Case Study: CRISPR 101 - a novel online learning course harnessing innovative ways to teach a complex biomolecular technology.

Complex biomolecular technologies revolutionise scientific research. Fully embedding scientific advances in the community requires innovative ways to educate learners on the molecular foundations upon which these technologies are based. In this case study, we present the conception and design of Walter and Eliza Hall Institute of Medical Research (WEHI's) inaugural wholly online learning course focussed on explaining the revolutionary genome-editing technology, clustered regulatory interspaced palindromic repeats (CRISPR). Utilising WEHI's strength in bringing science educators and world-leading CRISPR scientists together, we designed a multimodal online resource that introduces learners, without an extensive background in either science or genome editing, to the fundamental concepts of CRISPR technology. Using the online course creation tool, Articulate 360, we guided learners through three modules containing targeted lessons designed to focus on specific learning outcomes. Integrated videos, research articles, interviews, and other resources, allowed for self-paced learning that met various learning style needs. The extensive resources provided opportunities to delve deeper into the content for advanced learners. The effectiveness of the course, evaluated with survey responses collected upon completion of the course, highlighted the ease of use and functionality of the course, and an increased understanding of CRISPR technology after course completion. We anticipate future online learning course development to showcase complex molecular technology that will be valuable for tertiary education, as well as for those in the wider community interested in understanding important advances in biomedicine.

Antioxidant and Anti-Inflammatory Properties of Recombinant Bifidobacterium bifidum BGN4 Expressing Antioxidant Enzymes.

Bifidobacterium bifidum BGN4-SK (BGN4-SK), a recombinant strain which was constructed from B. bifidum BGN4 (BGN4) to produce superoxide dismutase (SOD) and catalase, was analyzed to determine its antioxidant and anti-inflammatory properties in vitro. Culture conditions were determined to maximize the SOD and catalase activities of BGN4-SK. The viability, intracellular radical oxygen species (ROS) levels, intracellular antioxidant enzyme activities, and pro-inflammatory cytokine levels were determined to evaluate the antioxidant and anti-inflammatory activities of BGN4-SK in human intestinal epithelial cells (HT-29) and murine macrophage cells (RAW 264.7). Antioxidant enzymes (SOD and catalase) were produced at the highest levels when BGN4-SK was cultured for 24 h in a medium containing 500 µM MnSO4 and 30 µM hematin, with glucose as the carbon source. The viability and intracellular antioxidant enzyme activities of H2O2-stimulated HT-29 treated with BGN4-SK were significantly higher (p < 0.05) than those of cells treated with BGN4. The intracellular ROS levels of H2O2-stimulated HT-29 cells treated with BGN4-SK were significantly lower (p < 0.05) than those of cells treated with BGN4. BGN4-SK more significantly suppressed the production of interleukin (IL)-6 (p < 0.05), tumor necrosis factor-α (p < 0.01), and IL-8 (p < 0.05) in lipopolysaccharide (LPS)-stimulated HT-29 and LPS-stimulated RAW 264.7 cells compared to BGN4. These results suggest that BGN4-SK may have enhanced antioxidant activities against oxidative stress in H2O2-stimulated HT-29 cells and enhanced anti-inflammatory activities in LPS-stimulated HT-29 and RAW 264.7 cells.

Morphological and behavioral limit of visual resolution in temperate (Hippocampus abdominalis) and tropical (Hippocampus taeniopterus) seahorses.

