Lauren J. Manderfield

Cardiac neural crest orchestrates remodeling and functional maturation of mouse semilunar valves

Congenital anomalies of the aortic valve are common and are associated with progressive valvular insufficiency and/or stenosis. In addition, aneurysm, coarctation, and dissection of the ascending aorta and aortic arch are often associated conditions that complicate patient management and increase morbidity and mortality. These associated aortopathies are commonly attributed to turbulent hemodynamic flow through the malformed valve leading to focal defects in the vessel wall. However, numerous surgical and pathological studies have identified widespread cystic medial necrosis and smooth muscle apoptosis throughout the aortic arch in affected patients. Here, we provide experimental evidence for an alternative model to explain the association of aortic vessel and valvular disease. Using mice with primary and secondary cardiac neural crest deficiencies, we have shown that neural crest contribution to the outflow endocardial cushions (the precursors of the semilunar valves) is required for late gestation valvular remodeling, mesenchymal apoptosis, and proper valve architecture. Neural crest was also shown to contribute to the smooth muscle layer of the wall of the ascending aorta and aortic arch. Hence, defects of cardiac neural crest can result in functionally abnormal semilunar valves and concomitant aortic arch artery abnormalities.

Plasticity of Hopx+ Type I alveolar cells to regenerate Type II cells in the lung

The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar Type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging Type I cells. In vivo, Type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here, we show that Hopx becomes restricted to Type I cells during development. However, unexpectedly, lineage-labeled Hopx+ cells both proliferate and generate Type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx+ Type I cells generate organoids composed of Type I and Type II cells, a process modulated by TGFβ signaling. These findings demonstrate unanticipated plasticity of Type I cells and a bi-directional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single cell culture.

Notch Activation of Jagged1 Contributes to the Assembly of the Arterial Wall

Background— Notch signaling in vascular smooth muscle precursors is required for smooth muscle differentiation. Jagged1 expression on endothelium activates Notch in vascular smooth muscle precursors including those of neural crest origin to initiate the formation of a smooth muscle layer in a maturing blood vessel. Methods and Results— Here, we show that Jagged1 is a direct Notch target in smooth muscle, resulting in a positive feedback loop and lateral induction that propagates a wave of smooth muscle differentiation during aortic arch artery development. In vivo, we show that Notch inhibition in cardiac neural crest impairs Jagged1 messenger RNA expression and results in deficient smooth muscle differentiation and resultant aortic arch artery defects. Ex vivo, Jagged1 ligand activates Notch in neural crest explants and results in activation of Jagged1 messenger RNA, a response that is blocked by Notch inhibition. We examine 15 evolutionary conserved regions within the Jagged1 genomic locus and identify a single Notch response element within the second intron. This element contains a functional Rbp-J binding site demonstrated by luciferase reporter and chromatin immunoprecipitation assays and is sufficient to recapitulate aortic arch artery expression of Jagged1 in transgenic mice. Loss of Jagged1 in neural crest impairs vascular smooth muscle differentiation and results in aortic arch artery defects. Conclusions— Taken together, these results provide a mechanism for lateral induction that allows for a multilayered smooth muscle wall to form around a nascent arterial endothelial tube and identify Jagged1 as a direct Notch target.

Notch signaling regulates murine atrioventricular conduction and the formation of accessory pathways

Ventricular preexcitation, which characterizes Wolff-Parkinson-White syndrome, is caused by the presence of accessory pathways that can rapidly conduct electrical impulses from atria to ventricles, without the intrinsic delay characteristic of the atrioventricular (AV) node. Preexcitation is associated with an increased risk of tachyarrhythmia, palpitations, syncope, and sudden death. Although the pathology and electrophysiology of preexcitation syndromes are well characterized, the developmental mechanisms are poorly understood, and few animal models that faithfully recapitulate the human disorder have been described. Here we show that activation of Notch signaling in the developing myocardium of mice can produce fully penetrant accessory pathways and ventricular preexcitation. Conversely, inhibition of Notch signaling in the developing myocardium resulted in a hypoplastic AV node, with specific loss of slow-conducting cells expressing connexin-30.2 (Cx30.2) and a resulting loss of physiologic AV conduction delay. Taken together, our results suggest that Notch regulates the functional maturation of AV canal embryonic myocardium during the development of the specialized conduction system. Our results also show that ventricular preexcitation can arise from inappropriate patterning of the AV canal-derived myocardium.

