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 September 30, 2004
doi: 10.1242/10.1242/dev.01300

Development 131, 5139-5152 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Rowan, S.
Right arrow Articles by Cepko, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rowan, S.
Right arrow Articles by Cepko, C. 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?

Transdifferentiation of the retina into pigmented cells in ocular retardation mice defines a new function of the homeodomain gene Chx10

Sheldon Rowan1, C.-M. Amy Chen1,*, Tracy L. Young1, David E. Fisher2 and Constance L. Cepko1,{dagger}

1 Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
2 Department of Pediatric Oncology, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA



View larger version (100K):

[in a new window]
  		Fig. 1. Progressive pigmentation in orJ retinas. (A-H) Eyes were sectioned to analyze for pigmentation in orJ/+ heterozygous (A,C,E) or orJ/orJ homozygous (B,D,F,G,H) littermates. Ages are indicated on the figure. (A,B) Insets show a full section of the eye (note scale bar differences). (B,D,F) Arrows indicate the central limit of ectopic pigmentation in the retinas of orJ mutants. (D) The lens is marked by a thin black line in the orJ eye at E17.5. (G,H) The peripheral retina in orJ mutants shows ectopic pigmentation, increasing over time until most of the retina appears pigmented at P15. (H) The presumptive retina is marked with arrowheads. Scale bar is 100 µm.

 


View larger version (85K):

[in a new window]
  		Fig. 2. Direct transdifferentiation of orJ retinal cells into pigmented cells. (A) Schematic of the transgenic mice used for fate mapping experiments. The Chx10 BAC transgenic mouse directs expression of GFPCre and AP under control of Chx10 regulatory sequences. This reporter is integrated in the first exon of Chx10 (exons are indicated by solid vertical bars) within the Chx10 RNA (black horizontal line) such that the 5' most reporter utilizes the Chx10 start methionine (large arrow). Cre recombinase mediates recombination (curved arrows) at loxP sites (triangles) in the R26R allele. This removes a transcriptional stop cassette allowing lacZ expression from the Rosa26 promoter (small arrow). (B-I) Sections through Chx10 BAC/R26R transgenic eyes from orJ/+ (B,C,F,G) or orJ/orJ (D,E,H,I) P0 littermates. (B,G) ß-gal expression is observed throughout the retina as well as in the nonpigmented ciliary body epithelium (npcbe), but not in pigmented cells, in orJ/+ eyes as detected histochemically (B) or with anti-ß-gal antibody (G). (C) GFP reporter activity is detected in the outer neuroblastic layer (ONBL) as well as npcbe. (D,I) ß-gal expression is observed throughout the retina as well as in many pigmented cells in the peripheral pigmented regions. (E) GFP reporter activity is observed throughout the retina as well as in lightly pigmented regions (arrows in (D,E)). (J-L) Sections through Chx10 BAC transgenic orJ mutant eyes at P0 (J,K) and P17 (L,M). (K,M) GFP reporter activity is detected throughout the peripheral retina including regions of the retina that are lightly pigmented (J,L) and in pigmented cells themselves (arrows). (M) DAPI nuclear counterstaining confirms that the GFP-expressing nucleus is within a pigmented cell. (N-S) Dissociated cells from an E17.5 Chx10 BAC/R26R transgenic orJ mutant eye antibody stained for ß-gal (N) or GFP (P) with DAPI nuclear counterstaining. Arrows indicate cells that express ß-gal (N) or GFP (P) in unpigmented cells (O,Q), while arrowheads indicate pigmented cells. (Q) Inset shows higher magnification image of cell marked with arrowhead. (R,S) Histochemical ß-gal staining showing pigmented cells also expressing ß-gal. All scale bars are 100 µm.

 


View larger version (114K):

[in a new window]
  		Fig. 3. In situ hybridization analysis of genes affected in orJ mutants. (A-G) Sections through E14.5 or E17.5 orJ/+ heterozygous or orJ/orJ homozygous littermates, as indicated, were analyzed for the following RNAs: (A) Chx10, (B) Fgf15, (C) Gap43, (D) Gas1, (E) Wfdc1, (F), Mitf, (G) Tfec. Full expression patterns are discussed in Results and summarized in Fig. 4. Note scale bar differences between orJ/+ and orJ/orJ sections. (E) Arrows indicate boundaries of Wfdc1 expression. (E) Inset shows peripheral section from the same eye. All scale bars are 100 µm.

 


View larger version (104K):

[in a new window]
  		Fig. 4. Model of progressive transdifferentiation in the orJ mutant. (A) Representative sections through E14.5 or E17.5 embryos that were orJ/+ heterozygotes or orJ/orJ homozygotes, as indicated, pseudocolored for expression of peripheral genes in zone 1 (green), zone 2 (red), zone 3 (blue), or zone 4 (magenta). Regions of tissue co-expressing zone 1 and zone 2 markers are pseudocolored as yellow/orange. (B) Tabular summary of some expression patterns shown in Figs 3, 4, S1 and S2 (supplementary material).

 


View larger version (130K):

[in a new window]
  		Fig. 5. In situ hybridization analysis of Mitf target genes in orJ mutants. (A-C) Sections through E17.5 orJ/+ heterozygous or orJ/orJ homozygous littermates, as indicated, were analyzed for the following RNAs: (A) Trpm1, (B) Dct, (C) Tyrosinase. Full expression patterns are discussed in Results and summarized in Fig. 4. Note scale bar differences between orJ/+ and orJ/orJ sections. All scale bars are 100 µm.

 


View larger version (98K):

[in a new window]
  		Fig. 6. Misexpression of Chx10 by in ovo electroporations. (A-U) HH Stage 10 embryos were electroporated with GFP and Chx10 (A,D,G,J,M,P,S) or GFP alone (C,F,I,L,O,R,U) and allowed to develop an additional day. (B,E,H,K,N,Q,T) Images are from the left non-electroporated eye of the same embryo shown in (A,D,G,J,M,P,S) respectively. (A-C) HH16-18 embryos were harvested and photographed for GFP to observe transfected regions. (D-U) HH16-18 embryos were harvested and whole-mount in situ hybridization was performed to detect the following RNAs: (D-F) Mmp115, (G-I) Mitf, (J-L) Tfec, (M-O) Dct, (P-R) Six3, (S-T) Chx10 3'UTR, (U) Chx10. (V-Y) Sections through the Chx10 and GFP electroporated embryo detected for Mmp115 (D,E). (V) Mmp115 RNA was detected in the RPE of the eye that was not electroporated (arrow, high magnification image shown in (X)), but was highly reduced in the eye electroporated with GFP and Chx10 (arrowhead, high magnification image shown in (Y)). (W) Section from (V) further stained with an antibody against GFP to show the region of the embryo that was electroporated (brown stain), including the retina and RPE in the bottom eye.

 

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 2004