Molecular mechanisms of optic vesicle development: Complexities, ambiguities and controversies

AbstractOptic vesicle formation, transformation into an optic cup and integration with neighboring tissues are essential for normal eye formation, and involve the coordinated occurrence of complex cellular and molecular events. Perhaps not surprisingly, these complex phenomena have provided fertile ground for controversial and even contradictory results and conclusions. After presenting an overview of current knowledge of optic vesicle development, we will address conceptual and methodological issues that complicate research in this field. This will be done through a review of the pertinent literature, as well as by drawing on our own experience, gathered through recent studies of both intra- and extra-cellular regulation of optic vesicle development and patterning. Finally, and without attempting to be exhaustive, we will point out some important aspects of optic vesicle development that have not yet received enough attention

Cytoskeleton proteins previously considered exclusive to Ganglion Cells are transiently expressed by all retinal neuronal precursors

<p>Abstract</p> <p>Background</p> <p>Understanding the mechanisms governing cell fate specification remains one of the main challenges in the study of retinal development. In this context, molecular markers that identify specific cell types become crucial tools for the analysis and interpretation of these phenomena. In studies using the developing chick retina, expression of the mid-size neurofilament (NF-M) and a chick-specific microtubule associated protein recognized by the RA4 antibody (MAP(RA4)), have been broadly used to selectively identify ganglion cells and their committed precursors. However, observations in our laboratory suggested that the expression of these proteins may not be restricted to cells of the ganglion cell lineage. Because of its potential significance in the field, we pursued a detailed analysis of the expression of these two molecules in combination with an array of proteins that allowed precise identification of all retinal cell-type precursors throughout the development of the chick retina.</p> <p>Results</p> <p>Both, NF-M and MAP(RA4) proteins, showed a dynamic pattern of expression coincident with the progression of retinal cell differentiation. Both proteins were coexpressed spatially and temporally in postmitotic neuronal precursors throughout development. Expression of both proteins was seen in ganglion cell precursors and adult differentiated ganglion cells, but they were also transiently expressed by precursors of the photoreceptor, horizontal, bipolar and amacrine cell lineages.</p> <p>Conclusions</p> <p>We have clearly demonstrated that, contrary to the generally accepted paradigm, expression of NF-M and MAP(RA4) proteins is not exclusive to ganglion cells. Rather, both proteins are transiently expressed by all neuronal retinal progenitors in a developmentally-regulated manner. In addition, MAP(RA4) and NF-M are the first molecules so far characterized that may allow unambiguous identification of postmitotic precursors from the pool of mitotically active progenitors and/or the differentiated cell population during retinogenesis. These results are of significant impact for the field of developmental biology of the retina, since they provide novel and important information for the appropriate design and interpretation of studies on retinal cell differentiation, as well as for the reinterpretation of previously published studies.</p

Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

<p>Abstract</p> <p>Background</p> <p>The use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. SPF eggs are certified free of infection by specific pathogen viruses of either exogenous or endogenous origin, including those belonging to the ASLV family. Based on this, SPF embryos are considered to be free of ASLV viral protein expression, and consequently in developmental research studies RCAS infected cells are routinely identified by immunohistochemistry against the ASLV viral proteins p19 and p27. Contrary to this generally accepted notion, observations in our laboratory suggested that certified SPF chicken embryos may endogenously express ASLV viral proteins p19 and p27. Since these observations may have significant implications for the developmental research field we further investigated this possibility.</p> <p>Results</p> <p>We demonstrate that certified SPF chicken embryos have transcriptionally active endogenous ASLV loci (<it>ev loci</it>) capable of expressing ASLV viral proteins, such as p19 and p27, even when those <it>loci </it>are not capable of producing viral particles. We also show that the extent of viral protein expression in embryonic tissues varies not only among flocks but also between embryos of the same flock. In addition, our genetic screening revealed significant heterogeneity in <it>ev loci </it>composition even among embryos of the same flock.</p> <p>Conclusions</p> <p>These observations have critical implications for the developmental biology research field, since they strongly suggest that the current standard methodology used in experimental studies using the chick embryo and RCAS vectors may lead to inaccurate interpretation of results. Retrospectively, our observations suggest that studies in which infected cells have been identified simply by pan-ASLV viral protein expression may need to be considered with caution. For future studies, they point to a need for careful selection and screening of the chick SPF lines to be used in combination with RCAS constructs, as well as the methodology utilized for qualitative analysis of experimental results. A series of practical guidelines to ensure research quality animals and accuracy of the interpretation of results is recommended and discussed.</p

Controlling Retinal Pigment Epithelium Injury after Experimental Detachment of the Retina