Islet1 Derivatives in the Heart Are of Both Neural Crest and Second Heart Field Origin

Rationale: Islet1 (Isl1) has been proposed as a marker of cardiac progenitor cells derived from the second heart field and is utilized to identify and purify cardiac progenitors from murine and human specimens for ex vivo expansion. The use of Isl1 as a specific second heart field marker is dependent on its exclusion from other cardiac lineages such as neural crest. Objective: Determine whether Isl1 is expressed by cardiac neural crest. Methods and Results: We used an intersectional fate-mapping system using the RC::FrePe allele, which reports dual Flpe and Cre recombination. Combining Isl1Cre/+, a SHF driver, and Wnt1::Flpe, a neural crest driver, with Rc::FrePe reveals that some Isl1 derivatives in the cardiac outflow tract derive from Wnt1-expressing neural crest progenitors. In contrast, no overlap was observed between Wnt1-derived neural crest and an alternative second heart field driver, Mef2c-AHF-Cre. Conclusions: Isl1 is not restricted to second heart field progenitors in the developing heart but also labels cardiac neural crest. The intersection of Isl1 and Wnt1 lineages within the heart provides a caveat to using Isl1 as an exclusive second heart field cardiac progenitor marker and suggests that some Isl1-expressing progenitor cells derived from embryos, embryonic stem cultures, or induced pluripotent stem cultures may be of neural crest lineage.

Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts

Making cardiomyocytes In the heart, multiple cell types work together. Cardiac progenitor cells give rise to cardiomyocyte, endothelial, or smooth muscle lineages. However, the identity of a marker specific to cardiomyocyte formation has been elusive. Jain et al. now identify a specialized progenitor population that is committed exclusively to forming cardiomyocytes. They also identify the niche signals that promote lineage commitment and the mechanisms involved in making cardiomyocytes. The findings may help in the development of future cell-based regenerative therapeutics for heart disease. Science, this issue 10.1126/science.aaa6071 Identification of the committed cardiomyoblast that retains proliferative potential may inform cardiac regenerative therapeutics. INTRODUCTION Cardiac progenitor cells are multipotent, and lineage analyses of murine and chick cardiac development have demonstrated that these cells give rise to the cardiac endothelium, smooth muscle, and cardiomyocytes. However, the mechanisms governing commitment to the myocyte lineage in vivo remain largely unknown. Further understanding of these mechanisms, and of the identity of progenitors committed to the myocyte lineage, may advance cardiac regenerative therapies. RATIONALE Hopx is an atypical homeodomain expressed in cardiac mesoderm shortly after cardiac progenitor cells are first evident. Previous studies have demonstrated that Hopx functions as a nuclear transcription co-repressor and is expressed in adult, +4 intestinal stem cells and hair follicle bulge stem cells. We compare lineage tracing of multipotent cardiac progenitor cells marked by Islet1 and Nkx2-5 expression with lineage tracing of Hopx+ cells. We also perform functional studies of Hopx from endogenous tissue and differentiated embryoid bodies to identify mechanisms promoting commitment and myogenesis. RESULTS We define and characterize a Hopx-expressing cardiomyoblast intermediate that is committed to the cardiomyocyte fate. Hopx+ is initially expressed in a subset of cardiac progenitor cells residing in the precardiac mesoderm prior to the expression of troponin T, a component of the contractile sarcomere apparatus of myocytes. Lineage-tracing experiments demonstrate that Hopx+ cells give rise to cardiac myocytes exclusively. Early Hopx+ cardiomyoblasts expand during cardiogenesis. Overexpression of Hopx in cardiac progenitor cells leads to an increase in myocytes, whereas Hopx deficiency compromises myogenesis. Whole-genome analysis reveals that Hopx occupies regulatory regions of multiple Wnt-related genes, and Hopx–/– cardiac tissues are characterized by an expansion of Wnt signaling. Restoration of Wnt levels during differentiation of Hopx–/– embryoid bodies partially rescues myogenesis. Wnt signaling is a potent regulator of stemness of cardiac progenitor cells, and our data suggest that Hopx promotes myogenesis by repressing Wnt signaling. Cardiac progenitor cells down-regulate Wnt signaling as they enter the cardiac outflow tract, coincident with the expression of Hopx. The outflow tract is also enriched for bone morphogenetic protein (Bmp) signaling, known to influence differentiation of myocytes. Hopx physically interacts with activated Smad complexes in vitro and in vivo. Exogenous Bmp4 represses Wnt signaling in cardiac explants, and Bmp4-mediated Wnt repression requires Hopx. Thus, Hopx functions to couple Bmp signaling to repression of Wnt. CONCLUSION Our work defines an intermediate cardiac progenitor that expresses Hopx and is committed exclusively to the myocyte fate. Therefore, akin to an erythroblast in hematopoietic differentiation, we have termed these committed cardiac progenitor cells “cardiomyoblasts.” The ability to identify committed, but undifferentiated, cardiomyocyte precursors may facilitate development of cardiac regenerative therapies, including those using embryonic stem cells and induced pluripotent stem cells. Hopx functions to promote myogenesis by physically interacting with Smad proteins to repress Wnt signaling. Our findings raise the possibility that Hopx-mediated integration of Bmp signaling to repress Wnt may be active in other progenitor populations and may potentially underlie the tumor suppressor function of Hopx. Lineage tracing of Hopx+ cells. Images depicting lineage tracing of early Hopx+ cardiomyoblasts that give rise to myocytes in the left ventricle and atria. Some images are duplicated and pseudocolored. Cardiac progenitor cells are multipotent and give rise to cardiac endothelium, smooth muscle, and cardiomyocytes. Here, we define and characterize the cardiomyoblast intermediate that is committed to the cardiomyocyte fate, and we characterize the niche signals that regulate commitment. Cardiomyoblasts express Hopx, which functions to coordinate local Bmp signals to inhibit the Wnt pathway, thus promoting cardiomyogenesis. Hopx integrates Bmp and Wnt signaling by physically interacting with activated Smads and repressing Wnt genes. The identification of the committed cardiomyoblast that retains proliferative potential will inform cardiac regenerative therapeutics. In addition, Bmp signals characterize adult stem cell niches in other tissues where Hopx-mediated inhibition of Wnt is likely to contribute to stem cell quiescence and to explain the role of Hopx as a tumor suppressor.

