Published online before print July 8, 2004, doi: 10.1161/01.RES.0000138302.02691.be
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Transcriptional Regulation of Cardiac Progenitor Cell Populations
From the Departments of Internal Medicine (A.M.M., T.D.G., D.J.G.) and Molecular Biology (E.N.O., D.J.G.), University of Texas Southwestern Medical Center, Dallas; Department of Oncology (C.A.W.), Presbyterian Hospital of Dallas, Texas; and Department of Medicine (R.S.W.), Duke University Medical Center, Durham, NC.
Correspondence to Daniel J. Garry, MD, PhD, NB11.118A 5323 Harry Hines Blvd, University of Texas Southwestern Medical Center Dallas, Dallas TX 75390-8573. E-mail daniel.garry{at}utsouthwestern.edu
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Abstract |
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Transcriptome-wide analysis of dynamically regulated progenitor cell populations has the potential to elucidate key aspects of cardiac development. The heart, as the first organ to develop in the mammal, is a technically challenging but clinically relevant target for study. To define the transcriptional program of the cardiac progenitor, we used a novel transgenic strategy and fluorescence-activated cell sorting to reliably label and isolate cardiac progenitors directly from mouse embryos. Pure populations of cardiac progenitor cells were isolated from the cardiac crescent and 2 subsequent stages of heart development: the linear heart tube and the looping heart. RNA was isolated from stage-specific cardiac progenitors and subjected to transcriptome analysis by oligonucleotide array hybridization. The cardiac transcriptional regulatory programs were compared with the molecular programs of age-matched noncardiac embryonic cells, embryonic stem cells, adult cardiomyocytes, and each other to identify sets of genes exhibiting differential expression in the cardiac progenitor cell population. These results define the transcriptional profile of mammalian cardiac progenitor cells and provide insight into the molecular regulation of the earliest periods of heart development.
Key Words: cardiac development • gene array • progenitor cells • transcriptome • transgenic mice
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Introduction |
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Mammalian heart development is initiated shortly after gastrulation, when cells of the anterior lateral plate mesoderm become specified to a cardiac fate.1 At embryonic day 7.75 (E7.75), bilaterally paired bands of these cardiac progenitors are apparent at the junction between the cranial and lateral mesoderm. The shape of this region resembles an inverted crescent, with its cranial apex located at the midline where the 2 bands of progenitors meet. By E8.0, progenitors from both halves of the cardiac crescent coalesce at the midline to form the linear heart tube, a structure consisting of an inner endocardial and outer myocardial cell layer. The heart tube subsequently undergoes rightward looping, a process that transforms the linear arrangement of the future chambers into a configuration, evident by E9.5, in which the atria are cranial to the ventricles. At approximately the same time, cells from a secondary heart field contribute to regions of the outflow tract, atria, and right ventricle.2–4 Further cardiac development is characterized by chamber maturation, migration of neural crest cell populations into the heart, and formation of the valves, septa, conduction system, and epicardium.1
Our understanding of the molecular events underlying the complex and precise processes of mammalian cardiac development has been enhanced through gene disruption studies. Using this technology, several transcriptional activators have been shown to have an essential role during cardiogenesis, including the homeobox transcription factor Nkx2.5, the zinc-finger transcription factor GATA4, and myocardin, a potent transcriptional cofactor of serum response factor. Nkx2.5, whose Drosophila ortholog is required for cardiac specification, is essential for ventricular myocardial development but dispensable for the specification of cardiac progenitors in mice.5,6 GATA4 null mice exhibit severe yolk sac and cardiac defects but are similarly capable of cardiac specification.7,8 Thus far, no single gene has been shown to be responsible for cardiac specification in mammals, suggesting functional redundancy and the need for identification of additional cardiac genes that direct pathways to promote cardiogenesis. Further definition of these molecular pathways requires a comprehensive study of the transcriptional program of the developing heart.
The clinical relevance of studying the transcriptome of the early heart is 2-fold. First, a comprehensive strategy serves to identify novel genes involved in cardiac development that are candidate factors in congenital heart disease. Numerous studies have shown that heterozygous mutations in Nkx2.5 and GATA4 lead to a variety of defects, including atrial and ventricular septal defects, tetrology of Fallot, persistent truncus arteriosus, and conduction system defects.9–12 Novel genes may reveal similar involvement. Second, increasing use of stem cell transplantation strategies for treatment of heart disease and myocardial infarction in animal models would benefit from detailed knowledge of the early cardiac transcriptional program.13 An extensive transcriptional profile will allow for detailed assessment of transplanted cell phenotype and perhaps affect selection of transplant cell populations.
