Outer Neuroblastic Layer Research Articles

Neuronal differentiation in the early human retinogenesis

AimOur study investigates the differentiation of retinal stem cells towards different neuronal subtypes during the critical period of human eye development. MethodsExpression of the neuronal marker neurofilament 200 (NF200), tyrosine hydroxilase (TH) and choline acetyltransferase (ChAT) was seen by immunofluorescence in the 5th–12th – week stage of development in the human eye. Data was analysed by Mann–Whitney, Kruskal–Wallis and Dunn’s post hoc tests. ResultsNF200, TH and ChAT cells appeared in the 5th/6th week and gradually increased during further development. The proportion of TH positive areas were distributed similarly to NF200, with a higher proportion in the outer neuroblastic layer. The proportion of a ChAT positive surface was highest in the 5th/6th – week whilst from the 7th week onwards, its proportion became higher in the optic nerve and inner neuroblastic layers than in the outer layer, where a decrease of ChAT positive areas were seen. ConclusionsOur study indicates a high differentiation potential of early retinal cells, which decreased with the advancement of development. The observed great variety of retinal phenotypic expressions results from a large scale of influences, taking place at different developmental stages.

RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina.

A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression.

Interplay of proliferation and differentiation factors is revealed in the early human eye development.

Eye development is a consequence of numerous epithelial-to-mesenchymal interactions between the prospective lens ectoderm, outpocketings of the forebrain forming optic vesicles, and surrounding mesenchyme. How different cell types forming eye structures differentiate from their precursors, and which factors coordinate complex human eye development remains largely unknown. Proper differentiation of photoreceptors is of special interest because of their involvement in the appearance of degenerative retinal diseases. Here we analyze the spatiotemporal expression of neuronal markers nestin, protein gene product 9.5 (PGP9.5), and calcium binding protein (S100), proliferation marker (Ki-67), markers for cilia (alpha-tubulin), and cell stemness marker octamer-binding transcription factor 4 (Oct-4) in histological sections of 5-12 -week human eyes using immunohistochemical and immunofluorescence methods. While during the investigated developmental period nestin shows strong expression in all mesenchymal derivatives, lens, optic stalk and inner neuroblastic layer, PGP9.5 and S100 expression characterizes only neural derivatives (optic nerve and neural retina). PGP9.5 is co-localized with nestin and S100 in the differentiating cells of the inner neuroblastic layer. Initially strong proliferation in all parts of the developing eye gradually ceases, especially in the outer neuroblastic layer. Proliferating Ki-67 positive cells co-localize with nestin in the retina, lens, and choroid. Strong Oct-4 and alpha-tubulin immunoreactivity in the retina and optic nerve gradually decreases, while they co-localize in outer neuroblastic and nerve fiber layers. The described expression of investigated markers indicates their importance in eye growth and morphogenesis, while their spatially and temporally restricted pattern coincides with differentiation of initially immature cells into specific retinal cell lineages. Alterations in their spatiotemporal interplay might lead to disturbances of visual function.

Loss of citron kinase affects a subset of progenitor cells that alters late but not early neurogenesis in the developing rat retina.

To understand how loss of citron kinase (CitK) affects retinal progenitor cells (RPCs) in the developing rat retina. We compared knockout (KO) and wild-type (WT) retinae by immunohistochemistry. The TdT-mediated dUTP terminal nick-end labeling (TUNEL) assay was performed to determine cell death. Pulse-chase experiments using 5-ethynyl-2'-deoxyuridine (EdU) were carried out to interrogate RPC behavior and in turn neurogenesis. Reverse transcription-polymerase chain reaction analysis showed that CitK was expressed at embryonic day (E)12 and was turned off at approximately postnatal day (P)4. Immunohistochemistry showed CitK being localized as puncta at the apical end of the outer neuroblastic layer (ONBL). Analyses during embryonic development showed that the KO retina was of comparable size to that of WT until E13. However, by E14, there was a reduction in the number of S-phase RPCs with a concomitant increase in TUNEL+ cells in the KO retina. Moreover, early neurogenesis, as reflected by retinal ganglion cell production, was not affected. Postnatal analysis of the retina showed that ONBL in the KO retina was reduced to half the size of that in WT and showed further degeneration. Immunohistochemistry revealed absence of Islet1+ bipolar cells at P2, which was further confirmed by EdU pulse-chase experiments. The CitK KO retinae underwent complete degeneration by P14. Our study showed that CitK is not required for a subset of RPCs before E14, but is necessary for RPC survival post E14. This in turn results in normal early embryonic neurogenesis, but severely compromised later embryonic and postnatal neurogenesis.

