Neuroblastic Layer Research Articles

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15–P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.

Evidence for an antiapoptotic role of dopamine in developing retinal tissue.

Inhibition of protein synthesis leads to apoptosis in the undifferentiated neuroblastic layer of the retina of newborn rats. We have shown previously that an increase in the intracellular concentration of cyclic AMP prevented apoptosis induced in the retinal neuroblastic layer by inhibition of protein synthesis. In this study, we tested the effects of dopamine on retinal apoptosis and related these effects to the intracellular concentration of cyclic AMP. Both dopamine (100 microM) and the D1-like agonists SKF-38393, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (6-Cl-PB), and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (100 microM) blocked apoptosis induced in the neuroblastic layer by the protein synthesis inhibitor anisomycin. The antiapoptotic effects of the D1-like agonists were not reversed by the D1-like antagonist SCH-23390 (5-100 microM). Both dopamine and D1-like agonists induced a five- to sevenfold increase in the intracellular concentration of cyclic AMP in the retina of newborn rats. The concentration of cyclic AMP induced by the D1-like agonists in the presence of 100 microM SCH-23390 was still at least two- to threefold as high as control values, showing that the activation of adenylyl cyclase by D1-like agonists was reversed only partially by the specific antagonist. The isoquinolinesulfonamide H-89 (20 microM), an inhibitor of cyclic AMP-dependent protein kinase, partially prevented the antiapoptotic effect of 6-Cl-PB. The data show that an early effect of dopamine in the developing retina is the control of programmed cell death. The antiapoptotic effect of dopamine is mediated, at least in part, through an atypical D1-like receptor coupled to stimulation of adenylyl cyclase, followed by activation of cyclic AMP-dependent protein kinase.

Characterization of the cell death promoter, Bad, in the developing rat retina and forebrain

Neuronal programmed cell death, or apoptosis, occurs during development, following injury or in certain disease processes, and is regulated by members of the B-cell leukemia-2 (Bcl-2) protein family. These molecules include both positive and negative regulators of cell death and act by selective dimerization that results in permissive or inhibitory effects on a cascade of cellular events, including mitochondrial release of cytochrome c, stimulation of cysteine protease activity and subsequent cellular deterioration. Here, we have characterized the expression of the cell death agonist, Bad, in the postnatal rat retina and forebrain. Isolation, subsequent amplification by RT-PCR and DNA sequence analysis revealed that retinal Bad was identical to Bad expressed in the developing and adult rat brain. Using a polyclonal antibody to Bad, we determined that, in the retina, on the day of birth (postnatal day-0, PND-0) Bad immunoreactivity was expressed primarily by retinal ganglion cells, some cells in the inner neuroblastic layer (NBL) and an indistinct plexus of processes in the inner plexiform layer (IPL). On PND-7, Bad immunoreactivity was observed in most cells in the ganglion cell layer (GCL), numerous cells scattered throughout the inner nuclear layer (INL), a lightly stained IPL and in a distinct band of immunostained fibers in the forming outer plexiform layer (OPL). By PND-15, Bad immunoreactivity was present in cells in the GCL, in some cells in the proximal INL and in horizontal cell processes in the OPL. The IPL was only faintly labeled. In the adult retina, specific Bad immunostaining was confined to large cells in the ganglion cell layer (presumed ganglion cells), occasional lightly stained horizontal cells and their processes in the OPL and to occasional small, lightly stained cells in the proximal INL (presumed amacrine cells) and GCL (presumed displaced amacrine cells). Again, the interposed IPL was faintly labeled. In the brain, Bad immunoreactive cells were scattered throughout the forebrain parenchyma but were particularly concentrated in neurons of the cerebral cortex, hippocampus and amygdala. Bad immunoreactivity was heaviest in these cells at PND-7, distinctly weaker at PND-10 and absent by PND-24. At all time points examined, Bad immunoreactivity was present in epithelial cells of the choroid plexus, as previously reported in the adult rat brain. These data suggest that Bad is transiently expressed by various cell types in the perinatal retina, particularly ganglion cells, and in discrete forebrain regions. In the context of corroborative observations, Bad expression may be regulated in response to acute ischemia and may act as a control point for retinal neuronal apoptosis.

