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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Dev Dyn. 2014 Dec 9;244(3):457–467. doi: 10.1002/dvdy.24233

Persistent Noggin arrests cardiomyocyte morphogenesis and results in early in utero lethality

Olga Simmons 1, Paige Snider 1, Jain Wang 1, Robert J Schwartz 2, YiPing Chen 3, Simon J Conway 1,#
PMCID: PMC4344892  NIHMSID: NIHMS645319  PMID: 25428115

Abstract

Background

Multiple BMP genes are expressed in the developing heart from the initiation to late-differentiation stages, and play pivotal roles in cardiovascular development. In this study, we investigated the requirement of BMP activity in heart development by transgenic over-expression of extracellular BMP antagonist Noggin.

Results

Using Nkx2.5-Cre to drive lineage-restricted Noggin within cardiomyocyte progenitors, we show persistent Noggin arrests cardiac development at the linear heart stage. This is coupled with a significantly reduced cell proliferation rate, subsequent cardiomyocyte programmed cell death and reduction of downstream intracellular pSMAD1/5/8 expression. Noggin mutants exhibit reduced heartbeat which likely results in subsequent fully penetrant in utero lethality. Significantly, confocal and electron micrographic examination revealed considerably fewer contractile elements, as well as a lack of maturation of actin-myosin microfilaments. Molecular analysis demonstrated that ectopic Noggin-expressing regions in the early heart’s pacemaker region, failed to express the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4), resulting in an overall decrease in Hcn4 levels.

Conclusions

Combined, our results reveal a novel role for BMP signaling in the progression of heart development from the tubular heart stage to the looped stage via regulation of proliferation and promotion of maturation of the in utero heart’s contractile apparatus and pacemaker.

Keywords: mouse embryo, transgenic overexpression, Noggin, BMP, cardiomyocyte, congenital heart defects, proliferation, contractile apparatus, bradycardia

Introduction

Regulated cardiomyocyte proliferation and differentiation are indispensable for normal cardiac development and embryo growth. Equally important, the initiation and maintenance of a spontaneous heartbeat is essential for normal in utero development and viability (Conway et al., 2003; Monfredi et al., 2013). Moreover, the mammalian heart is the first organ to form in the mammalian embryo and functions even before it is fully developed (Koushik et al., 2001). In mice, spontaneous contractions initiate early in embryogenesis at the 3-somite stage, a detectable heartbeat is present at the 5-somite stage, and vascular blood flow is observed at the 7-somite stage (Ji et al., 2003; Nishii and Shibata, 2006). Thus, by embryonic (E) day 8, the cardiomyocytes contain a full set of proteins required for contraction (Nishii and Shibata, 2006), with structural development of myofibrillar transverse striation enabling macroscopic changes of the cardiomyocyte's shape (Navaratnam et al., 1986). During embryonic and fetal development, multiple regulatory pathways control differential rates of cardiomyocyte proliferation necessary for proper cardiac chamber morphogenesis and function (Sedmera and Thompson, 2011).

The Bone Morphogenetic Protein (BMP) family of Transforming Growth Factorβ (TGFβ) molecules represents one class of cell–cell signaling molecules that plays a critical role in myocardial differentiation (Schultheiss et al., 1997; Chen et al., 2004a; Song et al., 2007). At least six BMP ligands (Bmp2, Bmp4, Bmp5, Bmp6, Bmp7, Bmp10), three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (Noggin) are all expressed during the initial steps of cardiac organogenesis (Chen et al., 2004a; Danesh et al., 2009). Regulatory R-SMADs (SMAD1/5/8 for BMPs) are activated upon phosphorylation by specific receptors, and associate with Smad4 to trigger transcriptional responses and drive differentiation (reviewed by Massague, 1998; Qi et al., 2007; Beyer et al., 2013). In addition, inhibitory SMADs negatively regulate signaling (Smad6 for BMP and Smad7 for both BMP/TGFβ) and several antagonists (including Noggin) can inhibit BMP signaling (Song et al., 2011; Beyer et al., 2013).

Previous studies have shown that Noggin is transiently expressed within the E8.75-10 mouse heart in the heart-forming region and is thought to act at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis (Danesh et al., 2009). Moreover, genetic deletion of Noggin results in perinatal lethality and numerous congenital defects including the somites, neural tube and skeleton (Brunet et al., 1998). Specifically, the loss-of-function of Noggin results in heart abnormalities in the cardiomyocyte and endocardial cushion lineages, namely increased cell numbers and thickened myocardial wall defects (Choi et al., 2007). Although the distinct cellular mechanism causing the thickened myocardium remains unknown, it was shown that reducing Bmp4 expression levels within Noggin nulls alleviated the cardiac defects (Choi et al., 2007). In order to further understand the cardiovascular effects of Noggin deregulation and to begin to address the potential lineage-specific gain-of-function effects of persistent ectopic Noggin expression in the cardiomyocytes, we generated binary mutant embryos that continue to express Noggin within the heart. Growth retarded Noggin transgenic embryos exhibit small unlooped hearts and bradycardia, which ultimately leads to fully penetrate in utero lethality by E12. Significantly, both confocal and electron micrographic examination revealed a dysfunctional contractile apparatus and absent Hcn4 expression within the dorsal aspect of mutant primitive pacemaker region. Taken together, these results indicate that a distinct level of BMP activity is necessary for cardiomyocyte proliferation and differentiation, and that suppression of BMP signaling results in loss of Hcn4 expression in the developing heart.

