Developmental Biology 381 (2013) 389–400

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Developmental Biology

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

Gabriel E. Rosenfeld a, Emily J. Mercer a, Christopher E. Mason b,c, Todd Evans a,n a Department of Surgery, Weill Cornell Medical College, NY 10065, USA b Department of Physiology & Biophysics, Weill Cornell Medical College, NY 10065, USA c Institute for Computational Biomedicine, Weill Cornell Medical College, NY 10065, USA article info abstract

Article history: Cardiac morphogenesis is a complex multi-stage process, and the molecular basis for controlling distinct Received 28 November 2012 steps remains poorly understood. Because gata4 encodes a key transcriptional regulator of morphogen- Received in revised form esis, we profiled transcript changes in cardiomyocytes when Gata4 is depleted from developing 17 June 2013 zebrafish embryos. We discovered that gata4 regulates expression of two small heat shock , hspb7 Accepted 24 June 2013 and hspb12, both of which are expressed in the embryonic heart. We show that depletion of Hspb7 or Available online 11 July 2013 Hspb12 disrupts normal cardiac morphogenesis, at least in part due to defects in ventricular size and Keywords: shape. We confirmed that gata4 interacts genetically with the hspb7/12 pathway, but surprisingly, fi Zebra sh we found that hspb7 also has an earlier, gata4-independent function. Depletion perturbs Kupffer's vesicle Gata4 (KV) morphology leading to a failure in establishing the left–right axis of asymmetry. Targeted depletion Heart development of Hspb7 in the yolk syncytial layer is sufficient to disrupt KV morphology and also causes an even earlier Cardiogenesis fi YSL block to heart tube formation and a bi d phenotype. Recently, several genome-wide association studies Kupffer's vesicle 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. & 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Introduction temperatures and induces transcriptional upregulation of heat shock proteins by heat shock factor 1 (HSF1; Westerheide et al., Cardiomyopathies contribute significantly to morbidity and 2009). During embryonic development, environmental stressors mortality, representing the most common congenital defects in can lead to abnormal aggregation of protein intermediates or newborns. Underlying most congenital cardiomyopathies are cause protein mis-function (Balch et al., 2008). Moreover, embry- believed to be defects in heart morphogenesis (Gittenberger-de ogenesis itself generates stress as germ layers are organized and Groot et al., 2005). The GATA4 encodes a key transcriptional subsequently organs are constructed through morphogenetic regulator that coordinates complex cardiac morphogenetic pro- rearrangements including gastrulation, convergent extension, grams. Congenital cardiomyopathies caused by in epithelial–mesenchymal transitions, and tissue deformation GATA4 are associated with a spectrum of common morphogenetic (Davidson, 2011). Members of the heat shock family with mole- abnormalities including septal, outflow tract, and valvular defects cular weights between 15 and 43 kDa are termed the small heat (Rajagopal et al., 2007). Yet it remains unclear how mutations in shock proteins (heat shock proteins-beta; HSPBs). HSPBs are GATA4 cause defects in heart development. expressed during embryonic development and may function as The family comprises a set of structurally chaperones to ensure protein homeostasis. In contrast to HSF1- similar chaperones regulating protein folding in response to mediated responses to stress, the regulation of HSPBs for mediat- environmental stress to maintain normal protein homeostasis. ing organogenesis during embryonic development is less well The classical heat shock response is activated by elevated investigated. Characterized by a C-terminal alpha-crystallin domain, several diverse biological roles for HSPBs have been described, including ☆This is an open-access article distributed under the terms of the Creative remodeling of the cytoskeleton, regulation of apoptosis, and as a Commons Attribution-NonCommercial-No Derivative Works License, which per- structural protein in the eye lens (Franck et al., 2004). Hspb1-9 is mits non-commercial use, distribution, and reproduction in any medium, provided evolutionarily conserved from mammals to teleosts. Hspb10 is the original author and source are credited. n restricted to mammals while only lower vertebrate genomes Correspondence to: Department of Surgery, Weill Cornell Medical College, 1300 York Ave., LC-708, NY, NY 10065, USA. encode Hspb11, Hspb12, and Hspb15 (Marvin et al., 2008). Muta- E-mail address: [email protected] (T. Evans). tions in HSPB proteins can cause several degenerative diseases

