A 2.2-kb Fragment Upstream of the vmhc Gene Is Sufficient to Drive Ventricle-Specific Reporter Expression
The vmhc transcript can first be detected in the heart primordium in the anterior lateral plate mesoderm around the 10-somite stage (see Supp. Fig. S1A [arrow], which is available online); this is later than that of titin, which occurs around the 5-somite stage (Seeley et al.,2007), but is earlier than that of essential or regulatory myosin light chains, both of which occur around 16-somites (Chen et al.,2008). Previously, vmhc expression was shown to be immediately restricted to the ventricle after its onset (Yelon et al.,1999), and we consistently detected vmhc expression in the ventricle at each stage of cardiogenesis: cardiac progenitor migration, tube formation, and chamber formation (Supp. Fig. S1A–F). In addition, we detected residual expression in the atrium at 36 hr post-fertilization (hpf) (Supp. Fig. S1E). In addition to cardiac expression, vmhc expression was also detected in the somites starting at 24 hpf (data not shown) and in the extraocular muscles and pharyngeal muscles after 3 days post-fertilization (dpf) (Supp. Fig. S1G).
The zebrafish vmhc gene (GenBank accession number: XM_001332905) consists of 39 exons on chromosome 2 and spans 13.3 kb. vmhc is located 6 kb downstream of myh1 (zgc:113832), another MHC homologue that exhibits a ventricle-restricted expression pattern, as revealed by whole mount in situ hybridization (data not shown). By searching the Fugu (Takifugu rubripes) genome, we identified a chromosomal region syntenic to the zebrafish vmhc gene; this region contains a pair of tandemly arranged MHC homologues (Supp. Fig. S2). We aligned and compared the intergenic sequences between these two pairs of mhc genes in the zebrafish (6.7 kb) and Fugu (6.5 kb) using rVista software (Frazer et al.,2004) and were able to identify several conserved regions across both species (Fig. 1A, white peaks), suggesting a regulatory element function for these genes.
Figure 1. Identification of a 2.2-kb fragment from a ventricle-specific promoter. A: Sequence comparison of upstream intergenic sequences between the zebrafish vmhc gene with its Fugu homologue. Red and blue peaks represent coding regions for myh1 and vmhc, respectively. White peaks represent inter-species conserved regions. B: Summary of promoter analysis by transient co-injection of naked DNA with an EGFP fragment. The full-length intergenic V-5.7∼+0.3k fragment can drive GFP expression in both the somites and ventricle, as can the V-4.7∼+0.3k, V-3∼+0.3k, and V-1.9∼+0.3k fragments. However, V-1.1∼+0.3k drives GFP expression in both the ventricle and atrium, and V-0.5∼+0.3k drives GFP expression in the heart but not in the somites. The yellow, blue, and red bars on the top line represent fragments required for chamber specificity, somite expression, and cardiac expression, respectively. The yellow or red bars below represent the minimal element sufficient for chamber-specific or cardiac expression, respectively. C: Representative pictures of 3-dpf embryos after co-injection of promoter DNA and the EGFP fragment. Left and middle panels are lateral views; anterior to the left. The right panel is a ventral view; anterior to the top. GFP-positive cells can be detected in single cells or in a group of cells in the somites (left and middle panel) or the heart. The ventricle (indicated by arrows) and the atrium (indicated by arrowheads) could be distinguished due to embryo transparency.
Download figure to PowerPoint
To experimentally dissect the chamber-specific vmhc promoter, we used transient co-injection assays (Muller et al.,1999), which are based on the principle that DNA fragments of different origin usually integrate together into a single breaking point on the chromosome (Bishop and Smith,1989). Therefore, when a promoter fragment is co-injected with a GFP reporter fragment into one-cell-stage embryos, the GFP expression pattern faithfully reflects promoter activity. We generated a series of promoter fragments derived from the 6.7-kb intergenic region upstream of the zebrafish vmhc gene. All fragments contained the basal promoter as well as 300 bp downstream of the transcription start site. We detected sporadic GFP-positive cells in the heart at 2–3 dpf (Fig. 1C) and in the somites at 3–4 dpf. It was possible to distinguish GFP-positive cells in the ventricle (arrow) from those in the atrium (arrowhead), due to the transparency of zebrafish embryos. In embryos that were co-injected with the full-length intergenic sequence, V-5.7∼+0.3k (Fig. 1B, line 1), GFP-positive cells were only detected in the somites and ventricle and not in the atrium, suggesting that this 6-kb fragment recapitulated endogenous vmhc expression. This transient co-injection assay appeared to be specific, as a similar ventricle-restricted expression pattern was also observed in embryos co-injected with three other fragments containing a series of 5′ deletions (until −1.9 kb) (Fig. 1B, line 2–4). Further deletions, however, disrupted chamber specificity or ablated expression in the somites. GFP-positive cells were detected in both the atrium and ventricle after co-injection of V-1.1∼+0.3k (Fig. 1B, line 5) or V-0.5∼+0.3k (Fig. 1B, line 6), while GFP-positive cells could be detected in the somites after co-injection of V-1.1∼+0.3k, but not V-0.5∼+0.3k. In summary, these studies indicated that transient co-injection assays are useful for dissecting chamber-specific promoters. Furthermore, we found that a 2.2-kb element was sufficient to recapitulate chamber-specific vmhc expression.
