Leonard M. Eisenberg

Molecular Regulation of Atrioventricular Valvuloseptal Morphogenesis

The majority of congenital heart defects arise from abnormal development of valvuloseptal tissue. The primordia of the valve leaflets and membranous septa of the heart are the cardiac cushions. Remodeling of the cushions is associated with a transitional extracellular matrix that includes sulfated proteoglycans and the microfibrillar proteins fibulin and fibrillin. Cushion formation is restricted to the AV canal and ventricular outflow tract regions of the primary heart tube. The proper placement of the cushions may be the result of the development of the primary heart tube as a segmented organ, as well as the subsequent looping of the heart. Segmentation of the heart tube may be demonstrated by the alternating molecular expression pattern along the longitudinal axis. In support of this hypothesis is the restricted expression of BMP-4 and msx-2 to the AV canal and ventricular outflow tract. The importance of looping for cushion positioning may imply that the iv and inv genes and retinoic acid are important for the proper patterning of the heart. The cells of the cushions evolve from endocardial cells that undergo an epithelial-to-mesenchymal transformation. This developmental event is regulated by the myocardium and is probably due to the production of protein complexes, present within the cardiac jelly of the cushion-forming regions, that consist of fibronectin and the ES proteins. Both the cushion mesenchyme and its endocardial cell antecedents express JB3, an ECM protein. JB3 expression is also featured within the heart-forming fields of the primary mesoderm, from which the endocardial progenitors of the cushion cells originate.(ABSTRACT TRUNCATED AT 250 WORDS)

Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of β-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.

WNT11 promotes cardiac tissue formation of early mesoderm

Cardiac tissue in the bird is derived from paired regions of lateral mesoderm within the anterior half of the embryo (Rawles [1943] Physiol. Zool. 16:22–42; Stalsberg and DeHaan [1969] Dev. Biol. 19:128–159). Previously, we reported that WNT11 is expressed in early avian mesoderm in a pattern that overlaps with the precardiac regions. To examine whether this molecule may play a role in promoting cardiogenesis, we cultured tissue explants from microdissected HH stage 4, 5, and 6 quail embryos. The isolated tissue consisted of both the mesoderm and endoderm layers from either anterior precardiac or posterior noncardiogenic regions of the embryo. As a necessary control for examining the ability of WNT11 to convert noncardiogenic mesoderm to cardiac tissue, we compared the cardiogenic potential of anterior and posterior regions. For stages 5 and 6, our results were consistent with what has been previously reported (Rawles [1943] Physiol. Zool. 16:22–42; Sugi and Lough [1994] Dev. Dyn. 200:155–162); as anterior mesoderm becomes contractile, while posterior mesoderm does not produce cardiac tissue. Surprisingly, when we examined stage 4 embryos both anterior and posterior regions gave rise to cardiac tissue in culture. To determine whether WNT11 could promote cardiac differentiation in tissue that was noncardiogenic, this molecule was ectopically expressed or added to mesoderm/endoderm explants obtained from stage 5 or 6 posterior tissue. Transfection of stage 5 posterior tissue with a WNT11 expression plasmid provoked the appearance of cardiomyocytes in 33% of the explants; half of which were contractile. Similarly transfected stage 6 posterior explants did not demonstrate cardiac differentiation. More dramatic results were obtained when noncardiogenic tissue was exposed to conditioned media containing soluble WNT11; as 63% and 33% of posterior stage 5‐ or stage 6‐derived explants underwent cardiac differentiation. Together, these results indicate that WNT11 can promote cardiac development within noncardiac tissue. The expression of WNT11 in anterior mesoderm of early gastrula stage embryos suggests it may play a role in the formation of the vertebrate heart. Dev Dyn 1999;216:45–58.

Wnt11 Signaling Promotes Proliferation, Transformation, and Migration of IEC6 Intestinal Epithelial Cells

Wnts are morphogens with well recognized functions during embryogenesis. Aberrant Wnt signaling has been demonstrated to be important in colorectal carcinogenesis. However, the role of Wnt in regulating normal intestinal epithelial cell proliferation is not well established. Here we determine that Wnt11 is expressed throughout the mouse intestinal tract including the epithelial cells. Conditioned media from Wnt11-secreting cells stimulated proliferation and migration of IEC6 intestinal epithelial cells. Co-culture of Wnt11-secreting cells with IEC6 cells resulted in morphological transformation of the latter as evidenced by the formation of foci, a condition also accomplished by stable transfection of IEC6 with a Wnt11-expressing construct. Treatment of IEC6 cells with Wnt11 conditioned media failed to induce nuclear translocation of β-catenin but led to increased activities of protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Inhibition of protein kinase C resulted in a decreased ability of Wnt11 to induce foci formation in IEC6 cells. Finally, E-cadherin was redistributed in Wnt11-treated IEC6 cells, resulting in diminished E-cadherin-mediated cell-cell contact. We conclude that Wnt11 stimulates proliferation, migration, cytoskeletal rearrangement, and contact-independent growth of IEC6 cells by a β-catenin-independent mechanism. These findings may help understand the molecular mechanisms that regulate proliferation and migration of intestinal epithelial cells.

