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doi: 10.1242/10.1242/dev.00430

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FRL-1, a member of the EGF-CFC family, is essential for neural differentiation in Xenopus early development

¹ Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
² Department of Life Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan
³ SORST Project, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan

View larger version (35K): [in a new window]   Fig. 1. (A) Overexpression of FRL-1 induced the expression of neural markers without mesodermal markers in animal caps. 1 ng of FRL-1 or 100 pg of chordin (chd) were injected into animal caps, which were dissected at stage 9, together with an uninjected control, and harvested at stage 13 with the whole embryos for RT-PCR analysis. FRL-1 induced the expression of zic3, soxD, Xngnr-1 without induction of Xbra and chordin, and inhibited Xmsx-1 and Xvent-1. `ND' indicated no data. (B) FRL-1 inhibits BMP signaling in animal caps. Animal caps injected with 1 ng of BMP-4 with or without 1 ng of FRL-1, were dissected at stage 9 and harvested at stage 10.5 for RT-PCR. The induction of Xvent-1, Xmsx-1 and Xbra induced by BMP-4 was inhibited by co-injection of FRL-1, and the inhibition of zic3 and soxD by BMP-4 was rescued by co-injection of FRL-1. (C) FRL-1 induced the expression of neural markers, N-CAM and otx2 in a concentration-dependent manner without induction of mesodermal markers, muscle specific-actin (ms-actin) and collagen type II (col II). 1 ng or 5 ng of FRL-1-injected animal caps were dissected at stage 9 with the uninjected control and harvested at stage 26 for RT-PCR.

View larger version (50K): [in a new window]   Fig. 10. (A) FRL-1 caused neural induction via FGF signaling in animal caps. The animal caps that were injected with 1 ng of FRL-1 alone or co-injected with 2 ng of XFD, were dissected at stage 9, together with an uninjected control and harvested at stage 25 with the whole embryos for RT-PCR analysis. FRL-1 induced the expression of N-CAM without induction of ms-actin, which was inhibited by XFD. (B) FRL-1 stimulated the MAPK activity. 1 ng of FRL-1 was injected into each animal blastomere of 4-cell-stage embryos, which were dissected at stage 9 with an uninjected control in Ca2+- and Mg2+-free MBS and harvested at stage 10.5 for western blotting. bFGF treatment (100 ng/ml) served as a positive control. FRL-1 induced MAPK activation to the same extent as bFGF, and to a greater extent than in uninjected caps. Total MAPK protein (ERK) was detected and used as a loading control. (C) Injection of FRL-1MO downregulated MAPK activity. Animal caps injected with 20 ng of FRL-1-MO or FRL-1-4misMO were dissected at stage 9 with the uninjected control and harvested at stage 10.5 for western blotting. (D,E) MAPK activity was enhanced in FRL-1-injected embryos. Activation of MAPK was detected by whole-mount immunohistology. 500 pg of lacZ mRNA was injected with (D) and without (E) 1 ng of FRL-1 into one blastomere of 2-cell-stage embryos. Injected embryos were grown to stage 9. The injected regions were visualized by red gal staining and the MAPK activated-region was visualised by blue staining. (F,G) Activated MAPKK rescued the phenotype caused by FRL-1MO. Head structure defects induced by 15 ng of FRL-1MO were rescued by co-injection of 2 ng of SESE-MAPKK (constitutively-activated MAPKK).

View larger version (23K): [in a new window]   Fig. 11. (A) FRL-1 enhances MAPK activity to cause neural induction in animal caps. Animal caps injected with 1 ng of FRL-1 were dissected at stage 8, together with an uninjected control (lane 1). FRL-1-injected animal caps were cultured in 100% Steinberg's solution (SS) with 0.1% bovine serum only (0.1% BSA in SS) (lane 2), or with LY294002 (20 µM), a specific inhibitor of phosphatidylinositol 3 kinase, in 0.1% BSA in SS (lane 3) or with PD98059 (20 µM), a inhibitor of MEK (MAPKK), in 0.1% BSA in SS (lane 4) or with dimethyl sulfoxide (DMSO) in 0.1% BSA in SS (lane 5) until sampling, then harvested at stage 25 with the whole embryos for RT-PCR. Since PD98059 and LY294002 were dissolved in DMSO, DMSO treatment is a control to exclude DMSO effects. FRL-1 induced the expression of N-CAM (lane 2), and this was inhibited by treatment with PD98059 but not LY294002, compared with DMSO alone. (B) FRL-1 inhibition of BMP signaling is required for MAPK activation. FRL-1 or uninjected animal caps were dissected at stage 8 and treated with or without PD98059 or DMSO only. The animal caps and control embryos were harvested at stage 12. The expression of BMP responsive genes, Xvent-1 and Xmsx-1 were inhibited by injection of FRL-1 whereas the inhibition of Xvent-1 and Xmsx-1 was rescued by treatment with PD98059.

