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First published online 28 February 2007
doi: 10.1242/dev.02816

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The nuclear envelope protein MAN1 regulates TGFß signaling and vasculogenesis in the embryonic yolk sac

Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick MD 21702, USA.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Mapping the gene-trap insertion in the Man1 locus. (A) Upper panel: the Man1 gene, showing exon organization (black boxes). Lower panel: the gene-trap vector, containing the engrailed2 intron (en2) and splice acceptor (SA), a ß-galactosidase-neomycin fusion cDNA (ß-geo), an SV40 polyadenylation site (SV40pA) integrated between exons 4 and 5. `Probe' indicates the location of the genomic probe used in Southern blotting. (B) Southern blot of genomic DNA from gene-trapped mice. Approximate size of detected DNA is indicated. (C) Northern blot, demonstrating 4.7 kb endogenous and 7.7 kb Man1/ß-geo transcripts in wild-type (+/+) or heterozygous (+/GT) MEFs. (D) The MAN1 911 amino acid polypeptide contains the LEM domain at the N-terminus, the RRM domain at the C-terminus and two transmembrane domains. The gene-trap insertion product retains the 567 N-terminal amino acids (arrow) linked to ß-galactosidase. (E) Western analysis on MEF whole cell extracts using an antibody raised to a C-terminus peptide shows that the C-terminus of MAN1 is absent in homozygotes (GT/GT). +/+, wild-type; +/GT, heterozygote; E, EcoRV.

View larger version (110K): [in this window] [in a new window]   Fig. 2. Expression of the Man1-lacZ reporter in embryos. (A-C) Whole-mount lacZ staining in heterozygotes at stages E8.5-11. (D-F) Sagittal sections showing expression of Man1-lacZ in both embryonic and extraembryonic compartments. (G) Frontal section showing expression of Man1-lacZ in the CNS. (H,I) High magnification views of sections through the yolk sac. Scale bars: 1 mm in A-G; 0.05 mm in H,I. al, allantois; bc, blood cells; ce, cerebellum; ec, endothelial cells; epc, ectoplacental cone; fb, forebrain; fg, foregut; fp, floor plate; fv, fourth vesicle; hb, hindbrain; ht, heart; mb, midbrain; me, mesothelium; mv, medial vesicle; nf, neural fold; nt, neural tube; pl, placenta; po, pons; s, somites; ve, visceral endoderm; ys, yolk sac.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Morphological analysis of Man1GT/GT embryos. (A,B) Sagittal sections of wild-type (+/+) and Man1GT/GT (GT/GT) E9.5 embryos stained with H&E. Left panels are low magnification views of sagittal sections. Right panels are high magnification views of frontal sections showing overall growth retardation and abnormal cardiac development in the Man1GT/GT embryos. (C) Man1GT/GT E9.5 embryo stained for lacZ expression with strong expression in the heart, amnion and allantois. (D) Sagittal section of an Man1GT/GT E10.5 embryo stained for lacZ expression. Inset is at higher magnification. (E) Yolk-sac section of Man1GT/GT E9.5 embryo shows disruption in the endodermal and mesodermal layers. (F) lacZ staining in cultured Man1GT/GT MEFs, showing strong localization to the nuclear envelope. Scale bars: 1 mm in A-E; 50 µm in E,F. ne, neuroectoderm.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Abnormal vasculogenesis in Man1GT/GT embryos. (A-C) Whole-mount views of wild-type (+/+, left) and Man1GT/GT (right) yolk sacs. (A) A blood pool formed in the mutant yolk sac at E10.5 instead of the distinct blood vessels in the wild-type at E9.5. (B) Benzidine staining at E9.5 indicates formation of hematopoietic cells within the blood vessels. (C) Whole-mount immunohistochemistry staining at E9.5 with a PECAM-1 antibody labels endothelial cells in the blood vessels. (D-F) Whole-mount immunohistochemistry in the embryo proper at E9.5 with a blood vessel marker, FLK-1. Arrows indicate a well-formed vasculature in the wild-type (D) and disorganized vasculature (E) and dorsal aorta (F) in the Man1GT/GT. (E,F) Man1GT/GT embryos appeared to be developmentally delayed by 24-36 hours. bp, blood pool; ht, heart; nf, neural fold; ve, blood vessels; ys, yolk sac. Scale bars: 1 mm.