Bone Marrow Cells Transdifferentiate to Cardiomyocytes When Introduced into the Embryonic Heart

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long‐term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co‐incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription‐polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker α‐cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial‐specific β‐galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of β‐galactosidase‐positive cells, indicating the presence of bone marrow‐derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.

Evaluating the role of Wnt signal transduction in promoting the development of the heart.

Wnts are a family of secreted signaling proteins that are encoded by 19 distinct genes in the vertebrate genome. These molecules initiate several signal transduction pathways: the canonical Wnt, Wnt/Ca2+, and Wnt/planar cell polarity pathways. Wnt proteins have major impact on embryonic development, tumor progression, and stem cell differentiation. Wnt signal transduction also influences the formation of the heart, yet many issues concerning the involvement of Wnt regulation in initiating cardiac development remain unresolved. In this review, we will examine the published record to discern (a) what has been shown by experimental studies on the participation of Wnt signaling in cardiogenesis, and (b) what are the important questions that need to be addressed to understand the importance and function of Wnt signal transduction in facilitating the development of the heart.

Wnt11 and Wnt7a are up‐regulated in association with differentiation of cardiac conduction cells in vitro and in vivo

The heart beat is coordinated by a precisely timed sequence of action potentials propagated through cells of the conduction system. Previously, we have shown that conduction cells in the chick embryo are derived from multipotent, cardiomyogenic progenitors present in the looped, tubular heart. Moreover, analyses of heterogeneity within myocyte clones and cell birth dating have indicated that elaboration of the conduction system occurs by ongoing, localized recruitment from within this multipotent pool. In this study, we have focused on a potential role for Wnt signaling in development of the cardiac conduction system. Treatment of embryonic myocytes from chick with endothelin‐1 (ET‐1) has been shown to promote expression of markers of Purkinje fiber cells. By using this in vitro model, we find that Wnt11 are Wnt7a are up‐regulated in association with ET‐1 treatment. Moreover, in situ hybridization reveals expression, although not temporal coincidence of, Wnt11 and Wnt7a in specialized tissues in the developing heart in vivo. Specifically, whereas Wnt11 shows transient and prominent expression in central elements of the developing conduction system (e.g., the His bundle), relative increases in Wnt7a expression emerge at sites consistent with the location of peripheral conduction cells (e.g., subendocardial Purkinje fibers). The patterns of Wnt11 and Wnt7a expression observed in vitro and in the embryonic chick heart appear to be consistent with roles for these two Wnts in differentiation of cardiac conduction tissues. Development Dynamics 227:536–543, 2003.

Belief vs. scientific observation: The curious story of the precardiac mesoderm

The transition of primary nondifferentiated mesoderm to definitive cardiac tissue is a process that occurs within a relatively narrow developmental window (Eisenberg and Eisenberg, 2001). In the developing chick, this interval encompasses Hamburger-Hamilton (HH) (Hamburger and Hamilton, 1951) stages 4–8, which generally corresponds to 18–30 hr after egg laying. In a recent issue of Development, Redkar et al. (2001) describe a newly generated fate map that defines the location of the precardiac regions within the early chick embryo. The experimentation consisted of microinjecting the fluorescent label DiI into precisely localized regions within the mesodermal layer of early gastrula-stage embryos. After allowing the embryos to develop to tubular heart stages, the fates of individual labeled mesoderm cells were compiled, with regard to whether heart or nonheart tissue was subsequently labeled. The compiled cardiogenic fate map extends back as early as HH stage 4—the point during gastrulation where the primitive streak is fully elongated—thereby providing the first definitive fate map for this early stage. Moreover, by labeling embryos ranging from HH stages 4–8, Redkar et al. (2001) generated fate maps that describe the location of precardiac cells throughout gastrulation. Thus, by identifying the cells that will contribute to the primary heart tube, this comprehensive study provides information that is fundamental for understanding how the vertebrate heart emerges from nondifferentiated mesoderm. In many ways, the key period in the conversion of primary nondifferentiated mesoderm to heart tissue is HH stage 5. This is the time during chick development when the primitive streak begins to recede and the notochord begins to form. At HH stage 5, the precardiac or heartforming regions (HFRs) comprise two mesodermal fields within the anterior half of the embryo, which are bilaterally distributed with respect to the embryonic axis (Rawles, 1943; Rosenquist and DeHaan, 1966; Stalsberg and DeHaan, 1969). This point in heart development either coincides with or is just prior to the initial expression of several cardiac-associated transcription factors, such as Nkx2.5, SRF, Tbx-5, GATA4-6, and MEF2C (Croissant et al., 1996; Evans, 1997; Black and Olson, 1998; Evans, 1999; Yamada et al., 2000). The mesoderm at HH stage 5 is a simple cell layer. However, by HH stage 6, the lateral mesoderm begins to separate into two layers, with the precardiac cells continuing their residence within the splanchnic mesoderm (DeHaan, 1965). This rearrangement serves as a prelude to subsequent fusion of the two HFRs and, with this, the formation of the primary heart tube. As reported by Redkar et al. (2001), the medial border of the HFRs within the HH stage 5 embryo is located 0.3 mm lateral to the primitive groove. The anterior border of the HFRs resides just rostral to Hensen’s node, with the posterior border extending one-fourth of the distance down the primitive streak. One of the authors’ major conclusions from this new fate map is that “the putative cardiogenic marker Nkx2.5 does not govern the boundaries of the HFR as described in the literature.” The conclusion that the precardiac mesoderm doesn’t coincide with the Nkx2.5 expression domain may seem surprising. After all, this transcription factor has been singled out for its importance for promoting the cardiac phenotype. At HH stage 5, expression of the Nkx2.5 gene first appears in a crescent pattern that hugs the anterior-most edge of the embryo (Schultheiss et al., 1995). However, no one should be astonished by the newly generated fate map. Why not? Because, the location of the precardiac mesoderm within the HH stage 5 embryo was well documented 32 years earlier. In an amazingly well-executed radiolabeling study, Stalsberg and DeHaan (1969) reported a cardiogenic fate map that is almost identical to that reported by Redkar et al. (2001). Yet, it has been the misfortune of the cardiac developmental biology field that this earlier study—one of the true landmarks in this field—has been increasingly ignored. Its dismissal had little to do with the quality of the experimentation and all to do with its conclusions differing from the recent belief that formation of the vertebrate heart was the result of a simple pattern of gene expression—a theory that was readily accepted de-

