spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 3 May 2006
doi: 10.1242/dev.02385

Development 133, 2201-2210 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hashimoto, T.
Right arrow Articles by Yang, X.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hashimoto, T.
Right arrow Articles by Yang, X.-J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation

Takao Hashimoto, Xiang-Mei Zhang, Brenden Yi-kuang Chen* and Xian-Jie Yang{dagger}

Jules Stein Eye Institute and Department of Ophthalmology, Molecular Biology Institute, University of California, David Geffen School of Medicine, 100 Stein Plaza Los Angeles, CA 90095, USA.


Figure 1
View larger version (131K):

[in a new window]
  		Fig. 1. Expression of VEGF and FLK1 during chicken retinogenesis. (A-F) Images of E5 and E6 retinal sections show double immunostaining against VEGF (A,D), Islet1/2 (B,E) and the respective merged images (C,F). (G-I) Images of E6 sections show in situ hybridization for FLK1 (G), and immunostaining for PCNA (H) and BrdU (I, in ovo labeling for 3 hours). Scale bars: 50 µm. gcl, ganglion cell layer; vz, ventricular zone.

 

Figure 2
View larger version (63K):

[in a new window]
  		Fig. 2. Influence of VEGF on retinal cell proliferation. (A) Effects of VEGF concentrations on BrdU incorporation in vitro. E5-E7 explants were labeled with BrdU for the last 6 hours (n=4 or 5). (B-D) Western blots show sFLK1 (B), VEGF (C) or FLK1-DN (D) expression. Culture media of transfected HEK cells (B,C) or infected DF-1 cell extracts (D) were probed with antibodies against AP (B), VEGF (C) or FLAG tag (D). Controls used were CMV-AP (B,C) or RCAS-AP virus (D). Arrowheads indicate bands with expected molecular weights. (E-G) Immunostaining show effects of AP (E), sFLK1 (F) or VEGF (G) in E5-E7 explants on BrdU labeling for the last 3 hours. Scale bars: 50 µm. (H) Effects of sFLK1- and VEGF-producing cells on BrdU incorporation (3 hours) at E5 in vivo. Black and white bars represent implanted and the contralateral non-implanted eyes, respectively (n=5 or 6; *P<0.03). (I) Effect of FLK1-DN expression on BrdU incorporation in vivo. Retinas infected at HH stage 10 with RCAS-AP (control) or RCAS-FLK1-DN virus were harvested at E5 and labeled with BrdU for 3 hours (n=5; *P<0.04).

 

Figure 3
View larger version (50K):

[in a new window]
  		Fig. 3. Influence of VEGF on retinal ganglion cell differentiation. (A) Effects of VEGF concentrations on development of NF+ cells in E5-E7 explants in vitro (n=5). (B) Effects of sFLK1- and VEGF-producing cells on percentages of NF+ cells in vivo at E5. Black and white bars represent implanted and the contralateral non-implanted eyes, respectively (n=6; *P<0.03). (C-E) Immunostaining show effects of AP (C), sFLK1 (D) or VEGF (E) on Islet1/2+ cells in E5-E7 explants. Scale bars: 50 µm.

 

Figure 4
View larger version (90K):

[in a new window]
  		Fig. 4. Effects of RNAi-mediated FLK1 knockdown. (A) A schematic represents the RCAS viral vector (white) encoding the U6 promoter and the CMV-GFP cassette. The shFLK1 RNA (FLK1i) contains a 29 nucleotide antisense (AS, red) and a complementary sense (S, blue) sequence of chicken FLK1 followed by transcription termination sequence (Term). (B-E) Efficiency of FLK1i knockdown in vitro. Fluorescence images show HEK cells co-transfected with the GFP-FLK1 chimeric target and the control U6 (B) or U6-FLK1i (C) plasmid. Western blots (D) show GFP levels in HEK cells co-transfected with the GFP-FLK1 chimeric target and control U6 or U6-FLK1i plasmid. Blots were probed against GFP (top) and AP (bottom), which serves as a transfection efficiency control. Optical densities of GFP signals (E) were normalized against AP signals (n=6; *P<0.03). (F-K) Effects of FLK1i knockdown on RGC development in vivo. Confocal images show retinas infected with the control RCAS (F-H) or RCAS-FLK1i (I-K) viruses at HH stage 17 and immunostained at E6.5 for GFP (F,I), Islet1/2 (G,J) and the respective merged images (H,K). White arrows indicate co-stained cells. Scale bars: 50 µm. (L-N) Quantification of FLK1i knockdown effects in vitro. Explants were electroporated at E5 with the control RCAS or the RCAS-FLK1i construct, and labeled with BrdU for 3 hours before dissociation at 48 hours. Percentages of BrdU+ (L), Islet1/2+ (M) or NF+ (N) cells among transected GFP+ cells are shown (n=6; *P<0.03).