Horizontal cell differentiation in the retina of the Brazilian opossum, Monodelphis domestica

Differentiation of many diverse neuronal phenotypes is an essential part of nervous system development. We have studied the differentiation of horizontal cells, one of the basic neuronal types in the vertebrate retina, in a small, easily maintained marsupial by immunocytochemistry using antineurofilament and antivimentin antibodies. At birth the retina consists of proliferating neural epithelial cells, with a few early ganglion cells. Horizontal cells were first detected in 12-day-old pups; somas were within the epithelial neuroblastic layer and processes extended radially. By 19days there were tangentially oriented dendrites and a few longer processes, the beginning of the outer plexiform (first synaptic) layer. By the time of eye opening (about 34days) the basic histological organization of the mature retina was established.In the mature retina and during development, horizontal cell neurites in the outer plexiform layer, as well as ganglion cell axons, reacted strongly with several antineurofilament antibodies and with antivimentin; horizontal cell somas were detected only with one antineurofilament antibody. Only one population was detected, which we identify as the short-axon subtype, by comparison with horizontal cells in other marsupials and in eutherian mammals.This is the first description of the putative absence in a marsupial of one of the two horizontal cell subtypes found in most amniotes, including mammals so far studied, except murid rodents, which have only the short-axon subtype. Absence of one subtype in Monodelphis supports the hypothesis that the short-axon cell is the basic conserved phenotype of this class and suggests that experimental analysis of differentiation of horizontal cells in Monodelphis and murid rodents, compared to marsupials and eutherian mammals which have the basic two subtypes, can help elucidate mechanisms for controlling differentiation of specific cellular phenotypes and the variations in neurons within and among species.

Fetuin in neurons of the retina and cerebellum duringfetal and postnatal development of the rat

Although long known to be a liver-derived fetal plasma glycoprotein, fetuin has morerecently been shown to be present in sub-populations of neurons in the developing nervous systemof a number of mammalian species. We have extended these observations to examine the fetuinimmunoreactivity (IR) in developing rat retina and cerebellum. Fetuin–IR was first seen in theretina on embryonic day (E)19 in a sub-population of cells in the retinal ganglion cell layer and asmall proportion of cells in the neuroblastic layer. The proportion of cells in the ganglion layerexhibiting fetuin-IR increased until postnatal day (P)10 when all cells in this layer were stronglyimmunoreactive. From P14 onwards fetuin–IR was absent or very weak and restricted to a smallproportion of ganglion cells. In the developing cerebellum, the outer and inner granule cell layers,the deep nuclei and cells in the sub-cortical white matter exhibited fetuin–IR from E19 to P10.There was little fetuin–IR in the cerebellum at ages P14 and older, and Purkinje cells did notexhibit fetuin–IR at any time. The results show that fetuin appears in many neurons in the retinaand cerebellum that are differentiating during the period from E19 to P10. The concentration offetuin in cerebrospinal fluid is at its highest in this same period which suggests that somesub-populations of neurons could obtain fetuin from extracellular fluid during this period ;however, the lack of fetuin–IR in other neuronal populations suggests that fetuin uptake is not ageneral property of developing neurons.

Spatial and Temporal Expression of the Cystathionine β-Synthase Gene During Early Human Development

We report the cystathionine-β synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.

Localization ofSix4/AREC3 in the Developing Mouse Retina; Implications in Mammalian Retinal Development

TheSix4/AREC3 gene was originally isolated as a regulatory factor which bound to the positive regulatory region of the Na, K-ATPase α1 subunit. It is a murine homologue of theDrosophila sine oculis (so)gene, which is essential for the development of the entire insect visual system. In this study, we attempted to determine the localization of theSix4/AREC3 gene product in the developing mouse retina in order to examine its role in retinal cell differentiation. Immunohistochemistry with anti-SIX4/AREC3 and anti-Na, K-ATPase α1 subunit antisera was performed on developing mouse retinas, and immunoblotting analysis with anti-SIX4/AREC3 was also performed. The localization ofSix4-like immunoreactivity (Six4-LI) showed a temporally regulated pattern: During embryonic development,Six4-LI was found in the nuclei of cells located at the inner neuroblastic layer of the retina as early as on ED12, nearly corresponding to the onset of retinal cell differentiation. In the PD1 retina,Six4-LI was observed in the nuclei of the ganglion cells, and increased its intensity until PD4, and thereafter kept its intensity until PD7 whenSix4-LI was often found in the cytoplasm. On PD4, the presumptive amacrine cells found in the inner portion of the inner nuclear layer appeared to be immunostained in their nuclei. On PD7, the presumptive bipolar cells located in the outer portion were immunostained in the nuclei. After that,Six4-LI gradually decreased, and in the mature retina no detectableSix4-LI was observed in the nuclei. This pattern ofSix4-LI localization during retinal development seemed to correlate with retinal cell differentiation, but did not correlate with the distribution pattern of Na, K-ATPase α1 subunit protein-like immunoreactivity. These results suggest that theSix4gene may play a role in the differentiation of neural retinal cells during mouse retinal development, rather than regulating the expression of the Na, K-ATPase α1 subunit gene.