Abnormal retinal development associated with FRMD7 mutations

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder, often associated with FRMD7 mutations. As the appearance of the retina is reported to be normal based on conventional fundus photography, IIN is postulated to arise from abnormal cortical development. To determine whether the afferent visual system is involved in FRMD7 mutations, we performed in situ hybridization studies in human embryonic and fetal stages (35 days post-ovulation to 9 weeks post-conception). We show a dynamic retinal expression pattern of FRMD7 during development. We observe expression within the outer neuroblastic layer, then in the inner neuroblastic layer and at 9 weeks post-conception a bilaminar expression pattern. Expression was also noted within the developing optic stalk and optic disk. We identified a large cohort of IIN patients (n = 100), and performed sequence analysis which revealed 45 patients with FRMD7 mutations. Patients with FRMD7 mutations underwent detailed retinal imaging studies using ultrahigh-resolution optical coherence tomography. The tomograms were compared with a control cohort (n = 60). The foveal pit was significantly shallower in FRMD7 patients (P < 0.0001). The optic nerve head morphology was abnormal with significantly decreased optic disk area, retinal nerve fiber layer thickness, cup area and cup depth in FRMD7 patients (P < 0.0001). This study shows for the first time that abnormal afferent system development is associated with FRMD7 mutations and could be an important etiological factor in the development of nystagmus.

Characterization of Progenitor Cells during Canine Retinal Development

We identify the presence of progenitor cells during retinal development in the dog, as this species represents a natural model for studying several breed-specific degenerative retinal disorders. Antibodies to detected progenitor cells (Pax6, C-kit, and nestin) and ganglion cells (BDNF, Brn3a, and Thy1) were used in combination with H3 for the purpose of identifying proliferating cells. Pax6, nestin, C-kit, and H3 were localized mainly in the neuroblastic layer of the retina during the embryonic stage. During the fetal stage, proteins were expressed in the inner neuroblastic layer (INL) as well as in the outer neuroblastic layer; BDNF, Thy1, and Brn3a were also expressed in the INL. During the neonatal stage only C-kit was not expressed. Proliferating cells were present in both undifferentiated and differentiated retina. These results suggest that, during canine retinogenesis, progenitor cells are distributed along the retina and some of these cells remain as progenitor cells of the ganglion cells during the first postnatal days.

Nuclear Receptor Rev-erb Alpha (Nr1d1) Functions in Concert with Nr2e3 to Regulate Transcriptional Networks in the Retina

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Analysis of Thyroid Response Element Activity during Retinal Development

Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRβ, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue.

