Schlueter et al., 2007 - Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival. Developmental Biology   305(1):377-387 Full text @ Dev. Biol.

Fig. 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.

Fig. 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 panels B and C are summarized in Table 1.

Fig. 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 overexpression of an antiapoptotic 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.

Fig. 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.

Fig. 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 panel 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, 200 x.

Knockdown Reagents:
Anatomical Term:
Stage: 20-25 somites

Fig. 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. 5, panels A–C). Magnification, 200x.

Fig. 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 Student′s t-test, P < 0.05).

Fig. S1 The igf1ra and igf1rb receptor subtypes are ubiquitously expressed in zebrafish embryos. (A–D) Whole-mount mRNA in situ hybridization for igf1ra (A, C) and igf1rb (B, D). Panels C and D present high magnification (400×) images of the respective genital ridge regions (indicted with boxes in panels A and B). Genital ridge boundaries are denoted by the dashed lines. (E–F) Primordial germ cells (PGCs) in genital ridges of the respective embryos, identified by whole-mount fluorescence immunohistochemistry with Vasa antibody. Panels G and H present merged images of C–E and D–F, respectively. Both receptor subtypes are expressed uniformly throughout the embryos, including the PGCs and genital ridges.

Anatomical Terms:
Stage: Prim-5
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Reprinted from Developmental Biology, 305(1), Schlueter, P.J., Sang, X., Duan, C., and Wood, A.W., Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival, 377-387, Copyright (2007) with permission from Elsevier. Full text @ Dev. Biol.