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A PCA plot was created of pooled filtered larval transcriptomes (total gene count >40 across all samples). Point colors are unique to copper concentrations and morphologies. Counts were normalized in DESeq2 and transformed with variance stabilizing transformation (vst) prior to plotting.
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One of the challenges facing efforts to generate molecular biomarkers for toxins is distinguishing between markers that are indicative of exposure and markers that provide evidence of the effects of toxicity. Phenotypic anchoring provides an approach to help segregate markers into these categories based on some phenotypic index of toxicity. Here we...
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... Following a 10-min incubation at room temperature, the beads were bound to a magnet, washed twice with 80% ethanol and dried for 10 min. The dried bead-bound total RNA was used directly as input to prepare 3′-tag RNAseq libraries using a protocol adapted from a single-cell RNAseq library construction protocol (Hall and Gracey 2021). Briefly, the bead-bound RNA was resuspended in 8 μL of a reverse-transcription reaction mixture with each well receiving a unique indexed anchored oligo-dT primer that contained the Illumina p7 sequence. ...
... In this study, we deployed recent technological improvements in single-cell RNA sequencing to sequence single larvae (Hall and Gracey 2021). We attempted to sequence eight individual larvae from each of the four to five replicates that were collected for each condition. ...
Anthropogenic carbon dioxide emissions have been increasing rapidly in recent years, driving pH and oxygen levels to record low concentrations in the oceans. Eastern boundary upwelling systems such as the California Current System (CCS) experience exacerbated ocean acidification and hypoxia (OAH) due to the physical and chemical properties of the transported deeper waters. Research efforts have significantly increased in recent years to investigate the deleterious effects of climate change on marine species, but have not focused on the impacts of simultaneous OAH stressor exposure. Additionally, few studies have explored the physiological impacts of these environmental stressors on the earliest life stages, which are more vulnerable and represent natural population bottlenecks in organismal life cycles. The physiological response of the ecologically and commercially important red sea urchin ( Mesocentrotus franciscanus ) was assessed by exposing larvae to a variety of OAH conditions, mimicking the range of ecologically relevant conditions encountered currently and in the near future along the CCS. Skeleton dissolution, larval development, and gene expression show a response with clearly delineated thresholds that were related to OAH severity. Skeletal dissolution and the induction of Acid‐sensing Ion Channel 1A at pH 7.94/5.70 DO mg/L provide particularly sensitive markers of OAH, with dramatic shifts in larval morphology and gene expression detected at the pH/DO transition of 7.71/3.71–7.27/2.72 mg/L. Experimental simulations that describe physiological thresholds and establish molecular markers of OAH exposure will provide fishery management with the tools to predict patterns of larval recruitment and forecast population dynamics.
... However, it must be considered that, when comparing the molecular and phenotypical responses observed at 48 hpf, gene expression analysis was carried out in pooled samples containing both normal and abnormal larvae, and the transcriptomic data do not reflect the same samples analyzed for larval phenotypes. This limitation, as recently underlined by Ref. [86] can be only overcome by gene expression analysis on single larvae. ...
Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.
