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Cell movements are essential for morphogenesis during animal development. Epiboly is the first morphogenetic process in zebrafish in which cells move en masse to thin and spread the deep and enveloping cell layers of the blastoderm over the yolk cell. While epiboly has been shown to be controlled by complex molecular networks, the contribution of reactive oxygen species (ROS) to this process has not previously been studied. Here, we show that ROS are required for epiboly in zebrafish. Visualization of ROS in whole embryos revealed dynamic patterns during epiboly progression. Significantly, inhibition of NADPH oxidase activity leads to a decrease in ROS formation, delays epiboly, alters E-cadherin and cytoskeleton patterns and, by 24 h post-fertilization, decreases embryo survival, effects that are rescued by hydrogen peroxide treatment. Our findings suggest that a delicate ROS balance is required during early development and that disruption of that balance interferes with cell adhesion, leading to defective cell motility and epiboly progression.

Tissue remodelling and organ shaping during morphogenesis are products of mechanical forces generated at the cellular level. These cell-scale forces can be coordinated across the tissue via information provided by biochemical and mechanical cues. Such coordination leads to the generation of complex tissue shape during morphogenesis. In this short review, we elaborate the role of cellular active stresses in vertebrate axis morphogenesis, primarily using examples from postgastrulation development of the zebrafish embryo.

Significance How do tissues self-organize to generate the complex organ shapes observed in vertebrates? Organ formation requires the integration of chemical and mechanical information, yet how this is achieved is poorly understood for most organs. Muscle compartments in zebrafish display a V shape, which is believed to be required for efficient swimming. We investigate how this structure emerges during zebrafish development, combining live imaging and quantitative analysis of cellular movements. We use theoretical modeling to understand how cell differentiation and mechanical interactions between tissues guide the emergence of a specific tissue morphology. Our work reveals how spatially modulating the mechanical environment around and within tissues can lead to complex organ shape formation., Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Three new aryldihydronaphthalene-type lignans, namely, 7′,8′-dehydropodophyllotoxin (1); 7′,8′-dehydro acetylpodophyllotoxin (2); and 7′,8′-dehydro trans-p-cumaroylpodophyllotoxin (3), were isolated from the stem bark of Bursera fagaroides var. fagaroides (Burseraceae), together with six known lignans, podophyllotoxin (4), acetylpodophyllotoxin (5), 5′-desmethoxy-β-peltatin A methylether (6), acetylpicropodophyllotoxin (7), burseranin (8), and hinokinin (9). The coumarin scopoletin (10) was also isolated from this bark. The chemical structure of all these compounds was determined by spectroscopic analyses including 2D NMR. We demonstrated that compounds 1–3 show different degrees of cytotoxic activity against human nasopharyngeal (KB), colon (HF-6), breast (MCF-7) and prostate (PC-3) cancer cell lines, with IC50 values ranging from 1.49 to 1.0 × 10−5 μM. In vivo studies of the effect of these natural lignans on the cell cycle, cell migration and microtubule cytoskeleton of developing zebrafish embryos, demonstrated their antimitotic molecular activity by disturbing tubulin. This is the first report on the occurrence of aryldihydronaphthalene lignans in the genus Bursera of the Burseraceae family, as well as on the determination of their cytotoxic activity and mechanism of action.

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), &beta, peltatin-A-methylether (2), 5&prime, desmethoxy-&beta, peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1&ndash, 7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2- to 4-cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo "waves" described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish.

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED50 = 9.6 × 10(-2) μg/mL), PC-3 (ED50 = 2.5 × 10(-1) μg/mL), MCF-7 (ED50 = 6.6 μg/mL), and HF-6 (ED50 = 7.1 × 10(-3) μg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5'-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED50= 1.0 × 10(-5) μg/mL), and KB (ED50 = 1.0 × 10(-5) μg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2- to 4-cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo "waves" described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish.