<em>Hand2</em> loss-of-function in <em>Hand1</em>-expressing Cells Reveals Distinct Roles In Epicardial And Coronary Vessel Development

Rationale

The bHLH transcription factors Hand1 and Hand2 are essential for embryonic development. Given their requirement for cardiogenesis, it is imperative to determine their impact on cardiovascular function.

Objective

Deduce the role of Hand2 within the epicardium.

Method &amp; Results

We engineered a Hand1 allele expressing Cre recombinase. Cardiac Hand1 expression is largely limited to cells of the primary heart field, overlapping little with Hand2 expression. Hand1 is expressed within the septum transversum (ST) and the Hand1-lineage marks the proepicardial organ and epicardium. To examine Hand factor functional overlap, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 mutants display defective epicardialization and fail to form coronary arteries, coincident with altered ECM deposition and Pdgfr expression.

Conclusion

These data demonstrate a hierarchal relationship whereby transient Hand1 ST expression defines epicardial precursors that are subsequently dependent upon Hand2 function.

Introduction

When cardiac gene programs are hobbled, there is rarely an ablation of the cardiac lineage, but more often defective cell differentiation and/or tissue morphogenesis, which manifests as congenital heart defects (CHDs). Multiple cell lineages collectively form the heart ¹. While myocyte derivatives ultimately provide the cardiac musculature that powers circulation, it is the extra-cardiac cell lineages (the cardiac neural crest cells (cNCC), coronary endothelium, and epicardium) that define the framework upon which these cardiomyocytes develop ²–5. Although the heart functions as an integrated whole, our understanding of its individual components is not uniform. For example, the second heart field (SHF) and cNCC have been extensively studied ⁶, while the differentiation programs of the primary heart field (PHF) and coronary vasculature are less well defined.

Both cNCC and epicardial cell lineages undergo Epithelial-to-Mesenchymal Transition (EMT) and directly interact with differentiating cardiomyocytes. cNCCs enable septation of the outflow track (OFT) into the aorta and pulmonary trunk. Epicardial mesothelium migrates from the proepicardial organ (PE) to cover the surface of the heart ⁷. Subsequently, an epicardial subpopulation undergoes secondary EMT, invades the heart, and differentiates into coronary smooth muscle and cardiac fibroblasts ⁸–11. Cardiac morphogenesis requires communication between cardiac and extra-cardiac cell populations ², 3, 12. The signaling cascades through which these cell lineages communicate to are just coming to light.

Expression of the Twist-family basic Helix-loop-Helix (bHLH) transcription factors Hand1 and Hand2¹³, 14 partially overlaps within the developing heart and cNCC ¹⁵–18. Hand2 expression marks the cardiac crescent and linear heart tube and is downregulated within the left ventricle at the onset of cardiac looping ¹⁸. At comparable developmental stages, the left ventricle and myocardial cuff express Hand1¹⁵, 16, 19. Understanding where and when Hand1 and Hand2 function during embryonic development is a challenge due to their dynamic spatiotemporal expression profiles ¹³.

Hand factors are critical for cardiac morphogenesis ¹³, 14. Systemic ablation of Hand2 results in a single ventricle, increased apoptosis, and lethality by E9.5 ¹⁸, 20. Hand1 knockout mice die by E9.5 from extraembryonic and vascular defects ¹⁶, 19, 21. Conditional NCC-specific Hand2 ablation (Hand2^{Δ) causes OFT defects, associated ventricular septal defects (VSD), Double Outlet Right Ventricle (DORV) and decreased SHF myocardial proliferation 22}, 23. Myocardial-specific Hand2 deletion causes right ventricular hypoplasia and death by E12.5 ²³. Reciprocally, myocardial-specific Hand1 deletion causes left-ventricular hypoplasia and although embryos survive until birth, Hand2 haploinsufficiency increases phenotypic severity suggesting genetic and functional overlap ²⁴, 25.

Here, we investigate the contributions of Hand1-expressing cells to cardiogenesis using a Cre recombinase-expressing Hand1 allele ²⁶. The Hand1 lineage robustly marks the left ventricular myocardium, a cNCC sub-population, and, unexpectedly, the epicardium and its derivatives.

To deduce functional/genetic interactions between Hand factors during cardiogenesis, we conditionally deleted Hand2 within the Hand1 lineage (H2CKO). H2CKOs embryos die by E14.5 and present Persistent Truncus Arteriosis (PTA), DORV and associated VSDs. Significantly, Hand2 expression lies temporally downstream of Hand1 within the septum transversum- (ST) derived cells that migrate into the proepicardial organ (PE), and is thus deleted from the PE, epicardium and the epicardial derivatives of H2CKOs. H2CKOs display defective epicardial EMT, decreased cardiac fibroblasts, and a nonfunctional coronary vasculature. Hand2 ablation within the WT1^-lineage phenocopies Hand1^Cre-mediated Hand2 ablation. Gene expression analyses reveal an altered ratio of Pdgfrα:Pdgfrβ mRNA, a decreased profile of fibroblast markers, and disorganized ECM. These changes are associated with increased epicardial cell apoptosis. Together, our data shows that Hand2 performs an essential role during epicardialization, which directly impacts epicardial cell differentiation, and formation of the coronary vasculature.

Methods

See the online supplement for detailed methods.

Targeting Hand1 and Generation of Mice

The construction of the targeting vector, generation of targeted ES cells, chimera and germline mice is described in detail in ²⁶. Tamoxifen was administered for the as described ⁴.

In Situ Hybridization

Section and Wholemount in situ hybridization (ISH) was performed essentially as previously described ²⁶, 28,29.

Primary Epicardial Culture

Primary epicardial cultures were isolated and cultured as previously described ³¹.

Immunohistochemistry

Embryos were fixed in 4% PFA overnight then embedded in paraffin or cryoprotected and sectioned at 7mm as previously reported ²⁶.

