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Fig. 1.
Molecular analysis and expression of FAM3C in the mouse and Xenopus retina. (A) Domain structure of mouse FAM3C. The gray box indicates a signal peptide (SP). The checkered box indicates a GG domain. Panel shows the percent amino acid identity and similarity of FAM3C among vertebrates. (B) Northern blot analysis of FAM3C transcript in adult mouse tissues. The size of the FAM3C transcript is approximately 2.7 kb. A strong band was detected in the retina. Lower, ethidium bromide staining of total RNA. The retina lane contained 5 μg of total RNA, and the other lanes contained approximately 10 μg of total RNA each. (C) Western blot analysis. FAM3C-FLAG was detected in 20-fold concentrated supernatant. The size of the FAM3C protein is approximately 25 kDa. (D–F) Section ISH for mouse FAM3C. The FAM3C signal was detected in the GCL (arrows) at E15.5 (D), P3 (E) and adult (F). The FAM3C signal was also detected in the ciliary body (arrowheads in D). (G) The XFAM3C signal was detected in the GCL of the Xenopus retina at stage 39. (H–K) Whole mount ISH of Xenopus embryos for XFAM3C. Lateral views with the anterior to the right at stage 24 (H), stage 32 (I), Stage 33/34 (J), Stage 39 (K). re, retina; le, lens; NBL, neuroblastic layer; PRL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars represent 0.1 mm (D–G) and 1.0 mm (H–K).
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Fig. 2.
XFAM3C overexpression disrupts retinal laminar formation and increases eye size. Control side (A, C, E, I, K) and XFAM3C + β-gal-injected side (B, D, F, H, J, L) of the Xenopus retinas at stage 37/38. (G, K) Control β-gal-injected retina at stage 37/38. The injected retinas were DAPI-stained (A, B). Immunostaining of the injected retinas with antibodies for Rhodopsin (C, D), Islet1 (G, H), and Phospho-histone H3 (red) with DAPI (blue) (K, L). Section ISH for Pax6 (E, F) and Rax (I, J). (B) Retinal architecture was disrupted in the XFAM3C-injected eyes. (D, F, H) Rhodopsin, Pax6 and Islet1 signals were dispersed throughout the retina. (J) Rax expression was not restricted to the CMZ in the XFAM3C-injected retina. (L) M-phase cells were dispersed throughout the retina. The retina is circled with a white line (K, L). (M) There was no significant difference of the proliferative rate between the control and XFAM3C-injected retina at both stage 31 and stage 37/38. (N) Retinal cell number per 10,000 μm2. Asterisks denote statistically significant changes (**P < 0.01). Error bars are ±SEM. White arrowheads indicate the CMZ (I, K). re, retina; le, lens; PRL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars represent 0.1 mm.
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Fig. 3.
Knocking down of XFAM3C results in the mislocalization of photoreceptor cells. Control side (AâC, GâI) and XFAM3C MO + β-gal-injected side (DâF, JâL) of the retinas at stage 37/38. Cryosection of the retinas followed by X-gal staining (A, D), DAPI-staining (B, E), or immunostaining for Islet1 (C, F), Rhodopsin (G, J). The control side and XFAM3C MO injected side retinas in section ISH with Otx2 (H, K) or Pax6 probes (I, L). (F) Islet1-positive cells are located on the basal side of the retina. (J) Rod photoreceptors form rosette-like structure. (K, L) Otx2 and Pax6 expression patterns were unaffected. (M, P) Control MO-injected retina at stage 33/34. (N, O, Q, R) XFAM3C MO-injected retinas at stage 33/34. (MâO) Injection efficiency is monitored by immunostaining of β-gal. (PâR) Immunostaining of the injected retinas with antibodies against phospho-histone H3 (red) with DAPI (blue). (P) M-phase cells were normally located on the apical surface of the retina at this stage. (Q, R) M-phase cells were mislocated at intermediate positions in the XFAM3C MO-injected retina. (N, Q) Mislocalization of phospho-histone H3-positive cells was observed only in the injected region. The injected region is circled with a white line. re, retina; le, lens; PRL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars represent 0.1 mm.
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Fig. 4.
Photoreceptor cell mislocalization caused by XFAM3C MO was partially rescued by mutated XFAM3C RNA-coinjection. (A, C) Retinas injected with XFAM3C MO and β-gal with XFAM3Cm6 RNA. (B, D) Retinas injected with XFAM3C MO + β-gal. (A, B) Immunostaining of the injected retinas with Rhodopsin (red) with DAPI (blue). (A) Rod photoreceptor location was partially rescued by XFAM3Cm6 RNA-coinjection. (B) XFAM3C MO caused rod photoreceptor mislocalization. (C and D) β-gal immunostaining of the injected retinas. C and D are adjacent sections of A and B, respectively. The injected part is circled with white line. re, retina; le, lens. Scale bars represent 0.1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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fam3c (family with sequence similarity 3 member C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
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