A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls.

The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs

Sex-specific Aging in Animals: Perspective and Future Directions

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age-associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer-lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well-characterized processes. In particular, understanding the role of sex-determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs

Sex-specific aging in animals: Perspective and future directions

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age‐associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer‐lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well‐characterized processes. In particular, understanding the role of sex‐determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs

Early Embryonic Exposure to Polychlorinated Biphenyls Disrupts Heat-Shock Protein 70 Cognate Expression in Zebrafish

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions

Directional Transport Is Mediated by a Dynein-Dependent Step in an RNA Localization Pathway

<div><p>Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in <i>Xenopus</i> oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.</p> </div

Model for vegetal RNA localization.

<p>The vegetal cytoplasm is depicted, with the vegetal cortex at the bottom. The oocyte nucleus is shown in gray and the perinuclear cup is indicated in gold. (A) The oocyte microtubules are shown in black with orientation indicated by plus and minus. The proposed arrangement of microtubules is based on the appearance of a subpopulation of microtubule plus-ends at the vegetal cortex following breakdown of the mitochondrial cloud <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001551#pbio.1001551-Messitt1" target="_blank">[12]</a>, which has been proposed to contain a microtubule organizing center <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001551#pbio.1001551-Kloc1" target="_blank">[52]</a>. (B) Vg1 mRNA enriched at the perinuclear cup is first transported by the dynein molecular motor in the upper vegetal cytoplasm in an initial highly directional step toward the vegetal cortex (blue). Microtubules are shown in grey. (C) Repeated cycles of bidirectional transport dependent on kinesin molecular motors occur in the lower vegetal cytoplasm (purple), until Vg1 mRNA exits the transport cycle by becoming anchored at the vegetal cortex (red).</p

Live imaging of RNA localization reveals RNA transport dynamics.

<p>(A) Diagram of VLE RNA (VLE-MS2) and nonlocalizing β-globin RNA (βG-MS2) tagged with multimerized MS2 binding sites, which recruit MS2 coat protein fused to mCherry (mCh-MCP). (B) Oocytes expressing mCh-MCP and injected with βG-MS2 RNA exhibit uniform cytoplasmic fluorescence. (C) Oocytes expressing mCh-MCP and injected with VLE-MS2 RNA exhibit strong vegetal fluorescence (red). (B–C) Images of live oocytes are shown, with vegetal poles towards the bottom; scale bars, 20 µm. (D) Diagram of oocyte showing regions used for analysis: cup region immediately adjacent to the nucleus on the vegetal side (Region 1), the upper vegetal cytoplasm (Region 2), the lower vegetal cytoplasm (Region 3), and the animal hemisphere (Region 4). The 5 µm circular regions for FRAP are indicated in red and are spatially defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001551#s4" target="_blank">Materials and Methods</a>. (E) Calculated half times of recovery and diffusion coefficients from FRAP analysis. βG-MS2 RNA mobility was measured in Regions 2–4 and VLE-MS2 RNA mobility was measured in Region 4. After nocodazole treatment, VLE-MS2 RNA mobility was measured in Regions 2 and 3. ± indicates standard error of the mean. (F) Averaged halftimes (t_½) of recovery for indicated regions in control oocytes (black bars) or oocytes with dynein function disrupted by expression of CC1 (white bars). Control (Region 1, <i>n</i> = 10; Region 3, <i>n</i> = 10), Disrupted dynein (Region 1, <i>n</i> = 21; Region 3, <i>n</i> = 22). Error bars show standard error of the mean. Half times of recovery (t_1/2) and diffusion coefficients were calculated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001551#s4" target="_blank">Materials and Methods</a>. The <i>p</i> values were generated using a two-tailed unpaired Student's <i>t</i> test; ** <i>p</i> = 0.0054, * <i>p</i> = 0.299.</p

Distinct regions of RNA transport directionality.

<p>Oocytes expressing PA-mCh-MCP were microinjected with VLE-MS2 RNA. (A) Prior to activation of PA-mCh-MCP in live oocytes (t = 0), minimal fluorescence is observed. The activation point is shown by the small white dot and the oocyte nucleus is outlined in white. Scale bar, 20 µm. (A′) By 7 s after activation of PA-mCh-MCP, robust fluorescence (red) is evident at and around the activation point (white dot). (A″–A′″) By 240–480 s after activation, PA-mCh-MCP tethered to RNA can be visualized asymmetrically around the activation point. (A′″) The four collection quadrants are indicated by white circles surrounding the activation point: V and A indicate the collection quadrants on the vegetal and animal sides of the activation point, respectively. L and R indicate the collection quadrants on the left and right sides. (B–C) After activation of PA-mCh-MCP in (B) the upper vegetal cytoplasm (Region 2) or (C) the lower vegetal cytoplasm (Region 3), corrected fluorescence intensities in the V (black) and A (grey) quadrants were plotted over time. (D, E) The ratios of V∶A (red) and L∶R (blue) intensities for oocytes activated in (D) the upper vegetal cytoplasm (Region 2) and (E) the lower vegetal cytoplasm (Region 3) were plotted over time.</p

Dynein and kinesin mediate distinct steps in vegetal RNA transport.

<p>(A–D) Fluorescently labeled VLE RNA was microinjected into oocytes expressing (A) kinesin-1 rigor mutant (K1r), (B) p150^Glued CC1 (CC1), (C) both K1r and CC1, or (D) no exogenous protein. Representative oocytes are shown with the vegetal pole towards the bottom. Scale bars, 50 µm. (E) Quantification of in vivo interference results for oocytes expressing no exogenous protein (control, <i>n</i> = 167), kinesin-1 rigor (K1r, <i>n</i> = 159), CC1 domain of p150^Glued (CC1, <i>n</i> = 209), and both K1r and CC1 (<i>n</i> = 208). Black bars indicate normal localization, gray denotes cup accumulation, and white bars indicate accumulation of RNA in the lower vegetal cytoplasm. Error bars indicate standard deviation.</p