Results

Hand1 expression within the ST marks the progenitors of the PE

As Hand1-lineage cells, but not Hand1-expressing cells, are observed within the epicardium at E10.5 (See online supplement Fig. I), we sought the Hand1-expressing progenitors of these cells. The PE is derived from the anterior ST and gives rise to the epicardium ³³. ISH was performed for both Hand1 and Tbx18, a marker of the PE, at E9.5 ³, 34 (Fig. 1A-E). Tbx18 is expressed throughout the PE but is not detectable within the ST. In contrast, Hand1 expression is not detectable within the PE but is expressed robustly throughout the ST, thus identifying the source of the Hand1-marked epicardium. X-Gal staining of Hand1^LacZ/+ embryos confirms expression within the LV and the ST (Fig. 1F). X-Gal staining of the Hand1-lineage shows Hand1-marked cells dispersed throughout the ST and within the more proximal Tbx18-expressing region, supporting established models that PE cells derive from migratory ST cells (Fig. 1G-I) ³, 34. These results establish that the Hand1-lineage cells observed throughout the cardiac fibroblasts, coronary vasculature, epicardium and PE originate from a Hand1-expressing ST cell population.

Figure 1

The Hand1 lineage gives rise to epicardial progenitors. (A) E9.5 illustration showing Hand1 expression (blue) and depicting the planes of section in B-E (B). Hand1 and Tbx18 section ISH of E9.5 embryos on adjacent sections through the ST and the PE (B-E). Hand1 expression is not detected within the PE but is expressed in the ST. X-gal staining of Hand1^LacZ (F) and Hand1 ^R26R activation (G-I) shows Hand1-lineage cells within the ST and in the PE.. lv, left ventricle; pe, proepicardium; st, septum transversum.

Distinct Hand factor expression during heart morphogenesis

E8.5 Hand1-lineage analysis reveals that the linear heart tube is not completely derived from Hand1-expressing cells, indicating that Hand1 expression initiates after heart tube fusion. To account for a possible temporal delay of Cre expression, we performed wholemount ISH for Hand1 at E7.5 (Fig. 2). Hand1 is detected throughout the extra embryonic mesoderm, chorion, and allantoic rudiment (Fig. 2A). As the chorion is directly adjacent to the cardiac crescent (Fig. 2A,B white arrow), we performed a double label ISH for both Hand1 and the cardiac marker Mlc2a. Hand1 expression does not directly overlap with Mlc2a expression at E7.5, indicating that, consistent with the Hand1-lineage analysis, Hand1 cardiomyocyte expression initiates subsequent to linear heart tube fusion (Fig. 2B).

Figure 2

Hand factor expression is distinct. Single and double-labeled Mlc2a and Hand1 wholemount RNA ISH of E7.5 embryos show that Hand1 is not expressed within the cardiac crescent (white arrow) but is expressed within extra embryonic mesoderm, allantoic rudiment, and the chorion (A, B). E8.5 wholemounts of Hand1 and Hand2 reveal complimentary expression during cardiac development (C-F). Hand2 expression is confined to the SHF and endocardium (black arrow). cc, cardiac crescent; eem, extraembryonic mesoderm; ht, heart tube; lm, lateral mesoderm; oft, outflow tract; v, ventricle.

Wholemount ISH at E8.5 (Fig. 2C-F) shows Hand1 expression within the posterior heart tube while Hand2 expression is observed throughout the entire cardiac field. Sagittal sections of wholemount-stained embryos confirm that Hand1 expression is restricted to the early ventricular chamber and cuff myocardium. Hand2 expression is robust throughout the endocardium, SHF and OFT. Interestingly, Hand2 expression is not detected within the ventricular myocardium at E8.5 and, in wholemount view, the Hand2 endocardial expression is visible through the thin myocardial wall (Fig. 2F, red arrow). At E9.5, Hand1 and Hand2 expression overlaps within the SHF-derived myocardial cuff and the left ventriclular myocardium (not shown). Collectively, these data reaffirm that Hand factor expression is dynamically regulated and that, during early heart morphogenesis, this expression overlaps solely within the SHF-derived myocardial cuff.

Hand1^Cre-mediated ablation of Hand2 within the, PE, and epicardium

To explore potential Hand factor functional overlap, we intercrossed Hand2^fx23 and Hand1^Cre mice to ablate Hand2 within the Hand1-lineage (H2CKO). Hand2 expression is robust throughout the PE and detectable in both E10.5 epicardial cells and isolated primary epicardial cell cultures (arrow, Fig. 3A,C; Online Fig. II). Hand2 expression was not detected within the epicardium or the PE of H2CKOs, confirming that Hand1-positive ST cells ultimately populate the PE and epicardium (Fig. 3B,D). Hand1 and Hand2 are not coexpressed within the ST or PE; rather, Hand1 expression within the ST precedes PE/epicardial upregulation of Hand2. Thus, Hand1^Cre conditionally ablates Hand2 function within the PE and epicardium.

Figure 3

Inactivation of Hand2 within the Hand1 lineage RNA ISH at E9.5 (A, B) shows Hand2 expression throughout the PE and in the epicardium (red arrow) at E10.5 (C, D). Consequently, Hand2 expression is ablated in the presumptive epicardial mesothelium in H2CKOs; pe, proepicardium.

Hand1-lineage H2CKO reveals novel phenotypes and embryonic lethality

H2CKO embryos were recovered at slightly below expected Mendelian ratios through E12.5 (Fig. 4). At E14.5, roughly 50% of H2CKOs collected were dead. E12.5 H2CKOs appeared phenotypically normal (data not shown). Cardiac patterning, and both Hand1 and cardiomyocyte marker expression were normal (Online Fig. IIIA-P).

Figure 4

Conditional Hand2 deletion within the Hand1 lineage results in extensive embryonic and cardiovascular defects. Histological (A, B, E, F, I-N) and wholemount (C, D, G, H) analysis of E14.5.H2CKO embryos. Mutants display pericardial hemorrhaging (C) and anasarca (D). Compared to WT mice (G), H2CKOs have hypoplastic livers with extensive hemorrhaging (H).. H2CKOs display either PTA or DORV (A, B, E, F, I, J). All H2CKOs exhibit VSDs (K, L). Myocardial examination shows abnormal trabeculae and non-compaction (M, N). ao, dorsal aorta; cz, compact zone; pt, pulmonary trunk; pta, persistent truncus arteriousus; tr, trabeculae.

