Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia
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The heart’s rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 −/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 −/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.
KeywordsArrhythmia Gene regulation Islet1 Shox2 Sinoatrial node Transcription factors
During embryogenesis most cells have to become specialized into one of many various cell types that build a whole organism. These patterning and differentiation processes are ensured by the temporal and spatial activation of different transcription factors . Homeobox genes, for example, regulate numerous processes during embryogenesis including heart development. Complex transcriptional regulation networks are important in many aspects of heart cell lineage development and morphogenesis .
The cardiac conduction system consists of electrically coupled cardiomyocytes that are responsible for impulse propagation and essential for the rhythmic and coordinated contraction of the heart. A specialized group of cells in the sinoatrial node (SAN) located at the junction of the superior caval vein and right atrium generates the electric impulse to activate the atrial myocardium (also termed primary pacemaker). Together with specialized cells in the atrioventricular node (termed secondary pacemaker) and conduction fibers in the interventricular septum, they form the cardiac conduction system [7, 19]. Abnormalities in the cardiac conduction pathway are responsible for various forms of arrhythmias [41, 44, 58]. Sinus node dysfunction, for example, is associated with sinus bradycardia, atrial tachyarrhythmias, sinus pause or arrest and sinoatrial exit block . Although the morphology, cellular components and electrophysiological properties of the SAN are rather well described [34, 38], knowledge of pathways and transcriptional regulation of the SAN formation are poorly understood. Unraveling the pathways that control pacemaker development and function is of crucial relevance to our understanding of pathologies associated with cardiac conduction.
So far, several transcription factors of the homeobox- and T-box gene families have been shown to be involved in cardiac conduction system specification, patterning, maturation, and function [17, 37]. Among these genes is the paired-related homeodomain transcription factor Shox2, which is known to play a crucial role in early cardiac formation, particularly in SAN development and specification [2, 11, 12, 30, 42]. Shox2 expression can first be detected in the posterior region of the primitive heart tube at murine embryonic stage E8.5 and as development progresses, Shox2 is specifically expressed in the sinus venosus myocardium comprising the SAN and the venous valves [2, 12]. These Shox2 expressing domains develop from myocardium that is added to the venous pole of the developing heart and contributes to the second heart field lineage [5, 52]. Newly recruited myocardium that is added to the arterial pole is divided into the anterior or secondary heart field, while the myocardium added to the venous pole is referred to as the posterior heart field . Loss of function studies revealed an essential role for Shox2 in the normal anlage of the posterior heart field myocardium [2, 12]. Shox2 −/− mice die between E11.5 and E17.5 due to heart failure and vascular defects. In particular, the sinus venosus myocardium including the SAN and venous valves show hypoplasia. Moreover, Shox2 has a crucial role in pacemaker function indicated by severe bradycardia with intermittent sinus exit block after morpholino-mediated knockdown in zebrafish embryos and markedly reduced heart beat rates of isolated Shox2 −/− hearts [2, 12]. To investigate the molecular processes underlying Shox2 function, different marker gene expression analyses have been performed. The aberrant expression of Cx40, Cx43, Nppa, and Nkx2.5 within the SAN of Shox2 mutant hearts as well as the decreased expression of the SAN-specific marker genes Tbx3 and Hcn4 has indicated an abnormal differentiation of pacemaker cells [2, 12]. It has also been shown that Shox2 prevents the SAN from atrialization by repressing Nkx2.5 expression [11, 12]. Recently, a further link between Tbx5, Shox2 and Bmp4 could be established in the developing heart, demonstrating that Tbx5 signaling in the cardiac pacemaker region controls the expression of Shox2, which in turn regulates Bmp4 in a direct manner . It has also been reported that Pitx2c represents an important susceptibility gene for atrial arrhythmias by suppressing left-sided sinus node formation via direct repression of Shox2 [25, 53]. Taken together, these findings illustrate the importance of Shox2 function in SAN development and highlight the need for further elucidation of the molecular networks involved.