Further Dissection of the vmhc Promoter by Transient Co-Injection Assays
Next, we quantified data from the transient co-injection assays by counting 4-dpf embryos with GFP-positive cells in either the ventricle or atrium. The results were represented as V:A ratios to reflect chamber specificity, where V represents the number of fish with GFP-positive cells in the ventricle and A represents the number of fish with GFP-positive cells in the atrium. As summarized in Figure 2, the V:A ratio for V-1.9∼+0.3k (Fig. 2A, line 1), V-1.1∼+0.3k (Fig. 2A, line 5), and V-0.5∼+0.3k (Fig. 2A, line 8) were V only, 2.4 and 0.9, respectively, suggesting that a repressor located between −1.9 to −1.1 kb was required for chamber specificity. We also calculated the percentage of fish that contained GFP-positive cells in either the somites or the heart (Fig. 2).
Figure 2. Dissection of the vmhc promoter using transient co-injection assays. A: Schematic summary of results of serial deletions (lines 1–8) or internal deletions (lines 9–12) to identify minimal cis-elements required for chamber specificity. A distal (V-1.7∼−1.5k) and a proximal (V-0.1∼+0.3k) cis-element were identified and are indicated by yellow bars. B: Schematic summary of results of the minimal cis-elements sufficient for chamber specificity. Both V-0.1∼+0.3k (line 1) and V-1.9∼−1.1k (line 5) drive GFP expression only in the ventricle (yellow bar), while V-0.5∼−0.1k (line 2) and V-1.1∼−0.7k (line 4) drive GFP expression in the entire heart (red bar). Four fragments drive GFP expression in skeletal muscle (blue bar), two are strong enhancers (V-0.7∼−0.5k, line 3 and V-1.1∼ −0.7k, line 4) and two are weak enhancers (V-0.1∼+0.3k, line 1 and V-1.9∼−1.1k, line 5). Note that lines 2–5 are fragments lacking the basal promoter. No., number of injected embryos that survived to 4 dpf; Somite/Heart ratio, number of fish with tissue-restricted GFP-positive cells over the total number of fish that survived to 4 dpf; Ventricle No. or Atrium No., number of fish with GFP-positive cells in the ventricle or atrium. A fish with GFP-positive cells in both chambers was counted in both categories. V:A, ratio of GFP-positive cells in the ventricle to that in the atrium.
Download figure to PowerPoint
To identify the minimal cis-elements needed to drive chamber-specific gene expression, we generated a series of fine deletion constructs that deleted every 200 bp from the distal end of the 2.2-kb fragment (Fig. 2A, lines 1–8). Deletion of a 200-bp region located between −1.7 and −1.5 kb resulted in reduction of the V:A ratio from completely ventricle to ∼2 (Fig. 2A, lines 2–3), suggesting that this region represents the minimal distal cis-element required for chamber specificity. We did not generate 5′-deletions from −0.5 kb to +0.3 kb, as the V:A ratio was 0.9 when the V-0.5∼+0.3k fragment was co-injected (Fig. 2A, line 8). Instead, we generated a series of four internal deletion constructs within this proximal region. Deletion of either −0.1 kb to +0.1 kb or +0.1 kb to +0.3 kb resulted in a marginal decrease in the V:A ratio to 8 or 5.9, respectively (compare Fig. 2A, lines 1 and 9–12), suggesting the involvement of a proximal cis-element for chamber-specific vmhc expression.
