The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3' untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers.
All data generated in this study are available in accompanying source data files. Transcriptome annotation files described in this study are available for download at zf-transcriptome.umassmed.edu. Raw and processed RNA-seq data generated in this study are available at GEO (GSE152759).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Zebrafish studies were performed in accordance with protocols #A2613 and #A2632 approved by the University of Massachusetts institutional animal care and use committee (IACUC).
© 2020, Lawson et al.
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The evolutionarily conserved Hippo (Hpo) pathway has been shown to impact early development and tumorigenesis by governing cell proliferation and apoptosis. However, its post-developmental roles are relatively unexplored. Here, we demonstrate its roles in post-mitotic cells by showing that defective Hpo signaling accelerates age-associated structural and functional decline of neurons in Caenorhabditis elegans. Loss of wts-1/LATS, the core kinase of the Hpo pathway, resulted in premature deformation of touch neurons and impaired touch responses in a yap-1/YAP-dependent manner, the downstream transcriptional co-activator of LATS. Decreased movement as well as microtubule destabilization by treatment with colchicine or disruption of microtubule-stabilizing genes alleviated the neuronal deformation of wts-1 mutants. Colchicine exerted neuroprotective effects even during normal aging. In addition, the deficiency of a microtubule-severing enzyme spas-1 also led to precocious structural deformation. These results consistently suggest that hyper-stabilized microtubules in both wts-1-deficient neurons and normally aged neurons are detrimental to the maintenance of neuronal structural integrity. In summary, Hpo pathway governs the structural and functional maintenance of differentiated neurons by modulating microtubule stability, raising the possibility that the microtubule stability of fully developed neurons could be a promising target to delay neuronal aging. Our study provides potential therapeutic approaches to combat age- or disease-related neurodegeneration.
CDK8 and CDK19 paralogs are regulatory kinases associated with the transcriptional Mediator complex. We have generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (iDKO) males, but not single Cdk8 or Cdk19 KO, had an atrophic reproductive system and were infertile. The iDKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single-cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star, and Fads) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes, and were likely associated with an impaired synthesis of steroids. Star and Fads were also downregulated in cultured Leydig cells after iDKO. The treatment of primary Leydig cell culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and a prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to the single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CCNC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CCNC stabilization.