Abstract
Rigorous exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here using multiple knockout mouse models and single- cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2as has a key, complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function, largely independent of Hand2as transcription and transcripts. Full-length deletion of Hand2as in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, resulting in profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose a cis-functional role for the Hand2as locus in dampening HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the need for complementary genetic and single-cell approaches to delineate the function and primary molecular effects of an lncRNA in animals.
Impact statement The long noncoding RNA Hand2as critically controls the precise expression of its neighboring gene HAND2, thereby balancing cardiac lineages and expression programs that are essential for heart development and function.
Introduction
Long noncoding RNAs (lncRNAs) have been implicated as an important layer of regulatory information in fine-tuning the spatiotemporal expression of pleiotropic developmental loci in their chromatin neighborhood, thereby modulating cell fate determination in various biological processes (Han et al., 2018; Luo et al., 2016; Morris and Mattick, 2014; Pauli et al., 2011; Ponjavic et al., 2009; Yin et al., 2015). Heart formation is tightly regulated during mouse embryogenesis and involves restriction of mesodermal precursor cells to the cardiac lineage and the subsequent formation of a primitive heart tube, which, in turn, undergoes looping, formation of the outflow tract and atrial and ventricular cavities, and septation to form the mature four-chambered heart (Bruneau, 2008; Olson and Schneider, 2003). Proper commitment of cardiac lineages during this complex process is required for normal development and function of the heart (Brade et al., 2013). Several lncRNAs have been reported to have roles in regulating heart development and function. For example, depletion of Chast/Wisper and overexpression of Mhrt, Tincr, or Carel protected the heart from hypertrophy in response to pressure overload following transverse aortic constriction surgery (Cai et al., 2018; Han et al., 2014b; Micheletti et al., 2017; Shao et al., 2017; Viereck et al., 2016). Inhibition of Fendrr led to embryonic lethality around E13.5 with cardiac hypoplasia (Grote et al., 2013).
The lncRNA Hand2as (also named Uph or lncHand2) is divergently positioned at −123 bp upstream of the transcription start site (TSS) of HAND2 (Anderson et al., 2016; Wang et al., 2018). HAND2, a transcription factor that promotes ventricular cardiomyocyte expansion and cardiac reprogramming, is a critical regulator of embryonic heart development (McFadden et al., 2005; Song et al., 2012; Srivastava et al., 1997). RNA in situ hybridization analysis of mouse embryos reveals that cardiac expression of HAND2 is initially detected in the cardiac crescent at E7.75 and continues throughout the linear heart tube at E8.5, and thereafter is specifically enhanced in the developing right ventricle (RV) and outflow tract (OFT), with lower levels of expression in the atrial and left ventricular chamber (Srivastava et al., 1997; Tamura et al., 2014). This pattern persists through E9.5-E10.0, after which HAND2 expression is downregulated in the cardiac mesoderm but is maintained in the neural crest-derived aortic arch arteries (Srivastava et al., 1997; Tamura et al., 2014).
Precise expression of HAND2 is essential for normal heart morphogenesis and function, and is tightly regulated at the transcriptional level by a network of cardiac transcription factors and upstream enhancers, and at the post-transcriptional level by microRNAs (Bruneau, 2005; Dirkx et al., 2013; McFadden et al., 2005; McFadden et al., 2000; Zhao et al., 2007; Zhao et al., 2005). Constitutive HAND2 knockout (KO) in mice caused right ventricle hypoplasia and embryonic lethality at E10.5 (Srivastava et al., 1997). Conditional ablation of HAND2 in specific sets of cardiac cells led to embryonic lethality at various stages prior to embryonic day E15.5 (summarized in Supplementary file 1) (Holler et al., 2010; Morikawa and Cserjesi, 2008; Morikawa et al., 2007; Tsuchihashi et al., 2011; VanDusen et al., 2014). Overexpression of HAND2 in transgenic mouse models also led to heart development defects and malfunctions (Supplementary file 1). For example, embryonic overexpression of HAND2 driven by the β-myosin heavy chain (MYH7) promoter prevented the formation of the interventricular septum in embryos (Togi et al., 2006), while overexpression driven by the α-myosin heavy chain (MYH6) promoter resulted in pathological myocardial hypertrophy and heart failure in adult hearts (Dirkx et al., 2013).
In a polyA-knockin (KI) mouse model of Uph/Hand2as reported previously, termination of transcription by insertion of a triple polyadenylation (polyA) stop sequence into intron 1 of Uph (- 644 bp upstream of the HAND2 TSS) abolished HAND2 expression and led to failed right ventricle formation and lethality at E10.5, partially phenocopying HAND2 KO mice (Anderson et al., 2016). In addition, compound heterozygous Uph and HAND2 mutant embryos lack HAND2 expression and recapitulate the HAND2 KO phenotype (Anderson et al., 2016). It was concluded that transcription of Uph/Hand2as is an essential switch for the activation of HAND2 and the onset of heart morphogenesis (Anderson et al., 2016). However, the functional role of Hand2as transcripts and the Hand2as DNA sequences in the heart remains elusive.
To delineate the role of Hand2as in heart development and function, we generated three deletion alleles of Hand2as in mouse (Han et al., 2018). Full-length deletion of the entire Hand2as sequence (Hand2asF/F KO) led to dysregulated cardiac gene expression programs, ventricular septal defects and heart hypoplasia and perinatal death, reminiscent of congenital heart diseases. A short distal deletion at the 3’ end of the Hand2as locus (Hand2asD/D KO) caused severe contraction defects in adult heart that progressively worsened with increasing age. In comparison, short deletion of the 5’ promoter and exons of Hand2as (Hand2asP/P KO) effectively diminished Hand2as expression, but failed to produce discernable heart phenotypes in either embryos or adults. These results indicate that the Hand2as DNA locus, rather than its transcription/transcripts, primarily controls heart development and function. To our surprise, cardiac expression of HAND2 was sustained in all three Hand2as KO mouse models we generated, in sharp contrast to the abolished expression of HAND2 in the Uph/Hand2as polyA KI embryos (Anderson et al., 2016). Importantly, single-cell transcriptomic analysis revealed subtle yet prevalent upregulation of HAND2 and concordant global gene expression changes in subsets of cardiac cells of Hand2asF/F embryos lacking the entire Hand2as DNA sequence. Altogether, these results illustrate a fine-tuning yet critical role for the lncRNA Hand2as locus in restricting the precise, spatial expression of HAND2, through which Hand2as modulates cardiac lineage development and heart function. This study reveals the unexpected complexity of lncRNA function in vivo, and also emphasize the usage of complementary genetic and single-cell approaches to delineate the primary molecular effects and elucidate physiological functions of an lncRNA in animals.