Seahorses are visually guided feeders that prey upon small fast-moving crustaceans. Seahorse habitats range from clear tropical to turbid temperate waters. How are seahorse retinae specialized to mediate vision in these diverse environments? Most species of seahorse have a specialization in their retina associated with acute vision, the fovea. The purpose of this study was to characterize the fovea of temperate Hippocampus abdominalis and tropical H. taeniopterus seahorses and to investigate their theoretical and behavioral limits of visual resolution. Their foveae were identified and photoreceptor (PR) and ganglion cell (GC) densities determined throughout the retina and topographically mapped. The theoretical limit of visual resolution was calculated using formulas taking into account lens radius and either cone PR or GC densities. Visual resolution was determined behaviorally using reactive distance. Both species possess a rod-free convexiclivate fovea. PR and GC densities were highest along the foveal slope, with a density decrease within the foveal center. Outside the fovea, there was a gradual density decrease towards the periphery. The theoretically calculated visual resolution on the foveal slope was poorer for H. abdominalis (5.25 min of arc) compared with H. taeniopterus (4.63 min of arc) based on PR density. Using GC density, H. abdominalis (9.81 min of arc) had a lower resolution compared with H. taeniopterus (9.04 min of arc). Behaviorally, H. abdominalis had a resolution limit of 1090.64 min of arc, while H. taeniopterus was much smaller, 692.86 min of arc. Although both species possess a fovea and the distribution of PR and GC is similar, H. taeniopterus has higher PR and GC densities on the foveal slope and better theoretical and behaviorally measured visual resolution compared to H. abdominalis. These data indicate that seahorses have a well-developed acute visual system, and tropical seahorses have higher visual resolution compared to temperate seahorses.

The cellular expression of antiangiogenic factors in fetal primate macula.

PURPOSE: To characterize the cellular expression patterns of antiangiogenic factors differentially regulated in the fetal human macula. METHODS: RNA was extracted from macular, nasal, and surround biopsies of three human fetal retinas at midgestation. Relative levels of expression of pigment epithelium-derived factor (PEDF), brain natriuretic peptide (BNP), collagen type IValpha2 (COL4A2), and natriuretic peptide receptors A and C (NPRA and NPRC) were determined with quantitative PCR. Cellular expression of PEDF and BNP was investigated by in situ hybridization on retinal sections from monkeys aged between fetal day 55 and 11 years. BNP, COL4A2, and NPRA proteins were localized by immunohistochemistry. Labeling was imaged and quantified by confocal microscopy and optical densitometry. RESULTS: Quantitative PCR confirmed higher levels of PEDF and BNP and lower levels of COL4A2 in the macula at midgestation. PEDF mRNA was detected in ganglion cells (GCs) and the pigment epithelium (RPE). BNP mRNA was detected in GCs and macroglia, although BNP immunoreactivity (IR) was predominantly perivascular. COL4A2-IR was detected in large blood vessels and NPRA-IR on the retinal vascular endothelium, GC axons in fetal retinas, and cone axons at all ages. Optical densitometry showed a graded expression of PEDF and BNP at all ages, with highest levels of expression in GCs in the developing fovea. CONCLUSIONS: Because the retinal vessels initially form in the GC layer, it is likely that PEDF has a key role in defining and maintaining the foveal avascular area. The precise role of BNP is unclear, but it may include both antiangiogenic and natriuretic functions.

Gradients of Eph-A6 expression in primate retina suggest roles in both vascular and axon guidance.

PURPOSE: Recently we identified high levels of expression of Eph-A6 in the macula of developing human retina and showed localization of Eph-A6 to ganglion cells (GC). In the present study we investigated the expression of some members of the ephrin family in developing primate retina, including the topography of Eph-A6 expression, and its ligands, in developing macaque retinas. METHODS: We extracted RNA from human fetal retinas and probed for Eph-A5-A7, Eph-B1, ephrin-B2, and ephrin-A1-A5 by RT-PCR, then prepared riboprobes for Eph-A5-A7, Eph-B1 and ephrin-A1, -A4 and -B2. Paraffin sections of fetal macaque retinas were used to localize expression of Ephs and ephrins by in situ hybridization and immunohistochemistry. RESULTS: We identified prominent gradients of Eph-A6 mRNA expression in the ganglion cell layer (GCL) of fetal macaque retinas of different ages. The gradient of Eph-A6 expression was high near the optic disc and low at the developing macula at fetal day (Fd) 55. At Fd 70 and 80, the gradient of Eph-A6 expression was reversed, being higher temporal to the macula, and low at the disc. By Fd 110, when the fovea begins to form, a pattern of expression was established that persisted into the postnatal period, in which the highest levels of expression were detected at the developing fovea, and progressively lower levels of expression were detected at increasing distance from the fovea. Beginning at Fd 70, we also detected a gradient of Eph-A6 expression running perpendicular to the retinal surface within the GCL of central retina that was high in the inner GCL and low in the outer GCL. This second pattern persisted into the neonatal period. We found the two ligands for Eph-A6, ephrin-A1 and ephrin-A4, expressed by Pax2-immunoreactive astrocytes, in the optic nerve head and in the retina, by in situ hybridization and immunohistochemistry. We propose that during development of the retinal vasculature, migration of ligand-bearing astrocytes is slowed along this Eph-A6 expression gradient through repellent Eph-A6 - ephrin-A1 and -A4 signaling. CONCLUSIONS: Patterns of Eph-A6 expression in the developing macaque retina suggest that Eph-A6 - ephrin-A1 and -A4 repellent signaling has a role in retinal vascular patterning, and in the postnatal maintenance of projections from macular and foveal GC.

