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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00450

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 Bäumer, N.
Right arrow Articles by Gruss, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bäumer, N.
Right arrow Articles by Gruss, P.
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?

Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6

Nicole Bäumer1, Till Marquardt1,*, Anastassia Stoykova1, Derek Spieler2, Dieter Treichel1, Ruth Ashery-Padan1,{dagger} and Peter Gruss1,{ddagger}

1 Department of Molecular Cell Biology, Max-Planck-Institute of Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
2 Department of Developmental Biology, Max-Planck-Institute of Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
* Present address: The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
{dagger} Present address: Sackler Faculty of Medicine, Department of Human Genetics and Molecular Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel



View larger version (62K):

[in a new window]
  		Fig. 5. Pax2 and Pax6 directly bind to and activate the MITF-A promoter in vitro. (A) Organization of the human MITF-gene (after Hallsson et al., 2000Go), position upstream promoter of exon A and of the predicted Pax2 and/or Pax6 binding sites (red boxes) in the MITF-A promoter construct, which is coupled to the luciferase reporter gene (Udono et al., 2000Go). (B) Sequences of the oligonucleotides used in the EMSAs in C and D. Red letters indicate the Pax6 or Pax2 consensus sequence (Epstein et al., 1994Go) in the Mitf promoter sequences. RE2 represents the Pax2 and Pax6 binding site (Schwarz et al., 2000Go). (C) EMSA with the sequences A1-A5 from (B), the control sequence RE2 and Pax2, Pax6 and `null' proteins (+Pax2, +Pax6, -Pax). Arrowheads indicate nonspecific binding. (D) The specificity of Pax2 and Pax6 binding to sequence A5 (lane 1 and 4, -ab) is confirmed by the addition of anti-Pax2 antibody (lane 2, +P2-ab) and anti-Pax6 antibody (lane 5, +P6-ab), which inhibits the formation of the complex. The addition of the reciprocal antibodies did not inhibit the binding (lanes 3 and 6). Without an overexpressed protein, only a faint band appears (lane 7, -Pax). The red arrowhead indicates the binding of Pax2 and of Pax6 by oligo A5. The green arrowhead marks the unbound oligos. (E) Pax2 and/or Pax6 activate the MITF-A promoter in cell culture experiments. Co-transfection of Cos-7 cells with the luciferase-coupled human MITF-A promoter and CMV-Pax2-cDNA and/or CMV-Pax6-cDNA lead to a strong increase in luciferase activity. CMV-Pax1-cDNA activates the MITF-A promoter only moderately.

 


View larger version (92K):

[in a new window]
  		Fig. 1. The expression domains of Pax2 and Pax6 entirely overlap with markers of the early OV progenitor domains. Immunohistochemical analysis of the expression of Pax6 (green, A,E,I) and of Pax2 (red, B,F,J) in serial, 6 µm sections. C, G and K show overlays of the respective Pax2 and Pax6 expression. D, H and L are at higher magnifications. Pax2 and Pax6 expression overlaps in the early OV at E9.5 (A-D) and at E10.0 (E-H). (I-L) At E12.5, Pax2 is restricted mainly to the optic stalk but Pax6 is still expressed in all distal eye components. (M-P) At E9.5, the segregation of RPE markers, such as Mitf (M) and Otx2 (N) is detectable, in contrast to the NR marker Chx10 (O). The adjacent section (P), stained for Pax2, reveals the overlap of Pax2 and Pax6 (see also A and B) with both RPE and NR markers. le, lens; nr, neural retina; os, optic stalk; ov, optic vesicle; rpe, retinal pigmented epithelium; se, surface ectoderm.

 


View larger version (98K):

[in a new window]
  		Fig. 4. Mitf expression and OV regionalization are lost in Pax2-/-; Pax6-/- embryos. Immunohistochemical staining of serial, 6 µm cryosections of E9.5 (A-B,E-F,I-J,M-N) and E11.5 (C,D,G,H,K,L,O,P) using antibodies against Pax2 and Pax6 (A-D), Mitf (E-H), Chx10 (I-L) and Otx2 (M-P). (A) Pax2 (green) and Pax6 (red) are expressed throughout the wild-type E9.5 OV. (C) At E11.5, Pax6 expression (green) is found in all compartments of the eye, but Pax2 (red) is mainly restricted to the ventral NR and RPE and the optic stalk. Both Pax2 and Pax6 are absent in Pax2-/-; Pax6-/- OVs at both stages (B and D). The E9.5 wild-type OV expresses Mitf predominantly in the proximal regions (E). At E11.5, Mitf expression is confined to the RPE (G). Mitf fails to be expressed in Pax2-/-; Pax6-/- OVs at both stages (F,H). The presumptive NR is marked by the Chx10-positive distal domain at E9.5 in both the wild-type (I) and the Pax2-/-; Pax6-/- OVs (J), but seems to be expanded dorsally in the mutant OV (J, arrow). At E11.5, Chx10 is confined to the NR region in the wild type (K) and to the distal OV in Pax2-/-; Pax6-/- OVs (L). The proximal expression of Otx2 in the wild type at E9.5 (M) appears expanded ventrally in the mutant OV (N, arrow). The Otx2 expression becomes restricted to the RPE domain at E11.5 in wild type (O), but remains co-expressed with the Chx10 positive distal OV in Pax2-/-; Pax6-/- mutants (P). lv, lens vesicle; ov, optic vesicle; os, optic stalk; nr, neural retina; rpe, retinal pigmented epithelium; se, surface ectoderm