 

Figure 5
View larger version (27K):

[in a new window]
  		Fig. 5. Effects of MEK-ERK inhibition on VEGF-dependent proliferation and differentiation. (A) Western blots of VEGF-induced phospho-ERK. E6 retinas were cultured with or without U0126 for 60 minutes before VEGF was added for 10 minutes. Blots were probed with phospho-ERK1/2 (top) and {alpha}-tubulin (bottom) antibodies. (B) Quantification of optical densities of phospho-ERK signals were normalized against {alpha}-tubulin signals (n=4). (C) RT-PCR detection of cyclin D1 transcripts in E6 retinas cultured for 24 hours in the presence or absence of VEGF. Ratios of cyclin D1 and GAPDH products are shown (n=8). (D-F) Quantifications of U0126 effects on VEGF-dependent cell proliferation (D) or differentiation (E,F). E5 explants were cultured for 24 hours and BrdU labeled for the last 3 hours (n=6; *P<0.03).

 

Figure 6
View larger version (26K):

[in a new window]
  		Fig. 6. Influence of HES1 activity on retinal proliferation and differentiation. E5 retinas were co-electroporated with HES1- or dnHES1-expressing construct and a GFP-expressing construct, and cultured as explants for 48 hours with BrdU present for the last 3 hours. Effects of HES1 (A,C) or dnHES1 (B,D) on Islet1/2+ (A,B) or BrdU+ (C,D) cells among transfected GFP+ cells are shown (n=6; *P<0.03).

 

Figure 7
View larger version (32K):

[in a new window]
  		Fig. 7. Requirements of HES1 in VEGF-dependent retinal proliferation and differentiation. E5 retinas were co-electroporated with dnHES1- and GFP-expressing constructs, and then cultured as explants (A,B) or dissociated cells in collagen gels (C,D). VEGF was added at 24 hours post transfection and BrdU was added for the last 3 hours. (A,B) Effects of dnHES1 on Brn3a+ cells at E8 (A) or BrdU+ cells at E7 (B) among transfected GFP+ cells (n=6; *P<0.03). (C,D) Effects of dnHES1 in collagen gels on Islet1/2+ cells (C, n=7; *P<0.02) or BrdU+ cells (D, n=5; *P<0.05) among transfected GFP+ cells.

 

Figure 8
View larger version (28K):

[in a new window]
  		Fig. 8. Effects of signaling inhibitors on cell proliferation and differentiation. (A-C) E5 explants were cultured without inhibitors (dark blue), with SU1498 (purple), with cyclopamine (yellow), or with SU1498 and cyclopamine (light blue) for 48 hours. Marker-positive cells per unit length retinal section were quantified and shown as ratios of BrdU+ (A), Islet1/2+ (B) or Brn3a+ (C) cells to the no inhibitor controls (n=9; *P<0.05, **P<0.01). (D,E) E5 retinal cells transfected with HES1-expressing construct (+) or the control plasmid (–) were cultured in collagen gels for 24 hours before treatment with SU1498, cyclopamine or both for another 24 hours. Bar graphs show BrdU+ (D) and Islet1/2+ (E) cells among transfected GFP+ cells (n=6; *P<0.05).

 

Figure 9
View larger version (22K):

[in a new window]
  		Fig. 9. A model of signal convergence in retinal progenitor cells. DELTA-NOTCH signaling yields the NOTCH intracellular domain (NICD) and activates HES1 transcription. SHH signaling through patched (PTC) and Smoothened (SMO) reduces Gli repressor (GliRep) and facilitates the accumulation of the Gli activator (GliAct). SHH signaling upregulates HES1 by unknown mechanism(s). VEGF stimulation causes the activation of the MEK-ERK cascade and enhances HES1 activity without the involvement of MEK1/2 function. The promotion of cell proliferation may require independent inputs from both MEK-ERK and HES1, whereas suppression of RGC specification mainly involves HES1 activity. Therefore, HES1 serves as a node of signal convergence to integrate inputs of multiple cell-extrinsic cues.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006