By E14.5, H2CKOs displayed pericardial hemorrhaging, anasarca, and liver hypoplasia (Fig. 4C, D, G, H). OFT septation defects, including PTA and DORV, were also observed, reflecting cNCC and/or SHF-derived myocardial defects (Fig. 4A, B, E, F, I-J). Hypertrabeculation/noncompaction and VSDs were also observed (Fig. 4K-N), although, H2CKO sarcomeric ultra-structure was indistinguishable from controls (Online Fig. IIIQ, R).

Hand2 ^{die from a sympathetic nervous system (SNS) norepinepherine deficit 22}, 23. As Hand1 and Hand2 are coexpressed in some NCCs, H2CKOs possibly die from SNS defects. Hand2 ^{lethality can be rescued by the β-adrenergic agonist isoproterenol 22}, 23. Unlike Hand2^NCC embryos, isoproterenol treated Hand1^Cre-generated H2CKOs fail to survive to birth (Fig. 4), indicating that SNS defects do not account for embryonic lethality.

Hand2 is required for a functional epicardium

Given the temporal relationship observed between Hand1 expression in the ST and Hand2 within the PE/epicardium, we examined H2CKO epicardial defects (Fig. 5A, B). E14.5 H2CKO display abnormal compaction, an absence of epicardium, and a lack of coronary lumens, indicating a possible epicardial phenotype (Fig. 5A,B). ISH analyses of epicardial markers at E10.5 (data not shown) and at E12.5 for Tcf21 suggest that specification and formation of the early epicardium occurs normally (Fig. 5C,D). To confirm direct disruption of H2CKO epicardial development, we generated a WT1 ^H2CKO4, in which Hand2 is ablated in the ST, PE and epicardium, but not the myocardium and NCC lineages (Fig. 5E-H). (See Online Fig. IV for a WT1^ERT2Cre, Hand1^Cre, and Hand2 cardiac lineage/expression comparison). Potentially complicating these analyses, WT1^ERT2Cre marks lateral mesoderm, where Hand2 is expressed, and early allelic WT1^ERT2Cre induction (E8.5) can produce ectopic Cre activity ⁴. HoxB6-Cre-mediated Hand2 deletion in lateral mesoderm generates viable neonates ²³. Tamoxifen-mediated WT1^ERT2Cre allelic induction at E9.5 resulted in no ectopic myocardial R26R reporter activity (Online Fig. V). At E13.5, Hand2^fx/−, WT1^ERT2Cre mutant embryos phenocopied H2CKO embryos, displaying a poorly organized epicardium and reduced WT1-marked cell lineage (Fig. 5E-H). Myocardial non-compaction was also evident, further suggesting that this phenotype is non-cell autonomous. These mutants appear to phenocopy the epicardial defects and E14.5 embryonic lethality associated with H2CKOs (Fig. 5A, B, G, H). These two independent epicardial Hand2 ablations indicate that Hand2 may play multiple roles during epicardiogenesis, the initiation of secondary epicardial EMT, and the terminal differentiation of post EMT epicardial cell populations.

Figure 5

H2CKOs display epicardial phenotypes. Histological examination at E14.5 shows a lack of epicardium and compaction abnormalities in H2CKOs (A, B). RNA ISH for Tcf21 at E12.5 shows initial establishment of the epicardium (C, D). X-gal staining and conditional deletion of Hand2 within the WT1 lineage at E13.5 photocopies H2CKO epicardial defects (E-H).

Loss of Hand2 disrupts epicardial gene expression and coronary vasculature patency

As conditional Hand2 loss-of-function leads to epicardial defects, we sought to further identify the mechanistic role for Hand2 within the epicardium. Platelet Derived Growth Factor Receptors (Pdgfr) play essential roles in cell fate and specification of the mature epicardium ³⁵, 36. E12.5 ISH reveals decreased Pdgfrα expression within the H2CKO epicardium (Fig. 6A,B). The number of cells expressing Periostin (Postn), a marker of the cardiac fibroblast cell fate ³², is visibly decreased in the myocardium of E12.5 H2CKO mutants (Fig. 6C-D). Together these data suggest that Hand2 functions in epicardial cell fate determination during or after secondary EMT.

Figure 6

Fibroblast lineage and secondary coronary defects in H2CKOs. RNA ISH at E12.5 for Pdgfrα and Postn (A-D). A decrease in cardiac fibroblasts is observed within the compact zone of H2CKOs. Immunohistochemistry for Flk1 (green) and F-actin (red) at E12.5 (E, F). A decrease in patent coronary vasculature is observed in H2CKOs.

We performed CD31 (Online supplement; Fig. VIA, B) and Flk1 immunohistochemistry (Fig. 6E, F) at E12.5 to examine coronary vessel formation. Flk1-positive cells running through the epicardium are absent. Phalloidin counterstaining indicates that the epicardium is intact, though absent of potential Flk1-positive lumens (Fig. 6F). The Hand1-lineage does not contribute to coronary endothelial cell populations (Online Fig. VIC-F) ²⁶, and therefore the absence of coronary vasculature within H2CKOs likely results from non-cell autonomous mechanism(s). To take advantage of induction, we administered tamoxifen at E11.5 CD31-positive coronary lumens are visible within H2CKOs when WT1^ERT2Cre is activated at E11.5, although they appear less prevalent than in embryos that lack the WT1^ERT2Cre allele (Online Fig. VC-D), suggesting that critical Hand2 epricardial function occurs between E9.5 and 11.5.