ATP controls cell cycle and induces proliferation in the mouse developing retina

Previous data suggest that nucleotides are important mitogens in the developing chick retina. Here, we extended the study on the mitogenic effect of ATP to newborn mouse retinal explants. Our results showed that P2Y1 receptors were widely distributed in C57bl/6 mice retina and that the majority of PCNA positive cells co-localized with P2Y1 receptor. To evaluate proliferation, retinal explants obtained from newborn mice were incubated with 0.5μCi [3H]-thymidine or 3μM BrDU 1h before the end of culture. Our data showed that ATP induced a dose-dependent increase in [3H]-thymidine incorporation, an effect that was mimicked by ADP but not by UTP and was blocked by the P2 antagonist PPADS in a dose-dependent manner. The increase in [3H]-thymidine incorporation induced by ATP was only observed in explants cultured for 3 days or less and was mimicked by the ectoapyrase inhibitor ARL 67156. It corresponded to an increase in the number of BrdU+ cells in the neuroblastic layer (NL) of the tissue, suggesting that ATP, through activation of P2Y1 receptors, induced proliferation of late developing progenitors in retinal explants of newborn mice. The increase in the number of BrdU+ cells was observed across the whole NL when explants were incubated with ATP for 24h and no increase in the number of p-histone H3 labeled cells could be noticed at this time point. In longer incubations of 48h with ATP or 24h with ATP followed by a period of 24h in fresh medium, an increase in the number of BrdU+ cells promoted by ATP was observed only in the middle and outer, but not in the inner NL. In these conditions, an increase in the number of p-histone H3 labeled cells was detected in the outer NL, suggesting that ATP induced cells to enter S and progress to G2 phase of the cell cycle in the first 24h period of incubation. ATP also induced an increase and a decrease in the expression of cyclin D1and p27kip1, respectively, in retinal progenitors of the NL. While the increase in the expression of cyclin D1 was observed when retinal explants were incubated for 3h or longer periods of time, the decrease in the expression of p27kip1 was noticed only after 6h incubation with ATP. Both effects were blocked by the P2 receptor antagonist PPADS. These data suggest that ATP induces cell proliferation in retinal explants by inducing late developing progenitors to progress from G1 to S phase of cell cycle.

Developmental expression of thyroid hormone receptor β2 protein in cone photoreceptors in the mouse

Thyroid hormone receptor beta2 (TRbeta2) controls the patterning of cone opsin photopigments that mediate colour vision. We raised an antiserum against TRbeta2 to study cone photoreceptor development by western blot and immunostaining analyses. TRbeta2-positive cells first appeared between embryonic day 10 (E10) and E12. Numbers increased until near birth, correlating with generation of the cone population. At birth, signals decreased until postnatal day 10, then declined to very low levels in adulthood. TRbeta2-positive cells were initially dispersed but became aligned at the edge of the outer neuroblastic layer by E15. Postnatally, these cells migrated inwardly until postnatal day 10, then outwardly to the edge of the outer nuclear layer, the location of mature cones. TRbeta2 represents a functionally unique marker for cone development.

Pias3-Dependent SUMOylation Directs Rod Photoreceptor Development

Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 binds the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod- and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identify Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.

Melanocortin-4 receptor messenger ribonucleic acid expression in rat cardiorespiratory, musculoskeletal, and integumentary systems.

We determined melanocortin-4 receptor (MC4-R) mRNA ontogeny in the rat using in situ hybridization and a rat MC4-R riboprobe and showed numerous peripheral sites of expression for MC4-R. The developing heart showed MC4-R mRNA expression as early as embryonic day (E) 14. In the lungs of E16-E20 fetuses, the cells surrounding developing bronchi expressed relatively strong in situ signal. Muscles associated with the respiratory system such as diaphragm and intercostal muscle expressed MC4-R mRNA as early as E14. Occipital and tongue muscles, in particular the genioglossus, showed diffuse signal at E15-E20. In the eye, a discrete signal was detected in an outer neuroblastic layer which may correspond to retina or extraocular muscle. Developing limb buds expressed relatively strong signal at E14, whereas skull bone and joint capsules of the paw of the forelimb showed signal at E18-E20. Using RT-PCR and ribonuclease protection assays, we determined that MC4-R mRNA is also expressed in adult rat heart, lung, kidney, and testis. The expression of the MC4-R in cardiorespiratory, musculoskeletal, and integumentary systems supports functional roles for the MC4-R in addition to its roles in appetite, weight control, and regulation of linear growth.

In Vitro Isolation and Expansion of Human Retinal Progenitor Cells

Human retinal development proceeds with temporal and spatial precision. Although differentiation starts around the beginning of the third month of gestation, the majority of cells in the outer neuroblastic layer of human neural retina are still proliferating, as evidenced by their Ki-67 immunoreactivity. In the present study, the proliferating human retinal progenitor cells (HRPCs) were isolated and expanded in culture. They were capable of dividing for multiple generations (with passage 8, the latest tested) and differentiating to several retinal cell phenotypes. These findings indicate that human retina at the 10th–13th week of gestation harbors progenitor cells that can be maintained and expanded in vitro for multiple generations. The availability of such cells may have important implications with respect to human degenerative retinal diseases, as these HRPCs have the potential to be used therapeutically to replace damaged retinal neurons.