Results

Persistent Noggin expression results in bradycardia and early in utero lethality

To assess the effects of persistent Noggin expression on cardiovascular morphogenesis and function, we crossed Nkx2.5Cre knockin Cre recombinase delete mice (Moses et al., 2001) to pMes-Noggin transgenic mice (Xiong at al., 2009) that have the mouse Noggin coding sequence cloned in front of the IRES-Egfp sequence under the control of the chick β-actin promoter, with a floxed STOP cassette inserted between the β-actin promoter and the Noggin cDNA. As Noggin is a known secreted extracellular BMP antagonist (McMahon et al., 1998; Liem et al., 2000) and Nkx2.5Cre initiates Cre expression around E7.5 in the cardiac progenitor pool (Moses et al., 2001). Nkx2.5Cre is thus expressed within cardiomyocytes, the endocardium, dorsal mesocardium, pericardial mesoderm, the epithelium of the first pharyngeal arch and second heart field (Moses et al., 2001; Stanley et al., 2002; Ma et al., 2008). Thus, over-expressing Noggin in Nkx2.5-Cre cells will suppress BMP signaling during early cardiac development throughout the heart.

These matings did not result in the birth of binary pMes-Noggin/Nkx2.5Cre over-expressor transgenic mutant mice (hereafter referred to as Nogoep mutants to distinguish them from published loss-of-function nulls), although other genotypes were present at Mendelian ratios and demonstrated no phenotypic abnormalities (n=4 litters). Genotyping of embryos during gestation allowed recovery of mutant embryos at the expected ratios between E8 and 11.5; however, no viable Nogoep mutant embryos were observed past E12.5 (n=4 litters). At E11 and 10, the Nogoep embryos were discernible grossly by their pale appearance, smaller size, and particularly by a lack of blood-filled vessels in the mutant yolk sac (Fig. 1A,B). PECAM-1 immunostaining revealed that despite some Nkx2.5Cre expression within the yolk sac vasculature (Stanley et al., 2002), that Nogoep yolk sac endothelial cells are present in empty mutant vessels. Significantly, the hearts of the E9.5 and 10 controls (wildtype, Nkx2.5Cre only or pMes-Noggin only) exhibited a rhythmic contraction but Nogoep mutants hearts had a much slower rate than control littermates (n=3 litters; controls= 113+/−14 beats/min; Nogoep = 31+/−8). The Nogoep mutant hearts were dysplastic and significantly undersized (Fig. 1B,G,I) when compared to age-matched litter mate controls. Moreover, the Nogoep embryos exhibited reduced/absent pharyngeal arches. Looking earlier, there was a significant difference in heart morphology between E9 controls and Nogoep mutants (Fig. 1C–E). Nogoep hearts did not contain an apparent right ventricle and cardiac looping (normally required to form a four-chambered heart) was arrested at the linear heart tube stage (Fig. 1C,E; n=12/15 mutants) or the Nogoep heart was looped abnormally leftwards (Fig. 1C; n=3/15 mutants). Histological analysis confirmed defects in the mutant hearts, including tissue degradation in both the outflow tract and sinus venous left and right mesocardial reflections (Fig. 1G,I). Thus, E8.5 and E9 mutant embryos exhibit negligible growth retardation but abnormal hearts, as growth retardation appears a day later in E9.5 mutant embryos and only becomes obvious at E10 and older stages. However, as somites continue to be added in both E9.5 and E10 mutants (Fig. 1B) and mutant primitive forebrain vesicle is subdivided into a separate third ventricle and two telencephalic vesicles similar to that of E10 control heads, our phenotype cannot be classed as wholesale developmental arrest but rather specific prevention of cardiac morphogenesis.

Fig. 1.

Fig. 1

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Phenotype of Nogoep mutant embryos. A: E11 control (left) and mutant (right) whole embryo littermates with yolk sac intact. Note a distinct lack of blood-filled vessels in mutant yolk sac as mutant has no heartbeat. Inserts are low power views of the blood-filled control (left) and empty mutant (right) yolk sacs immunostained with endothelial marker PECAM-1. B: Lateral right view of E10 control (left; 29 somites and mutant (right; 23 somites) embryos with yolk sac removed. Whilst the control has four distinct arches (numbered 1–4), the mutant arches are hypoplastic and its heart is undersized. C: Frontal view of an E9 control (left; 15 somites) and two mutants (right; 17 and 14 somites). The control presumptive looped left and right ventricles are easily identifiable but the mutant hearts are either unlooped (linear, middle embryo) or have looped abnormally (right embryo). D: Lateral right view of embryos in C, emphasizing lack of right ventricle (red arrow) and small arches. E: E8.5 control (left; 10 somites) and mutant (right; 12 somites) whole embryos illustrating that abnormal heart looping of Nogoep mutants occurs early in morphogenesis. F,G: H&E stained serial transverse sections of E9.5 control (F) and mutant (G) littermates revealing tissue degradation in mutant outflow tract (oft) and sinus venous regions (arrows). H,I: Higher magnification of control (H) and mutant (I) sections from F,G. Scale bars: B–E=100μm, H,I=20μm. Abbreviations: a, atria; c, endocardial cushion; h, heart; lv, left ventricle; sp, septum transversum; v, ventricle.

