Eric Rulifson

Wingless Repression of Drosophila frizzled 2 Expression Shapes the Wingless Morphogen Gradient in the Wing

In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. Here, we show that high levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation.

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. Here we show, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells.

Direct Evaluation of Myocardial Viability and Stem Cell Engraftment Demonstrates Salvage of the Injured Myocardium

RATIONALE The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs). OBJECTIVE To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging. METHODS AND RESULTS SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation. CONCLUSION The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium.

Serial specification of diverse neuroblast identities from a neurogenic placode by Notch and Egfr signaling

We used the brain insulin-producing cell (IPC) lineage and its identified neuroblast (IPC NB) as a model to understand a novel example of serial specification of NB identities in the Drosophila dorsomedial protocerebral neuroectoderm. The IPC NB was specified from a small, molecularly identified group of cells comprising an invaginated epithelial placode. By progressive delamination of cells, the placode generated a series of NB identities, including the single IPC NB, a number of other canonical Type I NBs, and a single Type II NB that generates large lineages by transient amplification of neural progenitor cells. Loss of Notch function caused all cells of the placode to form as supernumerary IPC NBs, indicating that the placode is initially a fate equivalence group for the IPC NB fate. Loss of Egfr function caused all placodal cells to apoptose, except for the IPC NB, indicating a requirement of Egfr signaling for specification of alternative NB identities. Indeed, both derepressed Egfr activity in yan mutants and ectopic EGF activity produced supernumerary Type II NBs from the placode. Loss of both Notch and Egfr function caused all placode cells to become IPC NBs and survive, indicating that commitment to NB fate nullified the requirement of Egfr activity for placode cell survival. We discuss the surprising parallels between the serial specification of neural fates from this neurogenic placode and the fly retina.

Paracrine Effects of the Pluripotent Stem Cell-Derived Cardiac Myocytes Salvage the Injured MyocardiumNovelty and Significance

Rationale: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. Objective: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. Methods and Results: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. Conclusions: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.

Interactions between wingless and frizzled molecules in Drosophila.

Over the past decade, our understanding of the mechanisms underlying growth and patterning of tissues has been enormously increased due to a convergence of the fields of developmental genetics and cancer research. Systematic screens in organisms such as Drosophila, Caenorhabditis elegans, and more recently the zebrafish have produced a wealth of genes and proteins that regulate development. At the same time, searches for genes implicated in cancer, either as dominant or as recessive (tumor suppressor) genes, have yielded an equally impressive list of important regulators of growth. There are now also numerous examples of genes found to control normal growth but to cause cancerous growth when misregulated. One of the best examples is the Wnt gene family. We will summarize our current view of how Wnt proteins signal, and the evidence that proteins of the Frizzled (Fz) family mediate Wnt signaling by acting as specific cell surface receptors.

In vivo multi-modality tracking of the regenerative effects of the human induced pluripotent stem cell-derived cardiomyocytes (iCMs)

Background In vivo multi-modality cellular and molecular imaging of the engrafted iCMs is necessary to characterize the engraftment and the regional effects on the viability of the injured myocardium. Zinc finger nuclease (ZFN)mediated integration of the reporter gene into the AAVS1 locus in the iCMs and manganese enhanced MRI (MEMRI) should allow precise in vivo detection of myocardial regeneration.

INCREASED MYOCARDIAL VIABILITY AND FUNCTION MEASURED BY MANGANESE-ENHANCED MRI (MEMRI) DEMONSTRATE MYOCARDIAL REGENERATION BY HUMAN PLURIPOTENT STEM CELL DERIVED CARDIOMYOCYTES (HPCMS)

background: Human pluripotent stem cell derived cardiomyocytes (hPCMs) hold the potential to regenerate the myocardium and enable restoration. Manganese-enhanced MRI (MEMRI) allows direct evaluation of myocardial viability. Persistent engraftment of the hPCMs associated with viability and LVEF increase suggests regenerative changes. This study evaluates whether the hPCMs generate regenerative changes in the murine model of myocardial injury.

Direct measurement of myocardial viability by manganese-enhanced MRI (MEMRI) tracks the regenerative effects by human pluripotent stem cell derived cardiomyocytes (hPCMs)

Background Human pluripotent stem cell derived cardiomyocytes (hPCMs) may regenerate the myocardium to restore the cardiac function. Manganese-enhanced MRI (MEMRI) enters the cardiomyocytes via calcium channel to generate viability signal directly. Persistent engraftment of the hPCMs associated with increased myocardial viability and LVEF suggests regeneration. This study tests the hypothesis that hPCMs regenerate the injured murine myocardium.