The aim of our current study was to identify Shox2 target genes during heart development. We used expression analysis to compare the transcriptomes in right atria of wildtype and Shox2 −/− mouse hearts, and Islet1 (Isl1) was identified as a direct transcriptional target. We have examined putative regulatory elements in the human ISL1 gene and show a pivotal role for sequences within intron 2 in activating transcription. Furthermore, a functional link between Shox2 and Isl1 in vivo was demonstrated using zebrafish as a model by rescuing the Shox2-mediated bradycardia via ectopic expression of Isl1.
Animals and tissue samples
Shox2 −/− mice were generated and genotyped as previously described . For maximal reproduction, we crossed our mice into the CD-1 outbred strain. To improve specificity of the microarray analysis, we dissected the right atria of E11.5 hearts that comprise the Shox2 expressing sinus venosus myocardium and venous valves. For total RNA isolation by TRIzol® (Invitrogen), samples from embryonic right atria (E11.5) of the same genotype (wildtype and Shox2 −/−) were pooled. For whole-mount in situ hybridization, complete hearts were dissected from E11.5 and E12.5 embryos and fixed overnight in 4 % paraformaldehyde at 4 °C.
Gene expression profiling was performed using the GeneChip Mouse Gene 1.0 ST array from Affymetrix (Santa Clara, CA) according to the manufacturer’s protocol. For each genotype (wildtype and Shox2 −/−), RNA from 6 right atria from embryos of 2 independent pregnancies was pooled. Purity and quality of isolated RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and showed a RIN >9. 200 ng RNA were used for production of end-labeled biotinylated ssDNA and subsequently hybridized to the arrays. Statistical comparisons of chip data based on ANOVA were performed using the software package JMP Genomics, version 4.0 from SAS (SAS Institute, Cary, NC, USA). Briefly, values of perfect-matches were log transformed, quantile normalized and fitted with log-linear models, with probe ID and genotype considered to be constant. Custom CDF version 14 with Entrenz gene-based gene definitions (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp) different from the original Affymetrix probe set definitions was used to annotate the arrays. The microarray data were deposited in the NCBI GEO database with accession number GSE39924.
cDNA was synthesized using the SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR Green ROX dye (Thermo Scientific). Primer sets used are presented in Supplemental Material (Table S1).
Generation of plasmid constructs
Cloning of the murine Isl1 in situ probe, the zebrafish shox2 and isl1 expression constructs, the human SHOX2a expression construct and generation of the ISL1 luciferase reporter constructs are described online in the Supplemental Material.
In situ hybridization
Shox2 −/− mice have been described previously . Whole-mount in situ hybridization on embryonic mouse hearts was performed as reported . Specific digoxygenin- or fluorescein-labeled RNA probes were as follows: Isl1 (see Supplemental Material) and Hcn4 (kindly provided by B. Bruneau, Gladstone Institute of Cardiovascular Disease, USA).
E11.5 embryos were fixed in 4 % paraformaldehyde at 4 °C overnight, embedded in OCT and transversely cryosectioned (10 μm). Tbx18 and Hcn4 stainings were enhanced using the ABC (Avidin–Biotin-Complex) reagent from Vector Laboratories (VECTASTAIN Elite Peroxidase ABC-Kit) and the TSA (Tyramide Signal Amplification) system (NEL741, Perkin Elmer) according to the manufacturers’ instructions. For double staining (Isl1/Tbx18 or Isl1/Hcn4), the respective fluorescent secondary antibody was added either during the biotinylated antibody incubation or in a second step afterwards. The following primary antibodies were used: mouse monoclonal antibody against Isl1 (1:100; 39.4D5, Developmental Studies Hybridoma Bank), goat polyclonal antibody against Tbx18 (1:250; C-20, Santa Cruz) and rabbit polyclonal antibody against Hcn4 (1:500; APC-052, Alomone). Alexa Fluor 568 rabbit anti-mouse (1:800; A-11061, Invitrogen), biotinylated horse anti-goat (1:500; BA-9500, Vector Laboratories) and biotinylated goat anti-rabbit (1:500; 550338, BD Pharmingen) were used as secondary antibodies. Nuclei were counterstained with Hoechst (Invitrogen). Imaging was carried out on a Nikon 90i upright epi-fluorescence microscope with a Nikon DS-Qi1 mc camera.