Lineage mapping ectopic Noggin expression

As histology demonstrated that overexpression of Noggin at the heart initiation stage results in small unlooped hearts, an absence of the normal four pharyngeal arches, and fully penetrant in utero lethality; we placed pMes-Noggin/Nkx2.5Cre mice onto the ROSA26r reporter background (Soriano, 1999) to follow the fate of the Noggin overexpressing lineage and determine whether BMP suppression resulted in abnormalities in lineage specification as well as morphogenesis. Significantly, lacZ staining is still present within E11.5 Nogoep hearts (Fig. 2B) and yolk sac vasculature (Fig. 2B insert). E9.5 and 8.5 Nogoep cardiomyocytes, as well as the epithelium of the pharyngeal arches, similarly retain lacZ expressing cells derived from the Nkx2.5Cre Cre progenitors (Fig. 2D,E). As previously reported, lacZ staining can be clearly seen in all four distinct arches of the control embryos as well as all cardiomyocytes and SHF of the four chambered, properly looped heart (Fig. 2C). Although the mutant cardiomyocytes also stain with X-gal, the E9.5 Nogoep heart is visibly smaller, remains unlooped, and contains only a single ventricle (Fig. 2D,E). Consistent with the gross defects observed, histology confirmed that the Nogoep mutant right ventricle was absent (Fig. 2F, lower panel), but that Noggin-expressing progenitors can contribute to the E9.5 cardiomyocyte lineage, both within the outflow and sinus venous pole regions, and persist until subsequent ~E12 embryo lethality.

Fig. 2.

Fig. 2

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R26r reporter analysis of Noggin overexpressing lineages. A,B: Right lateral view of X-gal stained E11.5 control (A) and Nogoep mutant (B) littermates, illustrating Nkx2.5Cre–driven Noggin expression within the cardiac progenitors/second heart field (SHF) results in severely growth retarded embryos, small heart (red arrow in B) and absent pharyngeal arches (B). Inserts are low power views of the X-gal stained control (A) and mutant (B) yolk sacs, indicating that Nkx2.5Cre derivative Noggin-expressing yolk sac vasculature persists in E11.5 mutants. C,D: Xgal stained E9.5 control (C; 24 somites) with lacZ evident in the first four arches and cardiomyocytes, but only the first arch and undersized heart are present in the mutant (D; 21 somites). E: E8.5 control (left; 9 somites) and mutant Xgal stained for lacZ (right; 9 somites) revealing the still linear heart tube in the mutant as opposed to a correctly looped control heart. F: E9.5 control (upper series) and mutant (lower series) serial sections X-gal stained and counterstained with eosin. Note, mutant abnormal outflow tract and absent right ventricle, and that Nkx2.5Cre–expression is present throughout the control and remaining mutant cardiomyocyte, endothelial and most of the SHF lineages (but there are some absences of lacZ-positive cells in the Nogoep pharyngeal arch; red arrow). Scale bars: A,B=200μm, C–E=100μm, F=50μm. Abbreviations: fore, foregut; nt, neural tube, sv, sinus venosus.

Molecular characterization of Nogoep phenotype

As endogenous Noggin is transiently expressed in the heart-forming region during gastrulation (Danesh et al., 2009), we used non-radioactive in situ hybridization analysis to evaluate the spatiotemporal extent of Noggin mRNA up-regulation following loxP/Cre recombination in binary transgenic Nogoep mutants from E8-9.5. As previously described (Danesh et al., 2009), endogenous Noggin is expressed in the E8 control notochord, but very little Noggin is observed in control embryo hearts (Fig. 3A). In contrast, robust Noggin expression is present within the E8 Nogoep heart, particularly the cardiomyocytes (Fig. 3B). Likewise, endogenous Noggin expression is absent in E9.5 control hearts (Fig. 3C), but ectopic transgenic Noggin mRNA expression is still seen in E9.5 littermate mutant cardiomyocytes and in the endocardium (Fig. 3D,E). Thus, in Nogoep mutants ectopic Noggin is consistently induced and is confined to the Nkx2.5Cre lineages. To determine when and to what extent BMP signaling is suppressed, we used immunohistochemistry to analyze phospho-SMAD1/5/8 expression, the downstream effector of BMP ligand signaling. Significantly, phospho-SMAD1/5/8 expression is reduced in both the E9 Nogoep pharyngeal arches (Fig. 3H) and cardiomyocytes (Fig. 3H,I) when compared to littermate controls (Fig. 3F,G). Although phospho-SMAD1/5/8 signaling is high in E10 control hearts, it remains suppressed in dysplastic E10 Nogoep hearts (Fig. 3K). Interestingly, the continuous phospho-SMAD1/5/8 suppression observed in cardiomyocytes does not appear to be present within Nogoep endothelium (Fig. 3I,K), within either the inflow, AVC or OFT. Combined, these data indicate that Nkx2.5Cre-mediated Noggin overexpression continually suppresses SMAD-dependent BMP signaling in only Cre-positive cardiomyocytes and pharyngeal arches.

Fig. 3.