Results
Hand2as transcripts are dispensable for heart development
Hand2as and HAND2 are divergently transcribed from the shared core promoter sequences, and are highly enriched in the heart compared to other tissue types analyzed (Figure 1A, Figure 1-figure supplement 1A-B). Interesting, within the heart, Hand2as and HAND2 exhibit an inverse expression pattern during embryonic heart development and postnatal growth (Figure 1-figure supplement 1C).
The DNA sequence of Hand2as is 17 kb in length and encompasses a super-enhancer element, and branchial arch (BA) and cardiac enhancers annotated previously (Figure 1A) (McFadden et al., 2000; Yanagisawa et al., 2003). In E12.5 embryonic hearts, the Hand2as locus as well as HAND2 and its downstream regions harbor multiple DNase I hypersensitive sites (DHS), and show strong binding signals of active histone H3K4me3 and H3K27ac marks (H3 Lys4 tri-methylation and Lys27 acetylation, respectively), and RNA polymerase II and master transcription regulators of cardiac development, including GATA4, NKX2-5 and HAND2 (E10.5) itself (Figure 1A) (He et al., 2014; Laurent et al., 2017; Ye et al., 2015; Yue et al., 2014). This suggests a possible involvement of multiple enhancers in regulating HAND2 expression. In support of this notion, we found that in mouse embryonic stem cells (ESCs), the HAND2 promoter interacts with two known upstream enhancers (BA and cardiac) in the Hand2as locus, and also with multiple downstream DNA elements embedded in the lncRNA 5033428I22Rik locus (a strong interaction within the first intron of 5033428I22Rik is marked by blue shading in Figure 1A), as shown by HiCap, a genome-wide promoter capture method to detect chromatin contacts (Sahlen et al., 2015).
We set out to dissect the function of Hand2as in the heart. To remove Hand2as transcription/transcripts with minimal manipulation of the genome, we first generated two mouse models carrying short genomic deletions at the 5’ or 3’ end of the Hand2as locus (Figure 1A) (Han et al., 2018). These deletions do not affect the two known enhancers for cardiac and branchial arch expression of HAND2 (McFadden et al., 2000; Yanagisawa et al., 2003). In the 5’ proximal knockout allele (Hand2asP KO), we deleted a 1-kb DNA sequence covering the core promoter and the first two exons of Hand2as (Figure 1A) (Han et al., 2018). The deletion starts at −61 bp and −62 bp upstream of the TSSs of Hand2as and HAND2 respectively. To avoid any direct effect of promoter alteration on HAND2 expression, we generated a 3’ distal knockout allele (Hand2asD KO) by deleting a 2.7-kb DNA sequence which spans exons 4 and 5 of Hand2as and is located 13-kb upstream of the HAND2 TSS (Figure 1A) (Han et al., 2018).
Both Hand2asP/P and Hand2asD/D mice from heterozygotes crosses were born at the expected Mendelian ratio and had no overt morphological defects in the heart (Figure 1B, Figure 1-figure supplement 1A; data not shown). Compared to heterozygous littermates, Hand2asP/P mutant mice showed residual expression (10~17%) of a truncated Hand2as RNA which lacks the first two exons and represents 64% of the full-length transcript, in hearts from E12.5 embryos or 8-week old adults (Figure 1-figure supplement 1A and 1D). Hand2asP/P KO mice therefore provide a partial loss-of- function model. However, cardiac expression of HAND2 was not altered in Hand2asP/P mice (Figure 1-figure supplement 1A and 1D).
In comparison, Hand2asD/D mice expressed a mutant Hand2as RNA that lacks exons 4 and 5 with 67% of its sequence remaining (Figure 1-figure supplement 1A). Embryonic expression of HAND2 in Hand2asD/D hearts was not affected (data not shown). However, Hand2asD/D adult cardiomyocytes showed moderate but significant increases of both Hand2as and HAND2 transcripts (~53% and ~34% up, respectively) (Figure 1C). It was reported that aberrant upregulation of HAND2 in the postnatal heart contributes to pathological hypertrophy (Dirkx et al., 2013). Interestingly, Hand2asD/D, but not Hand2asP/P mice, progressively developed heart contraction defects at 6-10 weeks old, with a 10~30% decrease in fractional shortening (Figure 1D). Consistent with the cardiac contraction defect, many genes involved in heart development, cardiac muscle contraction, and the cell cycle were dysregulated in Hand2asD/D cardiomyocytes (Figure 1-figure supplement 1E-F; Supplementary files 2 and 3). These results suggested that the Hand2as locus may exert a complex, pleiotropic influence on HAND2 expression and heart physiology. However, the lack of heart phenotype in Hand2asP/P mice indicates that Hand2as transcripts and perhaps its transcription may be largely dispensable for cardiac expression of HAND2 and embryonic heart development.
Deletion of the entire Hand2as locus causes congenital heart defects and perinatal lethality
Next, to rule out the possibility that residual activities of Hand2as might promote HAND2 expression and heart morphogenesis in the promoter and distal KO mouse models, we deleted a 17- kb sequence covering the entire Hand2as genomic region to completely eliminate Hand2as expression (Figure 1A) (Han et al., 2018). The deletion starts at −59 bp and −64 bp upstream of Hand2as and HAND2 TSSs, respectively, and encompasses the super-enhancer and two known enhancers of HAND2 expression (McFadden et al., 2000; Yanagisawa et al., 2003) (Figure 1A). This mutant allele is designated as Hand2as full-length knockout (Hand2asF KO).
Heterozygous Hand2asF/+ intercrosses failed to produce viable homozygous offspring (0 out of 77 pups) at the weaning stage (Figure 2A, Figure 1-figure supplement 1A). Viable Hand2asF/F embryos were observed during mid-gestation at the expected Mendelian frequency until E16.5, but thereafter, death occurred at varying times, ranging from E16.5 to just after birth (Figure 2A). Hand2asF/F newborns (7 out of 41 at P0) became cyanotic and invariably died shortly after birth (Figure 2B). Gross morphological examination of hearts revealed abnormal blood coagulation and fatal thrombosis in Hand2asF/F newborns (Figure 2C), indicating heart failure in response to increased demand for cardiac output upon birth. To uncover the heart defect causing the perinatal lethality, Hand2asF/F late-gestation embryos (E16.5) were subjected to necropsy and histological examination. Macroscopically, the most severe phenotype in homozygous embryonic hearts is the presence of ventricular septal defects (8 out of 12) (Figure 2D). These lesions may cause blood to leak from the left ventricle into the right ventricle and then a right-to-left shunt, consequently leading to immediate cyanosis and death (Minette and Sahn, 2006). Right ventricular (RV) hypoplasia was also frequently observed with significantly decreased chamber volume (10 out of 12) and slightly reduced thickness of the compact myocardium of the right ventricle (3 out of 12) in Hand2asF/F mutant hearts (Figure 2D). These defects are reminiscent of congenital heart diseases, and provide a morphological explanation for the heart failure of Hand2asF/F mice in response to cardiac stress at birth.