PURPOSE. Damage induced by detachment of the neural retina and the retinal pigment epithelium (RPE) can be reduced by dark adaptation. The authors evaluated the influence of the duration of dark adaptation, time of day, and modification of the melatonin-dopamine pathway on acute RPE lesions induced by mechanical detachment. METHODS. BALB/c mice were studied at different times of day and different periods of dark adaptation. Some mice were treated with melatonin or sulpiride, a D2 dopamine receptor antagonist. Enucleated eyes and different saline solutions were used in experiments ex vivo. Retinal detachments in vivo were made by subretinal injections of hyaluronic acid. RPE cell damage was quantitatively evaluated with a dye exclusion procedure, and their viability was tested by preservation of tight junctions in culture. Lectin histochemistry was used to examine the interphotoreceptor matrix (IPM). RESULTS. Significant propidium iodide (PI) incorporation in RPE cells was detected after ex vivo separation during daytime, but it was very low when detachment took place at night after 24 to 48 hours of dark adaptation. PI exclusion was achieved during daytime after a single hour of dark adaptation when mice were pretreated with melatonin or sulpiride. Reduction of RPE cell damage was accompanied by decreased lectin binding to cone sheaths. CONCLUSIONS. A combination of time of day and length of dark adaptation decreased damage induced by detachment of the retina ex vivo and in vivo. Melatonin or sulpiride could replace these environmental factors. Therefore, melatonin and dopamine pathways might be involved in the control of IPM properties and retina/RPE interactions. (Invest Ophthalmol Vis Sci. 2007;48:1348 -1354) DOI:10.1167/iovs.06-0964 A rtificial separation of the neural retina and the retinal pigment epithelium (RPE) is sometimes performed for specific purposes such as excision of a subretinal membrane, reapplication of a detached and folded retina, or performance of macular translocation surgery. 1,2 Recovery of visual function depends on the reestablishment of functional associations among these layers. Because of the close anatomic interdigitation of photoreceptor outer segments and RPE microvilli, epithelial cells may remain attached to the retina when RPE is mechanically separated. This phenomenon has been used to evaluate neural retina/RPE adhesion strength. 3,4 Attachment also depends on cone sheaths, a specialized region of the interphotoreceptor matrix (IPM) that encases outer and inner segments of cone (but not rod) photoreceptors. 10 Melatonin is rhythmically synthesized in the retina and acts as a neuromodulator imparting photoperiodic information to the retina. Although we have been able to obtain RPE laminae of high morphologic quality after an hour of dark adaptation, we found that epithelial structure rapidly deteriorated after a few hours in culture. Therefore, we used propidium iodide (PI) to detect submicroscopic damage to RPE cells that occurred before the appearance of overt necrotic or apoptotic signs. This dyeexclusion procedure has been found useful in several tissues, including RPE cells. MATERIALS AND METHODS Male BALB/c albino mice were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental Conditions Mice were bred under a light/darkness cycle of 12 hours each (7:00 am to 7:00 pm) with maximum illumination levels of 60 lux at the cage floor. At the beginning of experiments, mice were kept in complete darkness between 1 hour and 7 days. Animals were housed in a double enclosure with food and water ad libitum. Feeding and cage cleaning were carried out under a dim red light. One group of animals was submitted to a shifted light cycle (12-hour light/12-hour dark). Surgical procedures were carried out under chloral hydrate anesthesia (400 mg/kg, intraperitoneal) and bulbar topical application with 0.25% proparacaine (diluted Anestalcon; Laboratorios Alcon, Buenos Aires, Argentina). Melatonin (0.05 mg/kg; Sigma-Aldrich Chemical Co., St. Louis, MO) or the D2 dopamine receptor (D2R) antagonist sulpiride (50 mg/kg; Armstrong-Syncro, Buenos Aires, Argentina) was administered subcutaneously. Control animals received similar volumes of saline solution. Injections and experimental procedures were performed by different researchers. Ex Vivo Separation of Neural Retina and RPE Pharmacologic treatments, enucleation, and separation were performed under dim red light and aseptic conditions. Room temperature was set at 28°C because temperature can modify adhesion between retina and RPE. After enucleation, the cornea, lens, and vitreous were dissected through perilimbal incision, and the optic nerve stump was removed From th

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22–25 % of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner

Generation of three dimensional retinal tissue with functional photoreceptors from human iPSCs

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSC) hold great potential for the modeling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light-sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form 3-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment-disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modeling and open possibilities for future therapies

Rediscovering the chick embryo as a model to study retinal development

Abstract The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. This article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.</p

Focus on Molecules: Proinsulin in the eye: Precursor or pioneer?

4 páginas, 1 figuraAuthors’ research is supported by NIH grant EYO4859 (MVC-S) and Spanish Ministerio de Ciencia e Innovación Grant SAF2007-66175 (EJdlR)Peer reviewe