Lawrence J. Rizzolo

DIFFERENTIATION AND TRANSDIFFERENTIATION OF THE RETINAL PIGMENT EPITHELIUM

The retinal pigment epithelium (RPE) lies between the retina and the choroid of the eye and plays a vital role in ocular metabolism. The RPE develops from the same sheet of neuroepithelium as the retina and the two derivatives become distinguished by different expression patterns of a number of transcription factors during embryonic development. As the RPE layer differentiates it expresses a set of unique molecules, many of which are restricted to certain regions of the cell. PRE cells undergo both a loss of polarity and a loss of expression of many of these cell type-specific molecules when placed in monolayer culture. The RPE of many species, including mammals, can be induced to transdifferentiate by growth factors such as basic fibroblast growth factor. Under the influence of such factors the RPE is triggered to alter expression of a wide array of molecules and to take on a retinal epithelium fate, from which differentiated retinal cell types including rod photoreceptors can be produced.

Development and role of tight junctions in the retinal pigment epithelium.

The outer blood-retinal barrier is formed by the retinal pigment epithelium. In any epithelial monolayer, the tight junctions enable the epithelium to form a barrier by joining neighboring cells together and regulating transepithelial diffusion through the paracellular spaces. Tight junctions are complex, dynamic structures that regulate cell proliferation, polarity, and paracellular diffusion. The specific properties of tight junctions vary among epithelia, according to the physiological role of the epithelium. Unlike other epithelia, the apical surface of the retinal pigment epithelium interacts with a solid tissue, the neural retina. Secretions of the developing neural retina regulate the assembly, maturation, and tissue-specific properties of these tight junctions. The slow time course of development allows investigators to dissect the mechanisms of junction assembly and function. These studies are aided by culture systems that model different stages of development.

The distribution of Na+,K+-ATPase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture

The polarity of retinal pigmented epithelia (RPE) from chicken embryos was studied in primary cell culture. Since cultured RPE approximates the morphological polarity of RPE in vivo, we investigated whether this polarity extends to the distribution of plasma membrane proteins that are peculiar to RPE. In contrast to other epithelia, the Na+,K(+)-ATPase of RPE is located in the apical rather than basolateral plasma membrane. To examine this property, we cultured RPE on extracellular matrix-coated filters. Primary cultures were compared to embryonic RPE in situ using electron microscopy and indirect immunofluorescence of frozen sections. The viability and morphology of RPE was improved by using a serum-free medium containing a bovine pituitary extract in conjunction with an extracellular matrix coating derived from Engelbreth-Holm-Swarm tumors. Cultured RPE mimicked the morphology of RPE in vivo with microvilli and junctional complexes on the apical pole and infoldings along the basolateral plasma membrane. Functional tight junctions formed as demonstrated by an EDTA-sensitive, transepithelial electrical resistance, and by the retention of [3H]inulin added to the apical chamber. In 2 hr, only 4-6% of the [3H]inulin crossed the monolayer, compared to 24% in control filters. Despite these features of polarity, the Na+,K(+)-ATPase was detected in both apical and basolateral membranes by immunofluorescence. In embryonic eyes in which the neural retina was removed, the Na+,K(+)-ATPase was confined to the apical membrane. In addition, the polarity of cultured RPE was probed with vesicular stomatitis virus. In contrast to other epithelia, budding virus particles were observed emerging from the apical, as well as basolateral, domain further suggesting the cultured cells were only partially polarized. These data indicate that structural criteria are inadequate to determine if cultured RPE have become polarized in the same manner as the epithelium in vivo.

Integration of tight junctions and claudins with the barrier functions of the retinal pigment epithelium

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier by regulating the movement of solutes between the fenestrated capillaries of the choroid and the photoreceptor layer of the retina. Blood-tissue barriers use various mechanisms to accomplish their tasks including membrane pumps, transporters, and channels, transcytosis, metabolic alteration of solutes in transit, and passive but selective diffusion. The last category includes tight junctions, which regulate transepithelial diffusion through the spaces between neighboring cells of the monolayer. Tight junctions are extraordinarily complex structures that are dynamically regulated. Claudins are a family of tight junctional proteins that lend tissue specificity and selectivity to tight junctions. This review discusses how the claudins and tight junctions of the RPE differ from other epithelia and how its functions are modulated by the neural retina. Studies of RPE-retinal interactions during development lend insight into this modulation. Notably, the characteristics of RPE junctions, such as claudin composition, vary among species, which suggests the physiology of the outer retina may also vary. Comparative studies of barrier functions among species should deepen our understanding of how homeostasis is maintained in the outer retina. Stem cells provide a way to extend these studies of RPE-retinal interactions to human RPE.

Design, Implementation, and Evaluation of an Innovative Anatomy Course.

Starting in 2004, a medical school gross anatomy course faced with a 30% cut in hours went through an extensive redesign, which transformed a traditional dissection course into a course with a clinical focus, learning societies, and extensive on‐line learning support. Built into the redesign process was an extensive and ongoing assessment process, which included student focus groups, faculty development, surveys, and examinations. These assessments were used formatively, to enhance the course from year to year, and summatively, to determine how well the course was meeting the new learning objectives. The assessments from focus groups and faculty development prompted changes in support structures provided to students and the training and preparation of faculty. Survey results showed that, after student satisfaction declined the first year, satisfaction increased steadily through the fourth iteration as the course gained acceptance by students and faculty alike. There was a corresponding increase in the performance of students on course examinations. An additional examination given to students one and a half and three years after their anatomy course ended demonstrated the redesigned courses long‐term effectiveness for retaining anatomical knowledge and applying it to clinical cases. Compared to students who took the original course, students who took the shorter, more clinical course performed as well, or better, on each section of the examination. We attribute these positive results to the innovative course design and to the changes made based on our formative assessment program. Anat Sci Educ, 2010.