Progenitor cells of the rod-free area centralis originate in the anterior dorsal optic vesicle.

BACKGROUND: Nervous system development is dependent on early regional specification to create functionally distinct tissues within an initially undifferentiated zone. Within the retina, photoreceptors are topographically organized with rod free area centrales faithfully generated at the centre of gaze. How does the developing eye regulate this placement? Conventional wisdom indicates that the distal tip of the growing optic vesicle (OV) gives rise to the area centralis/fovea. Ectopic expression and ablation studies do not fully support this view, creating a controversy as to the origin of this region. In this study, the lineage of cells in the chicken OV was traced using DiI. The location of labelled cells was mapped onto the retina in relation to the rod-free zone at embryonic (E) 7 and E17.5. The ability to regenerate a rod free area after OV ablation was determined in conjunction with lineage tracing. RESULTS: Anterior OV gave rise to cells in nasal retina and posterior OV became temporal retina. The OV distal tip gave rise to cells above the optic nerve head. A dorsal and anterior region of the OV correlated with cells in the developing rod free area centralis. Only ablations including the dorsal anterior region gave rise to a retina lacking a rod free zone. DiI application after ablation indicated that cells movements were greater along the anterior/posterior axis compared with the dorsal/ventral axis. CONCLUSION: Our data support the idea that the chicken rod free area centralis originates from cells located near, but not at the distal tip of the developing OV. Therefore, the hypothesis that the area centralis is derived from cells at the distal tip of the OV is not supported; rather, a region anterior and dorsal to the distal tip gives rise to the rod free region. When compared with other studies of retinal development, our results are supported on molecular, morphological and functional levels. Our data will lead to a better understanding of the mechanisms underlying the topographic organization of the retina, the origin of the rod free zone, and the general issue of compartmentalization of neural tissue before any indication of morphological differentiation.

Photoreceptor and ganglion cell topographies correlate with information convergence and high acuity regions in the adult pigeon (Columba livia) retina.

The fovea and area dorsalis are high acuity vision regions in the pigeon retina. However, the degree of neural convergence (an important determinant of acuity) has not been quantified consistently in this bird. The purpose of the study was to determine the topographic density changes and degree of photoreceptor to ganglion cell convergence in the fovea and the area dorsalis. Total photoreceptor and ganglion cell densities were calculated on the horizontal and vertical meridia. In four eyes, retinal topography was mapped for photoreceptors and ganglion cells. Rod density was quantified by counting anti-rod opsin-stained outer segments across the retina. The ratio of cone photoreceptors to ganglion cells, a rough measure of information convergence, was calculated. The fovea and the red field contained significantly higher mean cone and ganglion cell densities compared with the yellow field. Rods were missing from the fovea. Outside the fovea, rods comprised 20% of the photoreceptor population, with no significant density changes across the retina. The ratio of photoreceptors to ganglion cells was highest in the yellow field, suggesting a high degree of information convergence and low acuity. Our data indicate that convergence of cones onto ganglion cells in the red field is similar to that observed in the fovea. Convergence ratios in both the fovea and red field suggest greater visual acuity compared to that of the surrounding yellow field, which is consistent with the higher visual acuities that have been reported in these regions.