To further address the impaired function of H2CKO epicardium, we generated Epicardial Primary Cultures (EPCs) from wild type and H2CKOs and isolated total RNA for microarray analyses (Online Fig. IIA-D). Gene ontology from our microarray analysis indicates significant differences in developmental, morphological, and cardiovascular gene programs within EPCs (Online Fig. VIG). Quantitative RT-PCR on RNA isolated from wild type and H2CKO EPCs was performed to validate changes in gene expression observed in the microarray (Online Fig. VIH). Quantitative RT-PCR confirms expression of Hand2 within the epicardium and its ablation within the H2CKO EPCs. Importantly, the ratio of Pdgfrα to Pdgfrβ is greatly altered in H2CKO EPCs. Expression of Pdgfrα is significantly reduced, whereas, Pdgfrβ expression is significantly upregulated (Online Fig. VIH). Hand2 regulation of Pdgfrα is direct (Online supplement; Fig. VI,I). In epicardial cells, Pdgfrβ-mediated signaling promotes a smooth muscle fate, while the role of Pdgfrα signaling is currently unclear ³⁵. The decrease in Postn-positive myofibroblasts within the compact zone suggests that Pdgfrα impacts fibroblast differentiation. Together, these data suggest that Hand2 directly impacts epicardial cell fate through a Pdgfr-dependent mechanism.

Hand2 alters Fn1 fibril assembly and organization

As H2CKO epicardial defects appear direct, we looked at the impact of a loss of Hand2 upon epicardial mesothelium integrity. Fn1 is downregulated in zebrafish hand2 mutants ³⁷. Fn1 fibril assembly regulates the organization and stability of ECM proteins, is capable of promoting EMT and adhesion-dependent growth, and is associated with integrin-mediated cell signaling ³⁸–40. Additionally, defects in Fn1 underlie integrin-mediated valve leaflet defects in the lymphatic system ⁴¹. Immunohistochemistry shows that Fn1 is expressed in the E12.5 epicardium and is neatly organized around the developing coronaries (Fig. 7A). H2CKOs retain epicardial Fn1; however, it appears disorganized (Fig. 7B). To look at Fn1 organization more closely, we compared EPCs from wild type and H2CKOs (Fig. 7C-F). Immunohistochemistry reveals that Fn1 fibrils form an organized lattice in wild type EPCs. Fn1 deposition appears abnormally uniform and sheet-like throughout the H2CKO explant (Fig. 7C-F). Fn1 dysfunction suggests a role for Hand2 in ECM assembly and epicardium homeostasis. Increased Alcian Blue staining indicates that ECM organization and/or deposition is altered in the H2CKO epicardium (Fig. 7G, H).

Figure 7

Abnormal Fn1 matrix assembly and function results in ECM related defects and impaired epicardial development. Immunohistochemistry at E12.5 for Fn1 (green) and F-actin (red) showing Fn1 expression in the epicardium and absence of patent coronary vessels (A, B). Primary epicardial cells isolated from H2CKOs show a disorganized Fn1 localization, where Fn1 appears to be distributed uniformly throughout the cells, further suggesting migratory defects in H2CKO epicardial cells (D, F). In contrast, wild type primary epicardial cells show Fn1 distributed in a series of well-organized bundles (C, E). An increase in Alcian Blue staining within the epicardium indicates increased deposition of ECM (G, H). Gene ontology analysis of microarray data from primary epicardial cells showing affected gene pathways within H2CKO isolated epicardium (I). qRT-PCR on wild type (blue bar) and H2CKO (black bar) RNA isolated from primary epicardial cells; n=4 (J). Cell proliferation (K) and cell apoptosis (L) on wild type and H2CKO embryos at E12.5; n=3. Asterisks indicate statistical significance: *p < 0.05; **p <0.01.

Gene ontology from microarray analysis to identify enriched biological processes indicates enrichment in ECM based processes, such as cell-cell signaling, assembly, connective tissue development, and motility (Fig. 7I). qRT-PCR on wild type and H2CKO EPCs detected no changes in Fn1 expression, as has been reported in mice (Fig. 7J) ⁴².

In addition to its role as an ECM component, Fn1 promotes intracellular signaling via interactions with cell surface integrins ⁴³. To see if the observed H2CKO Fn1 disorganization may alter cell signaling, we assessed integrin expression by qRT-PCR (Fig. 7J). Indeed, expression of the Fn1 receptor, Itga4, is significantly upregulated within H2CKO EPCs. Itgα4 and Itgβ1 together form an Fn1 receptor pair. Itgα4 influences epicardial Fn1 polymerization. Itgα4 overexpression impairs incorporation of new Fn1 into preexisting polymer structures ⁴⁴, while Itgα4 deletion causes embryonically lethal epicardial defects in mice ⁴⁵, 46. As Itgα4 and Fn organization are essential for epicardiogenesis, the observed dysregulation of these ECM components suggests that Hand2 plays an important role in maintaining a normal epicardial ECM environment.

Gene ontology data indicates enrichment for cell cycle regulation genes. Loss-of-function of Hand2 has been implicated in apoptosis ²⁰. Moreover, ECM disorganization and deposition are thought to be pro-apoptotic ⁴⁷. Activated-Caspase3 and Phospho-Histone H3 immunohistochemistry at E12.5 to assess H2CKO epicardial apoptosis and proliferation (Fig. 7K, L; Online Fig. VIIA-D) reveals no change in proliferation, but a significant increase in epicardial apoptosis, These data suggest that the process of epicardial EMT is not affected in H2CKO as EMT is linked with proliferation ⁴⁸, but the impaired function of the ECM in H2CKOs maybe tied both to the function, integrity, and survival of the epicardium.

Hand1 expression within the ST marks the progenitors of the PE

As Hand1-lineage cells, but not Hand1-expressing cells, are observed within the epicardium at E10.5 (See online supplement Fig. I), we sought the Hand1-expressing progenitors of these cells. The PE is derived from the anterior ST and gives rise to the epicardium ³³. ISH was performed for both Hand1 and Tbx18, a marker of the PE, at E9.5 ³, 34 (Fig. 1A-E). Tbx18 is expressed throughout the PE but is not detectable within the ST. In contrast, Hand1 expression is not detectable within the PE but is expressed robustly throughout the ST, thus identifying the source of the Hand1-marked epicardium. X-Gal staining of Hand1^LacZ/+ embryos confirms expression within the LV and the ST (Fig. 1F). X-Gal staining of the Hand1-lineage shows Hand1-marked cells dispersed throughout the ST and within the more proximal Tbx18-expressing region, supporting established models that PE cells derive from migratory ST cells (Fig. 1G-I) ³, 34. These results establish that the Hand1-lineage cells observed throughout the cardiac fibroblasts, coronary vasculature, epicardium and PE originate from a Hand1-expressing ST cell population.