We comprehensively characterized the gene expression profiles of cell populations from 3 early stages of cardiogenesis using a strategy that combined transgenic labeling of cardiac progenitors, fluorescence-activated cell sorting (FACS) isolation, and oligonucleotide array analysis. We engineered transgenic mice that express enhanced yellow fluorescent protein (EYFP) in the developing heart, with expression initiating in the cardiac crescent, the earliest cardiac structure, and the first defined location of the cardiac progenitor cell population.1 FACS was used to isolate pure populations of cells, whose gene expression profile was analyzed by oligonucleotide array. Using comparisons with embryonic stem (ES) cells, adult cardiomyocytes, and age-matched noncardiac embryonic cells, we generated a transcriptional profile for the cardiac progenitor that encompasses stage-specific and stage-independent markers of this population. We confirmed these results using semiquantitative RT-PCR and in situ hybridization of selected transcripts. These analyses have identified sets of genes uncharacterized previously in the heart, whose pattern of regulation implies key global and stage-specific functions during cardiogenesis.
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Materials and Methods |
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Transgenic Mice Generation and Analysis
The LacZ cassette of Hsp106 plasmid14 was excised and replaced with EYFP cDNA isolated from the pEYFP plasmid (Clontech) by NcoI-NotI digestion. The resulting transgene was injected into C57BL/6/C3H blastocysts. Southern analysis of founder animals used an EYFP probe. PCR genotyping of F1 and further offspring was performed using primers that amplify a 688-bp fragment of EYFP (fwd 5'-GGGCGAGGAGCTGTTCACCGGGG; rev 5'-GCCGAGAGTGATCCCGGCGGCGG) at a 60°C annealing temperature. Transgenic mice from 3 founder lines were mated to C57BL/6 wild-type mice for analysis of embryonic fluorescence. Pregnant females were euthanized between 7 and 15 days after plug appearance. Staging of embryos followed the criteria of Kaufman.15
Flow Cytometry
FACS samples were prepared in the following manner: stage-matched embryos were digested with 0.25% Trypsin/EDTA (Invitrogen), neutralized in DMEM (Invitrogen) containing 5% FBS (Hyclone) and 10 mmol/L HEPES (Invitrogen), rinsed and resuspended in PBS, and passed through a 70-µm nylon cell strainer (Falcon). Samples were sorted on a MoFlo flow cytometer (Cytomation, Inc.) using Summit software. Samples from wild-type stage-matched embryos were used for gating on a minimum of 10 000 events. Samples for RNA preparation were collected directly into Tripure (Roche), with a fraction of each collected into PBS for postsort assessment of purity.
RNA Amplification and GeneChip Analysis
RNA was isolated with Tripure (Roche) following the protocol of the manufacturer and subjected to 2 rounds of linear amplification, labeling, and analysis on the Affymetrix MGU74v2 GeneChip set as described previously.16,17 Microarray Suite 5.0 software was used for normalization, chip intensity scaling, calculation of presence/absence calls and fold change. User-definable parameters were kept at default settings. For each embryonic stage, a representative sample was used for analysis, with replicates used to confirm reproducibility. Transcripts were scored as differentially expressed if they exhibited a signal log ratio >2, a signal intensity >100 (for at least 1 side of the comparison), and the P value of the fold change determination fell outside of a 95% CI.
Tissue Immunohistochemistry and In Situ Hybridization
Embryos were fixed in 4% paraformaldehyde for 1 to 3 hours, paraffin embedded, and sectioned parasagittally. Deparaffinized tissue sections were permeabilized with 0.3% Triton X-100 for 3 minutes, blocked with 1.5% goat serum/1% BSA for 30 minutes, and then incubated in 1:250 mouse monoclonal desmin antibody (Affinity Biolabs). A 1:50 dilution of goat anti-mouse (Jackson ImmunoResearch) was used for detection. In situ hybridization was performed as described previously.18 35S-labeled probes correspond to the following Genbank accession numbers: NM009627 (Adr), AK030851 (Alcam), NM031161 (Cck), AF079565 (Capn6), NM018870 (Pgam2), and BC006926 (PFK-C).