View larger version (99K): [in this window] [in a new window]   Fig. 5. Expression of endothelial and smooth muscle cell markers in the yolk sacs of Man1GT/GT embryos. (A) H&E staining of paraffin sections of wild-type (+/+) and Man1GT/GT yolk sacs (GT/GT). (B) Immunohistochemistry for PECAM-1 reveals disorganized morphology of endothelial cells. Embryos were stained in whole mount followed by paraffin sectioning. (C) Immunohistochemistry for FLK-1 showing vascular cell disorganization in the mutant. (D) -smooth muscle actin staining shows a reduction in smooth muscle cells in the mutant. (E) Immunofluorescence staining for fibronectin, showing an apparent increase in signal in the mesoderm of the mutant with a reduction of expression in the endoderm. am, amnion; bc, blood cells; ec, endothelial cells; me, mesothelium; ve, visceral endoderm. Scale bar: 50 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Endothelial cell proliferation is suppressed in Man1GT/GT cells. (A,B) Immunohistochemistry of EBs, grown in suspension for 10 days then explanted for 6 days, using an antibody to PECAM-1. (A) Endothelial cells form extensive branches in the wild-type (+/+). (B) The Man1GT/GT explants form fewer and less organized branches. (C) FACS analysis of PECAM-1+ cells isolated from Man1GT/GT and wild-type EB explants, showing that in cultures of both genotypes the percentage of PECAM-low expressing cells was similar, but the percentage of PECAM-high expressing cells was strongly reduced in the Man1GT/GT cultures. (D) TGFß1 further inhibits cell proliferation in Man1GT/GT fibroblasts. Wild-type and Man1GT/GT MEFs and SVEC cells were treated with TGFß1 for 72 hours at the indicated concentrations. Mean±s.e.m. (n=6 wells per treatment). Asterisks indicate statistically significant decrease in the proliferation of Man1GT/GT MEFs compared with wild-type and SVEC cells. (E) Real-time gene expression analysis of EB explants. EBs were grown in suspension for 14 days then explanted for 4 days. RNA for each data point was derived from >20 EBs and assayed in triplicate. Data are shown as a ratio of Man1GT/GT relative to wild type and were normalized to an average of S18 and Gapdh. Mean±s.e.m.; *, P<0.05; **, P<0.005; Student's t-test. Scale bar: 100 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Upregulation of TGFß signaling in Man1GT/GT MEFs. (Aa-i) Immunofluorescence staining of TGFß-treated or untreated wild-type (wt) and Man1GT/GT MEFs with an antibody to SMAD2/3 (a-c). Cells were infected with a Flag-tagged MAN1 retroviral vector; Flag-positive cells are shown in insets, Flag-negative cells are indicated by arrows (d-f). Dapi was used to show nuclei (g-i). (a) In unstimulated MEFs SMADs2/3 are distributed between the nuclei and cytoplasm. (b) Following treatment with TGFß1 SMADs2/3 accumulate in the nucleus. (c) In Man1GT/GT MEFs, SMAD2 shows a strong nuclear localization in the absence of TGFß1 treatment. (b,e) In wild-type MEFs, overexpression of MAN1 reduces SMADs2/3 accumulation (inset). (c,f) In the Man1GT/GT MEFs, MAN1 similarly redistributes SMADs2/3 to the cytoplasm (inset). Scale bar: 20 µm. (B) Western blot of SMAD2 concentrations in the nucleus and cytoplasm. Upper panel: following TGFß1 treatment, wild-type (+/+) and Man1GT/GT (GT/GT) MEFs were fractionated into nuclear and cytoplasmic extracts and immunoblotted with an antibody to SMAD2. Both TGFß1 untreated and treated Man1GT/GT cells show enhanced nuclear accumulation of SMAD2. Bottom panel: immunoblot of nuclear and cytoplasmic fractions prepared from Man1GT/GT and wild-type MEFs expressing Flag-MAN1 showing a marked reduction in the nuclear accumulation of SMAD2. (C) Western blot of MEF whole cell lysates with antibodies to SMAD2 (upper) and SMAD1 (lower). SMAD2, but not SMAD1 is hyperphosphorylated in Man1GT/GT MEFs following treatment with TGFß1. (D) Man1GT/GT MEFs show higher levels of both basal and stimulated SMAD transcriptional activity. Man1GT/GT and wild-type MEFs were transfected with a TGFß-responsive reporter plasmid p3TP-Lux and co-transfected with pTK-RL. Transfected cells were stimulated with indicated concentrations of TGFß1 and analyzed for luciferase activity. Mean±s.e.m.; n=3. (E) Real-time PCR analysis of TGFß target genes. MEFs were treated with TGFß1 and quantitative PCR analysis performed on duplicate extracts. Means±s.e.m. are indicated. (F) Western analysis shows upregulated fibronectin (upper) and p21waf1 (lower) protein levels in TGFß1 treated Man1GT/GT MEFs. Actin was used as a loading control.

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