Applying Rho Pathway Inhibitors to Investigate Germ Plasm Localization.

The correct assembly, migration, and segregation of the mRNAs of the germ plasm during the first cell divisions are intimately connected to the cytoskeleton and cytokinesis.RhoA is a key regulator of germ plasm localization during the first two cell division cycles in zebrafish embryos. Pharmacological inhibition of RhoA and his effector ROCK affected the correct assembly of microtubules in the cleavage furrow with the concomitant abnormal localization of germ plasm mRNAs. The inhibition of RhoA/ROCK pathway caused a significant decrease in the germ cell population later in development.

Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

Protein Purification and Western Blot Detection from Single Zebrafish Embryo.

Characterization of a protein of interest during development is essential for functional studies. A general strategy for understanding the function of a particular protein involves the generation of null mutations, or treatment with drugs, that interfere with its activity. To demonstrate that the synthesis, stability, or activity of a protein has been affected, accurate and efficient detection of low amounts of protein is essential. This can be achieved by immunohistochemistry or by western blot. Here we describe a method for the detection of proteins from single de-yolked zebrafish embryos. This procedure includes a fixation step and the concomitant elimination of lipids from the yolk cell. We show that this approach allows the rapid analysis of proteins in embryos without having to manually remove the yolk. This method provides a convenient alternative for genotyping of mutant embryos as early as the 128 cell stage. In addition, in drug- or morpholino-treated embryos, the correlation between the penetrance of a phenotype and the concentration of a protein present may be established.

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

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.

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.

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.

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/ß-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.

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.

Swimming suppresses hepatic vitellogenesis in European female silver eels as shown by expression of the estrogen receptor 1, vitellogenin1 and vitellogenin2 in the liver.

BACKGROUND: When European silver eels (Anguilla anguilla) venture into the Atlantic Ocean for their 6,000 km semelparous spawning run to the Sargasso Sea, they are still in a prepubertal stage. Further sexual development appears to be blocked by dopaminergic inhibition of hypothalamus and pituitary activity. Recently, we found that swimming for several weeks in freshwater stimulated the incorporation of fat droplets in the oocytes. So, it was hypothesized that long term swimming in seawater would release the inhibition further and would also stimulate the production of vitellogenin by the liver. METHODS: For this study a swim-flume was constructed to allow simulated migration of migratory female silver eels for 3 months (1,420 km) in natural seawater at 20 degrees C. Primers were designed for polymerase chain reactions to measure the mRNA expression of estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) genes in the liver of European female silver eels. RESULTS: In comparison to resting eels, swimming eels showed a diminished expression of esr1, vtg1 and vtg2 in the liver. They also had lower plasma calcium (Ca; indicative of vitellogenin) levels in their blood. This showed that vitellogenesis is more strongly suppressed in swimming than in resting eels. However, when eels were subsequently stimulated by 3 weekly carp pituitary extract injections, the expression of the same genes and plasma levels of Ca strongly increased in both groups to similar levels, thus equalizing the initial differences between resting and swimming. CONCLUSIONS: It is concluded that vitellogenesis remains suppressed during resting and even more during swimming. The fact that swimming stimulates fat deposition in the oocytes but suppresses vitellogenesis indicates that these events are separated in nature and occur sequentially. Swimming-suppressed vitellogenesis may imply that in nature eels undergo vitellogenesis and final maturation near or at the spawning grounds.

Sexually dimorphic gene expression of the Zimp7 and Zimp10 genes in embryonic gonads.

Members of the PIAS (protein inhibitor of activated STAT) family perform essential functions in modulating the activity of transcriptional regulators. Zimp7 and Zimp10 are two proteins that together form a subfamily of the PIAS. Like the other members of this family, they contain the zinc-binding SP-RING/Miz domain, which confers SUMO-conjugating activity. Both proteins have been shown to stimulate androgen receptor-mediated transcription. Previously, we reported that both Zimp7 and Zimp10 genes are extensively expressed and dynamically regulated in the developing mouse embryo. In this work, we investigated the expression of these genes during gonadal development. We found that their expression is sex-specific. Both genes initiate their transcription at early stages in the embryonic male gonad, reaching their peak at 13.5days post coitum, which coincides with the process of sex-specific germ cell mitotic arrest. Zimp7 is expressed in germ cells of the embryonic gonad and the adult testis. Immunofluorescence of spermatogenic cells revealed that Zimp7 protein localizes to nuclear territories in meiotic spermatocytes, including the XY bodies. On the other hand, Zimp10 is found in somatic cells, outside the testis cords and ceases to be expressed in the adult testis.

Temporal expression of hepatic estrogen receptor 1, vitellogenin1 and vitellogenin2 in European silver eels.

