Identification of promoter elements responsible for gonad-specific expression of zebrafish Deadend and its application to ovarian germ cell derivation

Abstract

We discovered that a 150-bp region of zebrafish deadend (dnd) spanning the translation start codon, exon 1 and part of intron 1 is required to direct heterologous neomycin-resistance gene (neo) expression specifically in the gonad, similar to endogenous dnd. Using an 8.3-kb dnd promoter that contains this 150-bp region, we generated Tg(dnd:neo-dnd) transgenic zebrafish in which the expression of Neo was detected specifically in ovarian germ cells. The transgenic fish were used to initiate primary ovarian germ cell cultures with antibiotic G418 to select ovarian germ cells and eliminate ovarian somatic cells. RT-PCR results demonstrated that the drug-selected ovarian germ cells continued to express germ-cell markers nanos3, vasa and dnd. Growth assays demonstrated that recombinant zebrafish Lif had a significant mitogenic effect on the ovarian germ cells. When long-term ovarian germ cell cultures were transplanted into two-week-old infertile larvae, they successfully colonized and directed the formation of a truncated gonad in the recipient adult fish. Histological examination of the recipient adult fish revealed that 9 out of 34 individuals (26%) possessed donor-derived cells in their gonads. The identification of zebrafish dnd promoter and the use of this promoter to generate Tg(dnd:neo-dnd) led to the success of germ cell isolation through drug selection to generate homogenous germ cells that can be used to study zebrafish germ cell biology and may lead to a cell-mediated gene transfer strategy.

Sufficient Numbers of Early Germ Cells Are Essential for Female Sex Development in Zebrafish

“…However, on the basis of our results from the comparison of the expression patterns of the transgenic reporter gene ntr-egfp and endogenous dnd via in situ hybridization experiments (data not shown here), RT-PCR assays ( Fig. 4 ) and fluorescence protein observation (Figs 1 – 3 ), a much shorter region of the 5’-flanking region of genomic fragment (2.032-kb fragment at nucleotides 159830 to 161861 in BX890575.23, without the 150-bp critical region identified previously [ 29 ], is sufficient and efficient to promote the ntr-egfp expression in a gonad-specific manner, which could also be achieved without the presence of the well-recognized the 466-bp fragment of 3’UTR of zebrafish dnd [ 7 , 24 ]. According to our observations, this 3’UTR region is probably only critical for the stabilization of the dnd mRNA in developing embryos.…”

“…According to our observations, this 3’UTR region is probably only critical for the stabilization of the dnd mRNA in developing embryos. The difference between our observations and their results regarding the regulatory region may be due to the different lengths of both the 5’ and 3’ genomic DNA regions of dnd gene selected for promoter studies, the different 3’-UTR region, or the presence of Tol2 elements in their vector backbone [ 29 ]. However, taking advantage of our fluorescent protein reporter gene, an investigation of the expression patterns of the transgene in our transgenic fish was conducted in a more convenient and comprehensive manner.…”

“…An 8.3-kb genomic fragment of the 5’-flanking region of dnd (nucleotides 153781 to 162079 in BX890575.23) combined with a 980-bp 3’ UTR region of zebrafish dnd gene (nucleotides 172517 to 173493 in BX890575.23) was used in their transgene studies. The authors claimed that a fragment containing a 150-bp region of zebrafish dnd spanning the TAG, exon 1, and part of intron 1 (nucleotides 161929 to 162079 in BX890575.23) was essential to drive reporter gene transcription in a gonad-specific expression in their transgenic zebrafish [ 29 ]. However, on the basis of our results from the comparison of the expression patterns of the transgenic reporter gene ntr-egfp and endogenous dnd via in situ hybridization experiments (data not shown here), RT-PCR assays ( Fig.…”

Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems

“…The enriched cells were able to colonize the transplant recipient gonad and produce offspring. Wong et al [122,134] also showed that Percoll-enriched oogonia were able to proliferate in vitro.…”

“…The proliferation of FGSCs continued for more than 6 weeks in vitro with unchanged germ cell markers, and cells generated normal offspring after transplantation. Multiple germ cell cultures with decreased, or no, FBS for long-term maintenance have been reported [60,122,134]. Research should focus on development of a well-defined medium for mixed cell populations that inhibits growth of nontarget cells while stimulating proliferation of target cells.…”