During metamorphosis, the wings of Drosophila form from the wing imaginal discs of the larva and acquire a highly specific set of sensory neurons. The developing neurons leave the cell cycle, ...differentiate, and grow their axons in a fixed order. The axons follow stereotyped paths through nonneural tissue, fasciculate together, and ultimately reach the CNS, where they form regular arborizations. Neurons differentiating ectopically (in mutant flies) grow irregularly and often away from the CNS. We are analyzing the factors that generate order and appropriate polarity along the normal nerve paths; lack of order and of consistent polarity prevail elsewhere.
We have utilized enhancer trap markers to follow the development of ectopic sensillar precursors in the wings of Drosophila induced by the mutations hairy and Hairy wing. Normal sensilla are still ...present in these mutations, and can be distinguished from ectopic sensilla based upon both the position and the timing of their development. This correlates well with the development of ectopic achaete expression in these mutations: such staining is detected only after the appearance of normal staining. Thus, neither mutation appears to alter the specification of proneural clusters in the wing, as identified with anti-achaete, or the specification of sensillar precursors within these clusters. Rather, both act to induce the formation of a temporally and spatially distinct phase of sensillar development.
The development of new, adult-specific axonal pathways in the central nervous system (CNS) of insects during metamorphosis is still largely uncharacterized. Here we used axonal labeling with DiI to ...describe the timing and pattern of growth of sensory axons originating in the wing of Drosophila as they establish their adult projection pattern in the CNS during pupal life. The wing of Drosophila carries a small number of readily identifiable sensory organs (sensilla) whose neurons are located in the periphery and whose axons travel along specific routes within the adult CNS. The neurons are born and undergo axonogenesis in a characteristic order. The order of axon arrival in the CNS appears to be the same as that of their development in the periphery. Within the CNS, the formation of four prominent axon bundles leading to distant termination sites is followed by the formation of a compact axon termination site near the point of wing nerve entry into the CNS. This sensillum-specific pattern persists into adulthood without discernible modification. We also find a small number of axons filled with DiI prior to the formation of the four permanent bundles. We have only been able to fill them for a few hours in early pupal life and therefore consider them to be transient. The bundles of wing sensory axons travel within tracts that contain other axons as well. Using immunocytochemistry, the tracts start to be histologically identifiable at around 12 h after pupariation (AP), and grow substantially as metamorphosis proceeds. Wing sensory neurons are found in the tracts by 18-20 h AP and the full adult pattern is established by 48 h AP. When sensory axons first enter the CNS, they fan out in the region where their appropriate tracts are located, but they do not wander extensively. They quickly form bundles that become increasingly compact over time. Calculations show that the rate of axon extension within the CNS varies from bundle to bundle and is equal to or greater than that of the same axons growing through wing tissue.
Correlated anatomical and electrophysiological results demonstrate that sensory neurons, which differentiate de novo within the epidermis of regenerate abdominal cerci of crickets, enter the terminal ...ganglion and form functional central connections even when regeneration of the cerci is delayed through the greater part of postembryonic development. Stimulation of regenerate cerci evokes activity in giant interneurons which is normal by several physiological criteria.
In most studies of axon guidance in the peripheral tissues of insects, the ability of experimentally perturbed axons to pathfind was examined only along their normal pathways. This means that regions ...normally devoid of axons have not been sampled for their ability to influence axonal trajectories. To examine this question, we have induced the formation of single sensory neurons in a variety of abnormal locations in the developing wing of Drosophila and have examined the course taken by their axons. The axons of such ectopic neurons have a regionally varying tendency to grow in the normal, proximal direction. This proximal bias approaches 100% for neurons located in the distal part of vein L2 and 70% in distal vein L4 but falls to chance (50%) along vein L5. Thus, neurons forming in ectopic regions of the wing, especially those found near the normal axon pathways (veins L1 and L3), have a high probability of growing axons in the correct direction. We conclude that information relevant to axon outgrowth is not restricted to the normal pathways. Whether this information is intrinsic or extrinsic to the neurons, and why its strength shows such conspicuous regional variation, awaits further study.
