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 2 December 2004
doi: 10.1242/dev.01548

Development 132, 105-115 (2005)
Published by The Company of Biologists 2005
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 Seo, S.
Right arrow Articles by Kroll, K. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seo, S.
Right arrow Articles by Kroll, K. L.
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?

The SWI/SNF chromatin remodeling protein Brg1 is required for vertebrate neurogenesis and mediates transactivation of Ngn and NeuroD

Seongjin Seo, Genova A. Richardson and Kristen L. Kroll*

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA



View larger version (55K):

[in a new window]
  		Fig. 1. Cloning and expression profile of xBrg1. (A) Structure of hBrg1 and xBrg1. Domains are labeled after (Khavari et al., 1993Go) with percent amino acid identity shown. Asterisks mark the ATP binding pocket targeted by mutagenesis to generate a dominant-negative form. Lines under xBrg1 indicate probes used for in situ hybridization. (B) Phylogenetic analysis of Brg1 and Brm orthologs. Units indicate the number of substitutions. Distance between any two sequences is the sum of horizontal branch length separating them. (C-I) Expression profile of xBrg1. (C) Stage 8 and (D) stage 12, side views (vegetal pole toward bottom). (E) Dorsal view, anterior towards the top (stage 16). (F) Anterior view (stage 22). (G) Stage 25/26 and (H) stage 33/34, lateral views. (IJ) Cross-sectional views of stage 14 embryos stained for xBrg1 (I) or N-tubulin (J).

 


View larger version (14K):

[in a new window]
  		Fig. 8. Brg1 mediates transcriptional activities of Ngn3 and NeuroD2. (A) P19 cells were transfected with expression vectors for Ngn3, E12 and E1X3 E-box reporter together with or without DN-hBrg1 in triplicate and analyzed for luciferase activity. Data shown are a representative result from five independent experiments. Standard errors are indicated. (B) NeuroD2 was used, otherwise same as A. Data shown are a representative result from two independent experiments.

 


View larger version (86K):

[in a new window]
  		Fig. 2. DN-xBrg1 and xBrg1MO injections have similar effects on embryonic morphology. (A-C) Morphology of tadpoles (stage 37/38) injected with DN-xBrg1, xBrg1MO or standard MO. Embryos were injected in both blastomeres at the two-cell stage and raised to tadpoles. (A) Bottom three embryos were injected with DN-xBrg1, while the top embryo was uninjected. (B) xBrg1MO (20 ng) injected tadpoles. xBrg1MO-injected tadpoles display similar defects to DN-xBrg1, while standard MO (C) injected ones do not show apparent defects. (D) Reduction of endogenous xBrg1 protein by xBrg1MO. Embryos were injected with 25 ng of xBrg1 or standard MO in both blastomeres at stage 2 and harvested at the indicated stages. Ten embryos were used per sample, with one embryo-equivalent of lysate loaded per lane for western blotting.

 


View larger version (88K):

[in a new window]
  		Fig. 3. Embryos depleted of xBrg1 fail to produce primary neurons. Embryos were injected with DN-xBrg1 (A-C), xBrg1MO (D-F) or standard MO (G-I) in one blastomere at stage 2. ß-galactosidase mRNA was co-injected and X-Gal staining (blue) was performed to reveal distribution of injected materials. Embryos were analyzed for expression of Sox2 (A,B,D,E,G,H,J) and N-tubulin (C,F,I,K; stage 15). Embryos in J and K were injected with xBrg1MO and xBrg1 mRNA. Dorsal views with injected side facing rightwards.

 


View larger version (51K):

[in a new window]
  		Fig. 4. Loss of Brg1 increases cell proliferation. (A-D) Whole-mount stage 14 embryos immunostained (brown) to detect phosphorylated histone H3 (PH3) after injection of standard MO (A) or xBrg1MO (B-D). (C,D) Lateral views of an xBrg1MO-injected embryo: (C) uninjected side; (D) xBrg1MO-injected side. (E) Transverse section of embryo in B, showing the injected side facing rightwards. Black arrowheads mark PH3-positive cells. n, notochord; s, somite. (F-H) Cell division was blocked by adding hydroxyurea (HU) from stage 12.5 until fixation at stage 15. (F) PH3-immunostained embryo showing cell cycle is efficiently blocked by HU treatment (xBrg1MO+ß-galactosidase injection, blue; PH3 stain, brown). (G,H) xBrg1MO-injected embryos were raised in the presence of 30 mM HU from stage 12.5 and analyzed for Sox2 (G) and N-tubulin (H) expression. (I,J) Whole-mount TUNEL staining (brown) after injection of standard MO or xBrg1MO. A,B,F-J are dorsal views with injected side facing rightwards.

 


View larger version (71K):

[in a new window]
  		Fig. 5. Brg1 is required for the proneural activities of xNgnr1 and xNeuroD. Embryos were injected with xNgnr1 (A), xNeuroD (B) or xMyT1 (C) alone or together with xBrg1MO (D-F) and analyzed for N-tubulin expression (stage 15). Views are dorsal with injected side (X-Gal, blue) facing rightwards.

 


View larger version (55K):

[in a new window]
  		Fig. 6. The requirement of Brg1 for neurogenesis is conserved in mammalian P19 cells. P19 cells were transfected with plasmids encoding mouse NeuroD2 (A-C), mouse NeuroD2 and DN-hBrg1 (D-F), together with mE12 and GFP expression vectors. Three days after transfection, cells were analyzed by TuJ1 immunostaining (A,D,G) and GFP expression (B,E,H). C, F and I are overlays of images A and B, D and E, and G and H, respectively. (J) TuJ1-positive cells were scored as a percentage of GFP-positive transfected cells. A total of between 750 and 2200 GFP-positive cells were counted for each type of transfection. P19 cells transfected with GFP vector alone (G-I) or with GFP and E12 vectors (but not with NeuroD2) yielded less than 1 TuJ-positive cell per 1.8x105 cells assayed in each experiment. Data shown in J are the average of three experiments.

 


View larger version (46K):

[in a new window]
  		Fig. 7. Brg1 physically interacts with xNgnr1 and xNeuroD. (A) HeLa cells were transfected as indicated and applied to co-IP assay. Lysates were immunoprecipitated (IP) with anti-Myc antibodies and immunoblotted (IB) with anti-FLAG antibodies. Protein expression levels were monitored by western blotting of direct lysates. (B) Myc-xBrg1, FL-xBra, FL-xESR1, FL-xNgnr1 and FL-xNeuroD proteins were produced separately using reticulocyte lysate. After in vitro translation, an equal amount of lysate containing Myc-xBrg1 was added to FLAG-tagged proteins and subjected to co-IP with anti-Myc antibodies. Input of FLAG-tagged proteins was monitored by western blotting. Asterisks in B indicate specific bands corresponding to Ngnr1 and NeuroD.

 

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 2005