0012-1606/$ - see front matter & 2013 The Authors. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ydbio.2013.06.025 390 G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 including Charcot–Marie–Tooth Disease (Evgrafov et al., 2004), also generated two MOs that target hspb12 at the splice donor site Distal Hereditary Motor Neuropathy (dHMN, Irobi et al., 2004), of intron 2 (5′-GCGCTCCTCGCTTACTCTTTATGTG, called 12MO) or and cataract formation (Berry et al., 2001; Litt et al., 1998). the start codon (5′-CATATAGAGAGCCGCAGCAGTGCAT-3′), called Intriguingly, members of this family display tissue-specific expres- 12MO1, respectively. For single injection experiments, a concen- sion patterns, and not all are responsive to heat shock, suggesting tration of 0.25 mM and 0.2 mM was used for hspb7 and hspb12 specific developmental functions independent of traditional envir- splice site MOs, respectively. 7MO1 and 12MO1 generated cardiac onmental stressors (Marvin et al., 2008). defects at 0.5 mM and above. For injection of both 7MO and 12MO, Cardiogenesis occurs through a series of extensive tissue each was used at 0.2 mM. For dual knockdown experiments with morphogenetic orchestrations, from generation of a primordial 7MO1 at 0.7 mM, 7MO was used at 0.15 mM. For dual knockdown heart tube, through cardiac looping, chamber formation, valve experiments with G4MO1 at a sub-threshold concentration of formation, and septation. One of the earliest steps of cardiac 0.25 mM, 7M0 or 12MO were used at a concentration of 0.15 mM. morphogenesis is dependent on the establishment of left–right asymmetry. Left–right asymmetry in zebrafish initiates from Fluorescence activated cell sorting (FACS) Kupffer's vesicle (KV), analogous to the mammalian node, which generates a flow of signaling molecules toward the embryonic left The gata4 morphant embryos or controls were dechorinated at side (Essner et al., 2005). Asymmetric gene expression is initiated 24 hpf and batches of 200 embryos placed into 1.5 ml tubes. by the feed-forward activation of spaw expression on the embryo- Embryos were dissociated by manual agitation with a pellet pestle nic left side (Long et al., 2003). Downstream expression of the (Fisher) and trypsinized with pre-heated TrypLE (Life Technolo- nodal antagonists lft1 and lft2 results in propagation of left–right gies) at 32 1C for 15 min on a rotator. Trypsinized samples were asymmetry to the cardiac mesoderm (Long et al., 2003; Meno pipetted through a 35 μm cell strainer into a 5 ml tube, trypsin was et al., 1999). Previous work has implicated gata4 functioning inhibited by addition of 4 ml FACS buffer (L-15 medium supple- downstream of WNT/beta-catenin signaling to regulate the com- mented with 1% heat inactivated FCS, 0.8 mM CaCl2, 50 U/ml petence of cardiac mesoderm to express left-sided genes (Lin and penicillin, and 0.05 mg/ml streptomycin) followed by addition of Xu, 2009). FCS to 7.5% final concentration. Cells were pelleted at 300 RCF for We showed previously that gata4 regulates cardiac morpho- 5 min and then washed with FACS buffer. Dissociated embryonic genesis and that embryos depleted of gata4 develop with a cells were resuspended at 7.5 106 cells/ml in FACS buffer. FACS primordial heart tube that fails to loop or grow (Holtzinger and was performed on a Vantage cell sorter (BD) into Trizol LS (Life Evans, 2005). Here we investigated at the whole genome level the Technologies), and stored at 80 1C until RNA isolation. developmental program regulated by gata4 in myocardium prior to the looping stage of heart morphogenesis. When gata4 was RNA isolation and sequencing depleted, we identified among the most down-regulated tran- scripts those encoding the small heat shock proteins Hspb7 and RNA was isolated according to the Trizol protocol except that Hspb12. We analyzed loss-of-function phenotypes for these two after adding ethanol, the solution was transferred to an RNeasy HSPBs during zebrafish embryogenesis and discovered a pre- minElute column (Qiagen). On-column DNase digestion, subse- viously unknown role for them in controlling cardiac morphogen- quent washing, and RNA elution was performed according to the esis. Surprisingly, we also discovered earlier roles for Hspb7 in the manufacturer's protocol. Samples were prepared for RNA sequen- yolk syncytial layer (YSL) controlling precursor migration and left– cing using either the mRNA-Seq or TruSeq Kit according to the right asymmetry, functions that are independent of gata4. manufacturer's recommendations (Illumina) and sequenced using the Illumina GIIx platform.