Fig. 3

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Molecular analysis of Nogoep phenotypes. A–E: Non-radioactive in situ hybridization was used to verify Noggin induction and continued expression. Using an anti-Noggin DIG-labelled probe, endogenous Noggin is expressed in the early E8 control (A) and mutant (B) notochord. Very low level endogenous Noggin expression is detected in the E8 control heart (A) while transgenic Noggin is highly induced in the E8.0 mutant heart (B). Similarly, Noggin is not detectable in E9.5 control hearts (C; 20 somites), but Nogoep mutants continue to express robust Noggin mRNA (D,E; 22 somites) within the cardiomyocytes and endocardium (red arrowheads in E). F–K: Immunohistochemistry confirms that E9.0 control heart (F,G) expresses phospho-SMAD1/5/8 (a downstream effector of BMP ligand signaling) in cells within the arches and in cardiomyocytes, but pSMAD1/5/8 is diminished in cells in the mutant arches (H arrow) and within cardiomyocytes (H and I arrow) that express ectopic Noggin, when compared to littermate controls. Moreover, BMP signaling remains suppressed in E10 mutants (K) when compared with littermate controls (J). Note pSMAD1/5/8 levels are specifically diminished in mutant cardiomyocytes wherein Noggin is over-expressed (* in K), but remains relatively unchanged in the endothelium (arrows in I,K). Scale bars: A–D=20μm. Abbreviations: h, heart; n, notochord.

Analysis of Nogoep mutant growth and development

Since Nogoep mutant hearts are so much smaller than control littermates, we counted the total number of cells within H&E stained histological serial sections of both the control and mutant hearts at E8.5, 9 and 9.5 embryonic stages (Fig. 4A). As expected given the absence of any gross morphological anomalies, at E8.5 there is no significant difference in cell numbers between Nogoep mutants and controls. However, by E9.0, there is a drastic (~50%) reduction in total number of cells within the mutant heart. Moreover, analysis at E9.5, revealed Nogoep cell counts were reduced by ~75% compared to control littermates (n=3 for each age group and genotype). In order to determine if the reduction in Nogoep cell number was a result of decreased proliferation, increased apoptosis, or both we used phospho-histone H3 (pHH3) immunostaining and TUNEL analysis. Interestingly, proliferation within Nogoep mutants was not completely inhibited coincident with BMP suppression and the number of cells did continue to increase during development, although at a much reduced rate compared to control littermates. Meaningfully, pHH3 staining demonstrated that there was no difference in proliferation rates in E9 tissues where Noggin is not overexpressed (neural tube and somites), but in the Nogoep heart itself, proliferation was decreased ~50% in mutants (Fig. 4B). Although TUNEL did detect normal equivalent apoptotic cells in both the control and Nogoep pharyngeal arches (Fig. 4C,D), TUNEL did not detect wholesale apoptosis in the E9.5 control nor Nogoep cardiomyocytes, but did detect isolated apoptotic cells in Nogoep ventricles (Fig. 4D,F). Combined, the pHH3 and TUNEL data suggest that sustained ectopic Noggin within the cardiomyocyte lineage principally results in a smaller Nogoep heart size due to reduced cell proliferation rate, as well as subsequent elevated programmed cell death in isolated Nogoep cardiomyocytes.

Fig. 4.

Fig. 4

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Analysis of Nogoep mutant growth and development. A: Comparison of total number of cells within control (black) and mutant (red) hearts at E8.5, E9.0, and E9.5. At E8.5 there is no significant difference in cell numbers, but starting E9, the total number of cells in mutant hearts dramatically decreases by ~50%, compared to control littermates. By E9.5, the mutant heart contains ~1/3 the cells present in control hearts. Error bars represent SEM. B: pHH3 analysis of cell proliferation revealed that there is a specific decrease in cell proliferation within the E9.5 mutant hearts, but that cell proliferation rates are unaffected within the rest of the mutant embryos compared to control littermates. Statistical significance of observed differences was determined by Student's t test, where * denotes p_0.05. C–F: TUNEL analysis revealed Nogoep mutants exhibit elevated levels of apoptosis within the mutant hearts but that similar levels are present within non-Nkx2.5Cre lineages. Note that the E9.5 mutant (23 somites) contains several apoptotic cells in the common ventricle (arrows, F), that is not present in control littermate (26 somites). Scale bars: C,D=20μm; E,F=5μm.

Characterization of the Nogoep contractile apparatus and cardiac function regulation

To understand the basis for the Nogoep embryo bradycardia and cardiac failure leading to in utero lethality, we analyzed myofibril genes important for early cardiac contraction and generation of the heartbeat. As αSmooth muscle actin (αSMA) is one of the first and transiently expressed microfilament proteins in the embryonic heart (Clément et al., 2007) we examined its expression first. Significantly, αSMA was grossly unaltered in Nogoep mutant heart (Fig. 5B) and was not ectopically expressed. Similarly, sarcomeric myosin heavy chain expression (revealed by immunostaining with cardiomyocyte-specific MF20 antibody) was comparable in E9.5 and E10 Nogoep and control hearts (Fig. 5C–F), indicating that neither microfilament nor myosin heavy chain expression is perturbed via ectopic Noggin expression. To examine the contractile apparatus detail comprehensively, we used confocal analysis of F-actin (linear filament expression) and αActinin (localized to the Z-disk) expression in early hearts prior to growth retraction. Although both these cytoskeletal proteins were present in both E8.5 control (Fig. 5G) and mutant (Fig. 5H) cardiomyocytes, there was measurably less contractile fibrils within Nogoep mutant cardiomyocytes. Transmission electron microscopy confirmed E9 Nogoep cardiomyocytes (within remaining left ventricle/linear heart tube) contain fewer sarcomeres (as detected via reduced numbers of Z-lines), as well as abnormal myofibril alignment within the mutants (Fig. 5K,L) compared to control littermates (Fig. 5I,J). Analysis of Nogoep mitochondria numbers and appearance (i.e. abnormal swelling could be indicative of altered myocardial bioenergetics; Cao and Chen, 2009) did not reveal any differences compared to controls. These data suggest that Nogoep myofibrillar maturation and expansion of the contractile apparatus may be compromised via BMP suppression resulting in contractile dysfunction.