CDNA Cloning and Expression Analysis ofNeuroDmRNA in Human Retina

We have shown that bHLH proteins are involved in mammalian retinal development. Here we report the identification and analysis of the expression of a neurogenic differentiation gene,NeuroD,in human retina.In situhybridization and immunocytochemical analyses of adult retina showed thatNeuroDtranscripts andNeuroDimmunoreactivity are predominantly localized to the outer nuclear layer which contains the photoreceptors. Southern analysis of PCR-amplified cDNA revealed thatNeuroDmRNA is also expressed in fetal human retina. Fetal monkey retina was used to analyse the spatial distribution of NeuroD in the developing retina. BothNeuroDtranscripts and immunoreactivity are largely detected in the outer neuroblastic layer. Therefore,NeuroDmay be involved in the differentiation as well as maintenance of the differentiated properties of photoreceptors.

Storage of human fetal retina in optisol prior to subretinal transplantation

We have previously reported that Optisol, a commercially available medium (Chiron Vision, Irvine, CA), is a favorable fluid for the storage of neural retina. We now characterize the cell survival and histologic integrity of the tissue following storage and then transplantation. Neural retinas from human fetal eyes obtained within 1 h of pregnancy termination were stored in Optisol-GS medium (containing TC-199 and MEM medium, 2.5% chondroitin sulfate, 1% dextran, HEPES buffer, gentamicin sulfate, 0.1 mmol/L nonessential amino acids, sodium bicarbonate, 1 mmol/L sodium pyruvate, and additional antioxidants), at 4°C for the following time periods: 0, 1, 2, and 7 days, and then tested for viability. Retinas stored for 2 and 7 days were mechanically dissociated and grafted into the eyes of light-damaged Fischer 344 rats (17 animals, 28 eyes). Non-transplanted eyes received injections of vehicle only (5 animals, 6 eyes). All animals were immunosuppressed daily with cyclosporine (10 mg/kg). All experiments were conducted in strict accordance with institutional, federal, and ARVO guidelines. Cell viability averaged 94.8%, 90.2%, 83.2%, and 76.8% at 0, 1, 2, and 7 days storage, respectively. Light microscopy demonstrated that the fetal retina was preserved without evident changes for up to 48 h. Up to 7 days there was a good to very good preservation of the cells in the outer neuroblastic layer. One month after transplantation subretinal grafts of retinal tissue stored for 2 and 7 days in Optisol showed good integration with host retina and initial photoreceptor differentiation. Optisol is a widely used medium for the preservation of human corneas prior to transplantation. Our results show that this medium is also highly suitable for preserving human fetal retinas prior to transplantation.

Storage of human fetal retina in optisol prior to subretinal transplantation.

We have previously reported that Optisol, a commercially available medium (Chiron Vision, Irvine, CA), is a favorable fluid for the storage of neural retina. We now characterize the cell survival and histologic integrity of the tissue following storage and then transplantation. Neural retinas from human fetal eyes obtained within 1 h or pregnancy termination were stored in Optisol-GS medium (containing TC-199 and MEM medium, 2.5% chondroitin sulfate, 1% dextran, HEPES buffer, gentamicin sulfate, 0.1 mmol/L nonessential amino acids, sodium bicarbonate, 1 mmol/L sodium pyruvate, and additional antioxidants), at 4 degrees C for the following time periods: 0, 1, 2, and 7 days, and then tested for viability. Retinas stored for 2 and 7 days were mechanically dissociated and grafted into the eyes of light-damaged Fischer 344 rats (17 animals, 28 eyes). Nontransplanted eyes received injections of vehicle only (5 animals, 6 eyes). All animals were immunosuppressed daily with cyclosporine (10 mg/kg). All experiments were conducted in strict accordance with institutional, federal, and ARVO guidelines. Cell viability averaged 94.8%, 90.2%, 83.2%, and 76.8% at 0, 1, 2, and 7 days storage, respectively. Light microscopy demonstrated that the fetal retina was preserved without evident changes for up to 48 h. Up to 7 days there was a good to very good preservation of the cells in the outer neuroblastic layer. One month after transplantation subretinal grafts of retinal tissue stored for 2 and 7 days in Optisol showed good integration with host retina and initial photoreceptor differentiation. Optisol is a widely used medium for the preservation of human corneas prior to transplantation. Our results show that this medium is also highly suitable for preserving human fetal retinas prior to transplantation.