Notably, Hand2asF/F late-gestation embryos and newborns had cleft palate (Figure 2-figure supplement 1A), resembling the craniofacial defects observed in branchial arch enhancer KO mice, which reportedly, failed to suckle and died with an empty stomach 24 hours after birth (Yanagisawa et al., 2003). As Hand2asF/F pups died much earlier, within hours after birth, we reasoned that the suckling defect is not the cause of death in these animals. In addition, we found no gross abnormalities in other organs, including aortic arch arteries, liver and lung (Figure 2-figure supplement 1B-C). Moreover, Hand2asF/F pups showed normal floating lungs in a buoyancy test (data not shown), excluding the possibility of respiratory failure as the cause for their immediate death upon birth.
To reveal transcriptional changes that underlie the morphological defects and perinatal lethality, we performed RNA-seq analysis of embryonic hearts or ventricles isolated from littermates from Hand2asF/+ intercrosses. Interestingly, a subset of gene programs pertaining to cardiac muscle contraction, such as ACTA1, COX6C and MYL2 were upregulated in Hand2asF/F embryonic hearts at E11.5, implying abnormally increased cardiac myogenesis in the mutant heart (Figure 2E-F; Supplementary files 2 and 4). Further transcriptome analysis of E16.5 ventricles also revealed significant upregulation of genes related to hypertrophic cardiomyopathy in Hand2asF/F embryos (Figure 2-figure supplement 1D; Supplementary files 2 and 5). The transcriptomic defects offer a molecular explanation for heart morphological defects and function failure, which are most likely the cause of the perinatal death of Hand2asF/F newborns.
Sustained HAND2 expression in Hand2asF/F embryos
To study the direct effect of Hand2as deletion on HAND2 expression, we first confirmed the complete absence of Hand2as transcripts in Hand2asF/F mutant embryos by RNA-seq and RT-qPCR (reverse transcription and quantitative PCR) (Figure 3-figure supplement 1A; data not shown). In addition, we did not observe any RNA signals or transcripts downstream of the Hand2as locus (data not shown). Thus, Hand2asF/F KO mice provided a complete loss-of-function model, in which the transcription, transcripts and DNA sequences of Hand2as were simultaneously removed. However, to our surprise, the levels of cardiac HAND2 transcripts were not lost in Hand2asF/F mutant embryos. The coding sequence (CDS) of HAND2 showed comparable expression between homozygous and heterozygous littermates throughout heart morphogenesis from E9.5 to E16.5 (Figure 3A-B).
To confirm this finding, we performed RNA in situ hybridization (ISH) analysis of E9.5 embryos and found similar distribution and expression patterns of HAND2 mRNA between homozygous and heterozygous littermates (Figure 3C). Next we analyzed HAND2 protein levels by immunostaining analysis of transverse sections of E9.5 embryonic hearts. Again, comparable levels of HAND2 protein were detected in mutant embryos (Figure 3D, Figure 3-figure supplement 1B). We noted a 1-2-fold reduction in HAND2 RNA signals that fall into its 5’ untranslated region (UTR) in Hand2asF/F embryonic hearts, despite unchanged expression in the CDS of HAND2 (Figure 3A, Figure 3-figure supplement 1C). Shortening of the HAND2 5’ UTR might promote translation of HAND2 protein, as demonstrated in cultured cells (Figure 3-figure supplement 1D) (Curtis et al., 1995; Leppek et al., 2018). Nevertheless, the overall expression of HAND2 at both the RNA and protein levels was sustained in complete absence of Hand2as.
The mature four-chamber heart of an E16.5 embryo can be experimentally dissected into distinct compartments, in which HAND2 transcripts can then be analyzed by RT-qPCR. Interestingly, except for an insignificant and slight decrease in mutant right ventricles, HAND2 expression showed a tendency to be upregulated in all other compartments of E16.5 Hand2asF/F hearts, compared to those of heterozygous littermates (Figure 3E, Figure 3-figure supplement 1E). In particular, HAND2 expression in the mutants was significantly increased by ~40% in the septum and ~24% in the right atrium (RA) (Figure 3E, Figure 3-figure supplement 1E). This subtle yet significant upregulation of HAND2 in specific regions of mutant hearts suggests that the Hand2as locus might be involved in controlling the spatial expression of HAND2 during heart formation.
Single-cell transcriptomic profiling reveals four cardiac cell types
To reveal subtle alterations that are not readily detectable in population-based analysis due to the averaged expression of mixed cells, we performed high-throughput single-cell RNA-seq analysis of E11.5 embryonic hearts isolated from Hand2asF/F and wild-type littermates (Figure 4A). To delineate the primary transcriptional effects of Hand2as deletion, we chose to analyze embryos at E11.5, which is also the most convenient early time point that we could experimentally isolate enough cells for 10X Genomics cell sorting and library construction. After removing sequencing reads from hematopoietic cells, we obtained expression profiles for a total of 3,600 cardiac cells, including 2,108 for Hand2asF/F and 1,492 for the wild type, with an average of 0.06 million reads/~13,000 Unique Molecule Identifier (UMI) counts per cell and a median level of 3,492 expressed genes per cell (transcripts per million [TPM] > 0) (Figure 4A-B; Supplementary file 6).
Classification using the t-distributed stochastic neighbor embedding method (t-SNE) revealed four well-separated clusters of cells that differentially express distinct marker genes (Figure 4B-D; Supplementary files 6 and 7). About 43% (1550) of cells are cardiomyocytes (CMs), which specifically express NKX2-5 and MYH7 (Figure 4B-C; Supplementary files 6 and 7). About 31% (1126) are mesenchymal cells (MCs) expressing CTHRC1 and POSTN. About 12% (416) of cells are epicardial cells (EPs) marked by membrane proteins UPK1B and UPK3B and ~14% (508) are endothelial cells (ECs) expressing CDH5 and PECAM1 (Figure 4B-C; Supplementary files 6 and 7) (Li et al., 2016). Interestingly, altered proportions of cardiomyocytes and mesenchymal cells were observed in Hand2asF/F embryonic hearts compared to the wild-type littermates (Figure 4E). Mutant CMs and MCs increased ~7% and decreased ~8%, respectively, while ratios of mutant EPs and ECs remained similar (Figure 4E). Hundreds of single-cell profiles obtained for each cell type thus provided large numbers of cells for in-depth statistical comparisons of gene expression changes between mutant and wild-type hearts.