To identify elements sufficient for chamber-specific gene expression, we generated a series of short fragments within the 2.2-kb region. Most of these fragments (Fig. 2B, lines 2–5) lacked the vmhc basal promoter, but GFP signals could still be detected, possibly due to a stretch of sequences located immediately before the GFP reporter that mimicked the basal promoter function. We found that co-injection of the V-1.9∼−1.1k fragment (Fig. 2B, line 5), which covered the distal element, drove GFP expression only in the ventricle, although co-injection of the V-1.7∼−1.5k fragment did not result in any GFP-positive fish (data not shown). Co-injection of the V-0.1∼+0.3k proximal element (Fig. 2B, line 1) was also able to drive chamber-specific expression. In contrast, co-injection of V-1.1∼−0.7k (Fig. 2B, line 4) or V-0.5∼−0.1k (Fig. 2B, line 2) drove GFP expression in both heart chambers. We also identified four fragments that drove GFP expression in skeletal muscle. V-0.7∼−0.5k (Fig. 2B, line 3) and V-1.1∼−0.7k (Fig. 2B, line 4) drove GFP expression in the somites of 20.3 and 31.6% of embryos, respectively, while V-0.1∼+0.3k (Fig. 2B, line 1) and V-1.9∼−1.1k (Fig. 2B, line 5) drove GFP expression in the somites of 1.8 and 2.9% of embryos, respectively. These data suggest that multiple modular enhancer elements co-exist within the vmhc promoter to cooperatively regulate tissue-specific expression. Thus, both distal and proximal elements are involved in ventricle-specific expression.
Dissection of the vmhc Promoter Using Tol2-Based Transient Assays
To confirm transient co-injection assay results, we performed classic promoter analysis by cloning a promoter fragment and the GFP reporter gene into a single construct. We used the Tol2 transposon vector system, which was originally identified in Medaka fish (Koga et al.,1996) and later adapted as a vehicle to efficiently integrate ectopic DNA into the zebrafish genome (Kawakami et al.,2000,2004). Transient injection of Tol2-based plasmids was previously shown to be a valuable tool to analyze tissue-specific promoters in zebrafish (Fisher et al.,2006b; Korzh,2007). In our hands, adaptation of the Tol2 vector facilitated the identification of GFP-positive fish, due to dramatically increased intensity and larger number of GFP-positive cells within each injected embryo. We easily detected GFP-positive cells in the heart at 2 dpf (Fig. 3A, left panel) a nd in skeletal muscle at 3 dpf, both of which occurred earlier than in embryos from co-injection assays. Due to increased sensitivity and reduced mosaicism, we could detect GFP in several subsets of muscles, including the extraocular muscles (data not shown), muscle pioneer cells in the body midline (Fig. 3A, middle panel), and myocytes in the whole myotome (Fig. 3A, right panel). Switching to the Tol2 system did not affect the percentage of embryos with GFP-positive cells.
Figure 3. Dissection of the vmhc promoter by transient assays using Tol2-based vectors. A: Representative pictures of 3-dpf embryos injected with Tol2 transposon constructs containing vmhc promoter sequences (left and right panel, V-0.5∼+0.3k; middle panel, V-1.9∼+0.3k). Shown in the left panel is a ventral view with anterior to the top; right and middle panels are lateral views with anterior to the left. Multiple GFP-positive cells could be detected in the heart (left) and/or skeletal muscle, including the eye muscle, muscle pioneer cells located in the body midline (middle), and myocytes in the somites (right). B: Schematic summary of results from transient assays using the Tol2 transposon system. A distal (V−1.9∼−0.7k) and a proximal (V+0.1∼+0.3k) element required for the chamber specificity were identified, consistent with results from transient co-injection assays in Figure 2. A shorter distal element (V-1.7∼−1.3k, line 7) is sufficient to drive ventricle-specific expression in this assay. In contrast to results from transient co-injection assays, the proximal element (V-0.1∼+0.3k, line 5) drives GFP expression in the whole heart without chamber specificity in Tol2-based assays. When the distal and proximal elements are linked in tandem (line 8) or in reverse (line 9), the constructs drive GFP expression in the ventricle. However, the distal element cannot alter the expression of the cardiac cmlc2 promoter to be chamber-specific (line 11), which by itself drives GFP expression in the whole heart (line 10). The minimal elements required for chamber specificity (yellow bars, top line), for chamber-restricted expression (yellow bars, line 7) and for cardiac expression (red bars, lines 5–6) are indicated. No., number of injected embryos that survived to 4 dpf; Somite ratio/Heart ratio, number of fish with tissue-restricted GFP-positive cells over total number of fish that survived to 4 dpf; Ventricle No. or Atrium No., number of fish with GFP-positive cells in the ventricle or atrium. A fish with GFP-positive cells in both chambers was counted in both categories. V:A, ratio of GFP-positive cells in the ventricle over that in the atrium.
