Enrique Salas-Vidal

Function of reactive oxygen species during animal development : Passive or active?

Oxidative stress is considered causal of aging and pathological cell death, however, very little is known about its function in the natural processes that support the formation of an organism. It is generally thought that cells must continuously protect themselves from the possible damage caused by reactive oxygen species (ROS) (passive ROS function). However, presently, ROS are recognized as physiologically relevant molecules that mediate cell responses to a variety of stimuli, and the activities of several molecules, some developmentally relevant, are directly or indirectly regulated by oxidative stress (active ROS function). Here we review recent data that are suggestive of specific ROS functions during development of animals, particularly mammals.

Genetic and Transcriptome Characterization of Model Zebrafish Cell Lines

Compared with the increasing use of zebrafish as a model organism in many laboratories, zebrafish cell lines are still unexploited and limited in application, partly due to their unknown genetic and physiological properties. We characterize two zebrafish embryonic fibroblast cell lines, ZF4 and PAC2. We demonstrate the genetic stability of these two zebrafish cell lines and achieved genetic manipulation by either lipid-mediated transfection or an electroporation- based nucleofection method. Data from zebrafish chip analysis (Affymetrix) demonstrate unique characteristics of these two cell lines in gene expression levels, showing that different zebrafish cell lines can be classified by their transcriptome profile. Their transcriptional responses to serum growth factor exposure suggest that zebrafish fibroblast cell lines may be used to study processes related to wound-healing or cancer.

Cytotoxic Podophyllotoxin Type-Lignans from the Steam Bark of Bursera fagaroides var. fagaroides

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.

Cell Proliferation Patterns in Early Zebrafish Development

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. Anat Rec, 296:759–773, 2013.

Aryldihydronaphthalene-type lignans from Bursera fagaroides var. fagaroides and their antimitotic mechanism of action

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.

Spatial and temporal expression of zebrafish glutathione peroxidase 4 a and b genes during early embryo development.

Antioxidant cellular mechanisms are essential for cell redox homeostasis during animal development and in adult life. Previous in situ hybridization analyses of antioxidant enzymes in zebrafish have indicated that they are ubiquitously expressed. However, spatial information about the protein distribution of these enzymes is not available. Zebrafish embryos are particularly suitable for this type of analysis due to their small size, transparency and fast development. The main objective of the present work was to analyze the spatial and temporal gene expression pattern of the two reported zebrafish glutathione peroxidase 4 (GPx4) genes during the first day of zebrafish embryo development. We found that the gpx4b gene shows maternal and zygotic gene expression in the embryo proper compared to gpx4a that showed zygotic gene expression in the periderm covering the yolk cell only. Following, we performed a GPx4 protein immunolocalization analysis during the first 24-h of development. The detection of this protein suggests that the antibody recognizes GPx4b in the embryo proper during the first 24 h of development and GPx4a at the periderm covering the yolk cell after 14-somite stage. Throughout early cleavages, GPx4 was located in blastomeres and was less abundant at the cleavage furrow. Later, from the 128-cell to 512-cell stages, GPx4 remained in the cytoplasm but gradually increased in the nuclei, beginning in marginal blastomeres and extending the nuclear localization to all blastomeres. During epiboly progression, GPx4b was found in blastoderm cells and was excluded from the yolk cell. After 24 h of development, GPx4b was present in the myotomes particularly in the slow muscle fibers, and was excluded from the myosepta. These results highlight the dynamics of the GPx4 localization pattern and suggest its potential participation in fundamental developmental processes.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

Cytotoxic Activity and Chemical Composition of the Root Extract from the Mexican Species Linum scabrellum: Mechanism of Action of the Active Compound 6-Methoxypodophyllotoxin

The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC50 = 0.57 μg/mL), MCF7 breast (IC50 = 0.56 μg/mL), PC3 prostate (IC50 = 1.60 μg/mL), and SiHa cervical (IC50 = 1.54 μg/mL), as well as toward the normal fibroblasts line HFS-30 IC50 = 1.02 μg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC50 value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant K b = 7.6 × 106 M−1). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.

PIAS-like protein Zimp7 is required for the restriction of the zebrafish organizer and mesoderm development

The Zmiz2 (Zimp7) protein and its homolog Zmiz1 (Zimp10) were initially identified in humans as androgen receptor co-activators. Sequence analysis revealed the presence of an SP-RING/Miz domain, which is highly conserved in members of the PIAS family and confers SUMO-conjugating activity. Zimp7 has been shown to interact with components of the Wnt/β-Catenin signaling pathway and with Brg1 and BAF57, components of the ATP-dependent mammalian SWI/SNF-like BAF chromatin-remodeling complexes. In this work, we analyze the role of zygotic Zimp7 in zebrafish development. We describe evidence indicating that Zimp7 is required for mesoderm development and dorsoventral patterning. Morpholino-mediated reduction of zygotic Zimp7 produced axial mesodermal defects that were preceded by up-regulation of organizer genes such as bozozok, goosecoid and floating head at the onset of gastrulation and by down-regulation of the ventral markers vox, vent and eve1 indicating loss of the ventrolateral mesoderm. Consistently, embryos overexpressing zimp7 RNA exhibited midline defects such as loss of forebrain and cyclopia accompanied by transcriptional changes directly opposite of those found in the morphants. In addition, the patterning of ventralized embryos produced by the overexpression of vox and vent was restored by a reduction of Zimp7 activity. Altogether, our findings indicate that Zimp7 is involved in transcriptional regulation of factors that are essential for patterning in the dorsoventral axis.

NADPH-Oxidase-derived reactive oxygen species are required for cytoskeletal organization, proper localization of E-cadherin and cell motility during zebrafish epiboly

ABSTRACT 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. Graphical abstract Figure. No Caption available. HighlightsHigh levels of reactive oxygen species (ROS) are formed during zebrafish gastrulation.ROS show dynamic localization at the interstitial space among blastoderm cells.Nox inhibition disturbs the cytoskeleton and E‐cadherin localization, important for proper cell motility during epiboly.Effects of Nox inhibition on epiboly are rescued by hydrogen peroxide treatment.Hydrogen peroxide treated embryos advance earlier in epiboly compared to control embryos.