Materials and methods Quantitative RT-PCR

Zebrafish husbandry The cDNA was prepared with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) according to manufacturer's recom- All zebrafish strains were maintained as described (Westerfield, mendations. The qPCR was performed on a LightCycler 480 II 1993) and staged according to standard developmental time (Roche) using the LightCycler 480 Sybr Green I Master mix points (Kimmel et al., 1995). The myl7:gfp strain was originally (Roche). Primers used are listed in Supplementary Table S1. obtained from H.J. Tsai (Taiwan). The myl:actn3b-gfp and myl7- Primers used for 18S RNA were described (Sedletcaia and Evans, mKate-Caax lines were kindly provided by Deborah Yelon (UCSD, 2011). Relative gene expression was determined as described CA). The myl7:dsRed-nls; myl7:gfp line was generously provided by (Holtzinger et al., 2010). Nathalia Glickman Holtzman (Queens College, NY). The sox17:gfp strain was obtained from the Zebrafish International Resource Whole-mount in situ hybridization Center (Eugene, Oregon). Analysis was performed as described (Holtzinger and Evans, Morpholino design and embryo injection 2005) with the exception that overnight hybridization was per- formed at 68 1C. Briefly, embryos were treated with 0.003% Morpholinos (MOs) were obtained from Genetools, LLC (Philo- phenylthiourea (PTU) to inhibit pigment formation. Embryos were math, OR). The MOs targeting the 5′UTR of gata4 (G4MO) or the fixed in 4% paraformaldehyde (PFA) and those older than 24 hpf splice acceptor site of exon 3 of gata4 (G4MO1) were previously were treated with 10 μg/ml proteinase K. Hybridization was in 50% validated for specificity and were used as described (Holtzinger formamide buffer with digoxigenin-labeled RNA anti-sense probes and Evans, 2005). All injections used 2 nl of MO dissolved in water. unless otherwise stated. Hybridization was in 65% formamide A standard control MO (5′-CCTCTTACCTCAGTTACATTTATA) was buffer for hspb7 and hspb12 probes before 25 somites. The probes used at 0.25 mM. Two MOs were designed to target either the for lft1 and lft2 were as described (Yen et al., 2006). Spaw probe hspb7 sequence at the splice acceptor site of intron 1 (5′- was as described (Long et al., 2003). For the hspb7 probe, a full- CCTGTTCTGTCTGATGAAAAACATA, named 7MO) or at the start length clone was purchased (Open Biosystems), verified by codon (5′-AGAAGAATTGCTCGCGCTCATTAGT, named 7MO1). We sequencing, and the XhoI insert subcloned into pCS2+. To generate G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 391 the anti-sense RNA probe, the pCS2-hspb7FL plasmid was linear- Bioinformatics ized with SnaB1 and RNA transcribed with SP6 polymerase. The sense RNA control for hspb7 was generated in the same way using Chip-Seq data for input control, BirA control, and Gata4 a separate pCS2+ clone with the cDNA in reverse orientation immuno-precipitation was obtained from GEO Accession number (pCS2-hspb7FL-reverse). For the hspb12 probe, zebrafish cDNA was GSE21529 (NCBI). The SRA files were converted to Fastq format generated from 24 hpf embryos and used for a PCR reaction using the SRA toolkit (NCBI). Fastq files were uploaded to the according to manufacturer's guidelines (Phusion hot start flex, gobyweb application (http://gobyweb.apps.campagnelab.org/). NEB). The forward (5′-CCGTTAGGTCCTTCAGTGGA) and reverse Sequences were aligned to the mouse reference genome (mm9) (5′-CTGCTGGAAGCACAGACTTG) primers generated a 391 using the BWA alignment algorithm. Alignment files were visua- product that was cloned into pCRII Topo-blunt plasmid and lized with IGV 2.0 to determine the density of sequence reads confirmed by sequencing. For anti-sense hspb12 probe, the along the genome (Broad Institute). Zebrafish cardiomyocyte RNA pCRII-hspb12 plasmid was linearized with EcoRV and RNA tran- sample sequence reads were analyzed with gobyweb and aligned scribed with SP6 polymerase. For sense hspb12 probe, the same to the zebrafish reference genome (Zv9.61) with the BWA algo- plasmid was linearized with BamHI and transcribed with T7 rithm. Differential expression analysis was performed with the polymerase. 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 Immunohistochemistry from GEO (Accession number GSE44233).

