Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors

Abstract

The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl ؊/؊ retinas, the GFP؉ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP؉ photoreceptors from wild-type and Nrl ؊/؊ retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.gene profiling ͉ gene regulation ͉ neuronal differentiation ͉ retina ͉ transcription factor E volution of higher-order sensory and behavioral functions in mammals is accompanied by increasingly complex regulation of gene expression (1). As much as 10% of the human genome is presumably dedicated to the control of transcription. Exquisitely timed expression of cell-type-specific genes, together with spatial and quantitative precision, depends on the interaction between transcriptional control machinery and extracellular signals (2, 3). Neuronal heterogeneity and functional diversity result from combinatorial and cooperative actions of regulatory proteins that form complicated yet precise transcriptional networks to generate unique gene expression profiles. A key transcription factor, combined with its cognate regulatory cis-sequence codes, specifies a particular node in the gene regulatory networks that guide differentiation and development (4).The retina offers an ideal paradigm for investigating regulatory networks underlying neuronal differentiation. The genesis of six types of neurons and Müller glia in the vertebrate retina proceeds in a predictable sequence during development (5). Subsets of multipotent retinal neuroepithelial progenitors exit the cell cycle at specific time points and acquire a particular cell fate under the influence of intrinsic genetic program and extrinsic factors (5-7). Pioneering studies using thymidine labeling and retroviral vectors established the order and birthdates of neurons in developing retina (5,(8)(9)(10)). The current model of retinal differentiation proposes that a heterogeneous pool of progenitors passes through states of competence, where it can generate a distinct subset of neurons (5). One can predict that, at the molecular level, this competence is acquired by combinatorial action of specific transcriptional regulatory proteins. Genetic ablation studies of transcription factors involved in early murine eye specification are consistent with combinatorial regulation (11-13).Rod and cone photorecep...

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

“…This promoter fragment has been previously shown to drive faithful expression of GFP in rod photoreceptors in transgenic mice [19]. Using the new lines and following day 10 retinal progenitor differentiation and Rax.GFP FACS, we differentiated the cells to day 28 of culture before performing a second FACS analysis to determine the percentage of Nrl.RFP þ cells (Fig.…”

Regulation of photoreceptor gene expression by Crx-associated transcription factor network

“…This was further demonstrated by mouse studies showing that ectopic expression of Nr2e3 in photoreceptor precursors of Nrl −/− mice can transform developing cones to rod-like photoreceptors . Microarray analyses of the developing and mature retina of wild-type and Nrl −/− mice have identified clusters of Nrl target genes (Yoshida et al, 2004;Yu et al, 2004;Akimoto et al, 2006). These not only include genes coding for phototransduction and structural or membrane associated proteins as expected, but also transcriptional regulators, intracellular transport proteins and components of known signaling pathways.…”

Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL

“…Interestingly, ETS transcription factors are selectively expressed during motor and sensory neuron development but only after their axons reach the periphery, suggesting that these proteins confer postmitotic subtype identity during the establishment of selective connections (24,25). These findings indicate that neural identity can be specified even after the terminal cell cycle exit; however, direct in vivo evidence of postmitotic plasticity has not been reported.The Maf-family transcription factor Nrl is expressed specifically in postmitotic rod photoreceptors of the retina and in the pineal gland (26,27). NRL interacts with the homeodomain protein CRX (28), orphan nuclear receptor NR2E3 (29), and other retinal proteins (30-32) to regulate the expression of rod-specific genes (26, 33).…”

“…The Maf-family transcription factor Nrl is expressed specifically in postmitotic rod photoreceptors of the retina and in the pineal gland (26,27). NRL interacts with the homeodomain protein CRX (28), orphan nuclear receptor NR2E3 (29), and other retinal proteins (30-32) to regulate the expression of rod-specific genes (26, 33).…”

“…NRL interacts with the homeodomain protein CRX (28), orphan nuclear receptor NR2E3 (29), and other retinal proteins (30)(31)(32) to regulate the expression of rod-specific genes (26,33). NRL is essential for rod differentiation, because rods are transformed to functional S-cones in the Nrl Ϫ/Ϫ mouse retina (26,34,35). The apparent switch from rod to S-cone fate in the Nrl Ϫ/Ϫ mouse suggests that postmitotic photoreceptor precursors retain some degree of plasticity.…”