FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

Abstract

Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.

Graphical abstract

Figure 1

TGF-β-Signaling Pathways Are Active in Chicken PGCs and Needed for PGC Proliferation In Vitro (A and B) Localization of pSMAD2 and pSMAD1/5/8 to SSEA1⁺ migrating PGCs in stage 6 HH chicken embryos and stage 19 HH embryos is shown. Scale bar, 25 μm. (C) RT-PCR was carried out on cDNA from cultured chicken cells and embryonic tissues for TGF-β family receptors. Bl, Blank.

Figure 2

Medium Conditions for Derivation of Both Male and Female PGCs (A) Inhibition of ALK4/5/7 inhibits PGC growth in HiS medium on feeder cells. Approximately 500 PGCs were seeded in growth medium and cultured for 10 days in control condition or in the presence of inhibitor SB0431542 or SB505124. Values represent three independent experiments using three lines of PGCs (SEM; ^∗∗p < 0.01 versus HiS control). (B) PGC line derivation. Blood from 2.5-day (stage 16 HH) chicken embryos cultured in FAIcs medium for 3 weeks without feeder cells is shown. Scale bar, 75 μm. (C) Male PGCs cultured in FAIcs colonize the gonad in host embryos. Male GFP⁺ PGCs were injected into the vascular system of stage 16 HH embryos, incubated for 3 days, and visualized for GFP fluorescence. Scale bar, 200 μm. (D) (Top) PGC propagation requires the addition of chicken serum. PGCs (5,000) were seeded and cultured for 14 days in FAIcs medium (control) or in medium lacking chicken serum or heparin. Four male PGC lines were assayed in three independent experiments. Error bars, SEM; ^∗∗∗p < 0.001 with respect to FAIcs. (Bottom) PGC derivation rates are shown. Blood was isolated from single-sexed embryos (stage 16 HH) and cultured for 3 weeks. Cultures containing >50,000 PGCs were scored as positive. (E) Large adherent clusters form in blood cultured from female embryos in FAIcs medium. Scale bar, 75 μm. (F) Male PGC line cultured in HiS media and immunostained for E-cadherin or N-cadherin is shown. Scale bar, 20 μm. (G) PGC number over multiple passages at varying levels of calcium. Female cells (500) were re-plated on days 6 and 10. Data are from three independent experiments. Error bars, SEM; ^∗∗∗p < 0.001 with respect to other conditions. (H) (Top) Representative example of male and female PGC lines derived from GFP⁺ transgenic embryos is shown. Scale bar, 100 μm. (Bottom) PGC derivation rates in low-calcium medium are shown. Blood was isolated from single-sexed embryos (stage 16 HH) and cultured for 3 weeks. Cultures containing >50,000 PGCs were scored as positive.

Figure 3

FGF, Insulin, and Either Activin or BMP4 Are Essential for PGC Self-Renewal (A–D) PGCs were starved for 24 hr, pre-incubated with the indicated inhibitor for 30 min, and then induced with the given concentrations of growth factors for 15 min. Cells were lysed and proteins were analyzed by western blot analysis. Blots represent one of two independent experiments using a male or a female PGC line. FAIcs, control cells in FAIcs medium; HiS, control cells in full-serum medium; Inhib, Inhibitor. (A) Activin A induction of pSMAD2 in PGCs is inhibited by SB0431542. (B) FGF2 induction of pERK1/2 in PGCs is inhibited by PD173074. (C) Insulin induction of pAkt in PGCs is inhibited by BMS 536924. (D) BMP4 induction of pSMAD1/5 in PGCs is inhibited by LDN-193189. (E and F) PGC number over 10 days in culture (500 were plated on day one). Each cell treatment was assayed on three technical replicates on three different PGC lines (one male and two female). (E) Control medium containing FAIcs compared with FAIcs after removal of one component (−) is shown. Error bars, SEM; ^∗∗∗p < 0.001 with respect to FAIcs samples. (F) Control medium containing FGF2, IGF-1, and chicken serum (FIcs) compared with FIcs medium after the addition of 25 ng/ml Activin A, Activin B, or TGF-β1 is shown. Error bars, SEM; ^∗p < 0.05 and ^∗∗∗p < 0.001 with respect to −Activin samples.