Upregulation of HAND2 and nearby genes in Hand2asF/F embryos
HAND2 is ubiquitously expressed (TPM>0) in 60% of cardiac cells at E11.5 (Figure 5A). This observation was consistent with previous reports of robust expression of HAND2 detected in many types of cardiac cells using RNA ISH and immunostaining (Laurent et al., 2017; VanDusen and Firulli, 2012). Interestingly, the Hand2asF/F hearts appeared to have many more cells (70%) expressing HAND2, representing a 10% increase of HAND2-positive populations (Figure 5A). The observed increase of mutant CMs mainly resulted from an increase of HAND2-positive, but not negative, cardiomyocytes (p = 2E-04) (Figure 5B). In comparison, the observed decrease of mutant MCs mainly resulted from a decrease of HAND2-negative mesenchymal cells (p = 6E-04) (Figure 5B). Thus, the opposing changes in the percentages of mutant CMs and MCs appeared to be positively correlated with changes of HAND2 expression in these populations.
Although the overall percentage of ECs remained the same, mutant ECs exhibited a significant increase in HAND2-positive cells but a decrease in HAND2-negative cells (p = 4E-09) (Figure 5B). In mutant EPs, HAND2-positive cells also increased ~8% (Figure 5A). Thus, cell populations expressing HAND2 increased ~8-24% in all types of cardiac cell in Hand2asF/F mutant hearts, with the most significant gain in the ECs (1.4-fold increase from 59% to 83%, p = 4E-09) (Figure 5A). Moreover, the median levels of HAND2 expression went up significantly by 8-12% across four cardiac cell types (Figure 5A). In comparison, other master regulators of cardiac development, such as GATA6, GATA4 and NKX2-5, either showed unaltered or decreased expression in particular cardiac cell types (Figure 5-figure supplement 1A). These results indicate that the subtle yet global upregulation of HAND2 is specific to the loss of Hand2as, rather than sequencing variations between the wild-type and mutant samples.
Next we sought to determine whether complete removal of Hand2as might also affect the expression of other nearby genes. High-order chromatin structure analysis by Hi-C in mouse ESCs (Bonev et al., 2017) showed that the HAND2 and Hand2as locus resides at the boundary of two topologically associating domains (TADs) (Figure 5C). This boundary demarcates an upstream gene desert of ~0.65 Mb in length from a downstream gene-rich region. Among 10 genes located within ±1-Mb genomic regions surrounding the TSSs of Hand2as and HAND2, we found that 6 genes were expressed in at least one type of cardiac cell, and four of them exhibited very subtle but statistically significant upregulation in the mutant heart (Figure 5D-E).
Of the four genes with significantly altered expression, one (FBXO8) lies ~0.75 Mb upstream of Hand2as and three (SAP30, 5033428I22Rik and HMGB2) lie within the same TAD immediately downstream of HAND2 (Figure 5C). For SAP30 and 5033428I22Rik the numbers of cells expressing these genes were significantly upregulated in subsets of mutant cardiac cells (~9-12% increase of SAP30-positive CMs and ECs, p < 0.002; and ~21% increase of 5033428I22Rik-positive ECs, p = 2E-06) (Figure 5E). HMGB2 which is highly expressed in all cardiac cells, was specifically upregulated in its transcript abundance in mutant CMs and EPs (p < 0.01) (Figure 5D). The median expression level of the upstream gene, FBXO8 was slightly upregulated (p = 0.01) in mutant CMs (Figure 5D). For comparison, we also randomly selected two ubiquitously expressed genes, EZH2 and PSMD1, and found that they showed unaltered expression in both transcript abundance and expression frequency in the mutant hearts (Figure 5-figure supplement 1B). The combined results demonstrate a cis-regulatory role for the Hand2as locus in dampening the expression of HAND2 and several neighboring genes in cardiac cells.
Aberrant cardiac gene programs in Hand2asF/F embryos
HAND2 facilitates cardiomyocyte proliferation and the reprogramming of fibroblasts into functional cardiac-like myocytes in vitro and in vivo (Song et al., 2012). We next asked how excess amounts of HAND2 might affect cardiac gene expression programs. Gene ontology (GO) analysis of dysregulated genes showed a significant enrichment of the functional term related to multicellular organism development in all four types of cardiac cell. Interestingly, only non-CMs are specifically enriched in functional terms related to cardiac muscle contraction and heart morphogenesis (Figure 6A; Supplementary file 8). GSEA further revealed global upregulation of muscle contraction genes and downregulation of genes involved in cardiac septum development, both of which were specifically observed in non-CMs of the Hand2asF/F mutant, but not in mutant CMs (Figure 6B-C, Figure 6-figure supplement 1; Supplementary file 2). These global, opposing changes in genes related to muscle contraction and cardiac septum development were also confirmed by gene expression of averaged single-cell and bulk RNA-seq of the E11.5 hearts (Figure 6D). Consistent with GSEA, mutant non-CMs exhibited more dramatic expression changes than mutant CMs (Figure 6D).
Many CM marker genes regulating muscle contraction and heart development, such as MYL4, ACTC1, TNNC1, and TNNT2, were significantly upregulated in their expression levels and frequencies (Figure 6D-E). In contrast, marker genes enriched in non-CMs, such as HES1, SOX4 and FZD2, which are involved in septum development, were specifically downregulated (Figure 6D and 6F). For example, ratios of MYL4-positive cells in mutant hearts were increased 18-33% in non-CMs (p < 9E-06), and this was also accompanied by 17-22% increases (p < 0.0001) of MYL4 transcript abundance (Figure 6E). In comparison, 9~11% of non-CMs lost HES1 expression (p < 0.05), and HES1 RNA abundance went down 12-18% (p < 0.0001) in mutant hearts (Figure 6F). These molecular changes explain the ventricular septal defects and ventricle hypoplasia observed in Hand2asF/F embryonic hearts. The concurrent upregulation of CM marker genes and downregulation of non-CM marker genes in the non-CM cells of Hand2asF/F hearts indicated that mutant non-CMs are aberrantly reprogrammed towards cardiomyocytes.
To further support this finding, we performed correlation analysis to compare the expression similarity of a panel of 1750 differentially expressed genes (DEGs) (Figure 4D) in each of the four cardiac cell types (from wild type and mutant) to those of wild-type CMs. Mutant and wild-type CMs had the highest median levels of correlation coefficient compared to non-CM cells, indicating the robustness of this assay (Figure 6G). Interestingly, compared to their wild-type counterparts, mutant non-CMs (EPs, MCs and ECs) showed significantly higher correlations with cardiomyocytes (Figure 6G). Thus, the gene programs in the non-CMs of Hand2asF/F embryos may resemble the gene program in cardiomyocytes (Figure 6G). Our data support a model in which an apparently subtle but global increase in the HAND2 dose in all cardiac cell types may lead to pronounced changes in cardiac gene programs, and eventually result in morphological and functional abnormalities in Hand2asF/F mutant hearts.