View larger version (32K): [in a new window]   Fig. 2. (A) Both the EGF and CFC domain are necessary for FRL-1-mediated induction of expression of neural markers. 1 ng of FRL-1-6myc, which encodes for FRL-1 tagged with six c-myc-epitope (FRL-1), the EGF domain-deleted form of FRL-1-6myc (FRL-1EGF domain) or the CFC domain-deleted form of FRL-1-6myc (FRL-1CFC domain) was injected into each animal blastomere of 4-cell-stage embryos. Animal caps were dissected at stage 9 with an uninjected control and harvested at stage 25 for RT-PCR. Western blotting analysis showed FRL-1-6myc, FRL-1EGF domain-6myc and FRL-1CFC domain-6myc proteins were generated in animal caps at stage 10.5. Although FRL-1 induced the expression of N-CAM, it was not detected in the FRL-1EGF domain and FRL-1CFC domain mRNA-injected animal caps. (B) Activity of neural induction by FRL-1 was conserved among other members of the EGF-CFC family in vertebrates. Animal caps injected with 2 ng of zebrafish oep, mouse cripto or chick CFC (chick cripto) were dissected at stage 9 with an uninjected control and harvested at stage 25 with whole embryos for RT-PCR. The data showed that not only FRL-1 but also oep, chick CFC, and mouse cripto induced the expression of N-CAM without ms-actin.

View larger version (31K): [in a new window]   Fig. 3. FRL-1MO specifically inhibited the translation of FRL-1. 10 ng of FRL-1-6myc mRNA, which codes for FRL-1 tagged with six c-myc-epitopes was injected with or without 40 ng of FRL-1MO or FRL-1-4misMO into oocytes. 10 ng of FRL-15'UTR-6myc lacking the target sequence of FRL-1MO, was also injected with or without 40 ng of FRL-1MO. Lysates with uninjected controls were used for western blotting. FRL-1 proteins with the 6myc-epitope tag were detected by anti-c-myc antibody. The translation of FRL-1 was inhibited by FRL-1MO but not by FRL-1-4misMO, whereas that of FRL-15'UTR was not inhibited by FRL-1MO.

View larger version (107K): [in a new window]   Fig. 4. FRL-1MOs specifically induced head defect phenotypes. FRL-1MOs were injected into animal blastomeres of 8-cell-stage embryos, which were grown to stage 40. (A) Uninjected embryo. (B) Embryo injected with 15 ng FRL-1MO showing defects of the head including lack of eyes. (C) Embryo injected with 15 ng of FRL-1-4misMO showing no defect. (D) Head structure defect induced by 15 ng of FRL-1MO was rescued by co-injection of 20 pg of FRL-15'UTR. (E,F) Some other EGF-CFC genes can rescue the head defect phenotype caused by FRL-1MO. (E) Co-injection with 20 pg of oep rescued head defects caused by microinjection of 15 ng of FRL-1MO. (F) Co-injection with 20 pg of mouse cripto also rescued head defects. (G,H) FRL-1MO-second suppressed neural formation as well as FRL-1MO. (G) Embryo injected with 40 ng of FRL-1MO-second showing the same phenotype as the FRL-1MO-injected embryo. (H) Head structure defects caused by microinjection of 40 ng of FRL-1MO-second was rescued by co-injection with 20 pg of FRL-15'UTR.