Semiquantitative RT-PCR
RNA was isolated and amplified as described above. cDNA was generated from 2 µg of aRNA using Superscript II (Invitrogen) and random nonamers (Invitrogen) as primers during a 2-hour incubation at 42°C. PCR was performed on a dilution series of the RT reaction (1:5, 1:10, and 1:25); primer sequences and cycling conditions are listed in expanded Methods section in the online data supplement available at http://circres.ahajournals.org.
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Results |
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Cardiac Progenitor Isolation
To label cardiac progenitor cells, we engineered transgenic mice in which a 6650-bp cardiac enhancer located
9700 bp upstream of the homeobox gene Nkx2.5 was used to direct expression of EYFP in the embryonic heart (Figure 1A). This distal enhancer fragment directs EYFP reporter gene expression in the developing heart between E7.75 and E10.5.14 This enhancer region does not direct reporter gene expression in the tongue, stomach, thyroid, or tooth primordium; these extracardiac sites of Nkx2.5 expression are under the control of separate enhancer elements. In the heart, this enhancer marks a presumptive myocardial population, as described previously using a LacZ transgenic reporter.14 The early myocardial expression pattern of Nkx2.5 was recapitulated in embryos harboring the Nkx2.5-EYFP transgene, with fluorescence apparent in the cardiac crescent at E7.75 and throughout the developing myocardium of the linear and looping heart tube at E8.0 and E9.5 (Figure 1B). By midgestational age (E12.0), EYFP expression was not detectable using conventional microscopic techniques (data not shown). Fluorescent cells were distributed throughout the myocardium in parasagittal sections of E8.5 and E9.5 transgenic embryos (Figure 1C). Immunostaining of transgenic embryo sections for desmin, an early cardiac-expressed intermediate filament protein, demonstrated that EYFP was expressed uniformly throughout the E8.5 heart tube myocardium compared with heterogeneous desmin expression. At E9.5, desmin and EYFP colocalized in the ventricular myocardial cells of the looped heart (Figure 1C). EYFP expression did not significantly colocalize with CD31/platelet endothelial cell adhesion molecule (an endocardial19 and panendothelial marker), further supporting the use of this strategy to mark cardiac progenitors (supplemental Figure 1, available online at http://circres.ahajournals.org). Flow cytometric analysis of staged transgenic embryos revealed that EYFP+ cells comprised, on average, 1.8% of the live-gated cell population at E7.75, 2.5% at E8.5, and 5.45% at E9.5 (supplemental Table 1, available online at http://circres.ahajournals.org). A second round of flow cytometry was used to assess the purity of cell separation, and progenitor cell samples were typically found to contain 95% to 99% pure populations of EYFP+ cells (Figure 1D).
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Transcriptional Profiling of Cardiac Development
We used Affymetrix GeneChip analysis to characterize global patterns of gene expression in cardiac progenitor cell populations. Between 5000 and 25 000 Nkx2.5-EYFP+ cells were isolated from pools of staged transgenic embryos at E7.75, E8.5, and E9.5 and used for RNA isolation. Because cardiac progenitor cell numbers and RNA were limited, we used a T7-based linear amplification protocol to generate sufficient amounts of aRNA for subsequent analysis with the Affymetrix MgU74v2 GeneChip set. We have established previously that amplification of RNA from limited ES cell numbers (100 to 100 000 cells) using this T7-based protocol is linear and reproducible with minimal skewing of gene expression.16 Hybridization signals for cardiac progenitor cell samples were compared with those obtained from age-matched noncardiac (EYFP–) embryonic cells that were collected from the same embryonic sample and analyzed in parallel. In E7.75 cardiac crescent-stage progenitors, a total of 11 070 features were present at a statistical significance level correlating to a 95% CI-bound, with 599 of these (5.41%) exhibiting 
2-fold differential expression when compared with age-matched noncardiac embryonic cells (supplemental Table 2, available online at http://circres.ahajournals.org). At E8.5, 10 860 features were present in cardiac progenitors, with 844 (7.77%) differentially expressed, and at E9.5, 1254 (13.13%) features were differentially expressed of 9551 deemed present. The increase in the percentage of features regulated as developmental time progresses likely reflects both cardiac and noncardiac differentiation in the developing embryo because both EYFP+ and EYFP– populations exhibit the same stage-dependent trend when compared with a control ES cell population.