Discussion
Despite extensive reports of lncRNA functions in cell lines, rigorous exploration and dissection of their potential actions and effects in animals are still lacking. Precise expression of HAND2 is critical for heart formation. Transcription of the divergent lncRNA Hand2as was reportedly essential for HAND2 activation and heart morphogenesis. Using three knockout mouse models, we demonstrate that the Hand2as DNA locus, rather than its transcription/transcripts, controls HAND2 expression and normal heart development and function. Full-length deletion of Hand2as led to congenital heart defects and perinatal lethality. Importantly, in embryos lacking the entire Hand2as DNA sequence, single-cell transcriptomic analysis of the heart revealed subtle yet prevalent upregulation of HAND2 and dysregulated cardiac gene expression programs. These results illustrate a critical, fine-tuning function of the lncRNA Hand2as locus in accurately controlling the spatial expression of HAND2, through which Hand2as modulates cardiac lineage development and heart function.
Hand2asP/P mice lacked any discernable phenotype in the heart and in animal survival. Consistent with our finding, a recent study of a Hand2as (lncHand2) mutant mouse model reported that deletion of the first two exons of Hand2as (lncHand2) did not cause apparent heart abnormality and failed to affect hepatic expression of HAND2 and other neighboring genes despite the absence of lncHand2 transcripts in mouse livers (Wang et al., 2018). Although we cannot rule out a potential role for residual (10%) Hand2as transcription/RNA in Hand2asP/P embryos, it is most likely in this case that disruption of the Hand2as DNA locus rather than Hand2as transcription/transcripts primarily contributes to heart morphological defects and lethality. Upregulation of HAND2 and its nearby genes in Hand2asF/F mutant hearts mainly resulted from the loss of cis-regulatory DNA sequences embedded in the Hand2as locus, rather than being a trans-acting consequence of developmental defects observed in these mice. The combined results from three Hand2as KO mice argue against a switch-like role for Hand2as transcription, transcripts and DNA sequences in regulating HAND2 activation in the heart (Anderson et al., 2016). Instead, the Hand2as locus acts as a nudger and tweaker to fine-tune the spatiotemporal expression of cardiac HAND2 and lineage expression programs. Hand2as transcripts might serve as proxy signals for the activity of important regulatory DNA elements for HAND2 expression (Mowel et al., 2018). However, it remains possible that in other pathological or stress conditions yet to be revealed, Hand2as transcription and transcripts may play a role in defining the chromatin environment required for the precise regulation of HAND2 transcription.
Complete removal of the entire 17-kb Hand2as sequence abolishes Hand2as transcription and transcripts, but fails to attenuate HAND2 expression, leading to much weaker cardiac defects and delayed onset of death, in sharp contrast to the abolished expression of HAND2 and failed heart morphogenesis at E10.5 in Hand2as/Uph polyA KI embryos (Anderson et al., 2016). These discrepancies were unexpected, as polyA KI should minimally disrupt the genomic DNA compared to a large deletion. We postulate that the severe phenotypes observed in the polyA KI mice might result from polyA-induced aberrant silencing of the nearby HAND2 locus that occurs independently of the lncRNA’s function. Because of the close juxtaposition of Hand2as and HAND2, the proximal insertion of a transcription stop signal immediate upstream (−644 bp) of the HAND2 TSS might artificially recruit the transcription termination machinery to inhibit the removal of the inhibitory H3K27me3 mark in the HAND2 promoter of mesodermal precursor cells (Almada et al., 2013), consequently inhibiting HAND2 activation during the onset of cardiogenesis. One example to support this possibility is the study of the lncRNA ThymoD in activating its upstream gene BCL11b in developing T cells (Isoda et al., 2017). The BCL11b enhancer embedded in the ThymoD DNA locus repositions from the nuclear lamina to the interior prior to the activation of BCL11b. Artificial insertion of a polyA signal downstream of the TSS of ThymoD maintained the silenced local chromatin state and inhibited its repositioning into the nuclear interior, consequently leading to failed activation of BCL11b (Isoda et al., 2017).
It is possible that alternative enhancer usages may contribute to the sustained expression of HAND2 in Hand2asF/F embryos. In fact, enhancer redundancy, which is commonly observed for expression of essential developmental genes, has been suggested to provide phenotypic robustness in mammalian development (Osterwalder et al., 2018). Interestingly, HiCap analysis in mESCs (Sahlen et al., 2015) showed that the HAND2 promoter interacts with both upstream and downstream DNA elements embedded in the Hand2as and 5033428I22Rik lncRNA loci. Loss of the entire Hand2as locus might lead to alternative engagement of the HAND2 promoter with downstream enhancers, which may sustain but not precisely control HAND2 expression. A recent study of promoter competition between the lncRNA Pvt1 and its nearby oncogene MYC for engagement with intragenic enhancers embedded in the Pvt1 locus demonstrated the importance of proper enhancer-promoter interactions in regulating the precise level of MYC expression in breast cancer cells. Transcription interference of the Pvt1 promoter enhances MYC expression and cancer cell growth in vivo (Cho et al., 2018).
Expression changes of genes involved in muscle contraction that are distal to the Hand2as locus appeared to be more pronounced than HAND2 expression changes in bulk analysis of the whole hearts or ventricles from Hand2asF/F mutants. These subtle cis effects could be easily missed in conventional ensemble analysis, thus confounding the mechanistic interpretation of a lncRNA’s function in cis or in trans. Although the molecular effects of Hand2as on HAND2 expression in individual cells were moderate, these changes were prevalently and robustly detected in hundreds of cells across all four cardiac cell types, and were also observed in the septum and the right atrium of E16.5 Hand2asF/F embryos. These results emphasize the usage of single-cell approaches to delineate the primary molecular effects of lncRNA inhibition in heterogeneous cell populations. Like many lncRNAs such as Flicr and Fendrr (Grote et al., 2013; Zemmour et al., 2017), Hand2as deletion had subtle molecular effects which nonetheless resulted in profound biological consequences in vivo.
