PUF-8, a C. elegans ortholog of the RNA-binding proteins PUM1 and PUM2, is required for robustness of the cell death fate.

During C. elegans development, 1090 somatic cells are generated, of which 959 survive and 131 die, many through apoptosis. We present evidence that PUF-8, a C. elegans ortholog of the mammalian RNA-binding proteins PUM1 and PUM2, is required for the robustness of this 'survival and death' pattern. We found that PUF-8 prevents the inappropriate death of cells that normally survive, and we present evidence that this anti-apoptotic activity of PUF-8 is dependent on the ability of PUF-8 to interact with ced-3 (a C. elegans ortholog of caspase) mRNA, thereby repressing the activity of the pro-apoptotic ced-3 gene. PUF-8 also promotes the death of cells that are programmed to die, and we propose that this pro-apoptotic activity of PUF-8 may depend on the ability of PUF-8 to repress the expression of the anti-apoptotic ced-9 gene (a C. elegans ortholog of Bcl2). Our results suggest that stochastic differences in the expression of genes within the apoptosis pathway can disrupt the highly reproducible and robust survival and death pattern during C. elegans development, and that PUF-8 acts at the post-transcriptional level to level out these differences, thereby ensuring proper cell number homeostasis.

miRNAs cooperate in apoptosis regulation during C. elegans development.

Programmed cell death occurs in a highly reproducible manner during Caenorhabditis elegans development. We demonstrate that, during embryogenesis, miR-35 and miR-58 bantam family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene egl-1, which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of egl-1 is dependent on binding sites for miR-35 and miR-58 family miRNAs within the egl-1 3' untranslated region (UTR), which affect both mRNA copy number and translation. Furthermore, using single-molecule RNA fluorescent in situ hybridization (smRNA FISH), we show that egl-1 is transcribed in the mother of a cell programmed to die and that miR-35 and miR-58 family miRNAs prevent this mother from dying by keeping the copy number of egl-1 mRNA below a critical threshold. Finally, miR-35 and miR-58 family miRNAs can also dampen the transcriptional boost of egl-1 that occurs specifically in a daughter cell that is programmed to die. We propose that miRNAs compensate for lineage-specific differences in egl-1 transcriptional activation, thus ensuring that EGL-1 activity reaches the threshold necessary to trigger death only in daughter cells that are programmed to die.

Application of molecular endpoints in early life stage salmonid environmental biomonitoring.

Molecular endpoints can enhance existing whole animal bioassays by more fully characterizing the biological impacts of aquatic pollutants. Laboratory and field studies were used to examine the utility of adopting molecular endpoints for a well-developed in situ early life stage (eyed embryo to onset of swim-up fry) salmonid bioassay to improve diagnostic assessments of water quality in the field. Coastal cutthroat trout (Oncorhynchus clarki clarki) were exposed in the laboratory to the model metal (zinc, 40µg/L) and the polycyclic aromatic hydrocarbon (pyrene, 100µg/L) in water to examine the resulting early life stage salmonid responses. In situ field exposures and bioassays were conducted in parallel to evaluate the water quality of three urban streams in British Columbia (two sites with anthropogenic inputs and one reference site). The endpoints measured in swim-up fry included survival, deformities, growth (weight and length), vitellogenin (vtg) and metallothionein (Mt) protein levels, and hepatic gene expression (e.g., metallothioneins [mta and mtb], endocrine biomarkers [vtg and estrogen receptors, esr] and xenobiotic-metabolizing enzymes [cytochrome P450 1A3, cyp1a3 and glutathione transferases, gstk]). No effects were observed in the zinc treatment, however exposure of swim-up fry to pyrene resulted in decreased survival, deformities and increased estrogen receptor alpha (er1) mRNA levels. In the field exposures, xenobiotic-metabolizing enzymes (cyp1a3, gstk) and zinc transporter (zntBigM103) mRNA were significantly increased in swim-up fry deployed at the sites with more anthropogenic inputs compared to the reference site. Cluster analysis revealed that gene expression profiles in individuals from the streams receiving anthropogenic inputs were more similar to each other than to the reference site. Collectively, the results obtained in this study suggest that molecular endpoints may be useful, and potentially more sensitive, indicators of site-specific contamination in real-world, complex exposure scenarios in addition to whole body morphometric and physiological measures.

Photosystem II and pigment dynamics among ecotypes of the green alga Ostreococcus.

We investigated the photophysiological responses of three ecotypes of the picophytoplankter Ostreococcus and a larger prasinophyte Pyramimonas obovata to a sudden increase in light irradiance. The deepwater Ostreococcus sp. RCC809 showed very high susceptibility to primary photoinactivation, likely a consequence of high oxidative stress, which may relate to the recently noted plastid terminal oxidase activity in this strain. The three Ostreococcus ecotypes were all capable of deploying modulation of the photosystem II repair cycle in order to cope with the light increase, but the effective clearance of photoinactivated D1 protein appeared to be slower in the deepwater Ostreococcus sp. RCC809, suggesting that this step is rate limiting in the photosystem II repair cycle in this strain. Moreover, the deepwater Ostreococcus accumulated lutein and showed substantial use of the xanthophyll cycle under light stress, demonstrating its high sensitivity to light fluctuations. The sustained component of the nonphotochemical quenching of fluorescence correlated well with the xanthophyll deepoxidation activity. Comparisons with the larger prasinophyte P. obovata suggest that the photophysiology of Ostreococcus ecotypes requires high photosystem II repair rates to counter a high susceptibility to photoinactivation, consistent with low pigment package effects in their minute-sized cells.