Figure 4

Chicken PGCs Propagated in Activin Are Germline Competent (A) PGCs cultured in FAcs are germline competent. One male and one female GFP⁺ PGC line were mixed, injected into host embryos, hatched, and raised to sexual maturity. The photograph shows several offspring of a surrogate host hen imaged for GFP fluorescence. (B) Frequency of germline transmission from donor GFP⁺ male and female PGCs in male and female surrogate host chickens is shown. ^∗The actual transmission rate is double the observed number of GFP⁺ chicks due to meiotic reduction of the heterozygous GFP transgene.

Figure 5

PGCs in Defined Medium Can Be Cultured Clonally in Activin, but Not BMP4 (A) Ovotransferrin (OT) replaces chicken serum in culture medium. PGCs (1,000) were cultured in FA medium with the addition of 0.2% fetal bovine serum (FBS), 0.2% chicken serum (CS), and 10 μg/ml human transferrin (HuT) or 10 μg/ml OT for 10 days. Error bars, SEM; ^∗p < 0.05 and ^∗∗∗p < 0.001 with respect to FA samples. Each cell treatment was assayed in three independent experiments on a male and a female PGC line. (B) PGCs can be propagated in Activin or BMP4 in defined medium conditions. PGC number over 14 days in culture (500 were plated on day 1) is shown. Each cell treatment was assayed on six different PGC lines (four male and two female) in three independent experiments. Error bars, SEM; ^∗∗p < 0.01 and ^∗∗∗p < 0.001 with respect to FABot sample. (C) PGCs express pluripotency and germ cell markers in Activin- or BMP4-defined culture medium. RT-PCR analysis of cDNA prepared from a PGC line cultured in FABot, FAot, or FBot is shown. FAcs and HiS are shown as positive controls. Image represents one of two independent experiments using two male PGC lines. CEF, chick embryonic fibrobast; cES, chick embryonic stem cell. (D) Activin is sufficient for derivation and clonal growth of PGCs. Blood was isolated from single embryos (stage 16 HH) and cultured for 3 weeks. PGCs were counted and cultures containing >50,000 cells were scored as positive. Cells from a male or a female PGC line derived in FABot were diluted to one cell/2 μl in Fot, and single cells were plated and cultured for 3 weeks in FABot, FAot, or FBot. PGCs were counted and cultures containing >50,000 cells were scored as positive. ^∗∗∗p < 0.001 with respect to FBot samples using two-tailed Fisher’s exact test. (E–G) FGF2, insulin, and Activin can be replaced by the corresponding downstream effectors. PGCs (500), cultured in FABot, were seeded in a well and the indicated growth factors were removed. Doxycycline (dox) was added and cells were propagated for 10 days (A and B) or 12 days (C) and then counted. (E) PGCs containing a tet-inducible constitutively active MEK1 construct proliferate in the absence of FGF2. (F) PGCs containing a tet-inducible constitutively active AKT construct proliferate in the absence of insulin. (G) PGCs containing constitutively active SMAD3 and tet-inducible constitutively active SMAD5 constructs proliferate in the absence of Activin and BMP4. Each cell treatment was assayed using two different PGC lines (one male and two female) in three independent experiments. ^∗∗∗p < 0.001 and ^∗∗p < 0.01 with respect to the indicated samples.

Figure 6

Model for Avian Primordial Germ Cell Self-Renewal FGF2, insulin, and Activin growth factors signaling through their cognate receptors are sufficient for chicken PGC self-renewal. BMP4 can replace Activin A in non-clonal growth conditions.