Figure 1

The Hand1 lineage gives rise to epicardial progenitors. (A) E9.5 illustration showing Hand1 expression (blue) and depicting the planes of section in B-E (B). Hand1 and Tbx18 section ISH of E9.5 embryos on adjacent sections through the ST and the PE (B-E). Hand1 expression is not detected within the PE but is expressed in the ST. X-gal staining of Hand1^LacZ (F) and Hand1 ^R26R activation (G-I) shows Hand1-lineage cells within the ST and in the PE.. lv, left ventricle; pe, proepicardium; st, septum transversum.

Distinct Hand factor expression during heart morphogenesis

E8.5 Hand1-lineage analysis reveals that the linear heart tube is not completely derived from Hand1-expressing cells, indicating that Hand1 expression initiates after heart tube fusion. To account for a possible temporal delay of Cre expression, we performed wholemount ISH for Hand1 at E7.5 (Fig. 2). Hand1 is detected throughout the extra embryonic mesoderm, chorion, and allantoic rudiment (Fig. 2A). As the chorion is directly adjacent to the cardiac crescent (Fig. 2A,B white arrow), we performed a double label ISH for both Hand1 and the cardiac marker Mlc2a. Hand1 expression does not directly overlap with Mlc2a expression at E7.5, indicating that, consistent with the Hand1-lineage analysis, Hand1 cardiomyocyte expression initiates subsequent to linear heart tube fusion (Fig. 2B).

Figure 2

Hand factor expression is distinct. Single and double-labeled Mlc2a and Hand1 wholemount RNA ISH of E7.5 embryos show that Hand1 is not expressed within the cardiac crescent (white arrow) but is expressed within extra embryonic mesoderm, allantoic rudiment, and the chorion (A, B). E8.5 wholemounts of Hand1 and Hand2 reveal complimentary expression during cardiac development (C-F). Hand2 expression is confined to the SHF and endocardium (black arrow). cc, cardiac crescent; eem, extraembryonic mesoderm; ht, heart tube; lm, lateral mesoderm; oft, outflow tract; v, ventricle.

Wholemount ISH at E8.5 (Fig. 2C-F) shows Hand1 expression within the posterior heart tube while Hand2 expression is observed throughout the entire cardiac field. Sagittal sections of wholemount-stained embryos confirm that Hand1 expression is restricted to the early ventricular chamber and cuff myocardium. Hand2 expression is robust throughout the endocardium, SHF and OFT. Interestingly, Hand2 expression is not detected within the ventricular myocardium at E8.5 and, in wholemount view, the Hand2 endocardial expression is visible through the thin myocardial wall (Fig. 2F, red arrow). At E9.5, Hand1 and Hand2 expression overlaps within the SHF-derived myocardial cuff and the left ventriclular myocardium (not shown). Collectively, these data reaffirm that Hand factor expression is dynamically regulated and that, during early heart morphogenesis, this expression overlaps solely within the SHF-derived myocardial cuff.

Hand1^Cre-mediated ablation of Hand2 within the, PE, and epicardium

To explore potential Hand factor functional overlap, we intercrossed Hand2^fx23 and Hand1^Cre mice to ablate Hand2 within the Hand1-lineage (H2CKO). Hand2 expression is robust throughout the PE and detectable in both E10.5 epicardial cells and isolated primary epicardial cell cultures (arrow, Fig. 3A,C; Online Fig. II). Hand2 expression was not detected within the epicardium or the PE of H2CKOs, confirming that Hand1-positive ST cells ultimately populate the PE and epicardium (Fig. 3B,D). Hand1 and Hand2 are not coexpressed within the ST or PE; rather, Hand1 expression within the ST precedes PE/epicardial upregulation of Hand2. Thus, Hand1^Cre conditionally ablates Hand2 function within the PE and epicardium.

Figure 3

Inactivation of Hand2 within the Hand1 lineage RNA ISH at E9.5 (A, B) shows Hand2 expression throughout the PE and in the epicardium (red arrow) at E10.5 (C, D). Consequently, Hand2 expression is ablated in the presumptive epicardial mesothelium in H2CKOs; pe, proepicardium.

Hand1-lineage H2CKO reveals novel phenotypes and embryonic lethality

H2CKO embryos were recovered at slightly below expected Mendelian ratios through E12.5 (Fig. 4). At E14.5, roughly 50% of H2CKOs collected were dead. E12.5 H2CKOs appeared phenotypically normal (data not shown). Cardiac patterning, and both Hand1 and cardiomyocyte marker expression were normal (Online Fig. IIIA-P).

Figure 4

Conditional Hand2 deletion within the Hand1 lineage results in extensive embryonic and cardiovascular defects. Histological (A, B, E, F, I-N) and wholemount (C, D, G, H) analysis of E14.5.H2CKO embryos. Mutants display pericardial hemorrhaging (C) and anasarca (D). Compared to WT mice (G), H2CKOs have hypoplastic livers with extensive hemorrhaging (H).. H2CKOs display either PTA or DORV (A, B, E, F, I, J). All H2CKOs exhibit VSDs (K, L). Myocardial examination shows abnormal trabeculae and non-compaction (M, N). ao, dorsal aorta; cz, compact zone; pt, pulmonary trunk; pta, persistent truncus arteriousus; tr, trabeculae.

By E14.5, H2CKOs displayed pericardial hemorrhaging, anasarca, and liver hypoplasia (Fig. 4C, D, G, H). OFT septation defects, including PTA and DORV, were also observed, reflecting cNCC and/or SHF-derived myocardial defects (Fig. 4A, B, E, F, I-J). Hypertrabeculation/noncompaction and VSDs were also observed (Fig. 4K-N), although, H2CKO sarcomeric ultra-structure was indistinguishable from controls (Online Fig. IIIQ, R).

