Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

Epigenetic silencing of selected hypothalamic neuropeptides in narcolepsy with cataplexy.

Narcolepsy with cataplexy is a sleep disorder caused by deficiency in the hypothalamic neuropeptide hypocretin/orexin (HCRT), unanimously believed to result from autoimmune destruction of hypocretin-producing neurons. HCRT deficiency can also occur in secondary forms of narcolepsy and be only temporary, suggesting it can occur without irreversible neuronal loss. The recent discovery that narcolepsy patients also show loss of hypothalamic (corticotropin-releasing hormone) CRH-producing neurons suggests that other mechanisms than cell-specific autoimmune attack, are involved. Here, we identify the HCRT cell-colocalized neuropeptide QRFP as the best marker of HCRT neurons. We show that if HCRT neurons are ablated in mice, in addition to Hcrt, Qrfp transcript is also lost in the lateral hypothalamus, while in mice where only the Hcrt gene is inactivated Qrfp is unchanged. Similarly, postmortem hypothalamic tissues of narcolepsy patients show preserved QRFP expression, suggesting the neurons are present but fail to actively produce HCRT. We show that the promoter of the HCRT gene of patients exhibits hypermethylation at a methylation-sensitive and evolutionary-conserved PAX5:ETS1 transcription factor-binding site, suggesting the gene is subject to transcriptional silencing. We show also that in addition to HCRT, CRH and Dynorphin (PDYN) gene promoters, exhibit hypermethylation in the hypothalamus of patients. Altogether, we propose that HCRT, PDYN, and CRH are epigenetically silenced by a hypothalamic assault (inflammation) in narcolepsy patients, without concurrent cell death. Since methylation is reversible, our findings open the prospect of reversing or curing narcolepsy.

Spastin mutations impair coordination between lipid droplet dispersion and reticulum.

Lipid droplets (LD) are affected in multiple human disorders. These highly dynamic organelles are involved in many cellular roles. While their intracellular dispersion is crucial to ensure their function and other organelles-contact, underlying mechanisms are still unclear. Here we show that Spastin, one of the major proteins involved in Hereditary Spastic Paraplegia (HSP), controls LD dispersion. Spastin depletion in zebrafish affects metabolic properties and organelle dynamics. These functions are ensured by a conserved complex set of splice variants. M1 isoforms determine LD dispersion in the cell by orchestrating endoplasmic reticulum (ER) shape along microtubules (MTs). To further impact LD fate, Spastin modulates transcripts levels and subcellular location of other HSP key players, notably Seipin and REEP1. In pathological conditions, mutations in human Spastin M1 disrupt this mechanism and impacts LD network. Spastin depletion influences not only other key proteins but also modulates specific neutral lipids and phospholipids, revealing an impact on membrane and organelle components. Altogether our results show that Spastin and its partners converge in a common machinery that coordinates LD dispersion and ER shape along MTs. Any alteration of this system results in HSP clinical features and impacts lipids profile, thus opening new avenues for novel biomarkers of HSP.

Skeletal muscle regeneration after extensive cryoinjury of caudal myomeres in adult zebrafish.

Skeletal muscles can regenerate after minor injuries, but severe structural damage often leads to fibrosis in mammals. Whether adult zebrafish possess the capacity to reproduce profoundly destroyed musculature remains unknown. Here, a new cryoinjury model revealed that several myomeres efficiently regenerated within one month after wounding the zebrafish caudal peduncle. Wound clearance involved accumulation of the selective autophagy receptor p62, an immune response and Collagen XII deposition. New muscle formation was associated with proliferation of Pax7 expressing muscle stem cells, which gave rise to MyoD1 positive myogenic precursors, followed by myofiber differentiation. Monitoring of slow and fast muscles revealed their coordinated replacement in the superficial and profound compartments of the myomere. However, the final boundary between the muscular components was imperfectly recapitulated, allowing myofibers of different identities to intermingle. The replacement of connective with sarcomeric tissues required TOR signaling, as rapamycin treatment impaired new muscle formation, leading to persistent fibrosis. The model of zebrafish myomere restoration may provide new medical perspectives for treatment of traumatic injuries.

BCL2L13 at endoplasmic reticulum-mitochondria contact sites regulates calcium homeostasis to maintain skeletal muscle function.