Differential expression of anti-angiogenic factors and guidance genes in the developing macula.

PURPOSE: The primate retina contains a specialized, cone-rich macula, which mediates high acuity and color vision. The spatial resolution provided by the neural retina at the macula is optimized by stereotyped retinal blood vessel and ganglion cell axon patterning, which radiate away from the macula and reduce shadowing of macular photoreceptors. However, the genes that mediate these specializations, and the reasons for the vulnerability of the macula to degenerative disease, remain obscure. The aim of this study was to identify novel genes that may influence retinal vascular patterning and definition of the foveal avascular area. METHODS: We used RNA from human fetal retinas at 19-20 weeks of gestation (WG; n=4) to measure differential gene expression in the macula, a region nasal to disc (nasal) and in the surrounding retina (surround) by hybridization to 12 GeneChip microarrays (HG-U133 Plus 2.0). The raw data was subjected to quality control assessment and preprocessing, using GC-RMA. We then used ANOVA analysis (Partek) Genomic Suite 6.3) and clustering (DAVID website) to identify the most highly represented genes clustered according to "biological process." The neural retina is fully differentiated at the macula at 19-20 WG, while neuronal progenitor cells are present throughout the rest of the retina. We therefore excluded genes associated with the cell cycle, and markers of differentiated neurons, from further analyses. Significantly regulated genes (p<0.01) were then identified in a second round of clustering according to molecular/reaction (KEGG) pathway. Genes of interest were verified by quantitative PCR (QRT-PCR), and 2 genes were localized by in situ hybridization. RESULTS: We generated two lists of differentially regulated genes: "macula versus surround" and "macula versus nasal." KEGG pathway clustering of the filtered gene lists identified 25 axon guidance-related genes that are differentially regulated in the macula. Furthermore, we found significant upregulation of three anti-angiogenic factors in the macula: pigment epithelium derived factor (PEDF), natriuretic peptide precurusor B (NPPB), and collagen type IValpha2. Differential expression of several members of the ephrin and semaphorin axon guidance gene families, PEDF, and NPPB was verified by QRT-PCR. Localization of PEDF and Eph-A6 mRNAs in sections of macaque retina shows expression of both genes concentrates in the ganglion cell layer (GCL) at the developing fovea, consistent with an involvement in definition of the foveal avascular area. CONCLUSIONS: Because the axons of macular ganglion cells exit the retina from around 8 WG, we suggest that the axon guidance genes highly expressed at the macula at 19-20 WG are also involved in vascular patterning, along with PEDF and NPPB. Localization of both PEDF and Eph-A6 mRNAs to the GCL of the developing fovea supports this idea. It is possible that specialization of the macular vessels, including definition of the foveal avascular area, is mediated by processes that piggyback on axon guidance mechanisms in effect earlier in development. These findings may be useful to understand the vulnerability of the macula to degeneration and to develop new therapeutic strategies to inhibit neovascularization.

Rod photoreceptor differentiation in fetal and infant human retina.

Human rods and cones are arranged in a precise spatial mosaic that is critical for optimal functioning of the visual system. However, the molecular processes that underpin specification of cell types within the mosaic are poorly understood. The progressive differentiation of human rods was tracked from fetal week (Fwk) 9 to postnatal (P) 8 months using immunocytochemical markers of key molecules that represent rod progression from post-mitotic precursors to outer segment-bearing functional photoreceptors. We find two phases associated with rod differentiation. The early phase begins in rods on the foveal edge at Fwk 10.5 when rods are first identified, and the rod-specific proteins NRL and NR2e3 are detected. By Fwk 11-12, these rods label for interphotoreceptor retinoid binding protein, recoverin, and aryl hydrocarbon receptor interacting protein-like 1. The second phase occurs over the next month with the appearance of rod opsin at Fwk 15, closely followed by the outer segment proteins rod GTP-gated sodium channel, rod arrestin, and peripherin. TULP is expressed relatively late at Fwk 18-20 in rods. Each phase proceeds across the retina in a central-peripheral order, such that rods in far peripheral retina are only entering the early phase at the same time that cells in central retina are entering their late phase. During the second half of gestation rods undergo an intracellular reorganization of these proteins, and cellular and OS elongation which continues into infancy. The progression of rod development shown here provides insight into the possible mechanisms underlying human retinal visual dysfunction when there are mutations affecting key rod-related molecules.