Download figure to PowerPoint
In contrast to results from the transient co-injection assays, injection of the full-length 2.2-kb fragment (V-1.9∼+0.3k) resulted in a smaller percentage of GFP-positive cells in the atrium (Fig. 3B, line 1). This may be due to the increased sensitivity of the Tol2 system, which more accurately reflects endogenous vmhc expression. As shown in Supp. Figure S1D, residual vmhc mRNA could still be detected in the atrium by in situ hybridization at 36 hpf. Injection of V-0.7∼+0.3k (Fig. 3B, line 3) or V-0.5∼+0.3k (Fig. 3B, line 4), two fragments lacking the distal element, resulted in V:A ratios of less than 2, while injection of V-1.9∼+0.1k, a fragment lacking the proximal region (Fig. 3B, line 2), induced a V:A ratio of 6.4. The results from the Tol2 system experiments confirmed the existence of two cis-elements required for vmhc chamber-specific expression and supported the notion that the distal element plays a stronger role than the proximal element in determining ventricular specificity.
The distal element was sufficient to drive ventricle-specific expression using the Tol2 system, consistent with results from transient co-injection assays. Indeed, injection of the shorter distal V-1.7∼−1.3k element (Fig. 3B, line 7, compared to Fig. 2B, line 5) was sufficient to drive ventricle-specific GFP expression, most likely due to the increased sensitivity of the Tol2 system. However, in contrast to the co-injection assays, injection of the proximal V-0.1∼+0.3k element (Fig. 3B, line 5, compared to Fig. 2B, line 1) resulted in a low V:A ratio of 1.6. One explanation for the inconsistency between the two transient assays could be that variable copies and ratios of the proximal elements and GFP reporter fragments were examined in co-injection assays (Marini et al.,1988; Bishop and Smith,1989), while the 1:1 ratio of the proximal element and GFP reporter was examined in Tol2-based assays. Consistent with this hypothesis, the injection of a construct containing 3 copies of the proximal element located upstream from a GFP reporter increased the V:A ratio to 3 (Fig. 3B, line 6).
When we combined the minimal distal and proximal elements together in tandem (Fig. 3B, line 8) or in reverse (Fig. 3B, line 9), both constructs were able to drive ventricle-specific expression. This result suggested that the distal element, which may function as a repressor to inhibit gene expression in the atrium, imposed a dominant effect over the proximal element. To test whether the distal element may function as a universal repressor in the atrium, we generated a chimeric construct consisting of V-1.9∼−0.5k, which includes the distal element, and a 300-bp cardiac promoter from the cmlc2 gene (Huang et al.,2003). Much like that of the cmlc2 promoter alone, the chimeric construct exhibited whole heart expression without chamber specificity (Fig. 3B, lines 10–11). Therefore, we concluded that the repressor function of the distal element is not universal, but functions cooperatively with the proximal vmhc element to achieve chamber-specificity.
Dissection of the vmhc Promoter by Generating Stable Transgenic Fish Lines
To confirm conclusions from transient assays, we generated four transgenic fish lines using Tol2-based constructs. In the Tg(V-1.9k:egfp) line, the GFP reporter was detected only in the ventricle at both the embryonic (Fig. 4C,E,G) and adult stages (Fig. 4I), confirming the chamber specificity of this 2.2-kb fragment. In both the Tg(V-0.7k:egfp) and Tg(V-0.5k:egfp) lines, GFP was detected in the whole heart without chamber specificity (Supp. Figs. S3A, 4D,F,H,J). This result confirmed that the distal element is required for chamber specificity and supported the hypothesis that this element functions as a repressor in the atrium. In the Tg(V-0.1k:egfp) line, GFP was detected in the entire heart in both embryos and in adult fish (Supp. Fig. S3B and data not shown). These results were consistent with results from Tol2-based transient assays, but in contrast to those from co-injection assays. Of note, each transgenic line was expected to contain a single copy of the distal element with a GFP reporter in each insertion locus.
Figure 4. Results from stable transgenic lines are consistent with those from transient assays. A, C, E, G, I: Transgenic fish expressing GFP under the control of the V-1.9∼+0.3k fragment. B, D, F, H, J: Transgenic fish expressing GFP under the control of the V-0.5∼+0.3k fragment. GFP expression begins around the 21-somite stage (A, B). The Tg(V-1.9k:egfp) transgenic line expresses GFP only in the ventricle as well as in both embryonic (C, E, G) and adult stages (I), while the Tg(V-0.5k:egfp) line expresses GFP in the whole heart (D, F, H, J). Tg(V-1.9k:egfp) has an early onset and drives strong GFP expression in the extraocular muscles, pharyngeal muscles, and other muscle types (G; Supp. Fig. S3C, F), while Tg(V-0.5k:egfp) has a late onset and drives weak GFP expression in these muscles types (H; Supp. Fig. S3H). V, ventricle; A, atrium; E, extraocular muscles; P, pharyngeal muscles; BA, bulbus arteriosus. A,B: Dorsal view, anterior to the top. C,D: Head on view. E–H: Ventral view, anterior to the top. I,J: Dissected adult heart.