Embryos were fixed with 4% PFA and embryonic hearts were Transcription activation-like effector nuclease (TALEN) generation dissected with fine forceps and a micro-dissecting knife (Fine and injection Science Tools). Hearts were permeabilized and processed as described (Yang and Xu, 2012). For staining of anti-acetylated TALENs were generated as described (Cermak et al., 2011)to alpha-tubulin, whole-mount immunohistochemistry of embryos target the following sequence in the first exon of the hspb7 locus: was performed. Embryos were fixed in 4% PFA, washed (1 PBS 5′-acctctgcaaaccca-3′. TAL1 repeat variable diresidue (RVD) supplemented with 10 μg/ml BSA and 1% v/v DMSO) and permea- sequence is N terminus-HD-HD-NG-HD-HD-NG-HD-NI-NG-HD- bilized with PBT (the same buffer with 1% Tritonx-100) overnight NG-NG-HD-NI-NG-HD-HD-NG-HD-NG-C terminus while TAL2 at 4 1C. Blocking was performed at RT in blocking solution (wash RVD sequence is N terminus-HD-HD-NG-HD-NN-NN-HD-NG-HD- buffer with 5% goat serum). Primary and secondary antibody NG-NG-HD-NG-HD-HD-NI-NG-NN-NG-NI-C terminus. 100 pg of incubations were performed overnight in blocking solution at synthesized RNA was injected per embryo. Genomic DNA was 4 1C and all wash steps between incubations were performed in isolated from uninjected embryos or siblings injected with both wash buffer at RT. S46 supernatant (Developmental Studies TALEN RNAs using Trizol reagent (Invitrogen). PCR reactions were Hybridoma Bank) was used at 1:15 dilution. Polyclonal rabbit performed on the genomic DNA according to manufacturer's anti-dsRed antibody (Clontech) was used at 1:1000 dilution. Anti- recommendations (Phusion hot start flex, NEB) and PCR products TagRFP antibody (Evrogen) was used at 1:400 dilution. Anti- purified (Qiaquick PCR Purification Kit). 1 mg of purified PCR acetylated alpha-tubulin antibody (Invitrogen) was used at 1:200 product was digested with HpyCH4V (NEB) and run on a gel along dilution. Anti-GFP antibody (Invitrogen) was used at 1:1000 with undigested control. dilution.

Statistics Imaging analysis All statistical analyses were performed with Excel 2011 (Micro- Images of the sox17:gfp, myl7:dsRednls; myl7:gfp, and myl7: soft). Unless otherwise indicated, Student's two-tailed t-test was actn3b-gfp; myl7:mKate-caax strains were acquired on an LSM510 performed to determine statistical significance between standard confocal microscope. Processed hearts were embedded in glycerol control and experimental groups. 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 Results point agarose so that the dorsal side of the vesicle faced the camera during imaging. Whole-mount in situ hybridization and The hspb7 and hspb12 genes are regulated by gata4 during early fi morphogenetic defects in the myl7:gfp background were imaged zebra sh heart development on a Nikon SMZ1500 dissecting microscope. Images were analyzed fi with ImageJ (NIH) as previously described (Lin et al., 2012) except Zebra sh embryos depleted of gata4 or mouse embryos mutant confocal stacks were processed into single images using ImageJ. 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 Bead injection and movies control have been identified in the mouse heart (Rojas et al., 2008), it remains poorly understood why cardiogenesis fails in the Wildtype embryos at the 6-somite stage were dechorionated absence of gata4. We used the zebrafish model to define at the and embedded in 1% low melting point agarose with the lumen whole genome level the changes in transcripts that occur in visible dorsally. Embryos were injected with 0.5 μm FluoSpheres cardiomyocytes when gata4 is depleted during embryogenesis. (Invitrogen F8812) before the 10-somite stage. 10-somite stage For this purpose, cardiomyocytes were flow-sorted from either embryos were positioned so that the KV was imaged dorsally wildtype or gata4 morphant embryos derived from the myl7:gfp using an Axio Observer microscope fitted with an AxioCam mRm transgenic reporter strain. Three independent samples were pre- camera. Fifty frame movies were taken at 10 frames/s and analysis pared from hearts of embryos isolated at 24 h post fertilization of movies was performed with ImageJ and the module “Manual (hpf) and RNA-sequencing was carried out using the Illumina Tracking”. platform to define significant changes in transcript levels caused 392 G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400