Examination of the transcripts expressed differentially in Nkx2.5-EYFP+ cells when compared with stage-matched EYFP– cells confirmed the validity of our strategy in targeting cardiac progenitor cells. Cardiac-specific genes predicted to be present in the EYFP+ cell population were enriched strongly at all stages examined, including the transcription factors Nkx2.5, myocardin, MEF2C, and GATA4 (supplemental Table 3). Twenty-three cardiac structural genes were enriched in the EYFP+ cell population; most were significantly enriched only in progenitors from the heart tube and looped heart, although 5 genes (Myla, Mylc2a, Tncc, Tnni1, and Tnnt2) were also enriched in cardiac crescent-stage progenitors (supplemental Table 3 and supplemental Table 4, available online at http://circres.ahajournals.org). The array data are consistent with work published previously regarding the onset of expression20 or dynamic spatiotemporal regulation21,22 of known cardiac genes. For example, Mef2a is detectable in the cardiac region by in situ hybridization beginning at E8.521 and eHAND, although expressed in the cardiac crescent at E7.75, it is also expressed robustly in lateral mesoderm,22 explaining the apparent depletion of these transcripts in E7.75 progenitors. The pattern of transcripts depleted in this cell population also corroborated its cardiac identity. Consistently depleted transcripts included markers of noncardiac lineages, such as Gbx2, expressed in the neuroectoderm and pharyngeal endoderm23 and brachyury and Dl1l, expressed in early gastrulating but not differentiated mesoderm.24,25
Stage-Specific Gene Expression Patterns
Cardiac progenitor cell populations isolated at sequential stages of early cardiac development exhibit both distinct and shared patterns of transcript enrichment. After removal of the redundancy of array features, 318 transcripts were enriched 2-fold at 2 stages, whereas only 35 exhibited enrichment in cardiac progenitor cell populations at all 3 stages (Figure 2A). Pairwise comparisons revealed that progenitor cell populations from the linear and looped heart (E8.5 and E9.5) shared a much higher percentage of enriched transcripts with each other than either one compared with cells isolated from the cardiac crescent. Forty-one percent of the transcripts enriched in either the E8.5 or E9.5 populations were enriched in both populations. In contrast, only 4.5% of transcripts enriched at E8.5, and 4.0% of enriched E9.5 transcripts were also enriched at E7.75. We noted that the majority of transcripts (61.5%) enriched at the cardiac crescent stage were stage specific as they were enriched in crescent-stage progenitors but not in progenitors from the heart tube or looped heart. We found that approximately half of these transcripts were not present in either EYFP+ or EYFP– cell populations at E8.5 and E9.5, potentially indicating a set of genes involved in early cardiac specification but not differentiation.
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To further characterize this molecular program, we categorized transcripts enriched in cardiac progenitors using Gene Ontology annotations. Transcription factors comprised a greater percentage of the enriched transcripts at the crescent stage compared with later developmental stages (Figure 2B). Strikingly, 14 of the 28 transcription factors enriched at E7.75 were stage specific as they were not enriched at E8.5 or E9.5 (supplemental Table 4). This group of crescent stage-specific factors appeared to be enriched for markers of the hematopoietic and vascular programs (Figure 3A). For example, Tal 1 (T-cell acute lymphoblastic lymphoma 1), exhibited 25-fold enrichment in the cardiac crescent when compared with age-matched noncardiac embryonic cells. Tal1 (also known as SCL) is a basic helix-loop-helix (bHLH) transcription factor expressed in vascular endothelium and hematopoietic progenitors and is considered a marker of the hemangioblast population.26–29 Additional crescent stage-restricted enriched transcription factors are similarly implicated in hematopoietic and vascular developmental programs (Figure 3A). We also noted that the set of stage-restricted transcription factor genes in the looped heart (E9.5) contained multiple genes encoding homeodomain-containing proteins, including Msx2 (Msh-like homeobox 2), Prrx1 (Pair-related homeobox 1), Dlx2 (distalless homeobox 2), and Six2 (sine-oculis-related homeobox 2). Cardiac expression or involvement has been described for Msx2 and Prrx1.30,31 We confirmed the stage-specific cardiac enrichment of these genes by performing semiquantitative RT-PCR on independently collected and amplified EYFP+ cardiac progenitor cell populations and stage-matched EYFP– cells (Figure 3B).