Download figure to PowerPoint
The fluorescent GFP signal in the heart can be detected in all four transgenic lines starting from the 21-somite stage (Fig. 4A and B), while the GFP transcript can be detected at 16-somites by in situ hybridization (data not shown). In addition to cardiac-specific expression, both the Tg(V-1.9k:egfp) and Tg(V-0.7k:egfp) lines exhibited strong GFP expression in extraocular muscles, pharyngeal muscles, and other muscle types (Fig. 4G; Supp. Fig. S3A,C,D), reaffirming the endogenous vmhc expression pattern. In contrast, GFP signals were barely detectable in skeletal muscles in both of the Tg(V-0.5k:egfp) and Tg(V-0.1k:egfp) lines during early embryogenesis (Fig. 4H; Supp. Fig. S3B). Later, these two lines exhibited weak GFP expression in cephalic musculature and medium-to-strong GFP expression in the trunk musculature (Supp. Fig. S3E). The skeletal muscle expression pattern persisted in adult animals, but was restricted to muscles around the eye, jaw, operculum (Supp. Fig. S3F–H), and subgroups of muscles next to the body border and close to the fins (Supp. Fig. S3H).
Nkx2.5 Binding Sites Are Important for the Chamber-Specific Activity of the vmhc Promoter
To identify transcription factor(s) involved in regulating chamber-specific vmhc transcription, we used bioinformatics software to predict transcription factor-binding sites within both distal and proximal elements, focusing on those previously reported to be involved in either chamber specificity or MHC gene regulation. We identified multiple binding sites of Ets, Thr, Yy1, Nkx2.5, Tef1, Mef-2, and Srf, but not Tbx, Gata4/5/6, or Irx4 (Knowlton et al.,1995; Ross et al.,1996; Lee et al.,1997; Chen et al.,1998; Wang et al.,2001; Gupta et al.,2003; Small and Krieg,2003) (Fig. 5A). For the following reasons, we elected to further examine nkx2.5, a member of the NK homeobox gene family and one of the earliest cardiogenic factors. First, the expression of nkx2.5 was reported to be initially restricted to the ventricle at the 7-somite stage during early zebrafish embryogenesis (Schoenebeck et al.,2007). Expression of nkx2.5 then expands into the whole heart at 14-somites, the stage at which vmhc expression begins. Second, nkx2.5 has been previously suggested to be required for ventricular expression of Irx4 (Bruneau et al.,2000). Third, two Nkx2.5-binding sites (NKE) in either the proximal or distal element were identified. An NKE site has also been detected in the corresponding conserved region of the Fugu vmhc promoter (Supp. Fig. S4). We generated fine deletions in the zebrafish 2.2-kb fragment to examine the function of these NKE elements. A 20-bp deletion that eliminated both NKE sites in the distal element of the 2.2-kb fragment reduced the V:A ratio from 12 to 3, and a 15-bp deletion that eliminated both NKE sites in the proximal element resulted in a reduction of the V:A ratio to 4.1 (Fig. 5B). Taken together, this data suggest that NKX-binding sites are required for chamber specificity.
Figure 5. nkx2.5 is important for ventricle-specific vmhc gene expression. A: Shown are potential transcriptional factor-binding sites in distal and proximal elements, as predicted by bioinformatic analysis. Yellow diamonds represent the Nkx-binding sites (NKE); red bars represent the first two non-coding exons. B: Schematic summary of results from transient assays using the Tol2 transposon system. Deletion of the two NKE sites in the distal element (∼20 bp) or in the proximal element (∼15 bp) resulted in loss of ventricular specificity. No., number of injected embryos that survived to 4 dpf; Somite ratio/Heart ratio, number of fish with tissue-restricted GFP-positive cells over total number of fish that survived to 4 dpf; Ventricle No. or Atrium No., number of fish with GFP-positive cells in the ventricle or atrium. A fish with GFP-positive cells in both chambers was counted in both categories. V:A, ratio of GFP-positive cells in the ventricle over that in the atrium.
Download figure to PowerPoint