Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Here, using conditional gene ablation in mice, we show that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converted differentiating photoreceptor cells to amacrine-like neurons and led to a total lack of pinealocytes in the pineal gland. We also found that Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. Our results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development.

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2 ؉/؊ ; Crx ؊/؊ retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx ؊/؊ retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2 ؉/؊ ; Crx ؊/؊ retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cellspecific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.The vertebrate neural retina is comprised of six types of neurons and one type of glial cell, all derived from one population of multipotent progenitors (38,39,41). Transcription factors such as homeobox and basic helix-loop-helix factors have been known to play pivotal roles in the specification and development of retinal cell subtypes. Among the Otx-like homeobox genes, Otx2 and Crx play critical roles in retinal photoreceptor development. The expression of Otx2 covers most of the forebrain and midbrain neuroepithelium, including the eye domain, during development (32). Complete elimination of Otx2 functions in mice by gene targeting results in the absence of the forebrain and embryonic lethality (1,3,27). In a previous study, we have shown that Otx2 is essential and sufficient for the cell fate determination of retinal photoreceptors (29). Crx, on the other hand, is reported to be expressed abundantly in retinal photoreceptors and pinealocytes and also weakly in retinal bipolar cells (12,19). It has also been reported that Crx regulates various photoreceptor-specific genes (12,19,20). Mutations of human CRX are associated with three types of photoreceptor diseases: cone-rod dystrophy 2, retinitis pigmentosa, and Leber's congenital amaurosis (15,16,33,36). A gene-targeting study has revealed that Crx is essential for the terminal differentiation of photoreceptors and normal circadian entrainment (20). Thus, Otx2 and Crx have distinct roles in retinal photoreceptor development although their expression patterns in the retina overlap to some extent. However, they are structurally related transcription factors and can bind to a common DNA-bindin...

Immunosuppression strategies that selectively inhibit effector T cells while preserving and even enhancing CD4+FOXP3+ regulatory T cells (Treg) permit immune self-regulation and may allow minimization of immunosuppression and associated toxicities. Many immunosuppressive drugs were developed before the identity and function of Treg were appreciated. A good understanding of the interactions between Treg and immunosuppressive agents will be valuable to the effective design of more tolerable immunosuppression regimens. This review will discuss preclinical and clinical evidence regarding the influence of current and emerging immunosuppressive drugs on Treg homeostasis, stability, and function as a guideline for the selection and development of Treg-friendly immunosuppressive regimens.

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.