Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. Here we show that Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles, and co-localize with them in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. We propose a novel function for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins.

SUMMARY The heart has the ability to grow in size in response to exercise, but little is known about the transcriptional mechanisms underlying physiological hypertrophy. Adult cardiomyocytes have also recently been proven to hold the potential for proliferation, a process which could be of great importance for regenerative medicine. Using a unique RT-PCR based screen against all transcriptional components, we showed that C/EBPβ was down-regulated with exercise, while the expression of CITED4 was increased. Reduction of C/EBPβ in vitro and in vivo resulted in a phenocopy of endurance exercise with cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at least in part by the increased CITED4. Importantly, mice with reduced cardiac C/EBPβ levels displayed substantial resistance to cardiac failure upon pressure overload. These data indicate that C/EBPβ represses cardiomyocyte growth and proliferation in the adult mammalian heart and that reduction in C/EBPβ is a central signal in physiologic hypertrophy and proliferation.

SUMMARYNatural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.

The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.

Electrical gradients are critical for many biological processes, including the normal function of excitable tissues, left-right patterning, organogenesis, and wound healing1–4. The fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood. Using high-speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart, we have identified a gradient of electrical coupling across the developing ventricular myocardium. We excluded a role for cellular excitability, connexin localization, tissue geometry and mechanical inputs, but in contrast we were able to demonstrate that non-canonical Wnt11 signals are required for the genesis of this myocardial electrical gradient. Importantly, though the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca2+ conductance mediated via the L-type calcium channel. These data reveal a previously unrecognized role for Wnt/Ca2+ signaling in establishing an electrical gradient in the plane of developing cardiac epithelium through modulation of ion channel function. The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Here we show that Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens.

Background: Dissociation of the Wnt/β-catenin pathway effector Dishevelled from its complex with nucleoredoxin is a redox-sensitive process, yet the ROS sources remain elusive.Results: Mitochondrial Ca2+ influx stimulates endogenous ROS production and mediates Wnt/β-catenin pathway activity.Conclusion: Ca2+-mediated ROS production modulates the signaling efficiency of the Wnt/β-catenin pathway.Significance: Metabolic states influence fundamental and developmental signaling to drive cell differentiation.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.