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Fig. 1. Characterization of the Xenopus fam3b gene and purification of recombinant human FAM3B-His protein. (A) RT-PCR assay showing fam3b expression across different developmental stages. odc was used as a loading control. –RT served as a negative control. (B–F') WISH of fam3b showing its diffuse localization at two-cell (B and B'), blastula (C and C'), and gastrula (D and D') stages (in Xenopus WISH, uniform mRNAs appear stronger in the animal pole due to the accumulation of large yolk platelets in the vegetal pole). At neurula stages, fam3b is localized to the epidermis (E and F'). (Scale bar, 500 μm.) (G) Xenopus fam3b encodes a secreted protein. HEK293T cells were transfected with His-tagged human FAM3B or Xenopus Fam3b constructs. Twenty-four hours after transfection, cells were cultured in serum-free medium for further 20 h. The lysate and medium were subjected to immunoblotting with the indicated antibodies. α-Tubulin served as a loading control and was detected only in the lysate and not in the medium. (H) Flowchart showing purification of His-tagged human FAM3B protein from CM generated from transfected HEK293T cells (Materials and Methods). (I) SDS-PAGE and Coomassie Blue staining showing the purity of recombinant human FAM3B-His protein. Arrowhead indicates the molecular weight of FAM3B protein.
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Fig. 2. FAM3B protein microinjection inhibits head development and induces ectopic tail-like structures. BSA or rhFAM3B protein (40 nL at 15 μM) was injected into the blastocoele cavity of blastula-stage embryos. Embryos were cultured until the neurula or tailbud stages and subjected to phenotypic analysis or WISH as indicated. (A and B') rhFAM3B protein causes microcephaly and short A–P axis and induces ectopic tail-like structures. (A) Lateral view of control. (B) Lateral view. (B') Dorsal view. Arrowheads indicate ectopic tail-like structures. (C–F') rhFAM3B protein decreases expression of the forebrain marker bf1 (C and C'), forebrain–midbrain marker otx2 (D and D'), and eye marker rx2a (E and E') and induces ectopic expression (arrowhead) of the spinal cord marker hoxb9 (F and F'). (G–N') rhFAM3B protein-induced tail-like structures express tailbud markers xpo (G and G'), xbra (H and H'), fgf3 (I and I'), and cdx4 (J and J'), pan neural marker sox2 (K and K'), chordoneural hinge markers chd (L and L') and not (M and M') and somite marker myod (N and N'). Numbers of embryos analyzed were as follows: A, n = 74, 100%; B and B', n = 128, 97% with ectopic tail phenotype; C, n = 37, 100%; C', n = 44, 93%; D, n = 35, 100%; D', n = 42, 97%; E, n = 26, 100%; E', n = 36, 94%; F, n = 32, 100%; F', n = 40, 95%; G, n = 29, 100%; G', n = 41, 95%; H, n = 30, 100%; H', n = 33, 93%; I, n = 27, 100%; I', n = 39, 92%; J, n = 36, 100%; J', n = 40, 92%; K, n = 41, 100%; K', n = 45, 97%; L, n = 34, 100%; L', n = 47, 95%; M, n = 38, 100%; M', n = 42, 88%; N, n = 35, 100%; N', n = 48, 95%. (Scale bars for A and B', C–F', and G–N' all indicate 500 μm.)
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Fig. 3. Knockdown of fam3b expands anterior tissues, and the phenotype is rescued by low amounts of rhFAM3B protein. Embryos were first injected with control MO (120 ng) or fam3b MOs (60 ng fam3b.l MO and 60 ng fam3b.s MO) at the one-cell stage and subsequently BSA or rhFAM3B protein (40 nL at 3 μM) were injected into the blastocoele cavity at blastula stage, as indicated in the panels. Embryos were cultured until tailbud stage for phenotypic analysis or neurula stage for WISH with the indicated neural markers. (A–D) fam3b knockdown leads to enlarged heads and shorter A–P axis, which was rescued by injection of rhFAM3B protein. Arrowhead in B indicates enlarged cement gland. (E–P) fam3b knockdown expands forebrain markers bf1 (E–H), forebrain–midbrain marker otx2 (I–L), and eye marker rx2a (M–P), a phenotype that was rescued by injection of rhFAM3B protein at low doses. Numbers of embryos analyzed were as follows: A, n = 85, 100%; B, n = 96, 92% with enlarged cement glands; C, n = 90, 100%; D, n = 113, 86%; E, n = 53, 100%; F, n = 55, 96%; G, n = 49, 95%; H, n = 60, 88%; I, n = 38, 100%; J, n = 43, 95%; K, n = 50, 96%; L, n = 46, 91%; M, n = 37, 100%; N, n = 47, 93%; O, n = 39, 94%; P, n = 43, 86%. (Scale bars for A–D and E–P indicate 500 μm.)