Expression of Nedd2/ICH-1 (caspase-2) in the developing rat retina

Expression and localization of Nedd2/ICH-1 (caspase-2) were examined in the developing rat retina. The expression of Nedd2 mRNA was conspicuous in the prenatal period and down-regulated in the postnatal period. In contrast, the expression of an alternatively spliced form of Nedd2 encoding a truncated protein, which has been shown to protect against programed cell death (PCD), was faint in the prenatal period but increased gradually in the postnatal period. Nedd2 immunoreactivity was observed in almost all cells of the ganglion cell layer (GCL) during the perinatal period, when some nuclei in the GCL showed pyknosis or fragmentation. Nedd2-positive cells in the neuroblastic layer (NBL) were present in patches at embryonic day 19, and began to assume a stratified arrangement at postnatal day 3, when nuclei showing PCD were observed in places across the NBL. At postnatal day 14, only faint Nedd2 immunoreactivity was observed in the GCL and the inner nuclear layer, and no Nedd2 immunoreactivity was present in the outer nuclear layer. These findings demonstrate the cell type- and differentiation-specific patterns of the Nedd2 expression in the developing retina.

Developmental study of Müller cells in the rat retina using a new monoclonal antibody, RT10F7

We produced the monoclonal antibody RT10F7, characterized its antigenic specificity and expression in the adult and developing retina, in cultured retinal cells and in other parts of the central nervous system. In metabolically-labelled retinal cultures RT10F7 immunoprecipitated a protein of approximately 36 000 mol. wt. In the adult, RT10F7 stained endfeet of Müller cells in the ganglion cell layer, four horizontal bands in the inner plexiform layer, and radial fibres in the outer plexiform layer which terminated at the outer limiting membrane. In the inner nuclear layer, most somata were underlined by Müller processes that wrapped around them, but some cell bodies were immunoreactive for RT10F7 in the cytoplasm. During development, postnatal day 21 was the first age at which the adult pattern of immunoreactivity was present, although a fourth band in the inner plexiform layer was less clear than for the adult. By 14 and eight days after birth, the pattern of RT10F7 immunoreactivity approximated that of the adult; however, only three bands and one band were present, respectively, in the inner plexiform layer. At earlier ages, postnatal days 4, 1 and embryonic ages 19 and 15, the monoclonal antibody stained Müller cell endfeet and radial fibres, from the inner plexiform layer through the neuroblastic layer to the outer limiting membrane. At these ages, the immunoreactivity was more prominent at the level of Müller cell endfeet. The monoclonal antibody stained glia in preparations of dissociated retinal cells maintained in culture but not astrocytes or oligodendrocytes from optic nerve cultures. In brain sections, tanycytes exhibited RT10F7 immunoreactivity. The monoclonal antibody RT10F7 recognized a specific cell type in the retina, the Müller cell. In the adult and developing retina, RT10F7 recognized an antigen that is present primarily in Müller cell processes. This feature allowed us to follow the maturation of the Müller cell and correlate it with developmental events in the retina. RT10F7 is a specific marker for Müller cells in vivo and in vitro and may be useful for studies of function of Müller cells after ablation or after injuries that are known to activate Müller cells.

JAGGED2: a putative Notch ligand expressed in the apical ectodermal ridge and in sites of epithelial–mesenchymal interactions

The Drosophila Notch gene and its ligands, Delta and Serrate, are involved in cell fate determination in a variety of developing tissues. Recently, several Notch, Delta and Serrate homologues have been identified in vertebrates. We report here the cloning of the human and murine JAGGED2 (JAG2), a Serrate-like gene, and the analysis of its expression pattern during embryogenesis. Jag2 was found to be expressed as early as E9 in the surface ectoderm of the branchial arches and in the apical ectodermal ridge (AER) of the developing limb. At E12.5, Jag2 expression is upregulated in differentiated neurons of the central and peripheral nervous system and in the inner neuroblastic layer of the developing retina. Outside the nervous system, Jag2 is expressed in the developing vibrissae follicles, tooth buds, thymus, submandibular gland and stomach. Our findings suggest the involvement of Jagged2 in the development of the mammalian limb, branchial arches, central and peripheral nervous systems and several tissues whose development depends upon epithelial–mesenchymal interactions.