Fig. 5.

Fig. 5

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Characterization of Nogoep contractile apparatus. A,B: Immunohistochemical detection of _Smooth muscle actin expression in control (A) and mutant (B) E10 embryos shows that the mutant hearts correctly express αSMA. C–F: Similarly, E9.5 and E10 mutant hearts appropriately express the cardiomyocyte-specific MF20, indicating that myosin heavy chain expression is also unaffected in mutant cardiomyocytes despite poor cardiac function and dysmorphology (E,F). G,H: Confocal immunofluorescent imaging of F-actin (red), αActinin (green) with DAPI nuclear staining (blue), reveals that while these cytoskeletal proteins are present within both control (G) and mutant (H), there are far fewer mature contractile fibrils within the E8.5 mutant cardiomyocytes (red arrow) when compared to littermate controls. I–L: Electron microscopy of E9 control (I,J; 11 somites) and mutant (K,L; 12 somites) cardiomyocytes, confirms reduced presence of sarcomeres and improper myofibral alignment in mutant hearts. In higher magnification views, it is evident that the mutant cardiomyocytes contain a decreased number of Z-lines (arrows) compared to control littermate. M,N: Radioactive in situ hybridization detection of Tbx5 expression in E9 control (M; 16 somites) and mutant (N; 16 somites) embryos. Note, that the Tbx5 cardiac chamber progenitor marker is highly expressed in both normal and mutant cardiomyocytes, and is particularly robustly expressed within the dorsal and ventral sinus venous/atrial regions (early pacemaker). O,P: Radioactive in situ hybridization detection of Hcn4 expression in control (O; 16 somites) and mutants (P; 16 somites) revealed Hcn4 is down-regulated specifically in the dorsal aspect of the cardiac inflow in the mutant but persists in the ventral aspect of the ventral sinus venous/atrial region (arrow in P), compared to normal bilateral Hcn4 in control hearts (O). Q: Nkx2.5Cre lineage mapping at E9.5 (19 somites) confirms that the ventral aspect of the primitive pacemaker region (arrow) does not express Nkx2.5Cre (i.e no blue lacZ cells) and hence is not able to drive ectopic Noggin expression. Scale bars: A,B=10μm; I–L=500nm. Abbreviations: a, atria; oft, outflow tract; v, ventricle.

Because Nogoep mutants exhibit tissue degradation (Fig. 1) at both ends of the heart, including the developing sinus venosus myocardium/SAN region, we addressed the possibility that ectopic Noggin and suppression of BMP signaling may lead to early pacemaking and conduction deficiencies in Nogoep embryos. Using in situ hybridization we examined Tbx5 and Hcn4 mRNA expression in E9 Nogoep and control littermates. Tbx5 (T-box transcription factor 5) whose physical interaction with Nkx2.5 is known to control gene expression in cells of the cardiac conduction system (McCulley and Black, 2012) was highly expressed within both control (Fig. 5M) and mutant (Fig. 5N) cardiomyocytes within the sinus venous/atrial region and the ventricle. However, Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) which is a marker of the cardiac conduction system and whose expression pattern partially overlaps with Tbx5 and Nkx2.5 (Espinoza-Lewis et al., 2009; Später et al., 2013) was absent in the dorsal aspect of the cardiac inflow (ectopic Noggin-positive) region of the mutants (Fig. 5P) when compared to controls (Fig. 5O), while persisting in the ventral aspect that does not express Nkx2.5Cre (ectopic Noggin-negative region). Correspondingly, Nkx2.5Cre-driven Cre expression (and hence ectopic Noggin induction) is only present in the dorsal aspect of the cardiac inflow and is absent from the ventral aspect of the pacemaker region (i.e. no blue lacZ cells) and therefore cannot drive ectopic Noggin (Fig. 5Q). Thus, not only have we shown that the upstream regulator Tbx5 is equivalently expressed (i.e. sino tissue present, is correctly specified and has intact mRNA), we also demonstrate that Hnc4 is only absent within the Cre-positive pacemaker tissues. Collectively, the overall decrease in total cardiac Hcn4 mRNA levels within the mutant hearts could account for the observed bradycardia, and along with the reduced number of cardiomyocytes and delayed maturation of the contractile apparatus, may contribute to the poor Nogoep in utero cardiac function and eventual lethality.