Table 1 Gata4 regulates a specific developmental program in the cardiomyocytes. Downregulated genes from the RNAseq experiment at 24 hpf were submitted to DAVID for 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 adapter 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

by gata4 depletion. Cardiomyocytes were enriched by FACS to sub-threshold concentrations of MOs were injected targeting hspb7 70–80% purity, and approximately 75% of the sequence reads were or hspb12 that generate on their own a low percentage of looping successfully aligned to the zebrafish genome (Supplementary Fig. S1). defects. These were injected with or without a sub-threshold Genes down-regulated at least 2-fold were submitted to the concentration of MO targeting gata4 that also on its own does not Database for Annotation, Visualization, and Integrated Discovery cause looping defects. Combined knockdown of Gata4 with Hspb7 (DAVID) (Huang et al., 2009a,b) to identify those annotated or Hspb12 resulted in a much larger number of embryos with specifically for expression in the heart (Table 1). Genes encoding defective looping, demonstrating genetic interactions (Fig. 2C). small heat shock proteins Hspb7 and Hspb12 were identified in Injection of hspb7 RNA was not sufficient to rescue the looping this subset. Using independent sets of flow-sorted wildtype and defect in the gata4 morphant embryos (data not shown) suggesting morphant cardiomyocytes, RNA samples were analyzed by quan- that hspb7 is only one of multiple downstream components titative RT-PCR (qPCR) and this validated the RNA-seq data, mediating looping morphogenesis. showing that loss of gata4 causes a significant reduction in transcript levels for hspb7 and hspb12 (Supplementary Fig. S2). Loss of hspb7 or hspb12 disrupts ventricular cardiomyocyte During embryogenesis transcripts for these genes could be readily development detected by qPCR from the 11 somite (hspb7) or the 20 somite (hspb12) stage (Supplementary Fig. S3A and B). In situ hybridiza- The ventricles appear smaller and dysmorphic in the HSPB tion was used to define expression patterns for both genes. The morphants (Fig. 2A), which could be caused by a reduction in the hspb7 transcripts could be detected throughout the early embryo numbers of cells. Experiments were performed to evaluate mor- by five somites, and remained ubiquitous throughout the 18–20 phants derived from the myl7:gfp; myl7:dsRed-nls reporter line, somites stages (Fig. 1A–E). During these stages, hspb12 levels were which facilitates quantification of cardiomyocytes. To compare not reliably detected by in situ hybridization. However, by the 25 chambers, the embryos were also stained with S46 antibody, somite stage, transcripts for both genes are detected in the which labels specifically atrial cardiomyocytes. Ventricular cardi- developing heart, corresponding to the cardiac cone stage omyocytes were decreased in a statistically significant manner in (Fig. 1F). Both genes are expressed in the heart tube at 24 hpf both hspb12 morphants and double hspb7/12 morphants (Fig. 3). and 48 hpf (Fig. 1G and H), consistent with previous observations Interestingly, the number of atrial cardiomyocytes was not (Marvin et al., 2008). By 48 hpf, both hspb7 and hspb12 are affected, and cardiomyocyte numbers were not significantly expressed in the ventricular and atrial myocardium, coincident depleted in hspb7 morphants, suggesting that hspb12 modulates with the cardiac marker myl7 (Supplementary Fig. S3). ventricular cell number. Although cell number was not altered, the ventricles of hspb7 Loss of hspb7 or hspb12 disrupts normal heart tube looping morphants appear dysmorphic; therefore we investigated the possibility that size, shape, or sub-cellular structure of ventricular We designed splice-blocking MOs to target specifically hspb7 or cardiomyocytes are disturbed. Ventricular sarcomere formation hspb12 and showed that these successfully deplete, with specificity, and cardiomyocyte cell size was investigated in morphants endogenous RNA when injected into fertilized eggs (Supplementary derived from the myl7:actn3b-gfp; myl7:mKate-caax transgenic Fig. S4). Both splice-blocking MOs generate cardiac phenotypes reporter strain that labels both the sarcomeric Z-lines and cardi- when injected at or above a concentration of 0.2 mM, correlating omyocyte cell boundaries (Lin et al., 2012). By 48 hpf, sarcomere with significant knockdown of target transcripts. Knockdown of formation had proceeded normally in the hspb7 morphants hspb7 or hspb12 results with high penetrance in the development of (Supplementary Fig. S5). On the other hand, this analysis showed dysmorphic or non-looping heart tubes (Fig. 2A and B). The results that embryos injected with MOs targeting hspb7 or both HSPBs suggest that cardiac small heat shock proteins function downstream showed statistically significant defects in ventricular cardiomyo- of gata4 mediating cardiac morphogenesis. To test this hypothesis, cyte cell size, defined by measuring cardiomyocyte area (Fig. 4). G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 393

Fig. 1. 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 indicated. Upper panels (7-sense) are controls at each time points for comparison to anti-sense (hspb7), demonstrating ubiquitous staining pattern between 5 and 20 somites. (F–H) In situ hybridization expression data for hspb7 and hspb12 at 25-somites, 24 h post fertilization, and 48 h 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).

