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First published online 30 April 2003
doi: 10.1242/jcs.00452

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SFRP1 modulates retina cell differentiation through a ß-catenin-independent mechanism

Pilar Esteve, Françoise Trousse, Josana Rodríguez and Paola Bovolenta*

Departamento de Neurobiología del Desarrollo, Instituto Cajal, CSIC, Dr Arce 37, Madrid 28002, Spain



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  		Fig. 1. Localization of chick Sfrp1 transcripts during eye development. Embryos of indicated developmental stages were hybridised (A-E) in toto or (F-H,K,L) in frontal cryostat tissue sections. Embryos are viewed (A) dorsally or (C) laterally. Images in (B,D,E) represent (B) transversal and (D,E) frontal paraffin sections of whole mount processed embryos. Image in (I,J,K) are high magnification views of (E,F) and of the boxed area in (H), respectively. Note the early expression of Sfrp1 in the prospective neural retina, (arrow in B) lens placode ectoderm and (A-D) lens vesicle. cSfrp1 transcripts are first localized to (arrows in E,I) the centro-dorsal retina, expand later to (F,G,J) the periphery and become restricted to (H,K,L) the inl following neurogenesis gradients. inl, inner nuclear layer; lv, lens vesicle; pnr, presumptive neural retina; ppe, presumptive pigment epithelium. Bar: (A,F,G) 400 µm; (B) 94 µm; (C) 500 µm; (D,E,G) 50 µm; (K,L) 40 µm; (H) 625 µm.

 


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  		Fig. 2. Generation and purification of recombinant and soluble SFRP1. (A) The conditioned media (CM) from MDCK/control or MDCK/SFRP1 mass cultures (MC) and few of the derived clones were tested for the presence of secreted SFRP1 by immunoblot with anti-myc antibody. Clones 2, 3 and 10 secreted the highest yield of SFRP1 compared with the yield from MC or clone 4. (B) Partial purification of SFRP1. Concentrated CM from either clone 10 [see (A)] or MDCK/control were applied to Ni++-NTA agarose column and eluted with 100 mM imidazole. Eluted fractions were separated by 10% SDS-PAGE. The protein content of a representative fraction eluted from control (lanes 1 and 3) or clone 10 (lanes 2 and 4) media were compared. The identity of SFRP1 in the fraction was confirmed by immunoblot with anti-myc antibody.

 


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  		Fig. 3. SFRP1 affects cell differentiation in retina cell cultures. E5 chick retinas were dissociated and grown for 24 hours in the presence of CM from (A,C,E) MDCK/control or (B,D,F) MDCK/SFRP1. After fixation, cultures immuno-stained with antibodies against (A,B) anti-NF, (C,D) islet1/2, (E,F) visinin or phospho-histone3 (PH3, a mitotic marker) and counterstained with Hoescht (blue staining). (G) Final cell density (Hoescht+), the degree of differentiation (Tuj1+; islet1/2+; visinin+) and mitotic rate (PH3+) were evaluated in control (empty bars) and treated cultures (filled bars) by counting, in each experimental condition, the number of immuno-positive cells and the total number of cells in 14 randomly selected microscopic fields. Data represent a typical experiment performed in triplicate. Note how the presence of SFRP1 increases the number of differentiated cells without significant variations in the total number of cells or in the rate of cell division. *P<0.05; ***P<0.0001, Student's t-test. Bar, 185 µm.

 


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  		Fig. 4. DsRNA-mediated interference of endogenous Sfrp1 reduces cell differentiation. (A) To assess specificity of the designed dsRNAs, dsRNAs were introduced into MDCK/SFRP1. The ability of Sfrp1 dsRNAs to interfere with myc-SFRP1 protein levels was determined by western blots of the CM of control (con) and dsRNA treated cells using anti-myc antibodies. The Cterm Sfrp1 dsRNA [Cter, two independent evaluations in the upper and lower panels in (A)], and to a lower extent the Nterm Sfrp1 dsRNA (Nter) but not the Gfp dsRNA (Gfp), reduced the protein level in the CM of treated cells. (B) Similar reduction was observed in the mRNA content as determined by RT-PCR analysis. (C) RT-PCR showing chick mRNA levels of Sfrp1 in untreated, control dsRNA (Gfp) or Sfrp1 dsRNA (Cterm) treated retina cells. Cterm Sfrp1 dsRNA induced a decrease in the endogenous level of Sfrp1 mRNA. (B,C) Gapdh amplifications were used as controls. (D) E5 dissociated retina cultures were treated with dsRNAs and cultured for 24 hours. Cultures were immunostained with antibodies against islet1/2, or visinin and counterstained with Hoescht. The presence of Cterm Sfrp1 dsRNA decreases the number of both islet1/2 and visinin-positive cells. *P<0.05; **P<0.001, Student's t-test.