Discussion

Binary pMes-Noggin/Nkx2.5Cre mice were generated in order to determine effects of persistent Noggin and suppressed BMP function in embryonic cardiomyocytes during the early stages of heart chamber formation. Although Noggin is transiently expressed within the E8.75-10 mouse heart and is thought to establish conditions conducive to cardiogenesis (Danesh et al., 2009), the consequences of deregulated Noggin during early cardiovascular development are unclear. The major cardiac phenotypic consequences of persistent Noggin is that Nogoep mutants exhibit smaller hearts that fail to complete looping and are bradycardic. Mutants also have fewer contractile elements and lack of maturation of the actin-myosin microfilaments. Although Bmp4 is expressed throughout the early cardiac crescent and Bmp4 null hearts exhibit similar looping abnormalities and dysmorphic hearts (Conway et al., 2003), cTnTCre-mediated conditional deletion of Bmp4 does not affect heart size until fetal stages (Jiao et al., 2003). Similarly, myocardial expressed Bmp7 null mice are postnatal lethal (Jena et al., 1997) whilst trabecular Bmp10 knockouts die E10 with a hypoplastic myocardium (Chen et al., 2004b). Thus, suppression of BMP signaling can have wide ranging effects upon cardiomyocyte lineage, depending upon when and where BMP signaling is suppressed. As Noggin is a secreted extracellular BMP antagonist (McMahon et al., 1998; Liem et al., 2000) and Nkx2.5Cre initiates Cre expression in the E7.5 cardiac progenitors, as well as endocardium and SHF (Moses et al., 2001; Stanley et al., 2002; Ma et al., 2008), the Nogoep mutants provide a new model in which to assess global BMP signaling suppression throughout early heart development. As multiple BMP ligand and receptor combinations exist, and there is both genetic redundancy and functional compensation that obscures individual knockout phenotypes, the Nogoep mutants represent a unique tool to analyze cardiomyocyte morphogenesis and survival, and ultimately, study how downstream cardiac expression profiles may be altered by unbalanced TGFβ superfamily signaling.

Here we demonstrate that persistent Noggin overexpression in vivo in the primitive heart results in decreased cardiomyocyte cell proliferation with subsequent elevation in apoptosis, resulting in the small heart phenotype. Previous studies reported that Noggin null embryos exhibit a thicker myocardium and increased cell number, due to increased proliferation and decreased cell cycle exit (Choi et al., 2007). Thus, increased or decreased Noggin expression appears to differentially affect cardiomyocyte proliferation during the initial stages of heart chamber maturation. Likewise, we revealed that persistent Noggin suppresses SMAD-mediated BMP signaling and expression of both heart-specific Hcn4 channel and sarcomeric protein maturation. The in vivo Noggin-mediated alteration of contractile apparatus morphogenesis is consistent with in vitro studies using cultured chick precardiac mesoendoderm and exogenous Noggin (Nakajima et al., 2002), in which αSMA expression was unaltered but sarcomeric αactinin, titin, and sarcomeric myosin expression were mis-expressed. As Bmp signaling can directly regulate a miRNA-mediated effector mechanism that down-regulates cardiac progenitor genes and enhances myocardial differentiation (Wang et al., 2010) and Noggin has been recently demonstrated to regulate ameloblast and cartilage differentiation (Cao et al., 2013; Ning et al., 2013); it will be fascinating to examine whether persistent Noggin affects cardiomyocyte proliferation and/or differentiation via miRNA mis-regulation.

One intriguing aspect of the Nogoep phenotype is that Nkx2.5Cre-mediated Noggin overexpression does not affect endocardial phosphorylation of SMAD1/5/8. As continuous phospho-SMAD1/5/8 suppression is observed in cardiomyocytes this suggests that either ectopic endocardial Noggin is not expressed as highly as within Nogoep cardiomyocytes (our in situ data does not provide sufficient quantitative data to determine what amount Noggin is overexpressed in endocardium), that the Nogoep endocardium expresses a higher phospho-SMAD1/5/8 level than adjacent Nogoep cardiomyocytes or that supplementary signaling pathways are able to maintain phospho-SMAD1/5/8 levels. Precisely how this is achieved is presently unclear, but it is recognized that Noggin can exhibit concentration-dependent effects, including inducing downstream targets at low concentrations and repressing those same targets at high concentrations (Marchant et al., 1998). These data could also indicate that blocking BMP signaling may can disrupt/enhance other endocardial signaling pathways. For instance, although Noggin is usually known as a secreted extracellular BMP antagonist (McMahon et al., 1998; Liem et al., 2000), it has been shown that TGFβ can induce phospho-SMAD1/5/8 (Lechleider et al., 1996; Daly et al., 2008), which could drive redundant or cooperative endocardial signaling.

Nogoep mutants’ hearts fail to complete cardiac looping prior to overall embryonic growth delay, with the majority of the mutant hearts remain linear and the rest exhibit looping abnormally to the left. Thus, both aspects of abnormal heart looping, namely failure of heart looping and abnormal direction of heart looping result from the persistent expression of Noggin. Normally, the straight heart tube begins to elongate with simultaneous growth in the bulbus cordis and primitive ventricle, which forces the heart to bend ventrally and rotate to the right, forming a C-shaped loop with convex side situated on the right (Moorman and Christoffels, 2003; Sedmera and Thompson, 2011). Thus, as Nogoep E9 cardiac cell numbers and cell proliferation rates are both reduced, abnormal heart looping is most likely due to insufficient force generation as a consequence of too few new cells. As BMP signaling is known to be important for laterality pathways in the heart (Fujiwara et al., 2002; Smith et al., 2008), it will be important to determine whether there is a laterality defect, whether Nkx2.5Cre-marked SHF contribution is perturbed or whether failed looping is due to a specific BMP-mediated arrest of heart development. As Nogoep mutant looping, bradycardia and cardiomyocyte maturation defects all occur prior to appearance of paler yolk sacs and growth retardation, it is unlikely that these are secondary consequences but rather due to BMP suppression and consequent diminished cardiac output. Additionally, the newly added SHF-derived cardiomyocytes at either pole of the linear heart tube (Sedmera and Thompson, 2011), appear particularly sensitive to persistent Noggin, as tissue integrity is compromised in both the E9.5 Nogoep sinus venous and outflow tract regions. However, as the mutants do not die until E11-12, we think it unlikely that loss of tissue integrity is associated with death of the embryos but rather is a consequence of suppressed BMP signaling affecting homeostasis. Significantly, both Bmp2/4 and pSMAD1/5/8 are highly expressed within the left and right mesocardial reflections (Briggs et al., 2013) and various embryonic myosin’s are sharply expressed to define the cardiomyocyte/dorsal mesocardium boundary (Rutland et al., 2011), at the sites where new cardiomyocytes are added. While we cannot exclude the possibility that poor function may play a potential role within the looping abnormality, our previous data in which the sodium-calcium exchanger Ncx1 is deleted and results in an absent heartbeat and lethality at E10, the Ncx1 null hearts still undergo looping despite any cardiac function (Koushik et al., 2001).

