Jiandong Liu

Antioxidants protect PINK1-dependent dopaminergic neurons in Drosophila

Parkinsons disease (PD) is the most frequent neurodegenerative movement disorder. Mutations in the PINK1 gene are linked to the autosomal recessive early onset familial form of PD. The physiological function of PINK1 and pathological abnormality of PD-associated PINK1 mutants are largely unknown. We here show that inactivation of Drosophila PINK1 (dPINK1) using RNAi results in progressive loss of dopaminergic neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINK1). Expression of human SOD1 suppresses neurodegeneration induced by dPINK1 inactivation. Moreover, treatment of dPINK1 RNAi flies with the antioxidants SOD and vitamin E significantly inhibits ommatidial degeneration. Thus, dPINK1 plays an essential role in maintaining neuronal survival by preventing neurons from undergoing oxidative stress, thereby suggesting a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration.

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

Slit and Robo Control Cardiac Cell Polarity and Morphogenesis

Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. Here, we report a novel role of the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. We conclude that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium.

Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming

Rationale: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. Objective: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. Methods and Results: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. Conclusions: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.

Tbx5 and Bmp Signaling Are Essential for Proepicardium Specification in Zebrafish

Rationale: The proepicardial organ (PE) contributes to the cellular diversity of the developing heart by giving rise to the epicardium as well as vascular smooth muscle cells and fibroblasts. Despite the importance of these cells in cardiac development, function and regeneration, the signals required for the specification of the PE remain largely unexplored. Objective: We aim to identify the signaling molecules and transcription factors that regulate PE specification. Methods and Results: Here, we present the first genetic evidence that bone morphogenetic protein (Bmp) signaling in conjunction with the T-box transcription factor Tbx5a is essential for PE specification in zebrafish. Specifically, Bmp4 from the cardiac region, but not the liver bud, acting through the type I BMP receptor Acvr1l, is required for PE specification. By overexpressing a dominant-negative form of a Bmp receptor at various embryonic stages, we determined when Bmp signaling was required for PE specification. We also found that overexpression of bmp2b right before PE specification led to the ectopic expression of PE specific markers including tbx18. Furthermore, using loss-of-function approaches, we discovered a previously unappreciated PE specification role for Tbx5a at early somite stages; this role occurs earlier than, and appears to be independent from, the requirement for Bmp signaling in this process. Conclusion: Altogether, these data lead us to propose that Tbx5a confers anterior lateral plate mesodermal cells the competence to respond to Bmp signals and initiate PE development.

Transcription factor neuromancer/TBX20 is required for cardiac function in Drosophila with implications for human heart disease

neuromancer/Tbx20 (nmr) genes are cardiac T-box transcription factors that are evolutionarily conserved from flies to humans. Along with other known congenital heart disease genes, including tinman/Nkx2–5, dorsocross/Tbx5/6, and pannier/Gata4/6, they are important for specification and morphogenesis of the embryonic heart. The Drosophila heart has proven to be an excellent model to study genes involved in establishing and maintaining the structural integrity of the adult heart, as well as genes involved in maintaining physiological function. Using this model, we have identified nmr as a gene required in adult fly hearts for the maintenance of both normal myofibrillar architecture and cardiac physiology. Moreover, we have discovered synergistic interactions between nmr and other cardiac transcription factors, including tinman/Nkx2–5, in regulating cardiac performance, rhythmicity, and cardiomyocyte structure, reminiscent of similar interactions in mice. This suggests a remarkably conserved role for this network of cardiac transcription factors in the genetic control of the adult heart. In addition, nmr-tinman interactions also influence the expression of potential downstream effectors, such as ion channels. Interestingly, genetic screening of patients with dilated cardiomyopathy and congenital heart disease has revealed TBX20 variants in three sporadic and two familial cases that were not found in controls. These findings suggest that the fly heart might serve as an identifier of candidate genes involved in human heart disease.

Zebrafish in the Study of Early Cardiac Development

Heart development is a complex process that involves cell specification and differentiation, as well as elaborate tissue morphogenesis and remodeling, to generate a functional organ. The zebrafish has emerged as a powerful model system to unravel the basic genetic, molecular, and cellular mechanisms of cardiac development and function. We summarize and discuss recent discoveries on early cardiac specification and the identification of the second heart field in zebrafish. In addition to the inductive signals regulating cardiac specification, these studies have shown that heart development also requires a repressive mechanism imposed by retinoic acid signaling to select cardiac progenitors from a multipotent population. Another recent advance in the study of early zebrafish cardiac development is the identification of the second heart field. These studies suggest that the molecular mechanisms that regulate the second heart field development are conserved between zebrafish and other vertebrates including mammals and provide insight into the evolution of the second heart field and its derivatives.

Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming

Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK119ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

Embryonic cardiac chamber maturation: Trabeculation, conduction, and cardiomyocyte proliferation

Congenital heart diseases are some of the most common human birth defects. Though some congenital heart defects can be surgically corrected, treatment options for other congenital heart diseases are very limited. In many congenital heart diseases, genetic defects lead to impaired embryonic heart development or growth. One of the key development processes in cardiac development is chamber maturation, and alterations in this maturation process can manifest as a variety of congenital defects including non‐compaction, systolic dysfunction, diastolic dysfunction, and arrhythmia. During development, to meet the increasing metabolic demands of the developing embryo, the myocardial wall undergoes extensive remodeling characterized by the formation of muscular luminal protrusions called cardiac trabeculae, increased cardiomyocyte mass, and development of the ventricular conduction system. Though the basic morphological and cytological changes involved in early heart development are clear, much remains unknown about the complex biomolecular mechanisms governing chamber maturation. In this review, we highlight evidence suggesting that a wide variety of basic signaling pathways and biomechanical forces are involved in cardiac wall maturation.

High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.