Abstract Promising results are emerging in clinical trials focused on stem cell therapy for cardiology applications. However, the low homing and engraftment of the injected cells to target tissue ...continues to be a problem. Cellular glycoengineering can address this limitation by enabling the targeting of stem cells to sites of vascular injury/inflammation. Two such glycoengineering methods are presented here: i. The non-covalent incorporation of a P-selectin glycoprotein ligand-1 (PSGL-1) mimetic 19FcFUT7+ via lipid-protein G fusion intermediates that intercalate onto the cell surface, and ii. Over-expression of the α(1,3)fucosyltransferse FUT7 in cells. Results demonstrate the efficient coupling of 19FcFUT7+ onto both cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs), with coupling being more efficient when using protein G fused to single-tailed palmitic acid rather than double-tailed DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). This non-covalent cellular modification was mild since cell proliferation and stem-cell marker expression was unaltered. Whereas coupling using 19FcFUT7+ enhanced cell capture on recombinant P-selectin or CHO-P cell surfaces, α(1,3)fucosylation was necessary for robust binding to E-selectin and inflamed endothelial cells under shear. Pilot studies confirm the safety and homing efficacy of the modified stem cells to sites of ischemia-reperfusion in the porcine heart. Overall, glycoengineering with physiological selectin-ligands may enhance stem cell engraftment.
Abstract Background The time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and ...molecular remodeling may also play important roles. Objectives This study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction. Methods Swine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes. Results At 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post–percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit+ cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted. Conclusions Delayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.
Objective
We recently demonstrated that swine subjected to 2‐weeks of intermittent repetitive pressure overload (RPO) develop cardiac phenotypic characteristics of heart failure with preserved ...ejection fraction (HFpEF), including a reduction in left ventricular (LV) diastolic compliance, myocyte hypertrophy, and interstitial fibrosis without an increase in LV mass. The objective of the present study was to determine if this pattern of myocardial remodeling is accompanied by alterations in coronary microvascular function or structure that contribute to an impaired vasodilator response to adenosine (ADO).
Methods
Regional myocardial blood flow (BF) was assessed via administration of fluorescent microspheres at rest and during ADO vasodilation in swine subjected to 2‐weeks of RPO (300 µg/min phenylephrine (PE), 30 min/day; n=5) and normal controls (n=5). Adenosine (ADO)‐mediated vasodilation was initially assessed at a submaximal dose (0.15 mg/kg/min), followed by a maximal vasodilating dose (0.90 mg/kg/min) with concomitant PE to maintain arterial blood pressure. Post‐mortem histological evaluation of myocardial tissue samples was performed in separate groups of swine subjected to 2‐weeks of RPO (n=8) and normal controls (n=5) to assess arteriolar density (α‐smooth muscle actin staining) and structural remodeling (wall thickness/lumen diameter ratio).
Results
Submaximal ADO produced a significant ~60% rise in myocardial BF in normal controls, but this response was completely attenuated in the RPO group (Figure; A). The impaired vasodilator response to submaximal ADO in the RPO group was not explained by hemodynamic factors, as heart rate (RPO vs. control: 102±5 vs. 95±4 beats/min; p=0.29) and mean arterial pressure (71±3 vs. 67±5 mmHg; p=0.55) were similar between groups. However, group differences in ADO‐induced vasodilation were abolished by administration of maximal ADO, which elicited a comparable ~4‐fold rise in myocardial BF in normal controls and the RPO group. Arteriolar density (Figure; B) and arteriolar wall thickness/lumen diameter (Figure; C) were not different between groups, further supporting abnormal microvascular function as the primary mechanism underlying the impaired response to submaximal ADO after RPO.
Conclusions
These results indicate that RPO‐induced HFpEF is characterized by an impairment in ADO‐mediated coronary vasodilation that may compromise myocardial perfusion during episodes of increased metabolic demand. The absence of histopathological evidence of arteriolar structural remodeling along with a preserved vasodilator response to maximal ADO indicate that a functional abnormality is primarily responsible for the impaired responsiveness to ADO in this setting. Further studies are necessary to evaluate the contribution of endothelium‐dependent vs. endothelium‐independent factors, as well as whether therapeutic interventions targeting RPO‐induced coronary microvascular dysfunction may prevent or reverse adverse myocardial remodeling and diastolic dysfunction in HFpEF.
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