The Nogoep bradycardia phenotype was unexpected, as BMP suppression has not previously been linked with the generation of the heartbeat. Although the precise cause of the bradycardia is unclear, ultra-structural analysis of cardiomyocyte architecture suggest that Nogoep myofibrillar maturation and expansion of the contractile apparatus may be compromised via BMP suppression resulting in contractile dysfunction. These findings indicate that persistent Noggin has no effect on the formation of the thin filament and Z-disk in the embryonic heart, but it disrupts the normal maturation process (Sanger et al., 2000) of the actin-myosin microfilaments from cytoskeletal and cytoplasmic pools of proteins. Moreover, is was surprising that ectopic Noggin-expressing dorsal regions within the conduction system failed to express the Hcn4 pacemaker channel, while persisting in the ventral aspect. Although there are no known links between BMP signaling nor Noggin and Hcn4 induction, the absence of expression from only the dorsal sinus venous (which was still present and normally expressed the upstream Tbx5 transcription factor) suggests a direct interaction. Whether the observed myocyte proliferation defect secondarily alters Nogoep pacemaker morphogenesis is unknown, but precursors of ventricular conduction tissues are known to exit the cell cycle during looping (Sedmera and Thompson, 2011). Thus, bradycardia is likely a probable consequence of a diminished contractile apparatus combined with insufficient pacemaker activity. These data confirm that maintenance of BMP signaling homeostasis is essential for progression of heart development from the tubular to the looped stage, but is also required for preservation of cardiac function.

Our analysis of endogenous Noggin expression is in agreement with Danesh et al. (2009) who reported that Noggin appears at low levels in the myocardium of the heart E8.75-10. However, these data contrast the high level of Noggin expression previously reported in the early developing heart (Yuasa et al., 2005), but all three groups agree that Noggin expression is transient, very low at E9-10 and down-regulated as cardiomyocytes undergo differentiation. Indeed transient inhibition of BMP signaling by Noggin is being used to induce cardiomyocyte differentiation of mouse and human embryonic stem cells (Yuasa et al., 2005: Yamauchi et al., 2010). Although the cardiomyocyte inducer/s and regulators of Noggin expression are presently unidentified and it is unknown whether ectopic or persistent Noggin plays a role in congenital heart defect pathogenesis, these data highlight the essential role active extracellular regulation of BMP signaling plays in cell proliferation of cardiomyocytes, as well as the maturation of both the contractile apparatus and early pacemaker. Patients with bradycardia and Sick sinus syndrome have been shown to have mutations in their HCN4 gene, and our data may suggest that deregulation of BMP signaling may be a possible mechanism that could modify pacemaker morphogenesis and function.

Experimental Procedures

Mouse Models

pMes-Noggin floxed conditional overexpression mouse model (Xiong at al., 2009) was crossed with Nkx2.5Cre knockin mice (Moses et al., 2001) to generate mutant embryos expressing Noggin within the cardiomyocyte and SHF lineages. pMes-Noggin/Nkx2.5Cre mice were placed on R26r indicator background (Soriano, 1999) for lineage mapping and to assess lineage-restricted Cre-mediated induction of Noggin expression. For pregnant females, the day of observed vaginal plug was designated embryonic day 0.5 (E0.5) and mutant embryos E9 and older were also staged via counting somites to control for growth retardation and potential developmental arrest. Yolk sac or tail tissue genomic DNA was used for genotyping, using two sets of primers: for Cre (5’-CATTTGGGCCAGCTAAACAT; 3’-CCCGGCAAAACAGGTAGTTA) and Noggin (5’-CCCCCTGAACCTGAAACATA; 3’-GGCGGATGTGTAGATAGTGCT). All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee of Indiana University School of Medicine.

Histology, X-Gal staining and Immunohistochemistry

Tissue collection, 4% paraformaldehyde fixation, processing, paraffin embedding, whole mount staining for β-galactosidase, and H&E staining were performed using established protocols (Lindsley et al., 2007; Snider et al., 2008, 2009). Collected tissues were sectioned at 6μm thickness. Immunohistochemistry was performed using ABC kit (Vectorstain) with DAB and hydrogen peroxide as chromogens, as described (Olaopa et al., 2011). The following primary antibodies were used: phospho-SMAD1/5/8 (1:40000, Cell Signaling), α-Smooth muscle actin (1:5000, Sigma), MF20 (1:100, Developmental Studies Hybridoma Bank) and PECAM-1 (1:200, BD Biosciences Pharmingen). Both MF20 and phospho-SMAD1/5/8 antibodies required 10min antigen retrieval (DAKO). Antibody diluent (DAKO), without primary antibody, was used for negative controls. For each assay, whole embryos and/or serial sections were examined for at least three individual embryos of each genotype at each stage of development. Wildtype littermates and Nkx2.5Cre only embryos were always used as age-matched control samples.