Fig. 2. 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, n¼54), embryos injected with standard control MO (control, n¼46), hspb7 MO (hspb7, n¼67), or hspb12 MO (hspb12, n¼89). (C) Quantification of the cardiac phenotypes in wildtype embryos (wt, n¼20) or embryos injected with sub-threshold concentrations of MO specific for gata4 (n¼38), hspb7 (n¼36), or hspb12 (n¼29), or with the same concentrations combined (gata4+hspb7, n¼29) or (gata4+hspb12, n¼49). 394 G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400

Fig. 3. 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, n¼7) or embryos injected with standard control MO (control, n¼7), hspb7 MO (hspb7, n¼7), hspb12 MO (hspb12, n¼8), or both hspb7 and hspb12 MOs (hspb7+12, n¼6). npo0.05 by Student's t-test in comparison to standard control group. The bars represent 10 μm.

Fig. 4. Hspb7 regulates ventricular cardiomyocyte size. (A) 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 μm. (B) Wildtype embryos (wt, n¼3) or embryos injected with the standard control MO (control, n¼4), hspb7 MO (hspb7, n¼5), hspb12 MO (hspb12, n¼3), or hspb7+12 MOs (hspb7+12, n¼4) were evaluated with ImageJ to measure average cell area. At least seven cells were analyzed for each embryo to determine the average. The asterisk indicates po0.05 by Student's t-test in comparison to the standard control group.

Transmission electron microscopy was used to more closely at least grossly normal (Supplementary Fig. S6), indicating that evaluate sarcomere structures in hspb7 morphants. The results cardiac defects in the hspb7 morphants are not caused by a major suggest that overall myofiber formation and sarcomere structure is breakdown in muscle cell structure. G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 395

Fig. 5. Hspb7 is required for normal jogging of the heart tube. (A) 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, n¼110), standard control MO injected (control, n¼100), hspb7 morphants (hspb7, n¼75), or hspb12 morphants (hspb12, n¼64).

Hspb7 has an earlier, Gata4-independent role in cardiac of embryogenesis. One of the first steps in the pathway is left-side morphogenesis initiation of spaw expression, followed by downstream activation in the cardiac mesoderm of lft1 and lft2. In embryos injected with The primitive heart tube normally jogs to the left as an initial MOs targeting hspb7, all three markers were aberrantly expressed morphogenetic movement prior to overt looping. We noticed that on the right side (or both sides) in a significant number of over 30% of hspb7 morphants displayed aberrant heart tube jogging morphants (Fig. 6). Left–right asymmetry was not affected in (Fig. 5). This jogging defect was rarely seen in control-injected, gata4 control MO-injected or the hspb12 morphants (Fig. 6). Embryos or hspb12 morphants (Fig. 5 and data not shown). To confirm that depleted of Gata4 also correctly restrict spaw expression appro- this directional jogging defect was due specifically to targeting of priately to the left side, consistent with a gata4-independent hspb7, additional controls were performed. First, embryos were function of hspb7 (Supplementary Fig. S9). injected with sub-threshold concentrations of either a splice- Normally, left-side laterality markers are amplified specifically blocking or a translation-blocking morpholino, such that all embryos on the left side due to the function of Kupffer's vesicle (KV), which jogged hearts normally to the left. When the same concentrations of generates an asymmetric flow of signaling molecules to the left MOs were co-injected, over 30% of the embryos failed to jog normally side of the embryo (Essner et al., 2005). The disrupted expression to the left, consistent with an hspb7-specific defect (Supplementary of spaw in hspb7 morphants suggests that Hspb7 is involved at an Fig. S7). Second, as an independent test of hspb7 function, TALENS early stage in the generation of this left–right asymmetry, possibly were designed (Cermak et al., 2011)totargetthefirst exon of the at the level of the KV. The KV is generated from dorsal forerunner hspb7 locus. Injection of RNA encoding a matched pair of hspb7- cells (DFCs), a type of initially non-involuting surface epithelial specificTALENsresultsinasignificant percentage of embryos with cells that migrate at the dorsal blastomere margin during epiboly developing heart tubes that failed to loop normally towards the left stages (Oteiza et al., 2008). Unlike most embryonic cells, the DFCs (Supplementary Fig. S8AandB).PCR-amplified fragments from retain cytoplasmic bridges with the yolk cell. Therefore, it is genomic DNA of uninjected control or TALEN-injected siblings were possible to deregulate gene expression specifically in these KV subjected to restriction analysis with the enzyme HpyCH4V, which progenitors by targeting injections into the yolk syncytial layer specifically digests the non-mutated TALEN target site. Consistent (YSL) at the 1000-cell (1K) stage (Wang et al., 2013). At this point with successful targeting, a notable fraction of the PCR-amplified the YSL remains a syncytium that is non-contiguous with blas- genomic DNA from TALEN-injected embryos was resistant to tomeres of the embryo proper. HpyCH4V digestion (Supplementary Fig. S8C). We tested whether Hspb7 is required in the KV or YSL by targeting hspb7 MOs specifically in the YSL at the 1K stage, and Heart jogging defects in hspb7 morphants result from failure to examining morphants for cardiac morphology. Embryos were establish a normal left–right axis co-injected with fluorescent dextran beads in order to confirm proper targeting to the YSL. Knockdown of Hspb7 in the YSL Proper heart tube jogging to the left depends upon appropriate resulted in an early arrest in cardiac development, as most of the establishment of overall left–right asymmetry during early stages embryos developed with a non-looping partially fused heart tube, 396 G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400

