Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.     

FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium

The epicardium serves as a source of growth factors that regulate myocardial proliferation and as a source of epicardial-derived cells (EPDC), which give rise to interstitial cardiac fibroblasts and perivascular cells. These progenitors populate the compact myocardium to become part of the mature coronary vasculature and fibrous skeleton of the heart. Little is known about the mechanisms that regulate EPDC migration into the myocardium or the functions carried out by these cells once they enter the myocardium. However, it has been proposed that cardiac fibroblasts are important for growth of the heart during late gestation and are a source of homeostatic factors in the adult. Here, we identify a myocardial to epicardial fibroblast growth factor (FGF) signal, mediated by FGF10 and FGFR2b, that is essential for movement of cardiac fibroblasts into the compact myocardium. Inactivation of this signaling pathway results in fewer epicardial derived cells within the compact myocardium, decreased myocardial proliferation and a resulting smaller thin-walled heart.     

Mab21l2 is essential for embryonic heart and liver development

During mouse embryogenesis, proper formation of the heart and liver is especially important and is crucial for embryonic viability. In this study, we showed that Mab21l2 was expressed in the trabecular and compact myocardium, and that deletion of Mab21l2 resulted in a reduction of the trabecular myocardium and thinning of the compact myocardium. Mab21l2-deficient embryonic hearts had decreased expression of genes that regulate cell proliferation and apoptosis of cardiomyocytes. These results show that Mab21l2 functions during heart development by regulating the expression of such genes. Mab21l2 was also expressed in the septum transversum mesenchyme (STM). Epicardial progenitor cells are localized to the anterior surface of the STM (proepicardium), and proepicardial cells migrate onto the surface of the heart and form the epicardium, which plays an important role in heart development. The rest of the STM is essential for the growth and survival of hepatoblasts, which are bipotential progenitors for hepatocytes and cholangiocytes. Proepicardial cells in Mab21l2-deficient embryos had defects in cell proliferation, which led to a small proepicardium, in which α4 integrin expression, which is essential for the migration of proepicardial cells, was down-regulated, suggesting that defects occurred in its migration. In Mab21l2-deficient embryos, epicardial formation was defective, suggesting that Mab21l2 plays important roles in epicardial formation through the regulation of the cell proliferation of proepicardial cells and the migratory process of proepicardial cells. Mab21l2-deficient embryos also exhibited hypoplasia of the STM surrounding hepatoblasts and decreased hepatoblast proliferation with a resultant loss of defective morphogenesis of the liver. These findings demonstrate that Mab21l2 plays a crucial role in both heart and liver development through STM formation.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.     

心外膜的发育

心外膜是覆盖在心脏外层的间皮组织。心外膜来源于脏壁中胚层的前心外膜,后者位于心管流入极附近。心外膜的部分细胞通过上皮间充质转化进入心外膜下层,随后形成血管内皮、成纤维和平滑肌细胞,最终导致冠脉系统的形成。心外膜细胞可能形成心肌细胞,并且可能是心脏驻留干细胞的来源。因此,它在心脏修复治疗中发挥巨大作用。本文回顾了该领域的最新研究进展并且提出了目前存在的问题。     

An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.     

A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.     

FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation

Critical steps in coronary vascular formation include the epithelial-mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.     

Induction of proepicardial marker gene expression by the liver bud

Cells of the coronary vessels arise from a unique extracardiac mesothelial cell population, the proepicardium, which develops posterior to the sinoatrial region of the looping-stage heart. Although contribution of the proepicardial cells to cardiac development has been studied extensively, it remains unresolved how the proepicardium is induced and specified in the mesoderm during embryogenesis. It is known, however, that the proepicardium develops from the mesothelium that overlays the liver bud. Here, we show that the expression of proepicardial marker genes - Wt1, capsulin (epicardin, pod1, Tcf21) and Tbx18, can be induced in na?ve mesothelial cells by the liver bud, both in vitro and in vivo. Lateral embryonic explants, when co-cultured with the liver bud, were induced to express these proepicardial marker genes. The same induction of the marker genes was detected in vivo when a quail liver bud was implanted in the posterior-lateral regions of a chick embryo. This ectopic induction of marker gene expression was not evident when other endodermal tissues, such as the lung bud or stomach, were implanted. This inductive response to the liver bud was not detectable in host embryos before stage 12 (16-somite stage). These results suggest that, after a specific developmental stage, a large area of the mesothelium becomes competent to express proepicardial marker genes in response to localized liver-derived signal(s). The developmentally regulated competency of mesothelium and a localized inductive signal might play a role in restricting the induction of the proepicardial marker gene expression to a specific region of the mesothelium. The data might also provide a foundation for future engineering of a coronary vascular progenitor population.     

Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity

Abstract Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.     

Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.     

Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments

Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.     

Embryonic development of the proepicardium and coronary vessels

In the last few years, an increasing interest in progenitor cells has been noted. These cells are a source of undifferentiated elements from which cellular components of tissues and organs develop. Such progenitor tissue delivering stem cells for cardiac development is the proepicardium. The proepicardium is a transient organ which occurs near the venous pole of the embryonic heart and protrudes to the pericardial cavity. The proepicardium is a source of the epicardial epithelium delivering cellular components of vascular wall and interstitial tissue fibroblasts. It contributes partially to a fibrous tissue skeleton of the heart. Epicardial derived cells play also an inductive role in differentiation of cardiac myocytes into conductive tissue of the heart. Coronary vessel formation proceeds by vasculogenesis and angiogenesis. The first tubules are formed from blood islands which subsequently coalesce forming the primitive vascular plexus. Coronary arteries are formed by directional growth of vascular protrusions towards the aorta and establishing contact with the aortic wall. The coronary vascular wall matures by attaching smooth muscle cell precursors and fibroblast precursors to the endothelial cell wall. The cells of tunica media differentiate subsequently into vascular smooth muscle by acquiring specific contractile and cytoskeletal markers of smooth muscle cells in a proximal - distal direction. The coronary artery wall matures first before cardiac veins. Maturity of the vessel wall is demonstrated by the specific shape of the internal surface of the vascular wall.     

Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo

The epicardium regulates growth and survival of the underlying myocardium. This activity depends on intrinsic retinoic acid (RA) and erythropoietin signals. However, these signals do not act directly on the myocardium and instead are proposed to regulate the production of an unidentified soluble epicardial derived mitogen. Here, we show that Fgf9, Fgf16, and Fgf20 are expressed in the endocardium and epicardium and that RA can induce epicardial expression of Fgf9. Using knockout mice and an embryonic heart organ culture system, we show that endocardial and epicardial derived FGF signals regulate myocardial proliferation during midgestation heart development. We further show that this FGF signal is received by both FGF receptors 1 and 2 acting redundantly in the cardiomyoblast. In the absence of this signal, premature differentiation results in cellular hypertrophy and newborn mice develop a dilated cardiomyopathy. FGFs thus constitute all or part of the epicardial signal regulating myocardial growth and differentiation.     

BMP is an important regulator of proepicardial identity in the chick embryo

The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo.     

遗传谱系示踪技术揭示小鼠胚胎前体心外膜向心外膜转化过程

心外膜的形成是胚胎心脏发育的关键生理过程之一。利用遗传谱系示踪技术示踪观察前体心外膜向心外膜细胞转化过程,具有重要的科学研究价值。本研究拟利用Tbx18+前体/心外膜祖细胞遗传谱系示踪模型,揭示胚胎心外膜的起源及前体心外膜向心外膜转化的过程。利用整胚和切片原位杂交技术揭示,Tbx18 mRNA特异性表达于胚龄（E）9.5 d小鼠胚胎前体心外膜;故Tbx18是前体心外膜的特异性标记基因。利用整胚X-Gal染色,揭示报告基因Lacz在E9.5 d遗传谱系示踪模型鼠胚前体心外膜中大量表达,此时报告基因从前体心外膜逐渐迁移并开始少量表达于心外膜。Lacz在E10～E10.5 d双杂合鼠胚前体心外膜中表达逐渐减少,而在心外膜组织中逐渐增多;在E11.5 d,报告基因在前体心外膜中表达基本消失,而在心外膜组织中大量表达。切片进行X-Gal染色也揭示,报告基因Lacz定位于早期胚胎前体心外膜及心外膜。免疫荧光染色证实,早期胚胎心外膜细胞呈现未分化的祖细胞状态。通过报告基因的表达变化模式揭示,胚胎心外膜的形成经历了启动、转化、完成3个阶段;E9.5～11.5 d左右这个时间段发生的前体心外膜向心外膜转化,可能是心外膜形成的主要来源和形式。