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Stage-Independent Genetic Profile of the Cardiac Progenitor
Although stage-specific differential expression patterns identified genes likely to function in temporally restricted processes, we predicted that transcripts enriched in progenitor cell populations throughout all stages studied would highlight molecular pathways that functioned throughout early cardiac development. Of the 35 transcripts enriched in the cardiac progenitor cell population at all developmental stages studied, 28 represented annotated transcripts. (expressed sequence tags were excluded from this analysis). To clarify the relationships among these transcripts, we compared the expression level in cardiac progenitor populations of each transcript with SM1 ES cells and adult mouse cardiomyocytes (ACMs; Figure 4). As expected, cardiac transcriptional regulators (GATA4, Hop, Mef2C, and myocardin) and myocardial structural genes (Myla, Mylc2a, Tncc, Tnni1, and Tnnt2) were consistently enriched in the cardiac progenitor cell population when compared with ES cells. However, when compared with ACMs, several transcripts exhibited no change in expression level or, in the case of some structural proteins, were depleted, consistent with higher expression levels in the mature adult cardiomyocyte. This myocardial program was complemented by the expression of genes critical to vascular and endocardial development. Transcripts encoding the vascular endothelial growth factor receptors Kdr (kinase insert domain receptor, also called Flk-1) and neuropilin (Nrp), and the angiopoietin 1 and 2 receptor, Tek (endothelial-specific receptor tyrosine kinase, also called Tie2) were all enriched in the cardiac progenitor cell population. Enriched signaling genes include predicted and novel sequences. Bone morphogenetic proteins (BMPs) are implicated in the specification and maintenance of cardiac progenitors. Bmp2 transcripts were enriched in cardiac progenitor cells at all stages, reflecting the critical role of this morphogen in patterning and the heart-forming field.32 Bmp5 is also highly enriched across these comparisons. Additional genes not shown previously to function in cardiac development include Rgs5 (regulator of G-protein signaling 5), Ramp2 (receptor modifying calcitonin activity 2), and Capn 6 (calpain 6). This pattern of enriched genes serves to distinguish the early cardiac progenitor population from a diverse range of cell types (Figure 4).
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Microarray Analysis Accurately Identified Cardiac Transcripts
We verified GeneChip results by 2 methods. Selected transcripts that exhibited high levels of cardiac enrichment at E9.5 were analyzed through in situ hybridization. We confirmed cardiac expression and enrichment at E9.5 for transcripts encoding the neuroendocrine peptides adrenomedullin and cholecystokinin, the calcium-activated protease calpain 6, and the glycolytic enzymes phosphoglycerase mutase 2 and phosphofructokinase-C (Figure 5). Capn6 and Pfkc also exhibited enrichment at E8.5, as did Alcam, a cell surface receptor gene. Spatiotemporal expression patterns conformed to predictions on the basis of array data (supplemental Table 4). Low-abundance transcripts were verified with semiquantitative RT-PCR performed on independently collected samples of EYFP+ cardiac progenitors and EYFP– stage-matched cells from E7.75 embryos. Cardiac enrichment was confirmed for all transcripts tested (supplemental Figure 2, available online at http://circres.ahajournals.org). The robust early cardiac expression of these transcripts confirmed our methodology in targeting transcripts specific to the cardiac progenitor cell pool and identified several novel markers of this population.
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Discussion |
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We used transgenic technology and FACS to accurately isolate cardiac progenitor cells from murine embryos at 3 critical stages of early heart development: the cardiac crescent at E7.75, the linear heart at E8.5, and the looped heart at E9.5. RNA amplification followed by microarray analysis was used to define the dynamic transcriptional program of this progenitor cell population. We identified stage-specific and stage-independent sets of genes that were enriched in cardiac progenitor cell populations and used this information to identify novel cardiac genes and correlate waves of gene expression to key stages of cardiogenesis.
Gene expression patterns reflected a major shift in the transcriptional profile of the cardiac progenitor cell population between the cardiac crescent and heart tube stages. The expression profile of transcription factor genes in the crescent provided evidence of shared identity between cardiac crescent stage progenitors and a vascular/hematopoietic (or hemangioblast) cell type. Hemangioblasts comprise a transient embryonic cell population that serves as a precursor of hematopoietic and vascular cells.33,34 A related cell population that is relevant to this discussion is the mesoangioblast, an angioblast-derived progenitor with shared vascular and mesodermal potential.35 Work by Minasi et al36 demonstrated the cardiac myogenic differentiation capability of clonal populations of mesoangioblasts isolated from embryonic dorsal aorta. Both the crescent-stage cardiac progenitor and the mesoangioblast cell populations consist of mesodermally derived cells that give rise to endothelial (endocardium or aortic endothelium) as well as mesodermal (myocardium or pericytes) cell types, a differentiation capability potentially reflected in shared transcriptional identity.
