CRISPR/Cas9-Mediated Genome Editing in Zebrafish.

The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.

Early transcriptomic response of mouse adrenal gland and Y-1 cells to dexamethasone.

Glucocorticoids have short- and long-term effects on adrenal gland function and development. RNA sequencing (RNA-seq) was performed to identify early transcriptomic responses to the synthetic glucocorticoid, dexamethasone (Dex), in vitro and in vivo. In total, 1711 genes were differentially expressed in the adrenal glands of the 1-h Dex-treated mice. Among them, only 113 were also considered differentially expressed genes (DEGs) in murine adrenocortical Y-1 cells treated with Dex for 1 h. Gene ontology analysis showed that the upregulated DEGs in the adrenal gland of the 1-h Dex-treated mice were highly associated with the development of neuronal cells, suggesting the adrenal medulla had a rapid response to Dex. Interestingly, only 4.3% of Dex-responsive genes in the Y-1 cell line under Dex treatment for 1 h were differentially expressed under Dex treatment for 24 h. The heatmaps revealed that most early responsive DEGs in Y-1 cells during 1 h of treatment exhibited a transient response. The expression of these genes under treatment for 24 h returned to basal levels similar to that during control treatment. In summary, this research compared the rapid transcriptomic effects of Dex stimulation in vivo and in vitro. Notably, adrenocortical Y-1 cells had a transient early response to Dex treatment. Furthermore, the DEGs had a minimal overlap in the 1-h Dex-treated group in vivo and in vitro.

Transcriptional Profiles of Genes Related to Stress and Immune Response in Rainbow Trout (Oncorhynchus mykiss) Symptomatically or Asymptomatically Infected With Vibrio anguillarum.

Rainbow trout (Oncorhynchus mykiss) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum. Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.

Spatiotemporal expression profile of embryonic and adult ankyrin repeat and EF-hand domain containing protein 1-encoding genes ankef1a and ankef1b in zebrafish.

Recent human next-generation sequencing (NGS) studies indicate a correlation between ANKEF1 (ankyrin repeat and EF-hand domain containing protein 1) expression and cilia formation or function. Additionally, a single study conducted in the African clawed frog (Xenopus laevis) showed ankef1 is down-regulated after pharmacological fibroblast growth factor (FGF) inhibition and plays a role in protocadherin-mediated cell protrusion and adhesion. That study also revealed a critical role for ankef1 in the embryonic development of the frog, with morphants exhibiting phenotypes including spina bifida and a shortened body axis. Interestingly, while little is known about ANKEF1 function in other vertebrate systems, recent proteomic data has shown ANKEF1 enriched in ciliated cells. Likewise, publicly available EST profile databases imply ANKEF1 expression in multiple human tissues, including high levels in the testes. Together, these previous studies suggest an important role for ANKEF1 in ciliated tissues and during embryonic development. Here, we report cloning of zebrafish (Danio rerio) ankef1a, as well as its paralog, ankef1b, and expression analyses by whole-mount in situ hybridization (WISH) and quantitative polymerase chain reaction (qPCR) during embryonic development and in adult tissues. WISH shows both forms are ubiquitously expressed early in development, with more discrete expression of both transcripts in embryonic tissues known to precede or possess motile cilia, including dorsal forerunner cells (DFC) and the otic vesicles, respectively. Additionally, both transcripts are enriched in the developing pharynx and swim bladder. Our qPCR results indicate enhanced expression in the testes, along with increased expression in brain. Certainly, our experiments in the zebrafish model system with ankef1a and ankef1b provide a solid foundation for future studies to uncover the molecular pathways through which Ankef1 acts in both healthy and disease states.