Schisis-like rhegmatogenous retinal detachment associated with choroidal colobomas.

The breaks that cause retinal detachments in colobomatous eyes are often hidden within the lesion and difficult to find. To elucidate the pathoanatomy and possible pathomechanism of such detachments, histological sections of eight choroidal colobomas were reviewed. Sections of the margin showed central continuation of the inner neuroblastic layer (the intercalary membrane) and eversion and separation of the outer neuroblastic layer. The opposite direction of continuity of the neuroblastic layers created a schisis-like configuration between the intercalary membrane and the everted outer retina. The zone of duplication was a point of retinal adhesion, but also a locus minoris resistentiae due to vitreous attachments and variable glial support at the margin. The subset of coloboma-associated retinal detachments requires both a central break in the inner layer and a break in the outer layer at the margin of the coloboma. The inner layer break may be precipitated by retinovascular ischemia or scleral stretching; that in the outer layer may be caused by vitreous traction on the margin of the coloboma or extension of the formerly isolated detachment through the outer marginal zone of decreased glial support.

Development of the Retina in the Porcine Fetus A Light Microscopic Study

Morphogenesis of the porcine retina was studied using light microscopy from 4 weeks of gestation until birth (18 to 310 mm crown-rump length), and compared with the adult stage (6 months). Tissue samples were examined from the posterior and peripheral parts of the retina. At 18 mm the retina consists of an inner marginal layer and an outer layer of neuroblastic cells. At 18-40 mm the latter layer is divided into an inner and an outer neuroblastic layer by the transient layer of Chievitz. Subsequently, the development of the different retinal layers begins at the inner retinal border and moves progressively outwards; it also spreads from the posterior to the peripheral part of the neural retina. Many cells of the inner neuroblastic layer are prospective ganglionic cells which migrate inwards, thus forming the ganglion cell layer and the inner plexiform layer at 90 mm. At 120 mm, primitive horizontal cells appear within the outer neuroblastic layer. Separation of this layer into the inner nuclear, outer plexiform and outer nuclear layers is first evident at 180 mm. At this stage all retinal layers are present, except the layer of the photoreceptor cells which is not widespread until at 220 mm. The inner and outer segments of the photoreceptor cells lengthen considerably during the last month of gestation. During the late fetal stage the nerve fiber layer, the inner and outer plexiform layers and the layer of rods and cones all continue to increase in thickness. Concurrently, the ganglion cell layer and the inner and outer nuclear layers have reached their maximal thickness and become thinner. After the total thickness of the neural retina amounts to approximately 180 microns at two to three weeks before birth, it then thins to approximately 160 microns in the adult stage.

Retinal dysplasia of English springer spaniel dogs: light microscopy of the postnatal lesions.

The histologic features of retinal dysplasia were studied in 26 English springer spaniel dogs of one day to four years of age. Dysplastic lesions in neonatal pups occurred predominantly in the peripapillary tapetal area of sensory retina. They consisted of complex linear folds and rosettes which were composed of hyperplastic neuroblasts. Dysplasia usually was not associated with incomplete differentiation of the outer neuroblastic layer. The outer limiting membrane was absent locally and numerous patent subretinal capillaries existed in the region of folds. The dysplastic features may be related to aberrant development of Muller cells and of retinal capillaries. In adolescent dogs, dysplastic lesions were less complex and usually were not accompanied by aberrant capillaries. Focal or complete retinal separation was present in some adolescent dogs. In adult dogs, features of retinal atrophy were found within dysplastic areas. No significant histologic lesions were found in the extraocular tissues of 12 affected dogs which were surveyed extensively.