Fzr regulates silk gland growth by promoting endoreplication and protein synthesis in the silkworm.

Silkworm silk gland cells undergo endoreplicating cycle and rapid growth during the larval period, and synthesize massive silk proteins for silk production. In this study, we demonstrated that a binary transgenic CRISPR/Cas9 approach-mediated Fzr mutation in silkworm posterior silk gland (PSG) cells caused an arrest of silk gland growth and a decrease in silk production. Mechanistically, PSG-specific Fzr mutation blocked endoreplication progression by inducing an expression dysregulation of several cyclin proteins and DNA replication-related regulators. Moreover, based on label-free quantitative proteome analysis, we showed in PSG cells that Fzr mutation-induced decrease in the levels of cyclin proteins and silk proteins was likely due to an inhibition of the ribosome biogenesis pathway associated with mRNA translation, and/or an enhance of the ubiquitin-mediated protein degradation pathway. Rbin-1 inhibitor-mediated blocking of ribosomal biogenesis pathway decreased DNA replication in PSG cells and silk production. Altogether, our results reveal that Fzr positively regulates PSG growth and silk production in silkworm by promoting endoreplication and protein synthesis in PSG cells.

Transcription factor Sox3 is required for oogenesis in the teleost fish Nile tilapia.

Oogenesis is a complex developmental process responsible for the production of eggs from oogonia in fish and other animals. However, transcriptional regulation underlying oogenesis is not fully understood. In the present study, we demonstrated in the teleost fish Nile tilapia that the Sox transcription factor family member Sox3 was involved in regulating oocyte growth during oogenesis. Fluorescence in situ hybridization showed that Sox3 expression was enriched in growing oocytes of ovary but could not be detected in testes. CRISPR/Cas9-mediated homozygous mutation in the Sox3 gene disrupted oocyte growth. Further analysis revealed that Sox3 mutation caused a decrease in the contents of neutral lipids in oocytes and estradiol-17 beta (E2) production. RNA-seq-based transcriptome profiling and RT-qPCR analysis in ovaries demonstrated that the expression levels of genes involved in E2 production, lipid accumulation, and yolk formation were significantly downregulated following Sox3 mutation. Altogether, our findings indicate that Sox3 is required for oocyte growth in Nile tilapia and provides insights into transcriptional regulation underlying oogenesis in teleost fish.

The transcription factor Sox30 is involved in Nile tilapia spermatogenesis.

Spermatogenesis is a complex process in which spermatogonial stem cells differentiate and develop into mature spermatozoa. The transcriptional regulatory network involved in fish spermatogenesis remains poorly understood. Here, we demonstrate in Nile tilapia that the Sox transcription factor family member Sox30 is specifically expressed in the testes and mainly localizes to spermatocytes and spermatids. CRISPR/Cas9-mediated sox30 mutation results in abnormal spermiogenesis, reduction of sperm motility, and male subfertility. Comparative transcriptome analysis shows that sox30 mutation alters the expression of genes involved in spermatogenesis. Further chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), ChIP-PCR, and luciferase reporter assays revealed that Sox30 positively regulates the transcription of ift140 and ptprb, two genes involved in spermiogenesis, by directly binding to their promoters. Our data, taken together, indicate that Sox30 plays an essential role in Nile tilapia spermatogenesis by directly regulating the transcription of the spermiogenesis-related genes ift140 and ptprb.

Transcription of the Sox30 Gene Is Positively Regulated by Dmrt1 in Nile Tilapia.

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.

Dmrt1 directly regulates the transcription of the testis-biased Sox9b gene in Nile tilapia (Oreochromis niloticus).

The Nile tilapia Sox9b gene is characterized as a homolog of the mammalian Sox9 gene, which exhibits testis-biased expression and is involved in testis development. However, the transcriptional regulation of the Sox9b gene is poorly understood. In this study, we demonstrated that the male sex differentiation gene doublesex and mab-3 related transcription factor 1 (Dmrt1) was predominantly expressed in the Nile tilapia testis and that Dmrt1 knockdown in the Nile tilapia decreased the expression of the Sox9b gene in the testis. An in silico analysis predicted that the proximal promoter of the Nile tilapia Sox9b gene had two potential cis-regulatory elements (CREs) for the Dmrt1 transcription factor. Together, a luciferase reporter analysis and site-directed mutagenesis revealed that Dmrt1 increased the transcriptional activity of the Nile tilapia Sox9b promoter via a specific CRE near the translation start site. A chromatin immunoprecipitation (ChIP) analysis and an electrophoretic mobility shift assay (EMSA) confirmed that Dmrt1 could directly bind to this specific CRE for Dmrt1 in the Nile tilapia Sox9b promoter. Taken together, our results demonstrate that the male sex-differentiation factor Dmrt1 positively regulates the transcription of the Nile tilapia Sox9b gene by directly binding to a specific CRE within the Sox9b promoter.