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First published online 12 April 2006
doi: 10.1242/dev.02350

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Medaka simplet (FAM53B) belongs to a family of novel vertebrate genes controlling cell proliferation

¹ INRA MSNC Group, DEPSN, Institut A. Fessard, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
² Oncodesign, 20 rue Jean Mazen, 21000 Dijon, France.

View larger version (29K): [in a new window]   Fig. 1. Medaka smp belongs to a novel family of vertebrate genes. (A) Smp bears two highly conserved regions (HRI and HRII), two 14-3-3 consensus-binding domains and a nuclear-localization signal (NLS). (B) A phylogenetic tree of the smp family showing two smp groups: A and B. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio; Fr, Fugu rubripes; Tn, Tetraodon negroviridis; Ol, Oryzias latipes. (C) Western blot after co-immunoprecipitation of 14-3-3 or SKIIP with Ol-Smp and FAM53B. Co-immunoprecipitation reveals an interaction of SKIIP with human FAM53B, but not with Ol-Smp.

View larger version (67K): [in a new window]   Fig. 2. smp expression pattern throughout medaka embryonic development. WMISH at different embryonic stages. (A,B) Maternal smp transcripts were detected at the one-cell stage (A) and the four-cell stage (B). (C,D) At the 512-cell stage, transcripts were detected in central blastomeres but not in peripheral ones (D, arrow). (E,F) Transverse sections confirm that smp expression is not present in peripheral blastomeres (arrow), and that Ol-pou5f1 is expressed in both inner and marginal ones. (G,H) From 2-somite stage to 16-somite stage, smp is expressed at the anterior midline of the telencephalon (t), at the prospective midbrain-hindbrain boundary (mhb) and in the recently formed somites (s). (I) In 35-somite embryos, smp is detected in the progenitor region of the retina (r, arrow) and in a large portion of the CNS, including the forebrain (f), optic tectum (ot), mhb and rhombic lips (rl).

View larger version (121K): [in a new window]   Fig. 3. smp and PCNA transcript distributions showing that smp is expressed in CNS proliferating cells. (A) Drawing indicating section plans. (B-G) 34/35-somite embryos were sectioned after WMISH with a smp (B,D,F) or a PCNA (C,E,G) probe. smp and PCNA expression domains in the CNS were compared in the telencephalon (Tel), the diencephalon (Di) and the mesencephalon. Telencephalic (B,C) and diencephalic (D,E) sections show that the dorsal and peri-ventricular domains are positive for smp and PCNA. On diencephalic sections, eyes show ciliary marginal zones positive for both smp and PCNA. (F,G) Sections through the mesencephalon with smp and PCNA staining at the margin of the optic tectum (ot). Scale bars: 60 µm.

View larger version (80K): [in a new window]   Fig. 4. smp loss of function. Embryos were injected with smpMOI coupled with carboxyfluorescein. Dose-dependant phenotypes were observed at two developmental stages. (A-C) Lateral views of embryos at 25 hpf (corresponding to late gastrula stage). (D-F) Dorsal views of embryos at 44 hpf (corresponding to the 16-somite stage). (A,D) Control embryos. (B) A class II embryo displaying a strong delay of epiboly and abnormally large blastomeres (inset, arrowheads). (C) A class I embryo. Development is arrested at the beginning of epiboly. (D-F) Images shown at the same magnification to demonstrate the reduced size of abnormal embryos. (E) Mildly affected embryos that display 16 somites but have small eyes and poorly developed midbrain-hindbrain regions. (F) A severely affected embryo that lacks any diagnostic feature of the developmental stage. (G) Percentage of embryos of each type determined at 44 hpf, after sorting at 25 hpf into class I, II or III. Percentages were calculated from an average of eight independent experiments (smpMOI at 6-8 mg/ml, 190 embryos in total). Scale bars: 100 µm.