Because European silver eels have never been caught during or after their 6000-km reproductive migration to the Sargasso Sea, all existing knowledge on their sexual maturation comes from hormonal stimulation. Silver eels that start their oceanic migration are still immature with pre-vitellogenic oocytes. Hence we assumed that vitellogenesis should start with the expression of the estrogen receptor in the liver before the circulating 17beta-estradiol (E2) can have any effect. In this study we followed the hepatic vitellogenesis upon 4 weekly injections with carp pituitary extracts (CPE). New molecular primers for the expression of the estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) in the liver were developed. Sequences of vtg2 and esr1 were not previously described in Anguilla anguilla. All eels showed weekly increase of the eye size and pectoral fin length, which are signs of early maturation. The same occurred with the gonadosomatic index, the oocyte stage and diameter, and number of deposited fat droplets. Early vitellogenesis appeared as a 3-step process (1) E2-levels and esr1 expression were significantly increased already after one injection, (2) vtg1 and vtg2 expression were significantly increased after one and two injections, respectively, and (3) vtg1 and vtg2 expression increased further after three and four injections. Then also plasma calcium (corresponds with plasma vitellogenin) increased and yolk globuli appeared in the oocytes. These results show that esr1 is the first of the three genes examined that is expressed during the onset of hepatic vitellogenesis. Furthermore, ovarian vitellogenesis (appearance of yolk globuli in oocytes) occurs 1-2 weeks later than the onset of hepatic vitellogenesis.

Male silver eels mature by swimming.

BACKGROUND: If European silver eels are prevented from reproductive migration, they remain in a prepubertal stage by dopaminergic inhibition of pituitary activity. Because this inhibition is likely a requirement for an extended female growth stage, we tested if it is sex-specific by subjecting both sexes to stimulation by GnRHa (Gonadotropin-Releasing Hormone agonist) - injection or 3-months swimming in seawater. RESULTS: In contrast to females, males showed a two- to three-fold higher LHbeta (luteinising hormone beta subunit) - expression, a three- to five-fold higher GSI (Gonadosomatic index) and induced spermatogenesis when compared with the untreated control group. CONCLUSION: Dopaminergic inhibition is thus not effective in males and swimming results in natural maturation, probably via GnRH-release.

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.

Spatial and temporal expression of Zimp7 and Zimp10 PIAS-like proteins in the developing mouse embryo.

ZIMP7 and ZIMP10 are two novel human PIAS-like proteins that share a similarity beyond the SP-RING Zn-finger domain that characterizes the PIAS family. This extended similarity is conserved in proteins of several other species and define an independent subfamily. ZIMP10 has been shown to increase the sumoylation of the androgen receptor (AR) leading to a stimulation of AR-mediated transcription. The Drosophila tonalli (tna) is the ortholog gene of ZIMP7 and ZIMP10 and presents genetic interactions with the SWI-SNF complex. Mutations in the tna gene produce flies with homeotic phenotypes. In this study, we determined the spatial-temporal expression pattern of Zimp7 and Zimp10 in mouse embryos from embryonic day 7.5 (E7.5), to mid-gestation. We found that these two genes are extensively expressed during these embryonic days and present partially overlapping patterns with a predomination of the transcripts in the neural tissues at early stages and a drop of expression at E12.5. Unlike other PIAS proteins, the tonalli-related Zimp genes might be essential for development. Comparison of conserved motifs in Zimp7 and Zimp10 protein sequences identified characteristic family domains that might be related to their specific biological roles, besides their common role previously identified in the sumoylation pathway.

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death.

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.

Ectopic expression of KitD814Y in spermatids of transgenic mice, interferes with sperm morphogenesis.

Kit is a receptor tyrosine kinase that plays a fundamental role during the development of germ cells. Additionally, a truncated product, tr-kit, expressed in haploid spermatids and mature spermatozoa can induce parthenogenetic activation when microinjected into mouse eggs, through the activation of PLCgamma-1. In this work, we induced ectopic expression of a mutated Kit protein, Kit(D814Y) during germ cell development. The in vivo expression of this mutant in spermatids produced malformations in mature spermatozoa, and in the most severe cases, sterility. Ultrastructural analysis indicated that condensing spermatids in the transgenic mouse presented a mislocalization of the manchette; a structure that has a crucial role during the elongation steps of spermiogenesis. This morphogenetic phenotype was accompanied by an increased phosphorylation of PLCgamma-1 in spermatogenic cells. Interestingly, we also found that, in wild-type testis, PLCgamma-1 is specifically phosphorylated in condensing spermatids, coincident with the timing of expression of tr-kit in spermiogenesis. We propose that alterations of PLCgamma-1 activity artificially promoted by ectopic Kit(D814Y) expression are related to the abnormalities of spermiogenesis. Our observations suggest that PLCgamma-1 activity could be involved in the shaping of spermatozoa.