HAND2 expression undergoes a decrease after E10.5 and then remains low throughout the remaining course of heart development (Srivastava et al., 1997; Tamura et al., 2014). The observed upregulation of HAND2 in E11.5 and E16.5 Hand2asF/F embryos may reflect improper downregulation of HAND2 during cardiac development. Interestingly, studies of various mouse models have reported that aberrantly high levels of embryonic HAND2 led to ventricular septal defects (VSDs) (Supplementary file 1). For example, MYH7-driven overexpression of HAND2 prevented the formation of the interventricular septum in embryos (Togi et al., 2006). Overdosage of HAND2 is reportedly a major cause of perinatal lethality and heart phenotypes, including severe VSDs, in the Rim4 mouse model which mimics a human chromosomal disorder caused by partial trisomy of distal 4q (4q+), a region containing 17 genes including HAND2 (Tamura et al., 2013). Mice deficient in miRNA-1-2 expressed more HAND2 protein, and exhibited a VSD and embryonic death from E15.5 to just after birth with 50% penetrance (Zhao et al., 2007). Moreover, MYH6-driven overexpression of HAND2 in adult cardiac muscle cells caused pathological heart hypertrophy (Dirkx et al., 2013). Comparisons of the above mouse models with the three Hand2as KO mice we generated suggest a correlation between the level of HAND2 overexpression and the severity of heart defects. Subtle yet prevalent upregulation of HAND2 in Hand2asF/F embryonic hearts did not affect the overall formation of an organized four-chamber heart; however, the morphological defects in specific regions of the heart, including lesions in the interventricular septum and ventricle hypoplasia, were moderate but sufficient to impair heart function, leading to immediate death of the mutant animals upon birth.
Based on these lines of evidence, we interpreted the observed molecular changes in cardiac gene programs as well as the morphological and functional heart defects to be a result of excess amounts of HAND2 transcripts in Hand2asF/F embryos. We propose a cis-functional role for the Hand2as locus in dampening HAND2 expression to restrain cardiomyocyte proliferation, thereby leading to a balanced development of cardiac cell lineages. This study reinforces the notion that many lncRNAs act as local regulators to modulate the precise expression of genes of essential developmental importance, which may have profound physiological or pathological consequences in particular contexts upon lncRNA inhibition. Lastly, the fact that different knockout strategies produce distinct phenotypes underlines the requirement to utilize complementary genetic approaches to study the physiological functions of a lncRNA in mouse models (Han et al., 2018; Luo et al., 2016; Yin et al., 2015). We believe that careful genetic dissection coupled with single-cell analysis will lead to in- depth understanding of the functions and mechanisms of action of lncRNAs, thus truly impacting on our understanding of the noncoding genome in animal development, fitness and disease.
Materials and Methods
Animals
All mice we used were C57BL/6 background, with age described in the manuscript. Embryos were isolated at the developmental stages indicated in the manuscript. All animal experiments were conducted in accordance of institutional guidelines for animal welfare and approved by the Institutional Animal Care and Use Committee (IACUC) at Tsinghua University.
Cells
HEK 293T cells (CRL-3216, ATCC) were cultured in DMEM supplemented with 10% heat- inactivated fetal bovine serum (Hyclone) and 1% Penicillin/Streptomycin (Cellgro). ESCs (46C, Austin Smith Lab) were grown in DMEM (Cellgro) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone), 1% Glutamax (GIBCO), 1% Penicillin/Streptomycin (Cellgro), 1% nucleoside (Millipore), 0.1mM 2-mercaptoethanol (GIBCO), 1% MEM nonessential amino acids (Cellgro), and 1000U/ml recombinant LIF (leukemia inhibitory factor) (Millipore) on gelatin-coated plates. All cultured cells were maintained in a humidified incubator at 37°C with 5% CO2 (Luo et al., 2016).
CRISPR/Cas9-mediated genetic deletion
CRISPR/Cas9-meditated genetic deletion for lncRNA knockout mice generation was performed as previously described with minor modifications (Han et al., 2014a; Han et al., 2018). Briefly, Cas9 mRNA and sgRNAs were co-injected into mouse zygotes. For each genetic deletion, we used 2 sgRNAs (Hand2asP, sg1 and sg2; Hand2asD, sg3 and sg4; Hand2asF, sg1 and sg5) (Supplementary file 9). For Hand2asP, we deleted a 1-kb DNA sequence covering the core promoter and the first two exons of Hand2as, with 94% of Hand2as DNA sequences remaining intact. When genetic deletion was confirmed, the germline transmission was performed for 2 generations by mating with C57BL/6. F2 mice and later generations were used for heterozygote intercrosses.
Genotyping
KO primers (F and R) were designed outside of the deleted region. For WT bands amplification, we used one of the KO primers together with a primer designed inside of the deleted region (Supplementary file 9). KO bands were confirmed by Sanger sequencing (Han et al., 2018).
Echocardiography
Echocardiography was performed on Hand2asD/D, Hand2asP/P and their littermate control mice at 6-10 weeks old. Briefly, mice were gently restrained in the investigator’s hand during echocardiography detection. Two-dimensional, short-axis views of the left ventricle were obtained for guided M-mode measurements of the left ventricular (LV) internal diameter at end diastole (LVIDd) and end systole (LVIDs). LV internal diameter were measured in at least three beats from each projection and averaged. The fractional shortening (FS) was calculated by the following formula: FS (%) = [(LVIDd − LVIDs) / LVIDd] ×100, which represents the relative change of left ventricular diameters during the cardiac cycle.
Histology analyses
For hematoxylin-eosin staining (HE staining), E16.5 embryonic hearts were fixed in 4% PFA overnight at room temperature, dehydrated through a graded ethanol series (50%, 75%, 90%, 95%, 100%) and paraffin embedded. After section (7 μm), the tissues were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 75%), then stained with hematoxylin and eosin. Ratio of RV area was calculated as the RV chamber area divided by whole ventricle area. These data were measured in Adobe Photoshop CC2014 after selection of the image areas with myocardial color range. For immunostaining, E9.5 embryonic hearts were fixed, dehydrated, embedded as HE staining. After section (7 μm), the tissues were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 90%, 80%, 70%, 50%), then, the tissues were heated to retrieve epitope. E9.5 embryos were fixed in 4% PFA at 4 °C 2 h, dehydrated by 30% sucrose, embedded in OCT. Then frozen sections was cut at 7μm on a cryostat set at −20 to −25 °C. Immunostaining was performed with primary antibodies of TNNI3 (Abcam, ab56357), HAND2 (Abcam, ab200040). Primary antibodies were visualized by staining with Alexa-conjugated secondary antibodies: Alexa Fluor 488 donkey anti-goat (Life Technologies, #A-11055) and Alexa Fluor 555 donkey anti-rabbit (Life Technologies, #A-31572) with 200 fold diluted. All the slides were mounted in VECTASHIELD hardset antifade mounting medium (Vector Laboratories) and imaged on Zeiss Microsystems.