View larger version (121K): [in a new window]   Fig. 5. smp knockdown does not induce apoptosis. (A,B) At late blastula, the nuclear morphology of injected embryos was analyzed by DAPI staining (n=12 for injected-embryos, n=5 for control embryos). Flat-mounts of blastula displayed no evidence of apoptosis, although smpMOI-injected embryos have fewer nuclei. (C-G) TUNEL assay at later stages (dorsal views). No massive induction of apoptosis was detected in smpMOI-injected embryos at late gastrula (D, n=18) or at the 18-somite (G, n=23) stage, when compared with controls (C,F; n=7 and 6, respectively). (E) A few class II embryos displayed an increase in apoptosis, probably resulting from a secondary phenomenon due to epiboly delays. Scale bars: 100 µm.

View larger version (23K): [in a new window]   Fig. 6. DNA content at early gastrula stage. (A) Uninjected embryos (n=14). (B) smpMOI-injected embryos (n=14). The proportions of cells in each phase of the cell cycle are similar in A and B.

View larger version (74K): [in a new window]   Fig. 7. smp is involved in cell proliferation from MBT onwards. Clonal injections with either control MOs (smpMOIC and smpMOIIC) or specific MOs (smpMOI and smpMOII), coupled with carboxyfluorescein (-F) or lissamine (-L), respectively. (A-H) Injected embryos observed at early gastrula (A,D), mid gastrula (B,E) or late gastrula (C,F) stages. (A,B) Animal and dorsal views of control-injected embryos. (D,E) Animal and dorsal views of smpMO-injected embryos, showing smp-morphant cells that derived from the injected blastomere. Some of them remain aggregated during gastrulation (white dotted lines indicate the margin of the blastoderm). (C,F) At late gastrula stage, smpMO-containing cells are excluded from the embryonic axis (white dotted lines delimit both sides of the body axes). (G) Number of fluorescent cells per embryo, at early gastrula stage, within a single experiment (n, number of embryos). The average number of fluorescent cells is 67.3±4.3 for control embryos and 42.2±9.8 for smpMO-injected embryos. These values are significantly different (P<0.02). (H) Half of the smpMOII-L-injected embryos display a reduced number of isolated fluorescent cells (<50), always associated with a single cluster of cells. (I-N) Confocal microscope observation of flat mounts of early gastrula embryos after clonal injection with smpMOIIC-L (I-K, n=3) or smpMOII-L (L-N, n=5). The nuclear morphology of smpMO-containing cells (red) was analyzed by DAPI staining (blue). Control-injected cells showed normal mitotic (arrowhead) and interphasic (arrow and inset) nuclei (I-K). By contrast, smpMOII-L-injected blastomeres have descendants with abnormally large nuclei (L-N, arrows and inset).

View larger version (52K): [in a new window]   Fig. 8. smp regulates Ol-pou5f1 expression. (A,B) Transverse sections, after WMISH, showing Ol-pou5f1 expression in early gastrula embryos after smpMO injection (8 mg/ml). Decreased expression was observed in deep cells, but not in the EVL of injected embryos (arrowhead). (C-F) Embryos injected at the 32-cell stage with EGFP RNA (100 µg/ml), either alone (C,E) or together with smpRNA (500 µg/ml, D,F). At mid-gastrulation (C,D, dorsal view), no apparent modifications can be observed (white dotted lines indicate the blastoderm margin). At the 2-somite stage (E,F, dorsal view), fluorescent cells are excluded from the embryo axis and are found near the anterior body axis. (G) Lateral view of a 2-somite stage control embryo showing Ol-pou5f1 expression in telencephalon (t) and tailbud (tb). (H,I) Ol-pou5f1 expression at the 2-somite stage after smpRNA injection into two-cell stage embryos. (H) Ectopic expression is visible in the telencephalon (t), ventral mhb and rhombencephalic domains (rb). (I) A more widespread expression is observed in strongly affected embryos. (J-O) Tranverse sections of WMISH embryos. (J-L) Control embryos. Ol-pou5f1 CNS expression is restricted to the dorsal telencephalon (arrowhead). (M-O) After injection of smpRNA, ectopic Ol-pou5f1 expression was observed in telencephalon (M, arrowhead), and in the ventral mhb (N, arrowhead) and rhombencephalon (O). Scale bars: 30 µm.