Supporting Online Material


Abstract
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Tissue-Specific Regulation of Retinal and Pituitary Precursor Cell Proliferation
Xue Li, Valentina Perissi, Forrest Liu, David W. Rose, Michael G. Rosenfeld

Supporting Online Material

Materials and Methods

A targeting vector with 9-kb 5' arm and 3-kb 3' arm was constructed replacing both HD and SD encoding exon with IRESlacZ/pgk-neo sequence. Four independent R1 ES clones (JD Marth, University of California, San Diego), of which two were used for generating knockout mice, were identified by Southern blot screening with both 5' and 3' external probes which give rise to 13.5-kb wild-type and 9.5-kb or 8.5-kb mutant bands, respectively.

All in vitro expression plasmids were cloned into either the Gal4 expression vector or the pcDNA3.1 vector (Invitrogen®, CA) both of which have CMV promoter. Standard molecular cloning, tissue culture, and cell transfection experiments were performed as described by J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 3rd, 2001). The 3xUAS/p36 or TK luciferase reporter was described by K. Jepsen etal., Cell102, 753 (2000). The Six6 reporter plasmid contained a 4x Six responsive element (SE) (GTCGACATGAGCTT ACCTCCTGCATCAGTTGC). p27Kip1 promoter reporter constructs were described before Y. Zhang, S. C. Lin, Biochim. Biophys. Acta1353, 307 (1997). Both Six6 and Dach2 cDNA were cloned by PCR from murine pituitary cDNA library. The following genes were used in transfection studies: Eya1, Eya2 and Eya3 J. E. Zimmerman etal., Genome Res.7, 128 (1997), H. Ohto etal., Mol. Cell Biol. 19, 6815 (1999), and P. X. Xu, I. Woo, H. Her, D. R. Beier, R. L. Maas, Development124, 219 (1997); Dach1 R. J. Davis, W. Shen, T. A. Heanue, G. Mardon, Dev. Genes. Evol.209, 526 (1999) and K. L. Hammond, I. M. Hanson, A. G. Brown, L. A. Lettice, R. E. Hill, Mech. Dev.74, 121 (1998), and Sno S. Pearson-White, R. Crittenden, Nucleic Acids Res.25, 2930 (1997).

Apoptotic cells were detected with suggested protocol using ApoTag kit (Intergen). BrdU incorporation was detected on 10um cryostat sections as suggested (ICN) and at least four eyes from each Six6+/- and Six6-/- mice were analyzed. Three midline sections 100 Greek Letter Mum apart were analyzed for BrdU incorporation and all cells were counter-stained with DAPI. The statistic analyses with a student's t test were performed using Microsoft Excel®.

The mouse Six6 N-terminus and Dach2 N-terminus protein were expressed in bacteria using a standard His-tag expression vector according to the suggested protocols (Qiagen). Guinea pigs were immunized three times with 500 Greek Letter Mug of protein each. Immunohistochemistry and in situ hybridization procedure are essentially as described by K. Jepsen etal., Cell102, 753 (2000).

The T3-1 pituitary cells were grown up to 50% confluency for the ChIP assay performed as described Y. Shang, X. Hu, J. DiRenzo, M. A. Lazar, M. Brown, Cell103, 843 (2000). For the in vivo ChIP experiments, 20 wild-type mouse embryonic eyes were used for each immunoprecipitation. Retinas were microdissected at e13.5 in PBS, 1 mM PMSF and cross-linked with 1% formaldehyde for 6 hours at room temperature. Chromatin extraction and immunoprecipitations were performed following the same protocol described for the in vitro experiments. Each immunoprecipitation was repeated at least twice. PCR primer sets designed for the p27Kip1 promoter are: 5'- GAA ACC AGC CGA GAA TGA GG - 3'/5'- TTA GCC ACA TCT TTG CCA GG - 3' (over the Six6 response element), 5'- CAC TCG CGG CTC CGA GAC TGG- 3'/5'- ACC CCT CTT CGA AGT TCT GCG -3' (negative control, 300 bp amplicon located 2 kb downstream from Six6 response element.

The single cell nuclear microinjection assays were performed as described by Y. D. Wen et al., Proc. Natl. Acad. Sci. U.S.A.97, 7202 (2000). Each experiment was performed three times, with >300 injected cells per point. Preimmune antibody was used as a negative control in each experiment. Guinea pig IgGs against Six6 and Dach2, described above, were purified using affinity columns, all other antibodies used in this study have been previously described K. Jepsen et al., Cell102, 753 (2000). GST pull-down experiments were performed as previously described Y. D. Wen et al., Proc. Natl. Acad. Sci. U.S.A.97, 7202 (2000).


Supplemental Figure 1. (A) Schematic targeting strategy to replace both sine oculis domain (SD) and Homeodomain (HD) with IRES-LacZ reporter genes and pgk-Neo selection marker. (B) Southern blot analysis of tail DNA using 5'-probe as indicated in (A). (C) H&E histology of adult litter mate retinas with severe optic nerve defect indicate significant reduction of total cell numbers and dismorphogenesis.


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Supplemental Figure 2. Immunohistochemical analyses of adult retinas with severe (A) and mild (B) gross optic nerve phenotypes using cell-type specific antibodies indicating the presence of all cell types. Neurofilament 160 (NF160, ganglion and horizontal cells), Calbindin (ganglion, horizontal and a subset of amacrine cells); Calretinin (horizontal, ganglion and amacrine cells); Syntaxin (amacrine cells), TUJI (neurons), Glutamine Synthetase (GS, M�ller cells), Rhodopsin (rod photoreceptors).


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Supplemental Figure 3. BrdU analyses indicated a proliferation defect in the Six6 mutant. (A) Schematic diagram representing the differentiation time course of specific cell types and BrdU injection times for the analyses. The expression pattern of Six6 is indicated. The area under the solid lines represents total cells born at the time and the outer dashed line represents total number of ventricular cells undergoing DNA synthesis/day (adapted from figure 9, R. W. Young, Anat Rec 212, 199-205 1985). (B) BrdU injected at e11.5, analysis performed on p35. The brightness of BrdU positive cells at the GCL was analyzed using both ProImagine® program on the Spot® digital imagine and the analysis program on the Deconvoluting Confocal Microscopic imagine with same exposure time, setting intensity over 150 as strong labeling (S) and intensity of 50-150 as weak labeling (W).


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Supplemental Figure 4. (A) Deletional mutation analyses of Six6 repressive function in transient transfection experiments. Both sine oculis domain (SD) and homeodomain (HD) are required for full level repressive function. (B) Systematic deletional mapping indicated that the conserved N-terminus of Dach1 interacts with three independent regions of N-CoR in GST pull-down assay.


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