Hand2 ^{die from a sympathetic nervous system (SNS) norepinepherine deficit 22}, 23. As Hand1 and Hand2 are coexpressed in some NCCs, H2CKOs possibly die from SNS defects. Hand2 ^{lethality can be rescued by the β-adrenergic agonist isoproterenol 22}, 23. Unlike Hand2^NCC embryos, isoproterenol treated Hand1^Cre-generated H2CKOs fail to survive to birth (Fig. 4), indicating that SNS defects do not account for embryonic lethality.

Hand2 is required for a functional epicardium

Given the temporal relationship observed between Hand1 expression in the ST and Hand2 within the PE/epicardium, we examined H2CKO epicardial defects (Fig. 5A, B). E14.5 H2CKO display abnormal compaction, an absence of epicardium, and a lack of coronary lumens, indicating a possible epicardial phenotype (Fig. 5A,B). ISH analyses of epicardial markers at E10.5 (data not shown) and at E12.5 for Tcf21 suggest that specification and formation of the early epicardium occurs normally (Fig. 5C,D). To confirm direct disruption of H2CKO epicardial development, we generated a WT1 ^H2CKO4, in which Hand2 is ablated in the ST, PE and epicardium, but not the myocardium and NCC lineages (Fig. 5E-H). (See Online Fig. IV for a WT1^ERT2Cre, Hand1^Cre, and Hand2 cardiac lineage/expression comparison). Potentially complicating these analyses, WT1^ERT2Cre marks lateral mesoderm, where Hand2 is expressed, and early allelic WT1^ERT2Cre induction (E8.5) can produce ectopic Cre activity ⁴. HoxB6-Cre-mediated Hand2 deletion in lateral mesoderm generates viable neonates ²³. Tamoxifen-mediated WT1^ERT2Cre allelic induction at E9.5 resulted in no ectopic myocardial R26R reporter activity (Online Fig. V). At E13.5, Hand2^fx/−, WT1^ERT2Cre mutant embryos phenocopied H2CKO embryos, displaying a poorly organized epicardium and reduced WT1-marked cell lineage (Fig. 5E-H). Myocardial non-compaction was also evident, further suggesting that this phenotype is non-cell autonomous. These mutants appear to phenocopy the epicardial defects and E14.5 embryonic lethality associated with H2CKOs (Fig. 5A, B, G, H). These two independent epicardial Hand2 ablations indicate that Hand2 may play multiple roles during epicardiogenesis, the initiation of secondary epicardial EMT, and the terminal differentiation of post EMT epicardial cell populations.

Figure 5

H2CKOs display epicardial phenotypes. Histological examination at E14.5 shows a lack of epicardium and compaction abnormalities in H2CKOs (A, B). RNA ISH for Tcf21 at E12.5 shows initial establishment of the epicardium (C, D). X-gal staining and conditional deletion of Hand2 within the WT1 lineage at E13.5 photocopies H2CKO epicardial defects (E-H).

Loss of Hand2 disrupts epicardial gene expression and coronary vasculature patency

As conditional Hand2 loss-of-function leads to epicardial defects, we sought to further identify the mechanistic role for Hand2 within the epicardium. Platelet Derived Growth Factor Receptors (Pdgfr) play essential roles in cell fate and specification of the mature epicardium ³⁵, 36. E12.5 ISH reveals decreased Pdgfrα expression within the H2CKO epicardium (Fig. 6A,B). The number of cells expressing Periostin (Postn), a marker of the cardiac fibroblast cell fate ³², is visibly decreased in the myocardium of E12.5 H2CKO mutants (Fig. 6C-D). Together these data suggest that Hand2 functions in epicardial cell fate determination during or after secondary EMT.

Figure 6

Fibroblast lineage and secondary coronary defects in H2CKOs. RNA ISH at E12.5 for Pdgfrα and Postn (A-D). A decrease in cardiac fibroblasts is observed within the compact zone of H2CKOs. Immunohistochemistry for Flk1 (green) and F-actin (red) at E12.5 (E, F). A decrease in patent coronary vasculature is observed in H2CKOs.

We performed CD31 (Online supplement; Fig. VIA, B) and Flk1 immunohistochemistry (Fig. 6E, F) at E12.5 to examine coronary vessel formation. Flk1-positive cells running through the epicardium are absent. Phalloidin counterstaining indicates that the epicardium is intact, though absent of potential Flk1-positive lumens (Fig. 6F). The Hand1-lineage does not contribute to coronary endothelial cell populations (Online Fig. VIC-F) ²⁶, and therefore the absence of coronary vasculature within H2CKOs likely results from non-cell autonomous mechanism(s). To take advantage of induction, we administered tamoxifen at E11.5 CD31-positive coronary lumens are visible within H2CKOs when WT1^ERT2Cre is activated at E11.5, although they appear less prevalent than in embryos that lack the WT1^ERT2Cre allele (Online Fig. VC-D), suggesting that critical Hand2 epricardial function occurs between E9.5 and 11.5.

To further address the impaired function of H2CKO epicardium, we generated Epicardial Primary Cultures (EPCs) from wild type and H2CKOs and isolated total RNA for microarray analyses (Online Fig. IIA-D). Gene ontology from our microarray analysis indicates significant differences in developmental, morphological, and cardiovascular gene programs within EPCs (Online Fig. VIG). Quantitative RT-PCR on RNA isolated from wild type and H2CKO EPCs was performed to validate changes in gene expression observed in the microarray (Online Fig. VIH). Quantitative RT-PCR confirms expression of Hand2 within the epicardium and its ablation within the H2CKO EPCs. Importantly, the ratio of Pdgfrα to Pdgfrβ is greatly altered in H2CKO EPCs. Expression of Pdgfrα is significantly reduced, whereas, Pdgfrβ expression is significantly upregulated (Online Fig. VIH). Hand2 regulation of Pdgfrα is direct (Online supplement; Fig. VI,I). In epicardial cells, Pdgfrβ-mediated signaling promotes a smooth muscle fate, while the role of Pdgfrα signaling is currently unclear ³⁵. The decrease in Postn-positive myofibroblasts within the compact zone suggests that Pdgfrα impacts fibroblast differentiation. Together, these data suggest that Hand2 directly impacts epicardial cell fate through a Pdgfr-dependent mechanism.