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Fig. 4. FAM3B binds to FGFR and activates ERK through FGFR. (A) FAM3B binds to full-length FGFR1 in Xenopus embryos. Two-cell-stage embryos were injected with FAM3B-Flag mRNA (400 pg) and wild type (WT) or kinase-inactive mutant (KI) FGFR1-HA mRNA (400 pg) separately into different blastomeres. Gastrula-stage embryos were harvested for immunoprecipitation with Flag beads followed by immunoblotting with Flag and HA antibodies. Total protein expression was confirmed by immunoblotting of the input. (B) FAM3B binds to the ectodomain of FGFR1 in solution. CM for FAM3B-HA and ectodomain of FGFR1 (FGFR1-Ecto-Flag) were combined and allowed to bind, followed by immunoprecipitation with Flag beads and subsequent immunoblotting with Flag and HA antibodies. Protein expression in CM was confirmed by immunoblotting of the input. (C) FAM3B does not bind to β-Klotho ectodomain. CM for the ectodomain of β-Klotho (β-Klotho-Ecto-HA) and FAM3B-Flag were combined and allowed to bind. Total protein expression in the CM was confirmed by immunoblotting of the input. This experiment serves as a negative control for binding to the extracellular domain of FGFRs. (D–G) FAM3B binds to FGFR ectodomain in vitro. Purified rhFAM3B protein (1 μg) was incubated with Fc-tagged human FGFR ectodomain protein (1 μg), including FGFR1-Ecto-Fc (D), FGFR2-Ecto-Fc (E), FGFR3-Ecto-Fc (F), and FGFR4-Ecto-Fc (G) as indicated. Then the protein mixture was subjected to pull-down with Protein A magnetic beads and subsequent immunoblotting with Fc and His antibodies. Protein expression was confirmed by immunoblotting of the input. PD: pull-down; PA: Protein A magnetic beads. (H) FAM3B CM rapidly activates ERK. HEK293T cells were serum starved for 24 h and treated with serum-free control or FAM3B CM for the indicated times. Cells were harvested for immunoblotting with pERK and total ERK antibodies. α-Tubulin served as a loading control. (I) FAM3B CM-induced ERK activation is blocked by FGFR inhibition. HEK293T cells transfected with FGFR1 KI or not were serum starved for 24 h and pretreated with FGFR inhibitors SU5402 (20 μM), AZD4547 (1 μM), Erdafitinib (1 μM), or Ly2874455 (1 μM) for 2 h. Then cells were stimulated with serum-free control CM or FAM3B CM in the presence of these inhibitors for 20 min and harvested for immunoblotting with the indicated antibodies. α-Tubulin served as a loading control. (J) FAM3B CM activated an FGF reporter derived from the mouse Dusp6 promoter (40), which was blocked by FGFR1 inhibition. HEK293T cells were transfected with FGF Luciferase reporter together with Renilla and FGFR1 KI as indicated. Twenty hours after transfection, cells were serum starved for 20 h, followed by treatment with serum-free control or FAM3B CM for 8 h. Experiments were performed in triplicate. Data are mean ± SD. Statistical significance was assessed by unpaired two-tailed Student’s t test. ***P < 0.001. RLA, Relative Luciferase Activity.
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Fig. 5. FAM3B induces (A–P) axial patterning defects through FGFR in Xenopus embryos. Embryos were injected with FGFR1 KI mRNA (100 pg) at the one-cell stage and rhFAM3B protein (40 nL at 15 μM) into the blastocoele cavity at the blastula stage, followed by treatment with SU5402 (20 μM), Erdafitinib (2 μM), or Ly2874455 (20 nM) as indicated. Neurula or tailbud stage embryos were collected for WISH with bf1 (A–F), otx2 (G–L), sox2 (M–R), and xbra (S–X). rhFAM3B protein reduces the head markers bf1 and otx2 and induces sox2 and xbra in ectopic tails, and all phenotypes were rescued by FGFR1 KI mRNA, SU5402, Erdafitinib, or Ly2874455. Arrowheads indicate ectopic tail-like structures. Note that relatively low doses of FGFR1 KI mRNA and FGFR inhibitors were used here. Higher doses of SU5402 (50 to 100 μM) have been shown to cause defective blastopore closure and severe posterior truncation in Xenopus embryos by its own (9, 45). Higher doses of FGFR1 KI mRNA, Erdafitinib, or Ly2874455 had similar blastopore closure effects as those of high dose SU5402. (Scale bars, 500 μm.) Numbers of embryos analyzed were as follows: A, n = 34, 100%; B, n = 65, 98% with microcephaly; C, n = 40, 92%; D, n = 38, 73%; E, n = 41, 85%; F, n = 39, 82%; G, n = 54, 100%; H, n = 49, 100%; I, n = 47, 93%; J, n = 37, 89%; K, n = 56, 91%; L, n = 51, 92%; M, n = 35, 100%; N, n = 63, 95%; O, n = 45, 93%; P, n = 42, 90%; Q, n = 36, 91%; R, n = 40, 95%; S, n = 43, 100%; T, n = 55, 98%; U, n = 44, 90%; V, n = 66, 87%; W, n = 49, 83%; X, n = 53, 92%.
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