Knockdown of DEAD-box helicase 4 (DDX4) decreases the number of germ cells in male and female chicken embryonic gonads

Abstract

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways.

Transcriptome Profiling of the Ovarian Cells at the Single-Cell Resolution in Adult Asian Seabass

“…Likewise, the amplification of ribosomal genes allows synthesis of sufficient rRNA to supply the growing oocyte. Besides, most germ cell markers are RNA binding proteins, such as Vasa, Dazl, and Ziwi1 (Kehkooi et al, 2009;Leu and Draper, 2010;Aduma et al, 2019). RNA regulation is, therefore, crucial in the germ line of virtually all organisms and plays a variety of prominent roles.…”

“…Traditionally, light and electron microscopy have been used to discriminate PGCs from somatic cells during early embryonic stages and examine their morphological characteristics (Xu et al, 2010). In recent years, based on molecular and cellular techniques, several identified germ cell markers such as vasa, dazl, and ziwi are known to play essential roles in germ cell development across the animal kingdom (Kehkooi et al, 2009;Leu and Draper, 2010;Aduma et al, 2019). However, the identification of germ cells in many species was ambiguous due to the absence of germ cells or somatic marker genes, especially for female germ cells in ovary.…”

Vasa expression is associated with sex differentiation in the Asian yellow pond turtle, Mauremys mutica

“…Vasa (also known as DDX4 ), the best‐characterized germline regulator in metazoans, belongs to the DEAD‐box protein family of ATP‐dependent RNA helicases and it is majorly involved in germ cell specification and gametogenesis (Aduma et al, 2019; Lasko, 2013). The vasa gene was first identified as a maternal‐effect gene of Drosophila in which vasa was necessary for the formation of the perinuclear Nuage in nurse cells and for the differentiation of germ cells (Hay et al, 1988).…”

“…In zebrafish, vasa is required for germ cell development and meiosis in both females and males, but vasa mutants develop exclusively as sterile males (Hartung et al, 2014). Moreover, male and female gonads of DDX4 ‐knockdown chicken embryos contained a decreased number of primordial germ cells (PGCs), suggesting that Vasa plays an essential role in maintaining PGCs in chicken embryos of both sexes (Aduma et al, 2019). In the female mouse, disruption of the Vasa ( Mvh ) gene didn't affect the development of female germ cells and homozygous vasa mutant female mice were fertile and offspring delivered after mating with heterozygous males showed no difference in number compared with wild‐type mice (S. S. Tanaka et al, 2000).…”

Expression profiling of sexually dimorphic genes in the Japanese quail, Coturnix japonica

“…A number of transgenic chickens have been generated by retroviral infection 18 – 21 and transposon systems 22 , 23 . For several genes involved in gonadal sex differentiation, over-expression or knockdown experiments have been performed 4 , 5 , 8 – 10 , 24 , 25 . Genetic editing tools, such as transcription activator-like effector nuclease (TALEN) and clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9), have been applied to modify the chicken genome.…”