Figure 1.
Expression of neogenin during preimplantation mouse embryo development.
(A) Confocal microscopic images of neogenin expression in preimplantation mouse embryos at different developmental stages. DAPI, DAPI nuclear staining in blue; F-actin, F-actin staining in green; Neogenin, neogenin staining in red; Merged, superimposed images of DAPI, F-actin, and neogenin staining. (B) RT-PCR analysis of neogenin mRNA in preimplantation mouse embryos at different developmental stages. β-actin was used as an internal control.
Figure 2.
Effects of up-regulation and down-regulation of neogenin on embryo development.
2-PN mouse zygotes were microinjected with shRNA targeting neogenin (neogenin KD) or with neogenin cDNA vectors (neogenin OE) and were cultured to the blastocyst stage. To visually differentiate neogenin KD from neogenin OE embryos, GFP and RFP were co-expressed, respectively. Phase-contrast images of embryos at different developmental stages were seen.
Figure 3.
Neogenin overexpression favors ICM differentiation.
(A) ICM cells in a blastocyst were viewed by immunostaining for Oct3/4 in neogenin KD and neogenin OE embryos. DAPI was for nucleus staining. (B) The number of Oct3/4-positive ICM cells in a blastocyst of control, neogenin KD, and neogenin OE embryos was represented as the mean ± SEM from three independent experiments. *Significant differences from control at P<0.05. (C) RT-PCR analysis of Oct3/4, Sox2, Nanog, Cdx2, and Tead4 mRNAs from blastocysts of neogenin KD, neogenin OE, and control embryos. β-actin was used as an internal control.
Table 1.
Developmental potentials of 2-PN mouse zygotes after receiving neogenin shRNAs or neogenin cDNAs.
Figure 4.
Neogenin ligands netrin-1 and RGMc exert an antagonistic effect on ICM differentiation.
(A) Netrin-1-enriched conditioned media was added to the culture media at 20% (v/v), and the gross morphology of developing mouse embryos was observed over 96 hr-period. Control, normal embryos without netrin-1; 20% Netrin-1, normal embryos in 20% netrin-1; Neogenin KD+20% netrin-1, neogenin knock-down embryos in 20% netrin-1; Neogenin OE+20% netrin-1, neogenin overexpressing embryos in 20% netrin-1. (B) Recombinant RGMc was added to culture media at 10 ng/ml and the gross morphology of developing mouse embryos over 96 hrs in culture was observed. Control + RGMc, control embryos in culture media supplemented with recombinant RGMc; Neogenin KD + RGMc, neogenin knock-down embryos in culture media supplemented with recombinant RGMc; Neogenin OE + RGMc, neogenin overexpressing embryos in culture media supplemented with recombinant RGMc. (C) ICM cells in a blastocyst was counted from different treatments: Control, no treatment on normal embryos; Control + netrin-1, 20% netrin-1 treatment on normal embryos; Ng KD + netrin-1, 20% netrin-1 treatment on neogenin knock-down embryos; Ng OE + netrin-1, 20% netrin-1 treatment on neogenin overexpressing embryos. (D) ICM cells in a blastocyst was counted from different treatments: Control, no treatment on normal embryos; Control + RGMc, 10 ng/ml RGMc treatment on normal embryos; Ng KD + RGMc, 10 ng/ml RGMc treatment on neogenin knock-down embryos; Ng OE + RGMc, 10 ng/ml RGMc treatment on neogenin overexpressing embryos (E) Immunofluorescent staining for Oct3/4 after receiving different combinations of experimental treatment as described in C and D. *Significant differences from control at P<0.05.
Table 2.
Developmental potential of 2-PN zygotes cultured with netrin-1.
Table 3.
Development potential of 2-PN zygotes cultured with RGMc.
Figure 5.
A model for downstream signaling of neogenin and regulation of early cell fate determination in preimplantation mouse embryos.
The differential activation of neogenin signaling either by neogenin expression level or its ligation with ligands, which in turn receive thus relaying temporal and spatial input from various external stimuli during early embryo development leads to the establishment of the first cell fate specification.