gilliss B, Pu WT, Black BL. GATA4 is a direct transcriptional activator of cyclin D2 and Cdk4 and is required for cardiomyocyte proliferation in anterior heart field-derived myocardium. Mol Cell Biol. 2008;28:5420–5431. doi: 10.1128/MCB.00717-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sakaguchi T, Kikuchi Y, Kuroiwa A, Takeda H, Stainier DY. The yolk syncytial layer regulates myocardial migration by influencing extracellular matrix assembly in zebrafish. Development. 2006;133:4063–4072. doi: 10.1242/dev.02581. [DOI] [PubMed] [Google Scholar]
  37. Sedletcaia A, Evans T. Heart chamber size in zebrafish is regulated redundantly by duplicated tbx2 genes. Developmental Dynamics. 2011;240:1548–1557. doi: 10.1002/dvdy.22622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stark K, Esslinger UB, Reinhard W, Petrov G, Winkler T, Komajda M, Isnard R, Charron P, Villard E, Cambien F, Tiret L, Aumont M-C, Dubourg O, Trochu J-N, Fauchier L, DeGroote P, Richter A, Maisch B, Wichter T, Zollbrecht C, Grassl M, Schunkert H, Linsel-Nitschke P, Erdmann J, Baumert J, Illig T, Klopp N, Wichmann HE, Meisinger C, Koenig W, Lichtner P, Meitinger T, Schillert A, Koenig IR, Hetzer R, Heid IM, Regitz-Zagrosek V, Hengstenberg C. Genetic Association Study Identifies HSPB7 as a Risk Gene for Idiopathic Dilated Cardiomyopathy. Plos Genetics. 2010:6. doi: 10.1371/journal.pgen.1001167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sun XK, Fontaine JM, Rest JS, Shelden EA, Welsh MJ, Benndorf R. Interaction of human HSP22 (HSPB8) with other small heat shock proteins. J Biol Chem. 2004;279:2394–2402. doi: 10.1074/jbc.M311324200. [DOI] [PubMed] [Google Scholar]
  40. Suzuki YJ, Evans T. Regulation of cardiac myocyte apoptosis by the GATA-4 transcription factor. Life Sci. 2004;74:1829–1838. doi: 10.1016/j.lfs.2003.10.002. [DOI] [PubMed] [Google Scholar]
  41. Villard E, Perret C, Gary F, Proust C, Dilanian G, Hengstenberg C, Ruppert V, Arbustini E, Wichter T, Germain M, Dubourg O, Tavazzi L, Aumont MC, DeGroote P, Fauchier L, Trochu JN, Gibelin P, Aupetit JF, Stark K, Erdmann J, Hetzer R, Roberts AM, Barton PJR, Regitz-Zagrosek V, Aslam U, Duboscq-Bidot L, Meyborg M, Maisch B, Madeira H, Waldenstrom A, Galve E, Cleland JG, Dorent R, Roizes G, Zeller T, Blankenberg S, Goodall AH, Cook S, Tregouet DA, Tiret L, Isnard R, Komajda M, Charron P, Cambien F, Cardiogenics C. A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy. European Heart Journal. 2011;32:1065–1076. doi: 10.1093/eurheartj/ehr105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vos MJ, Zijlstra MP, Kanon B, van Waarde-Verhagen MA, Brunt ER, Oosterveld-Hut HM, Carra S, Sibon OC, Kampinga HH. HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones. Human molecular genetics. 2010;19:4677–4693. doi: 10.1093/hmg/ddq398. [DOI] [PubMed] [Google Scholar]
  43. Wang G, Yost HJ, Amack JD. Analysis of gene function and visualization of cilia-generated fluid flow in Kupffer’s vesicle. Journal of visualized experiments : JoVE. 2013 doi: 10.3791/50038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wang XJ, Osinska H, Klevitsky R, Gerdes AM, Nieman M, Lorenz J, Hewett T, Robbins J. Expression of R120G-alpha B-crystallin causes aberrant desmin and alpha B-crystallin aggregation and cardiomyopathy in mice. Circ Res. 2001;89:84–91. doi: 10.1161/hh1301.092688. [DOI] [PubMed] [Google Scholar]
  45. Watt AJ, Battle MA, Li J, Duncan SA. GATA4 is essential for formation of the proepicardium and regulates cardiogenesis. Proc Natl Acad Sci U S A. 2004;101:12573–12578. doi: 10.1073/pnas.0400752101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Westerfield M. The Zebrafish book : a guide for the laboratory use of zebrafish (Brachydanio rerio) University of Oregon Press; Eugene. Or: 1993. [Google Scholar]
  47. Westerheide SD, Anckar J, Stevens SM, Sistonen L, Morimoto RI. Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1. Science. 2009;323:1063–1066. doi: 10.1126/science.1165946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang J, Xu X. Immunostaining of dissected zebrafish embryonic heart. J Vis Exp. 2012:e3510. doi: 10.3791/3510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yen HJ, Tayeh MK, Mullins RF, Stone EM, Sheffield VC, Slusarski DC. Bardet-Biedl syndrome genes are important in retrograde intracellular trafficking and Kupffer’s vesicle cilia function. Hum Mol Genet. 2006;15:667–677. doi: 10.1093/hmg/ddi468. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS506598-supplement-01.pdf (5.9MB, pdf)

RESOURCES