Tetrodotoxin-resistant voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 are expressed in the retina.

Voltage-gated sodium channels (VGSCs) are one of the fundamental building blocks of electrically excitable cells in the nervous system. These channels are responsible for the generation of action potentials that are required for the communication of neuronal signals over long distances within a cell. VGSCs are encoded by a family of nine genes whose products have widely varying biophysical properties. In this study, we have detected the expression of two atypical VGSCs (Na(v)1.8 and Na(v)1.9) in the retina. Compared with more common VGSCs, Na(v)1.8 and Na(v)1.9 have unusual biophysical and pharmacological properties, including persistent sodium currents and resistance to the canonical sodium channel blocker tetrodotoxin (TTX). Our molecular biological and immunohistochemical data derived from mouse (Mus musculus) retina demonstrate expression of Na(v)1.8 by retinal amacrine and ganglion cells, whereas Na(v)1.9 is expressed by photoreceptors and Müller glia. The fact that these channels exist in the central nervous system (CNS) and exhibit robust TTX resistance requires a re-evaluation of prior physiological, pharmacological, and developmental data in the visual system, in which the diversity of VGSCs has been previously underestimated.

Evidence for an evolutionary conserved role of homothorax/Meis1/2 during vertebrate retina development.

During eye development in D. melanogaster, the TALE-homeodomain protein Homothorax (Hth) is expressed by progenitor cells ahead of the neurogenic wave front, promotes rapid proliferation of these cells and is downregulated before cells exit the cell cycle and differentiate. Here, we present evidence that hth function is partially conserved in vertebrates. Retinal progenitor cells (RPCs) in chicks and mice express two Hth-related proteins, Meis1 and Meis2 (Mrg1), in species-specific temporal sequences. Meis1 marks RPCs throughout the period of neurogenesis in the retina, whereas Meis2 is specific for RPCs prior to the onset of retinal differentiation. Transfection of Meis-inactivating constructs impaired RPC proliferation and led to microphthalmia. RNA-interference-mediated knock-down of expression indicated that progenitor cells expressing Meis1 together with Meis2 proliferate more rapidly than cells expressing Meis1 alone. Transfection of Meis-inactivating constructs reduced the expression of cyclin D1 (Ccnd1) in the eye primordium and co-transfection of cyclin D1 partially rescued RPC proliferation. Collectively, these results suggest that (1) Meis1 and Meis2, similar to hth, maintain retinal progenitor cells in a rapidly proliferating state; (2) they control the expression of some ocular-determination genes and components of the cell cycle machinery; and (3) together with the species-specific differences in Meis1/Meis2 expression, combinatorial expression of Meis family proteins might be a candidate mechanism for the differential regulation of eye growth among vertebrate species.

Molecular mechanisms of vertebrate retina development: implications for ganglion cell and photoreceptor patterning.

Although the neural retina appears as a relatively uniform tissue when viewed from its surface, it is in fact highly patterned along its anterior-posterior and dorso-ventral axes. The question of how and when such patterns arise has been the subject of intensive investigations over several decades. Most studies aimed at understanding retinal pattern formation have used the retinotectal map, the ordered projections of retinal ganglion cells to the brain, as a functional readout of the pattern. However, other cell types are also topographically organized in the retina. The most commonly recognized example of such a topographic cellular organization is the differential distribution of photoreceptor types across the retina. Photoreceptor patterns are highly species-specific and may represent an important adaptation to the visual niche a given species occupies. Nevertheless, few studies have addressed this functional readout of pattern to date and our understanding of its development has remained superficial. Here, we review recent advances in understanding the molecular cascades that control regionalization of the eye anlage, relate these findings to the development of photoreceptor patterns and discuss common and unique strategies involved in both aspects of retinal pattern formation.

Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina.