Fig. 6. Hspb7 is required for establishment of left–right asymmetry. (A–C) 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) Quantification of these phenotypes, and in each case n is at least 18 embryos.

Fig. 7. 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, n¼37) or embryos injected into the YSL with standard control MO (control, n¼29), hspb7 RNA (n¼30), hspb7 MO (n¼53), hspb7 MO +RNA (n¼74), or hspb12 MO (n¼21). npo0.05 comparing the embryos co-injected with RNA compared to those injected only with MO, according to Chi-squared test. G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 397

Fig. 8. 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 μm. (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 five embryos. The asterisk indicates po0.05 according to Student's t-test in comparison to the standard control group.

or cardia bifida (Fig. 7). Injection with lower doses of MO caused cilia-mediated KV flow was quantified. For this purpose, 0.5 μm laterality defects. As expected, these phenotypes were not found fluorescent beads were injected into the KV of YSL-targeted with similar injections using the hspb12 MO, since this gene is not morphants for hspb7 or (as control) hspb12. Clear defects in KV expressed at such early stages. The phenotype is relieved in a flow were evident in the hspb7 morphants. Average bead velocity significant number of embryos by co-injection with hspb7 RNA per embryo was decreased significantly (Fig. 9), confirming that into the YSL. These data support the hypothesis that the laterality Hspb7 regulates KV morphology and function; defects in this defects arise from specific loss of Hspb7 function in the KV and/ function therefore lead to failure in establishing normal laterality. or YSL. Finally, we considered if early laterality defects are an indirect cause of the cardiac morphogenic defects we had described earlier. YSL-dependent function of hspb7 is required for normal KV Hspb7 morphant embryos were separated at 30 hpf into cohorts development and left–right asymmetry demonstrating either normal (left-side) or abnormal jogging. A similar range of morphogenetic defects ensued, including Since the DFCs, precursors to the KV, are associated early on dysmorphic and non-looped heart tubes, regardless of whether with the YSL, we tested if KV development was affected by or not the early heart tube had jogged normally to the left knockdown of Hspb7 in the YSL. Hspb7-specific MOs were injected (Supplementary Fig. S10). Thus, the later morphogenetic heart into the YSL of embryos derived from the sox17:gfp reporter strain defects appear independent of the earlier role of Hspb7 in that marks the DFCs (Oteíza et al., 2008). Embryos were also controlling laterality. Taken together these results support an early stained with anti-acetylated alpha tubulin antibody to mark the role for hspb7 in the YSL or KV establishing KV morphology, and cilia. Overall KV morphology was found to be abnormal in thereby cardiac laterality. This early function is specifictohspb7 morphants, with overall reduced width, and in addition decreased and independent of gata4 and hspb12. Subsequent functions cilia length (Fig. 8). As a control, similar injections with the during cardiac morphogenesis for looping and chamber formation hspb12-specific MO did not disrupt KV development. To test are regulated by both hspb7 and hspb12 in a common pathway whether these morphological defects are functionally relevant, with gata4 (Fig. 10). 398 G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400

Fig. 9. Hspb7 is required for normal KV function. (A) Traces of fluorescent bead movement in the KV from a representative 5 s 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 μm. (B) Average bead velocity for each sample (n¼at least 5). The asterisk indicates po0.05 by Student's t-test in comparison to the standard control group. For each KV, the velocity of five beads was measured to determine the average.