 


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  		Fig. 5. Overexpression of SFRP1 in chick retinal progenitors increases the number of RGC and cone photoreceptors while decreases the number of amacrine cells. Frontal cryostat sections from retinas of E6 embryos infected with RCAS-SFRP1 viruses at stage 10. Adjacent sections from (A,C,E,G,I,K) infected and (B,D,F,H,J,L) contralateral uninfected central retinas were immunostained with antibodies against the (A,B) Gag viral protein, (C,D) Tuj1, (K,L) the mitotic marker PH3 and the cell-specific differentiation markers (E,F) islet1 (retinal ganglion cells), (G,H) visinin (cone photoreceptors) and (I,J) CRABP1 (amacrine cells). Note how the number of (C,E) cones and migrating (arrowheads) and (A) layered ganglion cells is increased in Gag-positive retinas, while amacrine cells are reduced, without apparent variations in the number of PH3-positive cells. Note also the thickness of the Tuj1-positive fibre layer in (C) the infected retina compared with the thickness of the fibre layer in (D) controls. GC/AM, ganglion cell/amacrine cells; FL, fibre layer; PE, pigment epithelium. Bar, 38 µm.

 


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  		Fig. 6. SFRP1 activities do not modify ß-catenin transcriptional activity. (A) Immunoblot analysis of cytosolic ß-catenin levels in MDCK/control or MDCK/SFRP1 treated retina cells harvested after three and five hours of culture. Cytosolic fractions were prepared and immunoblotted with antibodies against ß-catenin and {alpha}-tubulin (load control). Normalized density values (NDV) expressed in arbitrary units indicate no differences among the fractions. (B) Luciferase assay showing that SFRP1 does not modulate the endogenous level of ß-catenin/TCF-dependent transcription in E5 chick dissociated retinal cells. Cells were co-transfected with the reporter plasmid containing the Lef-1 responsive element (C4) or its mutated version (MUT), the control plasmid pRLTK and the effector plasmids in each case. Either 500 ng or 1 µg of Sfrp1 were transfected alone or in combination with 500 ng of Wnt8. 500 ng of Wnt8 or ß-catenin activate the reporter gene 240 and 130 fold, respectively. The two concentrations of Sfrp1 decreased Wnt8 activity 4 and 10 fold, respectively. 1 µg of dnGSK3ß did not activate the reporter. Luciferase activity is expressed in a logaritmic scale.

 


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  		Fig. 7. Treatment of retina cells with SFRP1 is associated with phospho-Ser9-mediated inhibition of GSK3ß activity. (A) SFRP1-inhibits GSK3ß activity by phosphorylation at Ser9. SFRP1- or control-treated retina cells, harvested three or five hours after the culture, were analysed by immunoblots with antibodies {alpha}-GSK3ß-phospho-Ser9 (inactive form), GSK3ß-phospho-Tyr216 (active form) and {alpha}-tubulin, as loading control. NDV indicate a clear increase in phospho-Ser9-mediated inhibition of GSK3ß activity. (B) The total and phospho-Ser9-fraction of GSK3ß was evaluated with specific antibodies by western blots in retina explants electroporated with either Gfp or Sfrp1-myc and collected five hours after protein expression. Total content of GSK3ß was normalised against {alpha}-tubulin. Phospho-Ser9 positive bands were normalised against total GSK3ß content. Anti-myc demonstrates Sfrp1-myc expression after electroporation.

 

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© The Company of Biologists Ltd 2003