Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish.

Drastic increases in myofiber number and size are essential to support vertebrate post-embryonic growth. However, the collective cellular behaviors that enable these increases have remained elusive. Here, we created the palmuscle myofiber tagging and tracking system for in toto monitoring of the growth and fates of ~5000 fast myofibers in developing zebrafish larvae. Through live tracking of individual myofibers within the same individuals over extended periods, we found that many larval myofibers readily dissolved during development, enabling the on-site addition of new and more myofibers. Remarkably, whole-body surveillance of multicolor-barcoded myofibers further unveiled a gradual yet extensive elimination of larval myofiber populations, resulting in near-total replacement by late juvenile stages. The subsequently emerging adult myofibers are not only long-lasting, but also morphologically and functionally distinct from the larval populations. Furthermore, we determined that the elimination-replacement process is dependent on and driven by the autophagy pathway. Altogether, we propose that the whole-body replacement of larval myofibers is an inherent yet previously unnoticed process driving organismic muscle growth during vertebrate post-embryonic development.

Skin cells undergo asynthetic fission to expand body surfaces in zebrafish.

As an animal's surface area expands during development, skin cell populations must quickly respond to maintain sufficient epithelial coverage. Despite much progress in understanding of skin cell behaviours in vivo1,2, it remains unclear how cells collectively act to satisfy coverage demands at an organismic level. Here we created a multicolour cell membrane tagging system, palmskin, to monitor the entire population of superficial epithelial cells (SECs) in developing zebrafish larvae. Using time-lapse imaging, we found that many SECs readily divide on the animal body surface; during a specific developmental window, a single SEC can produce a maximum of four progeny cells over its lifetime on the surface of the animal. Remarkably, EdU assays, DNA staining and hydroxyurea treatment showed that these terminally differentiated skin cells continue splitting despite an absence of DNA replication, causing up to 50% of SECs to exhibit reduced genome size. On the basis of a simple mathematical model and quantitative analyses of cell volumes and apical surface areas, we propose that 'asynthetic fission' is used as an efficient mechanism for expanding epithelial coverage during rapid growth. Furthermore, global or local manipulation of body surface growth affects the extent and mode of SEC division, presumably through tension-mediated activation of stretch-activated ion channels. We speculate that this frugal yet flexible mode of cell proliferation might also occur in contexts other than zebrafish skin expansion.

Whole-body clonal mapping identifies giant dominant clones in zebrafish skin epidermis.

Skin expansion during development is predominantly driven by growth of basal epithelial cell (BEC)-derived clonal populations, which often display varied sizes and shapes. However, little is known about the causes of clonal heterogeneity and the maximum size to which a single clone can grow. Here, we created a zebrafish model, basebow, for capturing clonal growth behavior in the BEC population on a whole-body, centimeter scale. By tracking 222 BECs over the course of a 28-fold expansion of body surface area, we determined that most BECs survive and grow clonal populations with an average size of 0.013 mm2. An extensive survey of 742 sparsely labeled BECs further revealed that giant dominant clones occasionally arise on specific body regions, covering up to 0.6% of the surface area. Additionally, a growth-induced extracellular matrix component, Lamb1a, mediates clonal growth in a cell-autonomous manner. Altogether, our findings demonstrate how clonal heterogeneity and clonal dominance may emerge to enable post-embryonic growth of a vertebrate organ, highlighting key cellular mechanisms that may only become evident when visualizing single cell behavior at the whole-animal level.

The ethyl acetate fraction of corn silk exhibits dual antioxidant and anti-glycation activities and protects insulin-secreting cells from glucotoxicity.

