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First published online 5 December 2007
doi: 10.1242/dev.011445

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Maternally derived FILIA-MATER complex localizes asymmetrically in cleavage-stage mouse embryos

Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Expression of Mater in mice with targeted mutation. (A) Northern blot of total RNA (0.6 µg) from 10-day-old ovaries isolated from wild-type, Matertm/+ and Matertm/tm mice. After separation by gel electrophoresis and transfer to nylon membrane, blots were hybridized with 32P-labeled Mater or Zp2 (control) cDNA, washed and imaged. (B) Immunoblot of ovulated eggs (10) isolated from wild-type, Matertm/+ and Matertm/tm mice. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with peptide affinity-purified antibody to MATER and visualized by chemiluminescence. (C) Two-dimensional gels of de novo synthesized proteins in one-cell (a-c) and two-cell (d-f) mouse embryos, collected from gonadotrophin-stimulated, mated females 24 and 43 hours after administration of hCG, respectively. After in vitro labeling with [35S]methionine/[35S]cysteine, 30 embryos were focused on isoelectric gels (pH 4-7) and separated by SDS-PAGE. After visualization with fluorography, radioactivity was rendered red (wild type, a,d) or green (Matertm/tm, b,e) and merged (c,f). Size (kb) is indicated on the left (A) and molecular masses (kDa) are indicated on the left (B) or right (C).

View larger version (40K): [in this window] [in a new window]   Fig. 2. Identification of FILIA. (A) SDS-PAGE of zona-free ovulated eggs (2000) isolated from wild-type or Matertm/tm mice. After fixation and staining, a protein (50 kDa) diminished in Matertm/tm eggs was designated FILIA and excised from the gel for analysis by mass spectrometry. Molecular masses (kDa) are indicated on the left. (B) RT-PCR of total RNA isolated from kidney, ovary, uterus, testis, brain, heart, lung, liver, spleen, stomach, small intestines, muscle and water using primers specific for Filia (top) or actin (bottom) to control for RNA integrity. Size (bp) is indicated on the left. (C) In situ hybridization of fixed, paraffin-embedded wild-type ovarian (2-week-old) sections probed with DIG-labeled antisense (a) and sense (b) synthetic Filia oligonucleotide probes. (D) Total RNA (0.6 µg) was isolated from ovaries of wild-type, Matertm/+ and Matertm/tm mice, separated by electrophoresis, transferred to a nylon membrane and probed with 32P-labeled FILIA cDNA. Molecular masses (kb) are indicated on the left. The two isoforms, FILIA 1.6 and FILIA 1.2, are indicated on the right. Scale bar: 50 µm. -, water; Br, brain; Hrt, heart; Ki, kidney; Li, liver; Lu, lung; Mu, muscle; Ov, ovary; Si, small intestines; Sp, spleen; St, stomach; Te, testis; Ut, uterus.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Alternative splicing of Filia in mice. (A) (a) Filia is a single-copy gene with three exons on chromosome 9 (73,235,088-73,237,040) that is transcribed as two isoforms. (b) The less abundant 1.6 kb transcript encodes a 440 amino acid protein with a smaller molecular mass (48 kDa) than predicted (70 kDa) by its mobility on SDS-PAGE. The tenfold repeat is indicated by double-arrow line, and peptides obtained by mass spectrometry (yellow rectangles) are aligned at the bottom. (c) Same as b for the 1.2 kb transcript that encodes a 346 amino acid protein with a smaller molecular mass (38 kDa) than predicted (50 kDa) by its mobility on SDS-PAGE. (B) Synthetic oligonucleotide primers and Taqman probes designed to distinguish the alternatively spliced 1.6 kb (upper) and 1.2 kb (lower) isoforms of Filia transcripts. E, exon.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Identification of potential protein isoforms of FILIA in mice. (A) Predicted primary structure of 48 kDa (upper) and 38 kDa (lower) isoforms with 440 and 346 amino acids, respectively. N-terminal peptide immunogen common to both isoforms is underlined (black) and regions (amino acids 120-350 and 120-340, respectively) containing a 23 amino acid repeat are indicated by a dashed line (black). Sequences unique to each are bold and italicized. The 50 kDa band (Fig. 2A) containing the presumptive 38 kDa isoform was digested with trypsin and microscale mass spectrometry identified peptides (red) common to both isoforms. (B) FILIA contains a novel, 23 amino acid tandem repeat (120-350 amino acids) defined by RADAR (Heger and Holm, 2000). Amino acids are color-coded: aliphatic, red; uncharged polar, green; acidic, blue; basic, yellow; neutral, magenta. (C) Fluorography of in vitro translation with [35S]methionine of FILIA cDNA encoding the 1.6 (lane 1) and 1.2 (lane 2) kb isoforms of FILIA protein after separation by SDS-PAGE. Molecular masses (kDa) are indicated on the left.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Developmental expression of Filia. (A) The relative abundance of Filia and Mater transcripts in mouse oocytes, eggs and preimplantation embryos. Poly (A)+ RNA was isolated from oocytes/eggs/embryos, reverse transcribed with oligo (dT) and aliquots were analyzed by qRT-PCR using synthetic oligonucleotide primers and Taqman probes specific to each of the two Filia isoforms and Mater. The amount of 1.2 kb Filia in mid-sized oocytes (50 µm) was set as 1.0. Inset shows primer efficiencies over four orders of magnitude of substrate, which were used to calculate PCR efficiencies for the two Filia isoforms and Mater. (B) The persistence of FILIA and MATER protein during oogenesis and preimplantation embryos. Immunoblot of lysates isolated from growing oocytes (50 µm, 75 µm), eggs, two-cell embryos, morulae and blastocysts (E3.5) was incubated with peptide-purified antibodies that bind to either MATER (upper) or to both isoforms of FILIA (lower).

