Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

Abstract

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.

Figure 1

Igf1rb is required to establish normal primordial germ cell (PGC) numbers at the prim-5 stage. Immunohistochemical identification of PGCs (Vasa-positive cells, arrows) in prim-5 stage zebrafish embryos, after ablation of Type-1 IGF receptors (igf1ra, igf1rb): (A) control (non-targeting) morpholinos; (B) igf1ra-MO; (C) igf1rb-MO; (D) igf1ra-MO + igf1rb-MO. Magnification 200x. Mean PGC numbers of treatment groups are summarized in Table 1.

Figure 2

(A) Western immunoblot analysis of zebrafish lysates demonstrating overexpression of dominant-negative IGF1R:GFP fusion protein (dnIGF1R:GFP). First lane, lysates from zebrafish embryos injected with synthetic mRNA encoding GFP only; second lane, lysates from zebrafish embryos injected with synthetic mRNA encoding dnIGF1R:GFP. Upper band in dnIGF1R:GFP corresponds to the predicted molecular weight (~60 kDa) of dnIGF1R:GFP after denaturation in reducing conditions. (B) Vasa immunostaining of PGCs (arrows) in zebrafish embryo overexpressing GFP only (control); (C) Vasa immunostaining of PGCs in zebrafish embryo overexpressing dnIGF1R:GFP fusion protein. Magnification (B–C), 200x. Mean PGC numbers in B and C are summarized in Table 1.

Figure 3

IGF1Rb is required for normal PGC migration. Ectopic PGCs (arrows) were detected rarely in Control-MO-injected embryos (A), whereas they were frequently observed, in both somatic tissues (B) and throughout the yolk cell (C) of igf1rb-MO-injected embryos. (D) Mean numbers of ectopic PGCs in zebrafish embryos after morpholino injections, and/or over-expression of an anti-apoptotic Bcl2-like protein (Bcl2l). Data represent means ± SEM, with sample size indicated in parentheses. Superscript letters denote significant differences between groups (ANOVA, Tukey’s, P < 0.05). Magnification (A–C), 200x.

Figure 4

Targeted knockdown of either IGF1R subtype results in increased apoptosis. In situ cell death (TUNEL) analyses of zebrafish embryos after injection with (A, D) control morpholinos; (B, E) IGF1Ra antisense morpholinos (igf1ra-MO); (C, F) IGF1Rb antisense morpholinos (igf1rb-MO). Embryos in upper panels are 18-somite stage embryos; embryos in lower panels are prim-5 stage embryos. Relative to control-MO-injected embryos (A, D), TUNEL-positive cells are more abundant in both igf1ra-MO- and igf1rb-MO-injected embryos, at both stages of development. Magnification 100x.

Figure 5

Ectopic PGCs undergo apoptosis. (A) Vasa immunostaining, and (B) TUNEL analysis of ectopic PGCs in dorsal trunk of igf1rb-MO-injected embryo; (C; merged image) colocalization of Vasa-positive (arrows) and TUNEL-positive (arrowheads) cells confirms DNA fragmentation in ectopic PGCs. (D) Vasa-positive, but (E) TUNEL-negative PGCs in genital ridge (circled) of igf1rb-MO-injected embryo; images merged in (F). An absence of DNA fragmentation in genital ridge PGCs confirms survival of the subpopulation of PGCs that successfully migrate to the genital ridge. Anterior is to the left in all images; magnification 200x.

Figure 6

Ectopic PGCs are rescued from apoptosis by overexpression of Bcl2l. (A) Vasa positive cells (arrows), and (B) rare TUNEL-positive cells (arrowheads) in dorsal trunk of igf1rb-MO/bcl2l mRNA co-injected embryo; (C; merged image) lack of colocalized Vasa and TUNEL signals (arrows, arrowheads) confirms absence of DNA fragmentation in ectopic PGCs (compare with Fig. 6A–C). Magnification 200x.

Figure 7

Relative mRNA levels of cxcl12a and cxcr4b in zebrafish embryos injected with morpholino oligonucleotides targeting IGF1Ra (igf1ra-MO) and IGF1Rb (igf1rb-MO). Values are normalized relative to odc mRNA levels (100%), and presented as means ± SEM (n=4). *denotes statistical difference from Control-MO-injected embryos (paired Students t-test, P < 0.05).