Myocardial Notch Signaling Reprograms Cardiomyocytes to a Conduction-Like Phenotype

Background— Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. Methods and Results— In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system–specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. Conclusions— Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.

Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest.

Notch signaling has well-defined roles in the assembly of arterial walls and in the development of the endothelium and smooth muscle of the vasculature. Hippo signaling regulates cellular growth in many tissues, and contributes to regulation of organ size, in addition to other functions. Here, we show that the Notch and Hippo pathways converge to regulate smooth muscle differentiation of the neural crest, which is crucial for normal development of the aortic arch arteries and cranial vasculature during embryonic development. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fail to produce vascular smooth muscle, and Notch target genes such as Jagged1 fail to activate normally. We show that Yap is normally recruited to a tissue-specific Jagged1 enhancer by directly interacting with the Notch intracellular domain (NICD). The Yap-NICD complex is recruited to chromatin by the DNA-binding protein Rbp-J in a Tead-independent fashion. Thus, Hippo signaling can modulate Notch signaling outputs, and components of the Hippo and Notch pathways physically interact. Convergence of Hippo and Notch pathways by the mechanisms described here might be relevant for the function of these signaling cascades in many tissues and in diseases such as cancer. Highlighted article: Direct interaction between the Hippo effector Yap and the Notch intracellular domain regulates Notch target gene expression during vascular smooth muscle differentiation from neural crest.

Epicardial YAP/TAZ orchestrate an immunosuppressive response following myocardial infarction

Ischemic heart disease resulting from myocardial infarction (MI) is the most prevalent form of heart disease in the United States. Post-MI cardiac remodeling is a multifaceted process that includes activation of fibroblasts and a complex immune response. T-regulatory cells (Tregs), a subset of CD4+ T cells, have been shown to suppress the innate and adaptive immune response and limit deleterious remodeling following myocardial injury. However, the mechanisms by which injured myocardium recruits suppressive immune cells remain largely unknown. Here, we have shown a role for Hippo signaling in the epicardium in suppressing the post-infarct inflammatory response through recruitment of Tregs. Mice deficient in epicardial YAP and TAZ, two core Hippo pathway effectors, developed profound post-MI pericardial inflammation and myocardial fibrosis, resulting in cardiomyopathy and death. Mutant mice exhibited fewer suppressive Tregs in the injured myocardium and decreased expression of the gene encoding IFN-&ggr;, a known Treg inducer. Furthermore, controlled local delivery of IFN-&ggr; following MI rescued Treg infiltration into the injured myocardium of YAP/TAZ mutants and decreased fibrosis. Collectively, these results suggest that epicardial Hippo signaling plays a key role in adaptive immune regulation during the post-MI recovery phase.

Coronary vasculature patterning requires a novel endothelial ErbB2 holoreceptor

Organogenesis and regeneration require coordination of cellular proliferation, regulated in part by secreted growth factors and cognate receptors, with tissue nutrient supply provided by expansion and patterning of blood vessels. Here we reveal unexpected combinatorial integration of a growth factor co-receptor with a heterodimeric partner and ligand known to regulate angiogenesis and vascular patterning. We show that ErbB2, which can mediate epidermal growth factor (EGF) and neuregulin signalling in multiple tissues, is unexpectedly expressed by endothelial cells where it partners with neuropilin 1 (Nrp1) to form a functional receptor for the vascular guidance molecule semaphorin 3d (Sema3d). Loss of Sema3d leads to improper patterning of the coronary veins, a phenotype recapitulated by endothelial loss of ErbB2. These findings have implications for possible cardiovascular side-effects of anti-ErbB2 therapies commonly used for cancer, and provide an example of integration at the molecular level of pathways involved in tissue growth and vascular patterning.