Cell culture, transfection and luciferase assay
HEK 293 cells were cultured at 37 °C in DMEM medium containing high glucose, supplemented with 10 % fetal calf serum and antibiotics. For luciferase assay analysis, the cells were cotransfected in triplicate with different constructs using polyethylenimine (Sigma-Aldrich). 24 h after transfection, luciferase activity was determined and normalized to Renilla luciferase activity with a dual luciferase assay kit (Promega). Experiments were repeated at least three times in triplicate with consistent results and representative data are shown.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed using standard protocols. For the binding reaction, 32P-labeled, double stranded ISL1 oligonucleotides (Oligo1 (+2364/+2418), Oligo2 (+2404/+2458), Oligo3 (+2447/+2502), Oligo4 (+2496/2561); Supplemental Material Table S1), were used together with purified, bacterially expressed recombinant GST-SHOX2 protein .
Zebrafish embryos and microinjections
Care and breeding of zebrafish, Danio rerio, were as described previously . For all plasmid and morpholino injection procedures, the TE4/6 wildtype strain was used.
Morpholino-modified antisense oligonucleotides (MO; Gene Tools) were directed against the translational start site (shox2-MO [5′-ACGCTGTAAGTTCTTCCATCACTGC-3′]) of zebrafish shox2, the splice donor site of exon 2 (isl1-MO [5′-TTAATCTGCGTTACCTGATGTAGTC-3′]) of zebrafish isl1 and the translational start site (isl1-MO2 [5′-GATCCCCCATGTCTCCCATGTCAAG-3′] of zebrafish isl1. A shox2-MO directed against the splice donor site of intron 3 causing the identical bradycardia phenotype has been described previously . shox2 and isl1 antisense oligonucleotides or a standard control oligonucleotide (MO-control), diluted in 0.2 mol/liter KCl, were microinjected into one-cell-stage zebrafish embryos . For rescue experiments 0.32 ng of pDestTol2CG2-shox2 or pDestTol2CG2-isl1 was microinjected into one-cell-stage embryos directly after injection of 2.8 ng of shox2-MO.
For histology, embryos were fixed in 4 % paraformaldehyde and embedded in JB-4 (Polysciences, Inc). Then, 5-μm sections were cut, dried, and stained with hematoxylin and eosin .
For immunoblotting, monoclonal mouse IgG1 anti-rat islet1 antibody (Developmental Studies Hybridoma Bank, DSHB, University of Iowa) and polyclonal anti pan-cadherin (Abcam) were used. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. Blots were probed with the mentioned antibodies, signals were detected by chemiluminescence (monoclonal anti-rabbit-Hrp and anti-mouse-Hrp) and imaging was performed with Image Quant LAS4000mini (GE Healthcare). Quantification of the immunoblot was performed using the Image Quant TL software.
Whole-mount antisense RNA in situ hybridization was carried out as described  using a digoxigenin-labeled antisense probe for zebrafish shox2 and islet1. For immunostaining, zebrafish embryos were fixed in Dent’s fixative and a monoclonal anti-acetylated tubulin antibody (Sigma) was used .
Data are presented as mean ± SEM from at least three independent experiments. Comparisons between experimental groups were performed using paired t test or 1-way ANOVA. Differences were considered significant if they showed a value of P < 0.05.
Shox2 has regulatory effects on Isl1 expression in the developing sinoatrial node
To identify novel transcriptional targets of Shox2, we performed microarray analysis and compared gene expression levels in right atria of wildtype and Shox2 −/− hearts. Only right atria comprising the Shox2 expressing sinus venosus myocardium and venous valves were dissected from murine E11.5 hearts to increase the specificity of the array analysis. Genes were considered to be up- or downregulated if they showed log2 ratios of ≥0.35 or ≤−0.4 with a significance of P ≤ 0.0001. Using these selection criteria, we identified 321 genes with significantly altered gene expression (80 up- and 241 downregulated genes). As expected, Shox2 was among the highest negatively regulated genes. Classification of the 321 genes into gene families using the Gene Set Enrichment Analysis (GSEA) revealed that the highest proportion of regulated genes belongs to the group of transcription factors. Among these, the LIM homeodomain transcription factor Isl1 showed a reduced expression in right atria of Shox2 −/− embryos.