The physical connection between mitochondria and endoplasmic reticulum (ER) is an essential signaling hub to ensure organelle and cellular functions. In skeletal muscle, ER-mitochondria calcium (Ca2+) signaling is crucial to maintain cellular homeostasis during physical activity. High expression of BCL2L13, a member of the BCL-2 family, was suggested as an adaptive response in endurance-trained human subjects. In adult zebrafish, we found that the loss of Bcl2l13 impairs skeletal muscle structure and function. Ca2+ signaling is altered in Bcl2l13 knockout animals and mitochondrial complexes activity is decreased. Organelle fractioning in mammalian cells shows BCL2L13 at mitochondria, ER, and mitochondria-associated membranes. ER-mitochondria contact sites number is not modified by BCL2L13 modulation, but knockdown of BCL2L13 in C2C12 cells changes cytosolic Ca2+ release and mitochondrial Ca2+ uptake. This suggests that BCL2L13 interaction with mitochondria and ER, and its role in Ca2+ signaling, contributes to proper skeletal muscle function.

Methodological advancements in organ-specific ectopic lipid quantitative characterization: Effects of high fat diet on muscle and liver intracellular lipids.

OBJECTIVE: Ectopic lipid accumulation is a hallmark of metabolic diseases, linking obesity to non-alcoholic fatty liver disease, insulin resistance and diabetes. The use of zebrafish as a model of obesity and diabetes is raising due to the conserved properties of fat metabolism between humans and zebrafish, the homologous genes regulating lipid uptake and transport, the implementation of the '3R's principle and their cost-effectiveness. To date, a method allowing the conservation of lipid droplets (LDs) and organs in zebrafish larvae to image ectopic lipids is not available. Our objectives were to develop a novel methodology to quantitatively evaluate organ-specific LDs, in skeletal muscle and liver, in response to a nutritional perturbation. METHODS: We developed a novel embedding and cryosectioning protocol allowing the conservation of LDs and organs in zebrafish larvae. To establish the quantitative measures, we used a three-arm parallel nutritional intervention design. Zebrafish larvae were fed a control diet containing 14% of nutritional fat or two high fat diets (HFDs) containing 25 and 36% of dietary fats. In muscle and liver, LDs were characterized using immunofluorescence confocal microscopy. In liver, intrahepatocellular lipids were discriminated from intrasinusoid lipids. To complete liver characteristics, fibrosis was identified with Masson's Trichrome staining. Finally, to confirm the conservation and effect of HFD, molecular players of fat metabolism were evaluated by RT-qPCR. RESULTS: The cryosections obtained after setting up the embedding and cryopreservation method were of high quality, preserving tissue morphology and allowing the visualization of ectopic lipids. Both HFDs were obesogenic, without modifying larvae survival or development. Neutral lipid content increased with time and augmented dietary fat. Intramuscular LD volume density increased and was explained by an increase in LDs size but not in numbers. Intrahepatocellular LD volume density increased and was explained by an increased number of LDs, not by their increased size. Sinusoid area and lipid content were both increased. Hepatic fibrosis appeared with both HFDs. We observed alterations in the expression of genes associated with LD coating proteins, LD dynamics, lipogenesis, lipolysis and fatty acid oxidation. CONCLUSIONS: In this study, we propose a reproducible and fast method to image zebrafish larvae without losing LD quality and organ morphology. We demonstrate the impact of HFD on LD characteristics in liver and skeletal muscle accompanied by alterations of key players of fat metabolism. Our observations confirm the evolutionarily conserved mechanisms in lipid metabolism and reveal organ specific adaptations. The methodological advancements proposed in this work open the doors to study organelle adaptations in obesity and diabetes related research such as lipotoxicity, organelle contacts and specific lipid depositions.

Mitochondria in Embryogenesis: An Organellogenesis Perspective.

Organogenesis is well characterized in vertebrates. However, the anatomical and functional development of intracellular compartments during this phase of development remains unknown. Taking an organellogenesis point of view, we characterize the spatiotemporal adaptations of the mitochondrial network during zebrafish embryogenesis. Using state of the art microscopy approaches, we find that mitochondrial network follows three distinct distribution patterns during embryonic development. Despite of this constant morphological change of the mitochondrial network, electron transport chain supercomplexes occur at early stages of embryonic development and conserve a stable organization throughout development. The remodeling of the mitochondrial network and the conservation of its structural components go hand-in-hand with somite maturation; for example, genetic disruption of myoblast fusion impairs mitochondrial network maturation. Reciprocally, mitochondria quality represents a key factor to determine embryonic progression. Alteration of mitochondrial polarization and electron transport chain halts embryonic development in a reversible manner suggesting developmental checkpoints that depend on mitochondrial integrity. Our findings establish the subtle dialogue and co-dependence between organogenesis and mitochondria in early vertebrate development. They also suggest the importance of adopting subcellular perspectives to understand organelle-organ communications during embryogenesis.