Remodeling of junctional complexes during the development of the outer blood-retinal barrier

The retinal pigment epithelium (RPE) forms the outer blood‐retinal barrier by separating the neural retina from fenestrated capillaries in the choroid. The barrier depends upon tight junctions within the apical junctional complexes that bind neighboring cells. During development, permeability decreases as the apical junctional complex gradually matures. To investigate this process, the composition of the apical junctional complex was monitored during RPE development in chicken embryos.

Regulation of glucose transporters during development of the retinal pigment epithelium

The retinal pigment epithelium (RPE) separates the outer retina from its blood supply. To satisfy the retinas large requirement for glucose, the RPE expresses high levels of glucose transporters. In most rat cells, the transporter GLUT3 provides a basal level of transport, but the expression of GLUT1 can be regulated. The opposite is true in chicken (P. Wagstaff, H.Y. Kang, D. Mylott, P.J. Robbins, M.K. White, Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts, Mol. Biol. Cell 6 (1995) 1575-1589). We examined chick RPE to determine which isoform is regulated during development, and if the neural retina regulates GLUT expression. By RT-PCR, RPE expressed GLUT1 and GLUT3, but not GLUT2. Only the level of GLUT1 increased between E5 and E18. A corresponding increase in GLUT1 protein was observed by immunoblotting. Most of the increase occurred between E14 and E18, which corresponds to the late stage of tight junction development. A culture model of development was used to examine the intermediate phase, which extends from E7 to E14. While medium conditioned by the neural retina decreased paracellular diffusion across the tight junctions, it increased diffusion through the glucose transporters. Unlike mammals, chick upregulates different isoforms in quiescent RPE and proliferating fibroblasts. Further, the upregulation of glucose transport is coordinated with the development of tight junctions in the blood-retinal barrier.

Claudin-19 and the Barrier Properties of the Human Retinal Pigment Epithelium

PURPOSE The retinal pigment epithelium (RPE) separates photoreceptors from choroidal capillaries, but in age-related macular degeneration (AMD) capillaries breach the RPE barrier. Little is known about human RPE tight junctions or the effects of serum on the retinal side of the RPE. METHODS Cultured human fetal RPE (hfRPE) was assessed by the transepithelial electrical resistance (TER) and the transepithelial diffusion of methylated polyethylene glycol (mPEG). Claudins and occludin were monitored by quantitative RT-PCR, immunoblotting, and immunofluorescence. RESULTS Similar to freshly isolated hfRPE, claudin-19 mRNA was 25 times more abundant than claudin-3. Other detectable claudin mRNAs were found in even lesser amounts, as little as 3000 times less abundant than claudin-19. Claudin-1 and claudin-10b were detected only in subpopulations of cells, whereas others were undetectable. Knockdown of claudin-19 by small interfering RNA (siRNA) eliminated the TER. siRNAs for other claudins had minimal effects. Serum affected tight junctions only when presented to the retinal side of the RPE. The TER increased 2 times, and the conductance of K(+) relative to Na(+) decreased without affecting the permeability of mPEG. These effects correlated with increased steady-state levels of occludin. CONCLUSIONS Fetal human RPE is a claudin-19-dominant epithelium that has regional variations in claudin-expression. Apical serum decreases RPE permeability, which might be a defense mechanism that would retard the spread of edema due to AMD.

Polarization of the Na+, K(+)-ATPase in epithelia derived from the neuroepithelium.

The neuroepithelium generates a fascinating group of epithelia. One of their intriguing properties is how they polarize the distribution of the Na+, K(+)-ATPase. Typically, this ion pump is concentrated in the basolateral membrane, but it is concentrated in the apical membranes of the retinal pigment epithelium and the epithelium of the choroid plexus. A comparison of their development with that of systemic epithelia yields insights into how cells polarize the distribution of this and other membrane proteins. The polarization of the Na+, K(+)-ATPase depends upon the interplay between different sorting signals and different types of polarity mechanisms. These include intracellular targeting signals that direct the delivery of newly synthesized proteins, and maintenance signals that stabilize proteins in the proper membrane domain. Conflicting signals appear to be arranged in a hierarchy that can be rearranged as cells respond to certain environmental stimuli. Part of this response is mediated by changes in the distribution and composition of the cortical cytoskeleton.

Barrier properties of cultured retinal pigment epithelium

The principal function of an epithelium is to form a dynamic barrier that regulates movement between body compartments. Each epithelium is specialized with barrier functions that are specific for the tissues it serves. The apical surface commonly faces a lumen, but the retinal pigment epithelium (RPE) appears to be unique by a facing solid tissue, the sensory retina. Nonetheless, there exists a thin (subretinal) space that can become fluid filled during pathology. RPE separates the subretinal space from the blood supply of the outer retina, thereby forming the outer blood-retinal barrier. The intricate interaction between the RPE and sensory retina presents challenges for learning how accurately culture models reflect native behavior. The challenge is heightened by findings that detail the variation of RPE barrier proteins both among species and at different stages of the life cycle. Among the striking differences is the expression of claudin family members. Claudins are the tight junction proteins that regulate ion diffusion across the spaces that lie between the cells of a monolayer. Claudin expression by RPE varies with species and life-stage, which implies functional differences among commonly used animal models. Investigators have turned to transcriptomics to supplement functional studies when comparing native and cultured tissue. The most detailed studies of the outer blood-retinal barrier have focused on human RPE with transcriptome and functional studies reported for human fetal, adult, and stem-cell derived RPE.