In both human and mouse retina development, the onset of expression for both Crx and IRBP mRNA is very similar. Bibb et al. [36] did not find Crx protein until Fwk 15—several weeks after the mRNA expression is detected. The developmental expression pattern and the in vitro work both suggest that Crx is a transcriptional activator of IRBP expression (positive transcription factor for IRBP).

A Crx knockout mouse was created by a targeted deletion of the homeodomain region of the Crx gene [97]. At P10, before the outer segments begin to form, there is no phenotypic difference seen in retinas of the different Crx null mouse genotypes. At P14, outer segments are not seen in Crx null mouse retinas, and in Crx hemizygotes the outer segments are shorter than those of wild-type mice. The expression levels of photoreceptor-specific genes were determined in Crx null mice mutant retinas by densitometry of northern blots from P10 wild-type, hemizygous, and Crx null mice. The expression levels for IRBP are the same in all three genotypes of mice, unlike other photoreceptor-specific genes such as rhodopsin and cone arrestin, which have expression levels that are tenfold lower when the hemizygous and null phenotypes are compared [97].

Transcription factors interact with specific sequences of DNA and with other proteins to regulate gene transcription/expression and cellular differentiation. The Crx null mice were used in a study designed to identify the Crx transcriptional networks [98]. Crx gene targets were identified by microarray analysis of gene expression patterns from normal (wt) and Crx null developing retinas. Photoreceptor-specific and enriched genes were divided into two categories based on the downregulation of their expression in the absence of Crx expression: genes that are downregulated more than 2.5-fold or genes downregulated less than 2.5-fold. IRBP is in the second category of genes, and this agrees with earlier work that the knockout of Crx does not have a large effect on the expression of IRBP [97].

In addition, comparison of the upstream sequences from genes that were downregulated in the absence of Crx led to the identification of a 11-base sequence motif that contains one strong Crx-binding element (CBE) upstream of a second, weaker CBE-like sequence [98]. The IRBP promoter contains two GATTA sequence motifs that are slightly similar to the CBE motif (Fig. 5). It contains the second weaker CBE-like sequence but not the first, stronger element, suggesting that Crx is not a main contributor to the activation of the IRBP gene. These two GATTA motifs are required for IRBP expression because mutations in both GATTA motifs, found in the −58 to −45 bp region and the second one just upstream (inverted), abolish expression of the reporter gene in transgenic mice photoreceptors [85].

The regulation by Crx may not contribute significantly to the overall transcriptional regulation of the IRBP gene, or the expression of IRBP may be regulated or coregulated by Otx2 because the consensus sequences for Crx and Otx2 are similar. Crx and Otx2 have an 86% homology in the homeodomain [99]. In Crx null mice, Otx2 activity may compensate for Crx activity in the regulation of IRBP gene expression, thus resulting in a minimal change in IRBP expression levels in these mice. Crx antisense knockdown suppresses p70 activity, suggesting that whereas Crx is not required for IRBP expression, it may in fact be used endogenously [100].

OTX2 Transcription Factor

Otx2 is another paired-type homeodomain protein, a member of the Otx homeobox gene family, and is expressed in the retina [101]. Otx2 is an essential developmental gene. It has

multiple roles in gastrulation and brain and retinal development. A targeted disruption of the Otx2 gene leads to embryonic lethality at midgestation, thus preventing the analysis of the influence of Otx2 expression retina development [102]. Conditional knockout mice show that Otx2 is required for neuronal specification of the retina and for pineal gland development. Absence of Otx2 results in the conversion of differentiating photoreceptor cells into amacrine-like neurons and to deficiency of pinealocytes in the pineal gland [103].

In adult mice, Otx2 immunoreactivity is found in several retinal cell types, including the nuclei of photoreceptors [99] or the cytoplasm of rod photoreceptors [101]. The subcellular location of the Otx2 protein appears to be antibody specific.

In situ hybridization experiments studying Otx2 expression in the developing mouse retina show that at E11.5 strong staining is seen in the RPE, while staining is weak in the neural retina [103]. At E12.5, expression of Otx2 in the neural retina has increased, and by E17.5 there is very strong staining for Otx2 in the outer region of the neuroblastic layer. At P6 and in the adult, faint staining is detected in the ONL, suggesting that there is weak expression of Otx2 in adult photoreceptor cells [103, 104]. This is in contrast to Crx (another member of the Otx gene family) expression. In the adult mouse retina, strong staining for Crx expression is found in the ONL [103].

Otx2 was identified as binding to the IRBP promoter in a yeast one-hybrid screen using a human retina library and an IRBP sequence that was protected in DNA protection experiments [90]. The Otx2 protein transactivates the IRBP promoter in vitro. When HeLa cells are cotransfected with a 123-bp hIRBP promoter-reporter gene construct and an Otx2 expression plasmid, IRBP transcription is activated fiveto sevenfold [99]. Cotransfection of the hIRBP reporter gene construct and a Crx expression plasmid increases IRBP promoter activity fourto fivefold, which is in agreement with the transactivation experiments of Chen et al. [74] in HEK293 cells. Transfection of HeLa cells with the 123-bp hIRBP reporter gene construct, Otx2 expression plasmid, and Crx expression plasmid does not increase IRBP promoter activity, suggesting that the Otx2 and Crx proteins do not act synergistically on the human IRBP promoter [99]. Otx2mediated transactivation of the IRBP promoter was also seen when a plasmid containing the region −66 to +68 bp of the IRBP promoter and a reporter gene was cotransfected with an Otx2 expression plasmid into HeLa cells [90]. This same plasmid was used to study the transactivation activity of different isoforms of Otx2 in 293T cells [104]. In these experiments, the B isoform had stronger stimulation of the IRBP promoter than the A or C isoforms; this was attributed to higher levels of translation of the B isoform of Otx2 in this in vitro model [104]. Transactivation activity of the IRBP promoter is enhanced when both Otx2 and Nrl expression plasmids are used [95]. Also, Otx2 binds to a fragment of the IRBP promoter in ChIP assays, indicating that Otx2 interacts with the IRBP promoter in vivo [95]. ChIP assays also show that Otx2 binds to the IRBP promoter in retinas from Crx null mice, so the interaction between the IRBP promoter and Otx2 is not dependent on Crx binding [95].

Otx2 is expressed before Crx in mouse retinal development. Experiments in which the Otx2 genes are inactivated under the control of the Crx promoter (in conditional knockout mice) show that Otx2 is a direct regulator of Crx gene expression [103].

The above-listed trans factors are some of those needed to achieve cell-type selectivity and specificity of expression in the retina. It is widely recognized that the rod photoreceptor [93] does express many other trans factors, as is indicated by microarray