Neurochemical development of the degenerating rat retina

The Royal College of Surgeons' (RCS) rat is an experimental model for a group of hereditary retinal diseases commonly called retinitis pigmentosa. We used postembedding immunocytochemistry to determine the localisation of glutamate, gamma-aminobutyric acid (GABA), glycine, aspartate, glutamine, taurine, and arginine in the RCS rat retina during postnatal development. In addition, we evaluated the uptake characteristics for the three dominant amino acid neurotransmitters, glutamate, GABA, and glycine. Whereas, cellular localisation of all amino acids was similar to control retinas, there were major changes in the level of immunoreactivity, even before eye opening, and well before the onset of visibly detectable photoreceptor degeneration. Two major patterns emerged. First, neurochemical changes evident before degeneration, involving the amino acids glutamate, GABA, aspartate, glutamine, and arginine. Second, neurochemical changes that become evident during photoreceptor degeneration involving the amino acids taurine and glycine. Anomalies in uptake characteristics also become evident during the degeneration phase and are likely to reflect changes in cellular function as a consequence of the degeneration process. Neurochemical changes evident before photoreceptor degeneration involve both glutamate and GABA manufacturing pathways. Müller's cells displayed elevated levels of glutamine and arginine from an early age, and the neuroblastic layer in the RCS retina showed high glutamate levels. Modified aspartate immunoreactivity began at postnatal day 11 and is consistent with altered metabolic activity. These results suggest that amino acid neurochemistry is different in the RCS rat retina from an early age, which may indicate an underlying metabolic defect affecting multiple cell classes.

Differential accumulation of DPTP61F alternative transcripts: regulation of a protein tyrosine phosphatase by segmentation genes

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. We have examined the spatial and temporal expression of the two alternative transcripts of dptp61F during Drosophila embryogenesis. Our observations indicate that the two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression in the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation in the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes.

Immunocytochemical localisation of neuronal nitric oxide synthase in developing and transplanted rabbit retinas.

Nitric oxide (NO) acts as a modulator of neuronal transmission in mature neuronal systems, including the retina. Recently, NO has also been suggested to have a trophic function during development. We examined immunocytochemically the distribution of NO-producing cells in developing and transplanted rabbit retinas. An antibody detecting the neuronal isoform of its biosynthetic enzyme, nitric oxide synthase (NOS), was used on normal developing retinas [starting at embryonic day (E) 15] and on rabbit retinal transplants after various survival times (1-139 days after surgery). Weakly stained cell bodies were first observed in the proximal margin of the neuroblastic layer at E 29. Stained processes projecting towards a developing inner plexiform layer were also visible at this time point. Immunoreactive cells were located at later stages in the innermost part of the inner nuclear layer and in the ganglion cell layer, and are likely to correspond mainly to amacrine cells. NOS-labelled cells were also found in retinal transplants. The first NOS-labelled cells appeared, as in normal developing retinas, in ages corresponding to E 29 and were still detected in transplants corresponding to postnatal day 123. NOS-labelled cells were seen in areas between rosettes, where amacrine cells are located. NOS-labelled processes were at times seen to project for long distances, forming very distinct plexuses. NOS-containing amacrine cells thus appear both in the transplants and in developing retinas in the embryonic stages, long before synaptic function involving these cells can be expected, suggesting a role for NO not only in neuromodulation but also in retinal development.

Protein kinases selectively modulate apoptosis in the developing retina in vitro

In the retina of newborn rats there is evidence for two mechanisms of programmed cell death. Apoptosis of ganglion cells (RGCs) following axotomy depends on protein synthesis. In contrast, inhibition of protein synthesis leads to apoptosis in the neuroblastic layer (NBL). The induction of apoptosis following translational arrest suggests that post-translational modifications of apoptosis-associated proteins may be crucial to the cell death programs in the developing retina. We investigated the possible role of protein kinases upon apoptosis in retinal explants in vitro. An increase in the intracellular concentration of cAMP produced either by the adenylyl-cyclase activator forskolin (10 μM) or by 8-Br-cAMP (1 mM), prevented apoptosis induced in the NBL by inhibition of protein synthesis, but had no statistically significant effect upon RGC death. In contrast, neither 8-Br-cGMP (1 mM) nor the specific cGMP-phosphodiesterase inhibitor zaprinast (10–100 μM) had significant effects on apoptosis in the retina. The cAMP-phosphodiesterase inhibitors isobutylmethylxantine (IBMX, 0.1–1 mM) and Ro-201724 (50–200 μM) also prevented apoptosis in the NBL. The isoquinolinesulfonamide H89 (20 μM), a specific cAMP-dependent protein kinase inhibitor, partially reverted the protective effect of either forskolin or IBMX within the NBL. Neither 12-O-tetradecanoyl phorbol-13-acetate (TPA, 10 nM) nor bisindolylmaleimide (0.2–0.5 μM), respectively an activator and an inhibitor of protein kinase C had significant effects upon the retinal explants. The protein kinase inhibitor 2-aminopurine (2-AP, 10 mM) prevented apoptosis of axotomized ganglion cells and induced apoptosis in the NBL. Forskolin prevented the apoptosis induced by 2-AP in the NBL, whereas TPA had no effect. The effects of 2-AP were, however, not dependent on inhibition of protein synthesis. The data indicate that modulation of the activity of both cAMP-dependent protein kinase and several protein kinases sensitive to 2-aminopurine selectively affect apoptosis in distinct cell layers of the developing retina.