Tangential cell dispersion in the retina is a spacing mechanism that establishes a regular mosaic organization among cell types and contributes to their final positioning. The present study has used the X-inactivation transgenic mouse expressing the lacZ reporter gene on one X chromosome. Due to X chromosome inactivation, 50% of early progenitor cells express beta-galactosidase (beta-Gal); therefore, all cells derived from a particular beta-Gal-expressing progenitor cell can be identified in labeled columns. The radial segregation of clonally related beta-Gal-positive and beta-Gal-negative cells can be used to determine whether single cells transgress a clonal boundary in the retina. We investigated the extent to which particular cell classes tangentially disperse by analyzing the placement of labeled cells expressing particular markers at several ages and quantifying their tangential displacement. Retinal neurons expressing cell markers at postnatal day (P) 1 have a greater degree of tangential dispersion compared with amacrine and bipolar cells at P5-6. We also studied whether there is a functional correlation with these dispersion patterns by investigating the emergence of functional ionotropic glutamate receptors. To determine the degree of functional glutamate receptor activation, agmatine (AGB) was used in combination with cell-specific labeling. AGB permeates functional glutamate receptor channels following activation with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate or N-methyl-D-aspartate (NMDA). Within these receptor groups, high concentrations of AMPA, kainate, and NMDA are associated with a high degree of tangential dispersion in the adult. Developmentally, functional kainate and AMPA receptors were detected by P1 and were associated with tangentially dispersed cells. Functional NMDA receptors were not detected as early as kainate and AMPA receptors. These results indicate that cells generated early during development are more likely to disperse tangentially compared with those generated later in development. Therefore, functional AMPA and kainate receptors may play a critical role in tangentially displacing cell types.

Expression of the homeodomain transcription factor Meis2 in the embryonic and postnatal retina.

Members of the Meis subfamily of homeodomain-containing transcription factors play important roles during development and disease. Here we report that the Meis family protein Meis2 is expressed by a subpopulation of gamma-aminobutyric acid (GABA)ergic amacrine (AM) cells in the adult and embryonic retina of different vertebrate species. In mice, Meis2-expressing (Meis2+) AM cells are not cholinergic or dopaminergic, but some are immunoreactive for neuronal nitric oxide synthase (bNOS). About 50% of the mouse Meis2+ AM cell population expresses the calcium-binding protein calretinin, and some Meis2+ AM cells show characteristics of Type II CD-15+ cells. AM cell expression of Meis2 is lost in a conditional knockout mouse model for Pax6, indicating a dependency upon Pax6. Bromodeoxyuridine pulse labeling experiments and immunohistochemical staining for the neuronal marker NeuN in embryonic mouse retinae indicate that Meis2 is an early marker for newly postmitotic AM cells. In addition, taking advantage of the protracted retinal development in humans, we show that newly generated AM cells express Meis2 before adopting the GABAergic or glycinergic neurotransmitter phenotype. As development proceeds, some AM cells lose Meis2 expression concomitantly with the appearance of glycine, while other AM cells retain Meis2 expression after they express GABA. These data identify Meis2 as a suitable marker for the study of AM cell diversity and development in addition to providing evidence for the stepwise specification of the glycinergic and GABAergic neurotransmitter phenotypes during AM cell differentiation.

Chick receptor protein tyrosine phosphatase lambda/psi (cRPTPlambda/cRPTPpsi) is dynamically expressed at the midbrain-hindbrain boundary and in the embryonic neural retina.

The isthmic organizer, located near the boundary between the developing midbrain and hindbrain, controls the patterning of adjacent brain regions. Here we describe the spatial and temporal expression of chick receptor-like protein tyrosine phosphatase lambda (cRPTPlambda, also known as cRPTPpsi) during the development of this structure. After an initial widespread expression throughout the caudal forebrain and midbrain region, expression of cRPTPlambda is confined to the ventral midline of the neural tube, the future neural retina and lens, and a sharp ring at the isthmic constriction, overlying the molecular mid-hindbrain boundary (MHB). MHB expression of cRPTPlambda borders at the caudal limit of the expression domain of the transcription factor Otx2, appears to partially overlap with that of the secreted protein Wnt1 and is similar to that of the Ig-CAM CEPU-1. In the neural retina, expression is restricted to the ventricular zone, where the cell bodies of retinal progenitor cells reside.

