Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch

Abstract

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.

Highly efficient genome modification of cultured primordial germ cells with lentiviral vectors to generate transgenic songbirds

“…To test the effect of in vitro propagation on PGC-specific gene expression we assessed germ and stem cell marker expression by zfPGCs at 10 DIV by performing immunofluorescent stainings and RT-PCR analyses. Stage-specific embryonic antigen 1 (SSEA-1), a carbohydrate epitope associated with cell adhesion and migration, is normally expressed by avian PGCs during migration to the gonadal anlage in turkeys (D´Costa and Petitte, 1999), as well as in zfgPGCs (Jung et al, 2019) and also by chPGCs (Macdonald et al, 2010) in vitro. Furthermore, epithelial membrane antigen 1 (EMA-1), a cell surface glycoprotein, is expressed by chPGCs at early embryonic stages and in vitro (Urven et al, 1988;Raucci et al, 2015).…”

“…During avian development, PGCs are located in the central area of the blastodisc until Eyal-Giladi and Kochav (EGK) stage X (Eyal-Giladi et al, 1976;Kochav et al, 1980); from here, PGCs translocate to the germinal crescent (Ginsburg et al, 1986) and finally migrate through the developing vascular system to reach the gonadal anlage (Fujimoto et al, 1976;Nakamura et al, 1988;Jung et al, 2019). Avian PGCs are easily isolated from the circulatory system by blood aspiration between Hamburger and Hamilton (HH) stages 14 and 17 (Hamburger and Hamilton, 1951).…”

“…Their ability to migrate to the gonadal anlage was unaffected by this treatment and the resulting hosts exhibited a high germline transmission rate (Van De Lavoir et al, 2006;Macdonald et al, 2010). For the zebra finch, it has been shown that primordial germ cells of the embryonic gonads (zfgPGCs) can be cultured for up to 30 days on a feeder cell layer in the presence of relative high concentrations of undefined fetal bovine serum, genetically modified with transposons and retain the ability to colonize embryonic host gonads (Jung et al, 2019).…”

“…Higher concentrations of calcium (medium with 100 % KnockOutTM DMEM containing 1.8 mM calcium) promoted stronger cell-cell interactions, leading to the formation of dense zfPGC clumps with indistinguishable cell boundaries. We also tested a culture medium previously developed for zfgPGCs (Jung et al, 2019) that contained higher serum concentrations. Using this medium condition, zfPGCs extracted from embryonic blood attached to the bottom of the culture plate ( Fig.…”

“…Cells were maintained in culture for up to 30 days and 80l of the medium was replaced every other day. Gonadal PGCs (gPGCs) were obtained and cultured as described byJung et al (2019).Histological stainingsFor periodic acid-Schiff (PAS) stain of blood smears, samples were extracted from zebra finch and chicken embryos at HH stage 14, dried on glass slides and fixed with 70% ethanol for 15 minutes. For staining, a PAS kit (Sigma Aldrich) was used according to the manufacturer's instructions, and cells were counterstained with hematoxylin solution Gill No.…”