 


View larger version (104K):

[in a new window]
  		Fig. 2. Pax2 and Pax6 activity is not required for the regionalization of the OV. Immunohistochemistry of serial, 6 µm sections at E12.5 (A-H) and E10.5 (I-P). In the wild-type eye at E12.5, Mitf expression is confined to the RPE and some cells in the dorsal optic stalk (A, arrowhead). By contrast, Otx2 is expressed in the RPE and the dorsal optic stalk, with a sharp, distal-proximal gradient in the distal NR arrowheads, (B). Chx10 is restricted to the NR (C) and Pax2 mainly to the optic stalk (D, arrowhead). (E) At E12.5, Mitf expression in the Pax6-/- mutant eye is confined to the distal tip of the OV. Otx2 is expressed highly in the same region (F) but less in the medial portion of the OV where Chx10 is expressed at a high level (G). (H) The proximal region of the Pax6-/- OV expresses Pax2 strongly (p), expression in the medial region is at a lower level (m) and the distal region is Pax2 negative (d). (F, inset) In the Pax6lacZ/lacZ OV, the ß-gal expression is restricted to the distal and the medial regions, which reflects the distribution of ß-gal expression in the RPE and NR in Pax6lacZ/+ eyes (B, inset). Broken lines in E-H indicate the borders between d, m and p regions. The comparison of the patterning of E10.5 wild-type (I-L) and Pax2-/- eyes (M-P) revealed in both genotypes the unchanged expression of Mitf (I and M) in the RPE and optic stalk, of Otx2 (J and N), which is found in both phenotypes in the RPE and optic stalk, gradually in the NR and some cells of the optic stalk, as well as the unchanged expression of Chx10 in the NR (K and O) and of Pax6 throughout all eye components (L and P). (I, inset) Expression of Pax2 in the wild-type optic disc and the missing Pax2 expression in the Pax2-/- eye (M, inset). le, lens; os, optic stalk; nr, neural retina; rpe, retinal pigmented epithelium.

 


View larger version (100K):

[in a new window]
  		Fig. 3. Pax2-/-; Pax6+/- eyes show transdifferentiation of the RPE into a second, inverted NR and reduced Mitf expression. Hematoxylin-Eosin (A,B,D,E,G,H) and immunohistochemical staining of serial, 6 µm sections using an antibody against the retinal ganglion cell marker Brn3b (C,F,I) and the RPE determinant Mitf (J-P). At E18.5, the wild-type eye differentiated to laminated NR, RPE and lens (A,B). The Pax2-/-; Pax6+/- eye did not form a lens, but the NR was much more expanded and folded at the expense of RPE development (D; detailed view in E). By contrast, at the same stage the Pax2-/- mutant eye displays a fully developed optic cup but the NR and the RPE are expanded towards the optic stalk (G,H). The expression of Brn3b in retinal ganglion cells marked the innermost layer of the NR in wild-type (arrow, C) and Pax2-/- NR (I). In the Pax2-/-; Pax6+/- eye, Brn3b expression (arrows) is confined to the inner layer of the inner NR (F: 1. nr) and to the outermost cell layer in the outer NR (F: 2. nr), indicating that the second NR is inverted compared to the inner NR. (J-P) At E9.5, Mitf expression is detected in the wild-type (J) and Pax2-/-; Pax6+/- OVs (M), predominantly in the proximal region. At E10.5, Mitf activity is confined to the outer layer of the optic cup in wild type (K, ol), but starts to be downregulated in the folded distal inner layer regions of Pax2-/-; Pax6+/- eyes (N, il). Mitf is still expressed the forming RPE in the wild-type E11.5 eye (L), whereas in the Pax2-/-; Pax6+/- optic cup, it is only found in regions between the newly formed, folded, NR regions and in the proximal RPE remnant (O, arrows). In the lens-CRE/Pax6flx/flx eye, Mitf-positive RPE surrounds the folded NRs, similar to wild-type RPE (P).

 


View larger version (63K):

[in a new window]
  		Fig. 6. pPax2Pax6 transgenic mice develop ectopic Mitf-positive RPE. (A) Construct used to establish the transgenic mouse line pPax2Pax6. A Pax2-genomic sequence (5 kb) driving expression in the optic nerve was coupled to full-length Pax6 cDNA (Schwarz et al., 2000Go). Bright-field photos (B,D,F,H) and immunohistochemical detection of Mitf (C,E,G,I) of 12 µm cryosections of E14.5 wild-type (B-E, wt) and transgenic (F-I, pPax2Pax6) eyes. Mitf is confined mainly to the iris in the wild-type (C, ir) and transgenic eye (G). Higher magnifications reveal that the ectopic RPE on the transgenic optic nerve is Mitf positive (I, arrows). Arrows in G indicate the ectopic Mitf+ RPE on the optic nerve; arrowheads in E indicate the endogenous Mitf expression in the RPE. ir, iris; le, lens; nr, neural retina; on, optic nerve; rpe, retinal pigmented epithelium.

 

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 2003