SHOX2 binds to regulatory elements in an intronic region of the ISL1 gene
Bradycardia caused by shox2 deficiency depends on isl1 expression
During embryogenesis, the proper development of the cardiac pacemaker is essential for a coordinated heart beat. Defects in the transcriptional network controlling SAN development may cause dysfunction of the primary pacemaker resulting in severe arrhythmias. The elucidation of the molecular mechanisms and key regulators involved in these developmental processes is therefore crucial for our understanding of arrhythmia-related diseases.
The LIM homeodomain transcription factor Isl1 is a prominent marker of second heart field derivatives comprising the right ventricle, outflow tract, inflow tract with the SAN and parts of the atria and plays a crucial role in cardiogenesis [6, 49, 51]. SAN development is initiated in the presence of Isl1 expressing second heart field-derived cells [35, 48, 51]. Homozygous loss of Isl1 results in embryonic lethality between E10.5 and E11.0 due to severe cardiac abnormalities. Besides heart looping defects, Isl1 −/− embryos show hypoplasia or complete absence of the right ventricle, the outflow tract and parts of the atria including the inflow tract . Early in embryogenesis, Isl1 positive cells, which exhibit multipotent properties [4, 36], contribute to both the arterial (outflow tract) and the venous pole (SAN) of the primary heart tube. Once these progenitor cells differentiate into cardiomyocytes, Isl1 expression becomes downregulated and almost extinguished from E12.5 to E18.5 . Small numbers of Isl1 positive cells can still be detected in the adult hearts, including the SAN [14, 24, 54].
Although signaling pathways up- and downstream of Isl1 have been investigated in the past [1, 6, 8, 26, 39], little is known about direct regulators controlling the transcriptional activity of this gene in cardiac cells. The only proteins known to regulate Isl1 expression so far via direct binding are ß-Catenin , Forkhead- and Gata4 transcription factors [22, 23] and the POU homeodomain transcription factor Oct1 . While the binding sites for these regulators reside in promoter and enhancer regions up- or downstream of the gene, we could identify a regulatory element within an intronic region of ISL1.
Based on the established functional data, SHOX2 as well as SHOX2 targets represent excellent candidates for human arrhythmias. While genetic variation in ISL1 is associated with congenital heart disease and cardiomyopathies [13, 47], SHOX2 has not been linked to any human phenotype yet. In a SHOX/Shox2 replacement mouse model, it was recently shown that a hypomorphic SHOX allele is not able to completely rescue the Shox2 −/− cardiac phenotype. Although the mice survive, they still show arrhythmias, suggesting that SHOX2 haploinsufficiency may lead to arrhythmia in humans . Thus, it will be interesting to see if both SHOX2 and ISL1 are involved in human heart disease, especially arrhythmias.
A possible connection between Isl1 expressing second heart field-derived cells and the formation of the cardiac conduction system has been previously proposed [35, 48, 54]. As Shox2 directly regulates Isl1 expression specifically in the embryonic SAN, our data show for the first time a direct functional consequence for Isl1 in the pacemaking system of the developing heart. Taken together, our data add another piece to the puzzle of regulatory transcriptional networks controlling the development of the pacemaker.
We thank the Developmental Studies Hybridoma Bank (DSHB, University of Iowa) for the Isl1 antibody and Maria Saile for help with the microarray analyses. This work was supported by the Deutsche Forschungsgemeinschaft [RA 380/14-2]; the Bundesministerium für Bildung und Forschung [01GS1104 NGFNplus]; by the DZHK (Deutsches Zentrum für Herz-Kreislauf-Forschung—German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research).
Conflict of interest
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