Localisation of amino acid neurotransmitters during postnatal development of the rat retina.

We used postembedding immunocytochemistry to determine the localisation of the amino acid neurotransmitters glutamate, gamma-aminobutyrate (GABA), and glycine, potential neurotransmitter precursors (aspartate and glutamine), and taurine in the rat retina during postnatal development. All amino acids investigated were present at birth; however, only the inhibitory neurotransmitters GABA and glycine displayed neuronal localisation. GABA was localised in a sparse population of amacrine cells, and glycine immunoreactivity was found in cells within the ventricular zone that appeared to migrate through the neuroblastic layer. Glutamate labelling was diffuse across the retina until postnatal day (PND) 8. Localisation of glutamine was evident within Müller's cells by PND 6, in agreement with the known age of onset of glutamine synthetase activity. Based on the findings of uptake of radiolabelled glutamate and GABA by PND 8 and changes in immunoreactivity, we propose that Müller's cells evolve at PND 6-8 from their precursor cells, the radial glial cells. Evidence for differences in glutamate turnover in the infant retina was seen on examination of aspartate and glutamine immunoreactivity. Aspartate labelling was weak until PND 11, when ganglion cells and some amacrine cells were labelled. Unlike the mature retina, a large number of amacrine cells were glutamine immunoreactive in the PND 6 retina. One reason for the observed differences in precursor pooling may be a lack of neuronal neurotransmitter release and overall low metabolic activity. We also investigated the response of the developing retina to ischaemic insult to test the physiological hypoxia model of vascular development. Our findings are consistent with the hypothesis that the developing retina has increased tolerance to ischaemic insult. Our findings suggest that, although the retina is morphologically adult like by PND 8, there are differences in neurotransmitter turnover in the immature rat retina.

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.

Changes in the immunoreactivity for inhibin in mouse retina during development of photoreceptor cells.

Inhibin, a widely distributed, multifunctional member of the transforming growth factor beta superfamily, was originally isolated from ovarian follicular fluid. This molecule was recently localized in bovine and rat retina by immunohistochemistry and the mRNA encoding the alpha-subunit of inhibin was detected in the retina by in situ hybridization and the reverse transcription polymerase chain reaction. In this study we have examined, by immunohistochemical methods, the distribution pattern of inhibin in developing mouse eyes on days 10, 11, 14, 16, 18 of gestation (E0 = coitus) and postnatal days 0, 2, 4, 6, 8, 12, 16 and 18 of age (P0 = day of birth). Immunoreactivity for inhibin appeared on E10 (the earliest day of gestation examined) in the lens and in migrating large cells between the lens and the inner layer of the optic cup (future neurons of retina). On E16, cells located in the neuroblastic layer were stained. At birth (P0), the ganglion cells were heavily stained and some amacrine cells were weakly stained. By P4, the amacrine and bipolar cells were positively stained. The inner plexiform layer was heavily stained and the outer plexiform layer and horizontal cells were also apparent and stained. From P6 on, inhibin-immunoreactive staining was localized at the inner segment of the photoreceptor layer, and to ganglion cells in the inner nuclear layer (INL). By P12, ganglion cells, INL, and inner segments of the photoreceptor layer were heavily stained. The outer segments of the photoreceptor layer, presumably the interphotoreceptor matrix, were strongly stained with antibodies against the alpha-subunit, whereas the outer and inner plexiform layers were also moderately stained. The staining in the INL generally decreased with progressing age. We conclude that inhibin is expressed in the eye cup as early as day 10 of gestation, migrates toward the direction of pigmented epithelial layers and is concentrated in the interphotoreceptor matrix by P12, which showed a distribution pattern similar to that of the adult mouse. These findings suggest that inhibin may play a role in the development of the eye cup and the retina.

Uptake of fluorescent probe DII-acetylated-LDL in microglia of embryonic mouse brain slices are spatially and temporally regulated

We report the cystathionine-β synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.

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.