Hand2 alters Fn1 fibril assembly and organization

As H2CKO epicardial defects appear direct, we looked at the impact of a loss of Hand2 upon epicardial mesothelium integrity. Fn1 is downregulated in zebrafish hand2 mutants ³⁷. Fn1 fibril assembly regulates the organization and stability of ECM proteins, is capable of promoting EMT and adhesion-dependent growth, and is associated with integrin-mediated cell signaling ³⁸–40. Additionally, defects in Fn1 underlie integrin-mediated valve leaflet defects in the lymphatic system ⁴¹. Immunohistochemistry shows that Fn1 is expressed in the E12.5 epicardium and is neatly organized around the developing coronaries (Fig. 7A). H2CKOs retain epicardial Fn1; however, it appears disorganized (Fig. 7B). To look at Fn1 organization more closely, we compared EPCs from wild type and H2CKOs (Fig. 7C-F). Immunohistochemistry reveals that Fn1 fibrils form an organized lattice in wild type EPCs. Fn1 deposition appears abnormally uniform and sheet-like throughout the H2CKO explant (Fig. 7C-F). Fn1 dysfunction suggests a role for Hand2 in ECM assembly and epicardium homeostasis. Increased Alcian Blue staining indicates that ECM organization and/or deposition is altered in the H2CKO epicardium (Fig. 7G, H).

Figure 7

Abnormal Fn1 matrix assembly and function results in ECM related defects and impaired epicardial development. Immunohistochemistry at E12.5 for Fn1 (green) and F-actin (red) showing Fn1 expression in the epicardium and absence of patent coronary vessels (A, B). Primary epicardial cells isolated from H2CKOs show a disorganized Fn1 localization, where Fn1 appears to be distributed uniformly throughout the cells, further suggesting migratory defects in H2CKO epicardial cells (D, F). In contrast, wild type primary epicardial cells show Fn1 distributed in a series of well-organized bundles (C, E). An increase in Alcian Blue staining within the epicardium indicates increased deposition of ECM (G, H). Gene ontology analysis of microarray data from primary epicardial cells showing affected gene pathways within H2CKO isolated epicardium (I). qRT-PCR on wild type (blue bar) and H2CKO (black bar) RNA isolated from primary epicardial cells; n=4 (J). Cell proliferation (K) and cell apoptosis (L) on wild type and H2CKO embryos at E12.5; n=3. Asterisks indicate statistical significance: *p < 0.05; **p <0.01.

Gene ontology from microarray analysis to identify enriched biological processes indicates enrichment in ECM based processes, such as cell-cell signaling, assembly, connective tissue development, and motility (Fig. 7I). qRT-PCR on wild type and H2CKO EPCs detected no changes in Fn1 expression, as has been reported in mice (Fig. 7J) ⁴².

In addition to its role as an ECM component, Fn1 promotes intracellular signaling via interactions with cell surface integrins ⁴³. To see if the observed H2CKO Fn1 disorganization may alter cell signaling, we assessed integrin expression by qRT-PCR (Fig. 7J). Indeed, expression of the Fn1 receptor, Itga4, is significantly upregulated within H2CKO EPCs. Itgα4 and Itgβ1 together form an Fn1 receptor pair. Itgα4 influences epicardial Fn1 polymerization. Itgα4 overexpression impairs incorporation of new Fn1 into preexisting polymer structures ⁴⁴, while Itgα4 deletion causes embryonically lethal epicardial defects in mice ⁴⁵, 46. As Itgα4 and Fn organization are essential for epicardiogenesis, the observed dysregulation of these ECM components suggests that Hand2 plays an important role in maintaining a normal epicardial ECM environment.

Gene ontology data indicates enrichment for cell cycle regulation genes. Loss-of-function of Hand2 has been implicated in apoptosis ²⁰. Moreover, ECM disorganization and deposition are thought to be pro-apoptotic ⁴⁷. Activated-Caspase3 and Phospho-Histone H3 immunohistochemistry at E12.5 to assess H2CKO epicardial apoptosis and proliferation (Fig. 7K, L; Online Fig. VIIA-D) reveals no change in proliferation, but a significant increase in epicardial apoptosis, These data suggest that the process of epicardial EMT is not affected in H2CKO as EMT is linked with proliferation ⁴⁸, but the impaired function of the ECM in H2CKOs maybe tied both to the function, integrity, and survival of the epicardium.

Discussion

Collectively, these data reveal a novel and essential function for Hand factors in epicardiogenesis (Online Fig. VIIIA-B). Transient ST expression of Hand1 marks cells that successively populate the PE, epicardium and their derivatives. Hand1 ST expression temporally precedes Hand2 PE expression. Hand2 ablation causes significant epicardial defects including directly impaired Pdgfrα regulation, abnormal differentiation and Fn1 organization, and Itga4 upregulation. Also evident in H2CKOs is increased epicardial apoptosis, potentially reflecting altered Fn-mediated signaling and/or cell migration. Fn1 and Itgα4, have been associated with similar mesodermal, epicardial, and cardiovascular decline ⁴⁵, 46, 49. Epicardiogenesis initiates normally in Itga4 ^{mice; however, the epicardium subsequently detaches from the myocardium and degrades 45}, 46. Itga4 overexpression alters Fn/integrins interactions and disrupts Fn deposition, demonstrating that integrin levels significantly impact Fn organization and function ⁴⁴. Our observations suggest that Hand2 is necessary to maintain the balance between this receptor/ligand pair. Ultimately, Hand2 ablation leads to compromised epicardial function and a failure to form a patent coronary vasculature, both phenotypes observed in other mouse models presenting coronary malformations ⁵⁰.

