A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rodspecific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.
Background:The transcription factor Nrl is required for rod photoreceptor development, but mechanisms governing Nrl transcription remain largely unknown. Results: The transcription factors CRX, OTX2, and ROR regulate Nrl by binding directly to its promoter region. Conclusion: These three factors combinatorially control Nrl expression in the developing mouse retina. Significance: This study elucidates a critical link in the photoreceptor cis-regulatory network.
Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (˜300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors 1-3 . They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs 4-5 . This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain.Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina 6-8 . While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko 6-7,9 , we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas 10 . Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells.When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production [11][12] . Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs. (Fig. 2A). The metal rails should be completely sealed to the bottom of the slide. 2. Use a Dremel tool to cut a handle off a plastic microcentrifuge tube rack. Cut the handle into 5 small rectangular pieces each with the following dimensions: length 0.8cm, height 0.6cm, width 0.3cm (Fig. 2B). These plastic pieces are reusable spa...
Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (˜300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors [1][2][3] . They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs [4][5] . This technique is time-consuming, costly and, on account of insertion site effects, largely nonquantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain.Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina [6][7][8] . While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko [6][7]9 , we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas 10 . Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells.When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production [11][12] . Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs. Video LinkThe video component of this article can be found at https://www.jove.com/video/2821/ Protocol 1. Construction of the electroporation chamber 1. Order a microslide, BTX Model 453 with a 3.2mm gap (Harvard Apparatus #45-0105) (Fig. 2A). The metal rails should be completely sealed to the bottom of the slide. ...
The RAS/RAF/MEK/ERK signaling pathway is one of the pillars of molecular cell biology. Decades of research have elucidated the components of this kinase cascade and its effects on cellular processes, including proliferation, motility, differentiation, and survival. In the era of translational medicine, the pathway is particularly relevant, as its dysregulation has been implicated in one-third of human cancers. Small-molecule inhibitors of BRAF and MEK have thus emerged as promising therapeutics for malignancies, including advanced melanoma, serous low-grade ovarian carcinoma, and others. Trametinib and cobimetinib are currently the only MEK inhibitors approved by the US Food and Drug Administration; each has been approved as combination therapy with BRAF inhibitors for the treatment of BRAF V600 mutant melanoma. Other MEK inhibitors, including binimetinib and selumetinib, are currently in advanced clinical trials.Adverse effects associated with MEK inhibitors include rash, diarrhea, fatigue, hypertension, and ophthalmic pathology, including retinal vein occlusion, uveitis, cystoid macular edema, and-most curiously-serous retinal detachments (Figure , A). The latter effect has received considerable attention in the oncologic and ophthalmic literature owing in part to its strikingly high incidence. Prospective 1 and post hoc 2 analyses published in 2016 report the development of subretinal fluid in more than 90% of patients receiving either bimetinib monotherapy or combination therapy with BRAF or phosphoinositide 3-kinase inhibitors. This retinopathy is thought to be a class effect of MEK inhibitors because of numerous case reports associated with other inhibitors, including trametinib, cobimetinib, and pimasertib, thus prompting the designation of MEK inhibitorassociated retinopathy (MEKAR). 1 Why does MEKAR matter? The most obvious reason to characterize this phenomenon is to learn whether it warrants discontinuation of MEK inhibitor therapy. Fortunately, most patients with subretinal fluid are asymptomatic or experience only mild visual disturbances. The symptoms and fluid typically resolve after several weeks or months even with continued therapy, 1 possibly due to tachyphylaxis, which has been previously demonstrated in MEK inhibition. While long-term studies are still necessary, the preponderance of clinical evidence suggests that it is safe from an ophthalmologic standpoint to continue MEK inhibitor therapy in patients who develop MEKAR.However, MEKAR should be intriguing to both clinicians and basic scientists for another reason: its similarity to central serous chorioretinopathy (CSCR), a poorly understood yet not uncommon cause of visual impairment. Acute CSCR typically presents with blurred or distorted central vision due to serous retinal detachment with underlying alterations of the retinal pigment
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