Measuring heartbeat

Individual whole E9.5 embryos (with deciduae and embryonic blood vessels left attached) were dissected from the mother in 37ºC DMEM medium supplemented with 5% fetal calf serum (Gibco-BRL), placed in a closed 12 well culture tray and allowed to recover for 10mins in incubator (37°C, 5% CO2), as previously described (Koushik et al., 2001). Each embryo was transilluminated and the heartbeat digitally recorded using an AxioCam MRc camera and dissecting scope (Zeiss) for 5mins and then PCR genotyped retrospectively. Heart rates were determined via calculating cardiac contractions/minute in 8 control and 4 pMes-Noggin/Nkx2.5Cre mutants (n=3 litters).

Cell proliferation, total cell counts, and Apoptosis

All cells were counted manually under 40X magnification. Apoptosis was evaluated using TdT-FragELTM DNA Fragmentation Kit (Calbiochem) following manufacture’s protocol. Proliferating cells were detected in serial sections of embryos (n=3 for each age group and genotype) using anti-phospho-Histone H3 primary antibody (1:500, Millipore) with antigen retrieval (DAKO). Sections were counterstained with weak hematoxylin. The outcome of pHH3 labeling was presented as percentage of labeled cells among total nuclei in the fixed region. Collected from nine continuous sections of three individual samples of wildtype controls and mutants, respectively, data were subjected to Student's t-test to determine the significance of differences. To insure that proliferation of only Cre-expressing cells was affected, the cardiomyocyte and non-cardiomyocyte proliferation rates were calculated. MF20 immunohistological staining on adjacent sections was used for cardiomyocyte detection. For total cell counts, serial sections of mutant and control hearts were Haematoxylin & Eosin (H&E) stained and statistical analysis of mutant and control cell counts and proliferation rates was performed using one-tailed t tests (p values were assigned, with <0.05 being significant).

Confocal Immunofluorescence

Embryos (n=3 for each age group and genotype) were collected in ice cold PBS, fixed in 4% paraformaldehyde, and placed in sequentially higher sucrose solutions. Specimens were embedded in Tissue-Tek OCT, frozen at −80oC and sectioned at 5μm. Immunostaining was performed using Vector Labs MOM fluorescent kit (FMK-2201) following the manufacturer’s protocol. Rhodamine phalloidin (1:100, Molecular Probes) and α-Actinin (1:400, Sigma) were used. Stained sections were photographed using Zeiss Axioskop-2 plus microscope and AxioVision Rel.4.8 software.

Transmission Electron Microscopy

Embryonic hearts (n=3 for each age group and genotype) were microdissected in ice cold PBS and fixed in 2% paraformaldehyde/2% gluteraldehyde in 0.1M cacodylate buffer overnight, followed by post fixation with 4% osmium tetroxide in PBS and subsequent ethyl alcohol dehydration. The infiltration process required 100% propylene oxide, and two changes of 50:50 100% propylene oxide:embedding resin (Embed 812, Electron Microscopy Sciences, Hatfield). Tissues were polymerized overnight in fresh embedding media at 60oC. Specimens were imaged on a Phillips400 microscope.

Radioactive and Non-radioactive in situ hybridization

Radioactive in situ hybridization for Hcn4 and Tbx5 (provided by YiPing Chen; Espinoza-Lewis et al., 2009) expression was performed as described (Simmons et al., 2014). The signal was detected only with hybridization of the anti-sense probe. cRNA Noggin probe (provided by Ondine Cleaver; Danesh et al. 2009) for non-radioactive in situ hybridization was labeled with the DIG RNA labeling mix (Roche). Control and pMes-Noggin/Nkx2.5Cre mutant paraffin sections (6μm) were treated with Proteinase K (10μg/mL), de-hydrated, air dried, incubated in hybridization solution, and hybridized with anti-sense probes (1μg/mL in hybridization solution) at 70oC overnight. Slides were washed with post-hybridization buffer, incubated in blocking solution for 2 hours at room temperature prior to addition of anti-DIG antibody (1:2000 in blocking solution). Slides were developed in BM Purple for several hours, followed by fixation in 4% paraformaldehyde, as described (Simmons et al., 2014). For all of these assays, serial sections were examined using at least three individual E8, E9 and E9.5 embryos of each genotype.

Bullet points.

  • Binary transgenic mouse embryos were generated to persistently express Noggin (BMP antagonist) within the Nkx2.5 cardiomyocyte lineage after initial transient endogenous Noggin expression was extinguished

  • 100% penetrant in utero lethality was observed by E12 in growth retarded Noggin transgenic embryos that exhibited small unlooped hearts and bradycardia

  • Persistent Noggin expression causes dysfunctional contractile apparatus maturation and diminished Hcn4 expression within the mutant primitive pacemaker region

  • Unaltered BMP activity is essential for early cardiomyocyte proliferation and expression of both heart-specific channel and sarcomeric proteins

Acknowledgments

We are grateful to the IU School of Medicine Electron Microscopy Center as well as the Indiana Center for Biological Microscopy for their assistance. The MF20 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported, in part, by American Heart Association 12PRE9430047 Pre-doctoral Fellowship (OS); as well as the Riley Children's Foundation, the Indiana University Department of Pediatrics and NIH HL60714 grant (SJC).

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