Fn1 is a multifunctional ECM protein that establishes cytoskeletal organization, motility, and cell signaling pathways required for proliferation and growth. As epicardial cells must migrate, alter morphology, and differentiate into functional cell types, the epicardial phenotypes observed in H2CKOs mechanistically fit a model of altered Fn1 function. Defects in Fn1 deposition are associated with increased fibrosis and apoptosis ⁵¹, all characteristic of the H2CKO epicardium. Fn1 mRNA expression is not altered within H2CKO epicardium, demonstrating that Hand2 regulation of Fn1 is indirect. Zebrafish hand regulates fn1 deposition and influences ECM deposition during lateral mesoderm remodeling ³⁷. Consistent with our data, hand regulates fn1 indirectly through ECM organization, rather than by directly regulating gene expression ⁵². These observations suggest a critical, evolutionarily conserved role for Hand2 during maturation of epicardial-derivatives.

Previous studies suggest that Hand factors have partially overlapping expression domains and are functionally redundant during cardiac patterning ²⁵. Detailed Hand1 and Hand2 expression profiles show less spatiotemporal myocardial overlap than previously indicated. Hand2 deletion within the Hand1 expression domain does not cause significant myocardial patterning or differentiation defects (Online Fig. III), suggesting that the H2CKO myocardial phenotypes are non-cell autonomous. Hand2 deletion using cardiac-specific Cre-drivers causes SHF defects and early embryonic lethality ²³. To completely rule out cell autonomous Hand2 function within PHF-derived cardiomyocytes, PHF-specific Hand2 deletion would be required.

WT1^- and Hand1^Cre-mediated Hand2 deletions produce epicardial phenocopies, indicating a novel Hand2 function during epicardial development. While the defects in the epicardium appear direct, WT1 is expressed in lateral mesoderm derivatives in addition to the epicardium ⁵⁰. Lineage analysis following E9.5 WT1^ERT2Cre induction detects no cardiomyocyte expression (Online Fig. V). Hand2 deletion using a lateral mesoderm and extraembryonic tissue-specific HoxB6^Cre23, which, as Hand2 is not detected within extraembryonic tissues ¹⁸, effectively generates a lateral mesoderm-specific Hand2 deletion, results in viable embryos ²³. Hand2 function in the lateral mesoderm is therefore not critical to embryonic survival, and we conclude that PE/epicardium-specific Hand2 ablation disrupts coronary vasculature maturation and thus contributes to H2CKO lethality.

The Hand1-lineage does not contribute to coronary vascular endothelium ²⁶. Current understanding identifies the origin of the coronary vessel endothelium as the sinus venosus ⁹, and presumes that the functional epicardium interacts with the coronary endothelium to both establish and maintain coronary vessel patency. H2CKOs display epicardial lineage-specific defects that impact both fibroblast and smooth muscle cell fates. No Hand1-lineage independent cells are detectable in the epicardium (Online Fig. I) ²⁶. Hand2 ISH similarly marks the entire epicardium, suggesting that Hand2 is not enriched within a subset of epicardial cells. Pdgfrs govern specific lineage subsets during epicardial development ³⁵. Pdgfrα is downregulated and, conversely, Pdgfrβ is upregulated within the H2CKO epicardium. Luciferase transactivation data suggests direct Pdgfrα regulation (Online Fig. VII,I). Although Pdgfrα’s epicardial function is unknown, it is essential in other EMT-derived cell populations ³⁶. This altered ratio could reflect a Pdgfrβ compensation for decreased Pdgfrα expression; however, Pdgfrβ drives epicardially-derived cell differentiation to smooth muscle cell ³⁵. As the epicardium gives rise to both fibroblast and coronary smooth muscle, and Postn is expressed in cardiac fibroblasts invading the myocardium, (Fig. 6), Pdgfrα may govern the fibroblast cell fate ⁵³. Hand2 appears to modulate the Pdgfr ratios that govern these divergent cell programs. Recently, it has been shown that Hand2 overexpression causes a significant increase in fibroblast marker expression, further implicating Hand2 in the cardiac fibroblast cell fate program ⁵⁴.

Hand1^Cre/+;Hand2^fx/− mutants also display cardiac OFT defects. Wnt1-Cre-mediated NCC-specific Hand2 deletion results in aortic arch defects, DORV, and associated VSDs ²², 23. As Hand1^Cre– mediated cNCC Hand2 ablation is temporally later and spatially more restricted then Hand2^{Δ but entails heterozygosity of} Hand1, the high penetrance of a more severe NCC-dependent PTA phenotype suggests that genetic and, possibly, functional interactions between Hand1 and Hand2 are critical for OFT septation. Established genetic interactions between Hand1 and Hand2 support this model ²⁴. It is also consistent with established dimer regulation mechanisms governing the biological output of Twist-family bHLH factors. Indeed, dysregulation of Twist1 dimerization causes the human disease Saethre Chotzen Syndrome and directly reflects molecular antagonism between Twist1 and Hand2 ⁵⁵. Thus, Hand1 and Hand2 dimer choice may prove crucial to OFT morphogenesis. Experiments to explore this possibility mechanistically are currently underway.

Our data defines unique Hand1 and Hand2 expression domains within the developing murine heart. Hand1 is largely restricted the left ventricle. SHF expression is restricted to the myocardial cuff ²⁶, and is the sole domain of co-expression with Hand2. At the onset of cardiac looping, Hand1 expression/lineage is detectable within the forming left ventricle. Although we observe a thin compact zone and hypertrabeculation within H2CKOs, the cell autonomy of these defects cannot be deduced. Cardiac-specific Hand2 ablation causes SHF defects and early embryonic death ²³. Although Hand1^Cre mice allow insight into a possible role for Hand2 in the PFH myocardium, a PHF-specific Cre will be required to address this directly. In summary, these studies demonstrate that, in addition to its established functions within the cNCC and myocardium, Hand2, modulates cell signaling mechanisms that dictate epicardial cell fates and ECM organization, thus playing a novel and critical role in the function and differentiation of the epicardium and, consequently, proper cardiac function.

Supplementary Material

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Acknowledgments