BACKGROUND: In this study, we aimed to develop a Stigmata Maydis (corn silk) fraction with dual bio-activities against oxidative stress and protein glycation to protect ß-cells from diabetes-induced failure. METHODS: Corn silk fractions were prepared by partition and chemically characterised by thin-layer chromatography. Free radical scavenging assay, glycation assay, and cell-based viability test (neutral red) were employed to decide the best fraction. Cell death analysis was executed by annexin V/ Propidium iodide staining. Cell proliferation was measured by WST-1. Finally, ß-cell function was evaluated by ß-cell marker gene expression (RT-PCR) and acute insulin secretion test. RESULTS: Four corn silk fractions were prepared from an ethanolic crude extract of corn silk. In vitro assays indicate ethyl acetate fraction (YMS-EA) was the most potent fraction. YMS-EA also attenuated the hydrogen peroxide- or methylglyoxal-induced induction of reactive oxygen species, reduction of cell viability, and inhibition of cell proliferation. However, YMS-EA was unable to prevent hydrogen peroxide-induced apoptosis or advanced glycation end-products-induced toxicity. Under hyperglycemic conditions, YMS-EA effectively reduced ROS levels, improved mRNA expression of insulin, glucokinase, and PDX-1, and enhanced glucose-stimulated insulin secretion. The similarity of bioactivities among apigenin, luteolin, and YMS-EA indicated that dual activities of YMS-EA might be derived from those compounds. CONCLUSIONS: We concluded that YMS-EA fraction could be developed as a preventive food agent against the glucotoxicity to ß-cells in Type 2 diabetes.

Appendage-resident epithelial cells expedite wound healing response in adult zebrafish.

Adult zebrafish are able to heal large-sized cutaneous wounds in hours with little to no scarring. This rapid re-epithelialization is crucial for preventing infection and jumpstarting the subsequent regeneration of damaged tissues. Despite significant progress in understanding this process, it remains unclear how vast numbers of epithelial cells are orchestrated on an organismic scale to ensure the timely closure of millimeter-sized wounds. Here, we report an unexpected role of adult zebrafish appendages (fins) in accelerating the re-epithelialization process. Through whole-body monitoring of single-cell dynamics in live animals, we found that fin-resident epithelial cells (FECs) are highly mobile and migrate to cover wounds in nearby body regions. Upon injury, FECs readily undergo organ-level mobilization, allowing for coverage of body surfaces of up to 4.78 mm2 in less than 8 h. Intriguingly, long-term fate-tracking experiments revealed that the migratory FECs are not short-lived at the wound site; instead, the cells can persist on the body surface for more than a year. Our experiments on "fin-less" and "fin-gaining" individuals demonstrated that the fin structures are not only capable of promoting rapid re-epithelialization but are also necessary for the process. We further found that fin-enriched extracellular matrix laminins promote the active migration of FECs by facilitating lamellipodia formation. These findings lead us to conclude that appendage structures in regenerative vertebrates, such as fins, may possess a previously unrecognized function beyond serving as locomotor organs. The appendages may also act as a massive reservoir of healing cells, which speed up wound closure and tissue repair.

Role of S-Palmitoylation by ZDHHC13 in Mitochondrial function and Metabolism in Liver.

Palmitoyltransferase (PAT) catalyses protein S-palmitoylation which adds 16-carbon palmitate to specific cysteines and contributes to various biological functions. We previously reported that in mice, deficiency of Zdhhc13, a member of the PAT family, causes severe phenotypes including amyloidosis, alopecia, and osteoporosis. Here, we show that Zdhhc13 deficiency results in abnormal liver function, lipid abnormalities, and hypermetabolism. To elucidate the molecular mechanisms underlying these disease phenotypes, we applied a site-specific quantitative approach integrating an alkylating resin-assisted capture and mass spectrometry-based label-free strategy for studying the liver S-palmitoylome. We identified 2,190 S-palmitoylated peptides corresponding to 883 S-palmitoylated proteins. After normalization using the membrane proteome with TMT10-plex labelling, 400 (31%) of S-palmitoylation sites on 254 proteins were down-regulated in Zdhhc13-deficient mice, representing potential ZDHHC13 substrates. Among these, lipid metabolism and mitochondrial dysfunction proteins were overrepresented. MCAT and CTNND1 were confirmed to be specific ZDHHC13 substrates. Furthermore, we found impaired mitochondrial function in hepatocytes of Zdhhc13-deficient mice and Zdhhc13-knockdown Hep1-6 cells. These results indicate that ZDHHC13 is an important regulator of mitochondrial activity. Collectively, our study allows for a systematic view of S-palmitoylation for identification of ZDHHC13 substrates and demonstrates the role of ZDHHC13 in mitochondrial function and metabolism in liver.