fiber formation. While other HSPBs also were capable of prevent- ing 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, tran- scripts 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 func- Fig. 10. Distinct functions for Hspb7 in YSL and cardiac morphogenesis. Hspb7 is tions for Hspb7 and Hspb12. First, Hspb7 is essential for establish- expressed at 5-somites and is required in the YSL for YSL-dependent processes ing the left–right axis of asymmetry, through controlling the including KV morphogenesis and migration of the cardiac precursors to the morphology of the key signaling center, Kupffer's vesicle. Hspb7 midline. By 25-somites, both hspb7 and hspb12 are increasingly restricted to the specifically regulates cilia length and KV size, which impacts KV myocardium and are under the regulation of gata4 during subsequent stages of – cardiac morphogenesis. 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. Discussion Hspb12, on the other hand, does not regulate KV development, and thus depletion of Hspb12 does not affect establishment of Hspb7 and Hspb12 are two small heat shock proteins each left–right asymmetry. Although Hspb7 is not present in the Ciliary containing a conserved α-crystallin domain characteristic of this Proteome Database (ciliaproteome.org), HSPB1 and numerous family. Little is known about the normal function of these proteins, other HSPs are found in the photoreceptor sensory cilia complex which are presumed to carry out chaperone activity. Hspb7 has (Liu et al., 2007), so it remains possible that Hspb7 has a direct role been shown to prevent the aggregation of polyQ proteins in a in KV ciliary morphogenesis. Although we discovered hspb7 as a manner dependent upon macroautophagy. However, overexpres- downstream target of gata4, this early role is independent of gata4 sion of Hspb7 does not activate autophagic pathways. Unlike other since depletion of Gata4 during embryogenesis has no effect on members of the HSPB family, Hspb7 is unable to facilitate the jogging, nor for establishment of left–right asymmetry. Targeted refolding of heat-denatured proteins, and is not dependent on the loss of Hspb7 in the YSL often led to a cardia bifid phenotype, ATP-dependent Hsp70 machine for its activity (Vos et al., 2010). caused when cardiac progenitors fail to migrate properly to Previous work characterized Hspb7 as most highly expressed in the midline, a process known to be regulated by the YSL cardiac muscle, and it has been referred to as cardiac heat shock (Langenbacher et al., 2012; Sakaguchi et al., 2006). This suggests protein (Krief et al., 1999). In a cardiac cell culture system, over- a wider (and perhaps unrelated) function for hspb7 in the YSL expression of Hspb7 prevented tachypacing-induced F-actin stress beyond KV morphology. This bifid phenotype was rarely noted G.E. Rosenfeld et al. / Developmental Biology 381 (2013) 389–400 399 when MOs were injected into the embryos, presumably because Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W., 2008. Adapting proteostasis for this is hypermorphic with respect to the YSL function, which would disease intervention. Science 319, 916–919. Berry, V., Francis, P., Reddy, M.A., Collyer, D., Vithana, E., MacKay, I., Dawson, G., be more fully targeted by injection directly into the YSL at the 1K Carey, A.H., Moore, A., Bhattacharya, S.S., Quinlan, R.A., 2001. Alpha-B crystallin stage. The range of phenotypes including bifid hearts is seen in gene (CRYAB) causes dominant congenital posterior polar cataract in embryos injected with RNA encoding hspb7-specific TALENs. humans. Am. J. Hum. Genet. 69, 1141–1145. Cappola, T.P., Li, M., He, J., Ky, B., Gilmore, J., Qu, L., Keating, B., Reilly, M., Kim, C.E., Second, we showed that Hspb12 is required to generate normal Glessner, J., Frackelton, E., Hakonarson, H., Syed, F., Hindes, A., Matkovich, S.J., cardiomyocyte numbers in the ventricle, while Hspb7 is required for Cresci, S., Dorn II, G.W., 2010. 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Most interestingly, several recent studies Evgrafov, O.V., Mersiyanova, I., Irobi, J., Van Den Bosch, L., Dierick, I., Leung, C.L., fi Schagina, O., Verpoorten, N., Van Impe, K., Fedotov, V., Dadali, E., Auer- identi ed HSPB7 in genome wide association analyses for advanced Grumbach, M., Windpassinger, C., Wagner, K., Mitrovic, Z., Hilton-Jones, D., heart failure and idiopathic cardiomyopathies (Cappola et al., 2010; Talbot, K., Martin, J.J., Vasserman, N., Tverskaya, S., Polyakov, A., Liem, R.K., Stark et al., 2010; Villard et al., 2011). Our identification of hspb7 as a Gettemans, J., Robberecht, W., De Jonghe, P., Timmerman, V., 2004. Mutant small heat-shock protein 27 causes axonal Charcot–Marie–Tooth disease and downstream target of gata4 is intriguing in this regard. Gata4 is known distal hereditary motor neuropathy. Nat. Genet. 36, 602–606. to be cardio-protective. 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