RNA in situ hybridization
Whole mount in situ hybridization was carried out with digoxigenin-labelled antisense RNA probes as previously described with some modifications (Anderson et al., 2016; Wei et al., 2011). In brief, RNA probe for HAND2 were amplified from cDNA of mouse embryonic heart and transcribed in vitro using T7 RNA polymerase (Roche, 10881767001) with DIG RNA labeling mix (Roche, 11277073910) (Supplementary file 9). Embryos were fixed in 4% PFA at 4 °C overnight, dehydrated through a graded methanol series (50%, 75%, 100%) and stored in 100% methanol at −20 °C. The embryos were bleached in a solution containing 30% H2O2: methanol 1:5 for 2 hr, then rinsed in methanol, rehydrated through a graded methanol series (100%, 75%, 50%), and then washed in PBS. The embryos were post-fixed 20 min in 4% PFA. After washed by PBS, embryos were transferred to the hybridization buffer (50% formamide, 5×SSC, 500 µg/ml yeast RNA, 50 µg/ml heparin and 0.1% Tween-20) and pre-hybridized 4 hr at 65 °C. Hybridizations were performed in fresh hybridization buffer containing 0.25 ng/µl digoxigenin-labelled antisense RNA probes overnight at 65 °C. Post- hybridization washes were performed at 65 °C by wash buffer 1 (50% formamide, 2× SSC), wash buffer 2 (2× SSC), wash buffer 3 (0.2× SSC), then performed by MABT (100 mM maleic acid, 150 mM NaCl and 0.1% Tween-20) at room temperature. After 1 hr blocking at room temperature in 10% sheep serum, 2% blocking reagent (Roche, 11096176001) (diluted in MABT), embryos were incubated overnight at 4 °C in blocking solution as above, with anti-DIG-AP antibody (Roche, 11093274910, 1:3,000). Then mouse embryos were washed in MABT at room temperature. After the post-antibody washes, embryos were washed in NTMT (100 mM NaCl, 100 mM Tris-HCl at pH 9.5, 50 mM MgCl2 and 0.1% Tween-20). Staining was realized in BM Purple AP Substrate (Roche, 11442074001).
Ink injection
Chinese ink (Yidege) was injected into the left ventricles of E16.5 embryos, to visualize the organization of the arteries.
Transfection
Plasmids were transfected into 293T or ESCs by lipofectamine 2000 (Life Technologies,#200059-61). For western blot, cells were harvested 24 hr after transfection. HAND2 cDNA with different length of 5’UTR or without 5’UTR were cloned into piggyBac vector. PiggyBac-GFP were co-transfected with HAND2 cDNA as control of transfection efficiency. For validation of HAND2 antibody, flag-tag was added to the N-terminal of HAND2.
Western Blot
Cultured cells were washed by PBS and boiled in 5× SDS sample buffer for 5 min at 95°C. After SDS-PAGE and transfer, membranes were blocked in 5% milk/TBS-Tween. Primary antibody were applied 2 hr and secondary HRP-conjugated antibodies were applied for 1 hr at room temperature. Membranes were washed for 3× 10 min in TBS/Tween after each antibody incubation, and incubated with ECL substrate before exposure to X-ray film. Primary antibodies of HAND2 (Abcam, ab200040), β-TUBULIN (Abmart, M30109), FLAG (EASYBIO, BE2005-100) and GFP (CWBIO, CW0086) were used. Secondary antibodies used included goat anti-mouse IgG (CWBIO, CW0102) and goat anti-rabbit IgG (CWBIO, CW0103). Antibodies were used following the manufacture recommended concentration.
RT-qPCR
Tissue were washed by PBS and harvested in TRIzol reagent (Life Techonologies, #15596018). Adult cardiomyocytes were isolated using type II collagenase in the Langendorff retrograde perfusion mode (O’Connell et al., 2007). Total RNA was extracted as the manufacture recommended procedure. 0.5 to 2 μg total RNA was used for reverse transcription by RevertAid First Strand cDNA Synthesis Kit (Fermentas, K1622) with random primers. Reverse transcription and quantitative PCR (RT-qPCR) were performed using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121) on a Bio-Rad CFX384 RealTime System. Error bars in RT-qPCR analysis represent standard error of mean expression relative to GADPH or 18S expression, or average fold changes compared to the indicated control (Supplementary file 9).
Bulk RNA-seq and data analysis
Adult (8-wk) cardiomyocytes of CTRL (Hand2as+/+ and Hand2asD/+) and Hand2asD/D, E11.5 hearts and E16.5 ventricles of Hand2asF/+ and Hand2asF/F from heterozygotes intercrosses were subjected for RNAseq following polyA purification. The RNA libraries were constructed by following Illumina library preparation protocols. High-throughput sequencing was performed on Illumina HiSeq 2500 or HiSeq X TEN. All RNA-seq data were mapped to the mouse reference genome (mm9) using TopHat (version 2.0.11) (Trapnell et al., 2012). Reads were assigned to their transcribed strand (Tophat parameter “--library-type=fr-firststrand”). The gene expression level was calculated by Cufflinks (version 2.0.2) (Trapnell et al., 2012) using the refFlat database from the UCSC genome browser. For visualization, the read counts were normalized by computing the numbers of reads per million of reads sequenced (RPM). Gene set enrichment analysis (GSEA) (version 2.2.4) was performed by comparing mutant samples to control samples (Subramanian et al., 2005). We used gene sets from KEGG V6.0 (Kanehisa and Goto, 2000) for GSEA (Supplementary file 2). First, genes with FPKM >10 and |log2 (fold change)| > 0.2 (fold change = (FPKM of KO+0.1)/(FPKM of CTRL +0.1)) were selected as candidates. To exclude the inconsistently dysregulated candidates, we further filtered genes with t.test >0.1 or |log10 (t.test)* log2 (fold change)| <0.5 (dysregulation score). For 8-wk cardiomyocytes of Hand2asD/D, 114 and 186 genes are upregulated and downregulated, respectively (Supplementary file 3). For E11.5 hearts of Hand2asF/F, 169 and 101 genes are upregulated and downregulated, respectively (Supplementary file 4). For E16.5 ventricles of Hand2asF/F, 31 and 19 genes are upregulated and downregulated, respectively (Supplementary file 5). Heatmaps were drawn by Cluster 3.0 and viewed by Treeview. The colors represent the fold change of gene expression which is relative to average FPKM of each gene across all analyzed samples.