View larger version (71K): [in a new window]   Fig. 5. Neural induction is suppressed in FRL-1MO-injected embryos. (A,B) FRL-1MO or FRL-1-4misMO was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 30. (A) Embryo injected with 10 ng of FRL-1-4misMO. Transcripts of otx2 were detected in the pineal gland and in the eye. (B) Embryo injected with 10 ng of FRL-1MO, showing no expression of otx2, confirming the loss of anterior neural tissue. (C-F) FRL-1MO, FRL-1-4mis-MO or FRL-1MO-second was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 30 for whole-mount in situ immunohistology analysis using Neu-1. (C) Uninjected embryo. (D) Embryo injected with 15 ng of FRL-1MO. (E) Embryo injected with 40 ng of FRL-1-4misMO. (F) Embryo injected with 40 ng of FRL-1MO-second. (G.H) Histological analysis shows that the embryos injected with FRL-1MO had neural tissue defects without loss of mesodermal tissue, such as notochord and pronephric tube. nc; notochord, pt; pronephric tube, sc; spinal cord. FRL-1MO or FRL-1-4misMO was injected into animal blastomeres of 8-cell-stage embryos, which were fixed at stage 40. (G) Histological section of 10 ng of FRL-1MO-injected embryos. (H) Histological section of embryo injected with 10 ng of FRL-1-4misMO.

View larger version (34K): [in a new window]   Fig. 6. FRL-1MO inhibits the expression of neural markers without inhibition of mesodermal and endodermal markers in Xenopus embryos. 10 ng of FRL-1MO injected into each animal or vegetal blastomere of 8-cell-stage embryos or FRL-1-4misMO injected into each animal blastomere of 8-cell-stage embryos, which were cultured until stage 31 for RT-PCR. FRL-1MO vegetally injected and FRL-1-4misMO animally injected did not affect the expression of any marker when compared with controls (uninjected), whereas FRL-1MO animally injected suppressed the expression of neural markers N-CAM, otx2, ß-crystallin and N-tubulin. The suppression of neural markers was rescued by 20 pg of FRL-15'UTR.

View larger version (60K): [in a new window]   Fig. 7. FRL-1MO inhibits an early step in the neural induction of Xenopus embryos. Albino embryos were injected with 20 ng of FRL-1MO (B,D,F,H), or FRL-1-4misMO (A,C,E,G) and 250 pg of lacZ into the dorsal marginal zone of a blastomere at the 4-cell-stage for analysis of chordin and a dorso-animal blastomere of 8-cell-stage embryos for analysis of zic3, soxD and Xngnr-1. They were cultured until stage 10.5 for whole-mount in situ analysis of chordin (A,B) and until stage 13 for analysis of zic3 (C,D), soxD (E,F) and Xngnr-1 (G,H) and then stained in the MO-injected region with red gal. The suppression of the neural markers zic3 (D, 77%, n=22), soxD (F, 84%, n=37) and Xngnr-1 (H, 86%, n=21) was observed in the FRL-1MO-injected region whereas chordin was not suppressed (B, 0%, n=29). However, there was no change in the FRL-1-4misMO-injected region (A, 100%, n=22; C, 96%, n=26; E, 93%, n=75; G, 91%, n=48). Arrows in G and H indicate trigeminal ganglia, where the expression of Xngnr-1 was suppressed in H.

View larger version (37K): [in a new window]   Fig. 8. FRL-1MO inhibits the induction of neural markers by chordin in animal caps. 100 pg of chordin was injected into the animal pole of 4-cell-stage embryos with 40 ng of FRL-1-4misMO (lane 3), FRL-1MO (lane 4), FRL-1MO and 500 pg of FRL-1 (lane 5), or without MO (lane 2). The animal caps were dissected at stage 9 and harvested at stage 22 for RT-PCR. chordin induces the expression of N-CAM, otx2 and N-tubulin without the induction of mesodermal markers ms-actin and collagen type II, which was inhibited by FRL-1MO but not by FRL-1-4misMO. The suppression of neural markers caused by FRL-1MO was rescued by the co-injection of FRL-15'UTR.

View larger version (47K): [in a new window]   Fig. 9. FRL-1 expression in neuroectoderm at the late gastrula stage. (A,B) Whole-mount in situ hybridization revealed the spatial expression patterns of FRL-1. (A) Lateral view of a stage 10 embryo; right side is dorsal. FRL-1 was ubiquitously expressed in the early gastrula. (B) Lateral view of a stage 12 embryo; right side is dorsal. Transcripts of FRL-1 are concentrated in the neuroectoderm. (C) RT-PCR analysis of chordin-injected ectoderm. Misexpression of chordin induced the expression of FRL-1 in the absence of the mesodermal marker, Xbra.

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