The Histone Methyltransferase Inhibitor BIX01294 Enhances the Cardiac Potential of Bone Marrow Cells

Bone marrow (BM) has long been considered a potential stem cell source for cardiac repair due to its abundance and accessibility. Although previous investigations have generated cardiomyocytes from BM, yields have been low, and far less than produced from ES or induced pluripotent stem cells (iPSCs). Since differentiation of pluripotent cells is difficult to control, we investigated whether BM cardiac competency could be enhanced without making cells pluripotent. From screens of various molecules that have been shown to assist iPSC production or maintain the ES cell phenotype, we identified the G9a histone methyltransferase inhibitor BIX01294 as a potential reprogramming agent for converting BM cells to a cardiac-competent phenotype. BM cells exposed to BIX01294 displayed significantly elevated expression of brachyury, Mesp1, and islet1, which are genes associated with embryonic cardiac progenitors. In contrast, BIX01294 treatment minimally affected ectodermal, endodermal, and pluripotency gene expression by BM cells. Expression of cardiac-associated genes Nkx2.5, GATA4, Hand1, Hand2, Tbx5, myocardin, and titin was enhanced 114, 76, 276, 46, 635, 123, and 5-fold in response to the cardiogenic stimulator Wnt11 when BM cells were pretreated with BIX01294. Immunofluorescent analysis demonstrated that BIX01294 exposure allowed for the subsequent display of various muscle proteins within the cells. The effect of BIX01294 on the BM cell phenotype and differentiation potential corresponded to an overall decrease in methylation of histone H3 at lysine9, which is the primary target of G9a histone methyltransferase. In summary, these data suggest that BIX01294 inhibition of chromatin methylation reprograms BM cells to a cardiac-competent progenitor phenotype.

Onset of a Cardiac Phenotype in the Early Embryo

Soon after fertilization, vertebrate embryos grow very rapidly. Thus, very early in gestation a sizeable yet underdeveloped organism requires circulating blood. This need dictates the early appearance of a contractile heart, which is the first functional organ in both the bird and mammalian embryos. Incipient heart tissue makes its arrival within the mesoderm layer during the onset of gastrulation. The process whereby nondifferentiated cells of primary mesoderm give rise to contractile cardiomyocytes is a subject that has greatly intrigued developmental biologists throughout the twentieth century. Since the early 1990s, a number of regulatory molecules have been identified that are important players in these events. Yet, how these molecular parts fit into the total story is still far from understood. In this chapter, we will discuss what is known about the morphological events that underlie the formation of the primitive heart, relate that information to the identification of candidate regulators of cardiogenesis in the early embryo (and description of their presumptive roles), and finally bring this information on cardiac development in context with the overall diversification of the primary mesoderm. Like many topics in biology, the study of cardiac development has profited both from tissue culture and in vivo experimentation. Because the onset of cardiogenesis occurs so early during embryogenesis, avian embryos have proven to be the most practical model system for studying these events in higher vertebrates, especially with regard to examining the behavior of precardiac tissue in isolation from the embryo. Thus, much of the discussion will be dominated by avian development. It has only been during recent years with the advent of transgenic and gene-targeted mice that mammalian models have made major contributions to our understanding of the primary events in cardiogenesis. Additionally, studies using frog and zebrafish embryos have contributed to this field. Surprisingly, an animal model that has yielded much information on early cardiogenesis is the fruit fly, Drosophila melanogaster. Despite the significant morphological differences between vertebrate and invertebrate hearts, there appears to be at least some homology of the molecular events that mold their respective cardiac tissue. Among vertebrate species, the molecular biology of early cardiogenesis seems to be totally conserved. This has allowed a fuller picture of early cardiogenesis to be compiled with information gathered from these various animal models, a cross-reference necessitated by the various strengths and weaknesses of each of the experimental systems.