Single-cell RNA-seq and data analysis
E11.5 hearts of wild type and Hand2asF/F from heterozygotes intercrosses were subjected for Single-cell RNA-seq. We harvested six embryonic hearts for each genotype. Embryonic hearts were trypsinized (0.25%, 5min, 37°C) individually, and subjected for Fluorescence-activated cell sorting (FACS) after 7-Aminoactinomycin D (AAT Bioquest, 17501) staining for collection of living single cells. Next, six embryonic hearts were combined as a single sample for 10X Genomics Single Cell 3’ library construction (10X Genomics, PN-120237). The RNA libraries were constructed by following the manufacture recommended procedure to get ~5,000 cells barcoded per sample. Sequencing data analyses including sample demultiplexing, barcode processing and single-cell 3’ gene counting were done by the Cell Ranger Single-Cell Software Suite (http://software.10xgenomics.com/single-cell/overview/welcome) (Zheng et al., 2017). We got ~190 million reads for 3,469 detected Hand2asF/F cells and ~181 million reads for 2,563 detected wild type cells, which indicate an average of 60,000 reads per cell. The cDNA insert was aligned to mouse reference genome (mm10). Cells with less than 1,000 detected genes were removed. We used RandomForest approach to discriminate a population of hemopoietic cells and excluded them from downstream analysis, and finally obtained 1,492 single cells from wild type heart and 2,108 single cells from Hand2asF/F heart. On the median, we detected 3,492 genes per cell. For comparative analysis of WT and mutant single-cell datasets, we took the union of the top 1,000 genes with the highest dispersion (var/mean) from both datasets (“WT” object and “Mutant” object) to perform the alignment procedure in the Seurat integration procedure (Butler et al., 2018). We run a canonical correlation analysis (CCA) to identify common sources of variation between the two datasets. Then we aligned the top 7 CCA subspaces (or dimensionalities) to generate a single new dimensional reduction integrated WT and mutant datasets used for subsequent analyses such as t-distributed stochastic neighbor embedding (t-SNE) visualization. Next we used the “FindClusters” function to identify four main cardiac cell types and verified them by known marker genes (Li et al., 2016). We achieved 43% cardiomycytes, 12% epicardial cells, 31% mesenchymal cells and 14% endothelial cells (Supplementary file 6). The dominant composition of CMs in our data is consistent with two previous single-cell studies which profiled 96 cells at E11.5 and 1,165 cells at E10.5 of embryonic hearts (Dong et al., 2018; Li et al., 2016). Increases percentages of MCs and EPs at E11.5 compared to those in the E10.5 heart reported previously are in accordance with increased proliferation of cushions and epicardium (Li et al., 2016). To identify unique cluster-specific marker genes and for heatmap plotting, we used the Seurat function “FindAllMarkers” (thresh.test = 0.5, test.use = “roc”) and define a group of differentially expressed genes (DEGs) containing 1750 genes (Supplementary file 7). Next, we used the function of “FindMarkers” to identify dysregulated genes between WT and mutant hearts in each cell type, which were used for Gene Ontology analysis (Supplementary file 8). Because the median UMI we detected in most of single cells did not reach one million UMIs, we used log2 (TPM/10 + 1) rather than log2 (TPM + 1), where TPM refers to transcripts per million, to normalize the expression levels for following analysis. Correlation coefficient of cardiac cells were analyzed by 1750 DEGs used for cell types clustering. We used average gene expression of all cardiomyocytes (591 single cells) from wild type sample as a standard gene expression profile, and calculated Pearson correlation of each single cell compared to the standard. GSEA was performed by comparing all single cells of Hand2asF/F hearts to wild type samples.
Published data collection
Published sequencing datasets used in this paper (Figure 1A) were collected from Encyclopedia of DNA Elements (ENCODE) and Gene Expression Omnibus (GEO), including DHS-seq of E11.5 heart (ENCSR932SBO), CTCF ChIP-seq of postnatal day 0 (P0) heart (ENCSR491NUM), H3K27ac ChIP-seq of E12.5 heart (ENCSR123MLY), H3K4me3 ChIP-seq of E12.5 heart (ENCSR688ZOR), polyA RNA-seq of E12.5 heart (ENCSR150CUE), total RNA-seq of P0 heart (ENCSR035DLJ) (Yue et al., 2014), Pol II (8WG16) ChIP-seq of E12.5 heart (GSM1260035) (He et al., 2014), GATA4 ChIP-seq of E12.5 heart (GSM1260026) (He et al., 2014), HAND2 ChIP-seq of E10.5 heart (GSM1891956) (Laurent et al., 2017), NKX2-5 ChIP-seq of E12.5 heart (GSM1724109) (Ye et al., 2015), and HiCap in mESC (GSE60494) (Sahlen et al., 2015).
Quantification and statistical analysis
Results for RT-qPCR, echocardiography and ratio of RV area are shown as mean values with error bars representing the standard error (SEM), except for Hand2as/HAND2 RT-qPCR results in cardiomyocytes from CTRL (Hand2as+/+, D/+) and Hand2asD/D (shown as median with range). Replicates are indicated in the figure legends. For each comparison between two groups, statistical analysis was performed and p values were calculated with an unpaired two-tailed Student’s t test by GraphPad Prism 5 software. Measurement of heart chamber area were performed by Adobe Photoshop CC2014. Imaging data analyses were done by Zen 2012. For single-cell RNA-seq analyses, scatter plots (gene expression and correlation coefficient) are shown as median and interquartile range. We used Mann Whitney test for statistical analysis of gene expression and correlation coefficient for single-cell RNA-seq results. Fisher’s test is used for significance test of gene expression frequency for single-cell RNA-seq results.
Author contributions
X.S. and A.H supervised the study. X.H., J.Z., X.F., S.A., Y.L., S.L., and Y.Y. performed the experiments. X.H., Y.L, X.F. and H.Z. performed bioinformatics analysis. X.S. wrote the manuscript with help from X.H, J.Z. and A.H.
Data availability
Bulk RNA-seq and single-cell RNA-seq data of embryonic hearts from progenies of Hand2asF/+ or Hand2asD/+ crosses have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE102935.
Supplementary files
Supplementary file 1. Summary of cardiac HAND2 knockout/overexpression mouse models
Supplementary file 2. Gene sets for GSEA
Supplementary file 3. Dysregulated genes in 8-wk cardiomyocytes of Hand2asD/D mice
Supplementary file 4. Dysregulated genes in E11.5 hearts of Hand2asF/F embryos
Supplementary file 5. Dysregulated genes in E16.5 ventricles of Hand2asF/F embryos
Supplementary file 6. Single-cell clustering
Supplementary file 7. 1750 differentially expressed genes in four types of cardiac cells
Supplementary file 8. Dysregulated genes in four cardiac cell types in E11.5 hearts of Hand2asF/F embryos
Supplementary file 9. Oligos (sgRNAs, genotyping primers, RT-qPCR primers, ISH probe)
Acknowledgements
We thank W. Pu, L. Yu, G. Ou, Y. Chen, F. Tang, Q. Xi, and Shen Laboratory members for insightful discussion and critical reading. X.S. is supported by grants from the National Natural Science Foundation of China (8141101062, 20161310854 and 31471219), the National Basic Research Program of China (2017YFA0504204), and the Center for Life Sciences (CLS) at Tsinghua University. A.H. is supported by grants from the National Basic Research Program of China (2017YFA0103402), and the National Natural Science Foundation of China (31571487 and 31771607).