Furthermore, it has been observed that the differentiation of mesoderm to cardiac or hematopoietic lineages may represent antagonistic fates.37 Anterior mesoderm becomes specified to a cardiac fate in response to inductive signals from the anterior endoderm (specifically BMP signaling38 and Wnt inhibition).39–41 Explant and transplant models have demonstrated that the potential of anterior mesoderm (which normally will differentiate into cardiac and head mesoderm) and posterior mesoderm (which forms blood and vasculature) are determined by the signals each receives from the adjacent endoderm, rather than intrinsic differences between the 2 tissues.42,43 When misexpressed in Xenopus, Wnt3a and Wnt8 ablated cardiac specification of anterior mesoderm and promoted the formation of primitive blood cells in the region. Similarly, expression of the Wnt inhibitor crescent in posterior mesoderm promoted cardiac cell differentiation at the expense of blood cell development.39,40 Features of the hemangioblast-like transcriptional program we observe in cardiac progenitors may reflect the apparent dual potential of the anterior mesoderm from which the cardiac mesoderm originates.
The specific role of these hematopoietic/vascular factors in the cardiac crescent is unclear. Several of these hemangioblast transcription factors function in vasculogenesis. For example, a compartment-specific rescue of the Tal1-null hematopoietic phenotype44 revealed that Tal1 also functioned in development of mature vessels from primitive endothelial precursors in the yolk sac.45 Tal1 may play an analogous role in early endocardial development. Specifically, Tal1 may function in inhibiting myogenesis; Tal1 suppressed myotube fusion in 10T1/2 cells transfected with MyoD via a C-terminal domain that interacts with other bHLH factors.46 Other factors may have broader roles. For example, GATA1 is highly expressed in the testis and may regulate early spermatogenesis,47 suggestive of a general role in progenitor cell function.
We also identified a set of homeodomain-containing proteins, some of which were previously uncharacterized in the looped heart. These genes clustered with known cardiac homeobox-containing genes such as Prrx1, Prrx2, and Msx2, and may function in the same processes, specifically, vessel or valve development.30,31 Three of the genes we found to be cardiac enriched (Dlx2, Hoxa9, and Hoxd9) have not exhibited cardiac phenotypes in gene disruption models.48,49 However, other studies have found that the high degree of redundancy among homeobox-containing paralogs may obscure the functional significance of 1 particular gene in a tissue. This is observed in the Prrx2 mutant, which exhibits no defects on its own but exacerbates the Prrx1 null phenotype.31 Because the induction of this homeobox transcriptional program correlates specifically to E9.5, a stage during which processes of cardiac differentiation such as cushion formation, endocardial, and epicardial differentiation, and conduction system development occur (and because some of these factors have already been described to play roles in these processes), it is likely that the novel cardiac homeobox genes and their interacting partners may prove to play key roles in cardiac morphogenesis.
Analysis of stage-independent enriched transcripts has identified several novel markers of the cardiac progenitor cell population, including signaling and cell surface proteins, the cardiac expression pattern of which had not been demonstrated previously by in situ hybridization. Previously published studies on the roles of these markers in other cell populations may provide clues as to their roles in cardiac development. Alcam is expressed in a variety of cell types, including metastatic tumors of multiple origin, epiblast tissue, and mesenchymal stem cells.50 Alcam expression occurs primarily in the migrating cell populations of these tissues. Several of these genes may also represent target genes of cardiac transcription factor pathways (for example, GATA- and Nkx2.5-binding sites are present in the 5' regulatory upstream region of the mouse PFK-C gene).51
In summary, this study has defined a stage-independent gene-specific transcriptional program of the cardiac progenitor cell population. Stage-specific enriched transcripts are implicated in the regulation of cardiac developmental processes. Further studies will focus on characterizing the mechanisms by which these factors participate in cardiac development.
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Acknowledgments |
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These studies were supported by grants from the National Institutes of Health (AR47850), Muscular Dystrophy Association, March of Dimes Association, and the D.W. Reynolds Foundation. The authors wish to thank N. Jiang and T. Macatee for assistance with flow cytometry and Dr J.A. Richardson, J. Shelton, C. Pomajzl, J. Stark, and D. Suttcliffe for their assistance with in situ hybridization, imaging, and histological techniques.
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Footnotes |
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Original received October 2, 2003; resubmission received May 27, 2004; revised resubmission received June 28, 2004; accepted June 28, 2004.
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