Expression of photoreceptor-specific nuclear receptor NR2E3 in rod photoreceptors of fetal human retina.

PURPOSE: To study the physiological function of NR2E3 and possible molecular mechanisms underlying enhanced short-wavelength cone syndrome (ESCS) pathogenesis in developing human retina, and to compare its expression to that of Neural Retina Leucine zipper (NRL), a transcription factor essential for rod differentiation. METHODS: Expression of NR2E3, a photoreceptor-specific orphan nuclear receptor, was examined in human retinas between fetal weeks (Fwk) 9 to 22 by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Both NR2E3 and NRL expression patterns were followed by immunocytochemistry. The human retina develops in a central to peripheral pattern, in which a protein may take weeks to be expressed throughout the entire retina. This allowed a detailed temporal analysis of NR2E3 and NRL expression. RESULTS: NR2E3 expression was detected shortly after the appearance of NRL in putative immature rods on the foveal edge at Fwk 11.7. Expression of both markers was maintained in rod opsin expressing fetal photoreceptors. NR2E3 expression was not detected in either long/medium- or short-wavelength cones. Its absence from cones was also supported by the position of labeled nuclei deep in the outer nuclear layer, and by the absence of NR2E3 from the fovea. CONCLUSIONS: A role for NR2E3 in the rod developmental pathway is suggested. The closely related expression patterns of NRL and NR2E3 supported an interactive function, where both transcription factors determine the rod fate and suppress immature rods from adopting the S-cone fate.

Expression of photoreceptor-associated molecules during human fetal eye development.

PURPOSE: A characteristic feature of the human retina is the early differentiation of foveal cells followed by a central to peripheral wave of maturation. This can obscure the true onset of differentiation when regions other that the fovea are sampled, or when methods based on whole retina or whole eye tissue are employed, such as reverse transcription-polymerase chain technique (RT-PCR). In order to assess the suitability of RT-PCR based approaches during human retinal development and to gain insight into the developmental progression of photoreceptor differentiation and maturation in the human, we analyzed the expression of several photoreceptor-associated genes by immunocytochemical labeling (ICC) of the foveal region as well as by RT-PCR of total RNA from whole fetal eyes from different developmental stages. METHODS: Expression of phosphodiesterase beta (PDEB), interphotoreceptor binding protein (IRBP), tubby-like protein (TULP), short wavelength specific (S) opsin, long and medium wavelength specific (L/M) opsin, rod opsin and the transcription factors Crx and Nrl were assessed by RT-PCR from total RNA prepared from snap frozen intact human fetal eyes ranging from fetal week 9 (Fwk 9) to Fwk 18. ICC labeling was performed in a large number of eyes within an age group for IRBP, TULP, Nrl, S opsin, L/M opsin and rod opsin on frozen sections that included the fovea centralis. RESULTS: All ICC markers appeared first in or around the fovea. We detected PDEB and Crx expression as early as Fwk 10, by RT-PCR. TULP and IRBP were first observed with ICC in a small number of foveal cones at Fwk 9, although the first transcripts were not detected until Fwk 12. Nrl-positive nuclei appeared around the fovea by Fwk 11 and S opsin-positive cones by Fwk 12. L/M opsin-positive cones and rod opsin-positive rods were first detected between Fwk 15-16. In general, ICC labeling in the fovea was present for most genes up to 2 weeks before the corresponding transcripts could be successfully amplified by RT-PCR from whole eye tissue. CONCLUSIONS: Our results indicate that in order to pinpoint exactly when and where a molecule appears, ICC labeling of the fovea is a more reliable indicator. RT-PCR was prone to underestimate the exact onset of expression of the molecules tested, yet it faithfully recapitulated the sequence in which they appeared. In addition, our data show that in the human fetal retina, Crx and Nrl are both expressed when the first rod photoreceptors are being generated. This agrees well with previous in vitro results suggesting a synergistic action of both proteins during differentiation of human rod photoreceptors.