View larger version (60K): [in this window] [in a new window]   Fig. 6. Co-localization of FILIA and MATER by confocal microscopy. Mouse oocytes, eggs, one-cell, two-cell and four-cell embryos, morulae and blastocysts (E3.5) were fixed, permeabilized and stained with peptide-purified antibodies to FILIA (green) and MATER (red). Co-localization of FILIA and MATER was determined by Merge (yellow) and oocytes/eggs/embryos were imaged by differential interference contrast (DIC) microscopy. Scale bar: 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 7. FILIA interacts with MATER. (A) Immunoprecipitation of MATER with antibodies to FILIA. Ovulated eggs (120) from wild-type (lanes 1,2,4) or Matertm/tm (lane 3) mice were lysed (0.5% Triton X100 in PBS) and either analyzed directly (lane 1) or immunoprecipitated with peptide-purified sheep antibodies to FILIA (lanes 2,3) or pre-immune sera (lane 4). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, incubated with peptide-purified rabbit antibodies to MATER and visualized with chemiluminescence. (B) Expression vectors containing full-length MATER (MYC-tagged) and FILIA were co-transfected into 293T cells (lanes 2,4). Controls (lanes 1,3) were co-transfected with the MYC vector lacking MATER and the FILIA expression vector. Whole cell lysates were analyzed before (left panel) or after (right panel) immunoprecipitation with antibodies to MYC. Proteins were resolved on SDS-PAGE gels, transferred to nitrocellulose and probed with antibodies to MATER (left) or FILIA (right panel). Molecular masses (kDa) are indicated at the left for both A and B.

View larger version (49K): [in this window] [in a new window]   Fig. 8. Plasticity of FILIA and MATER localization during preimplantation development. (A) Zona-free two-cell mouse embryos were incubated with [(+) Ca2+ (a-d)] or without [(-) Ca2+ (e-h)] calcium to induce disaggregation. After fixation and permeabilization, embryos were stained with antibodies specific to FILIA (a,e), MATER (b,f) and phalloidin, which binds to F-actin (c,g), before imaging by confocal (a-c,e-g) or differential interference contrast (DIC) microscopy (d,h). (B) Same as A except individual and clumps of blastomeres were isolated from single zona-free morulae in the absence of calcium for localization of FILIA (a), MATER, (b), actin (c) or visualization by DIC microscopy (d). (C) Cell cleavage of blastomeres can be parallel or orthogonal to the axis of polarity. If parallel, the daughter cells are equivalent; if orthogonal, the `outer' cells seemingly contain the FILIA-MATER complex and `inner' cells do not. The image is from Fig. 6. (D) The ICM of early two (a-c; d-f) blastocysts was isolated by immunosurgery and imaged as in A with antibodies to FILIA (a,d) and MATER (b,e) or visualized by DIC (c,f). Normal blastocysts served as positive imaging controls (data not shown). Scale bars: 20 µm in A,B,D.

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