Expression of axonin-1 in developing amacrine cells in the chick retina

This study focused on the temporal and spatial pattern of expression of the cell adhesion molecule axonin-1 in amacrine cells and the identification of these cells in the developing chick retina. We analyzed 5-20-day-old chick embryos. The antigen was localized and visualized by the indirect immunogold and the immunofluorescence technique. Colocalization studies with antibodies against tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, parvalbumin, calbindin, and calretinin served to characterize these cells further and to explore whether they have other properties in common. Axonin-1 was expressed in amacrine cells from E8 onward in the inner nuclear, in the inner plexiform, and in the ganglion cell layer. Their maturation showed a gradient similar to that found for amacrinogenesis. Expression was closely correlated with the period when the cells develop and shape their processes. The interneurons were classified with reference to Cajal, and most of the morphological types described by him were found. In addition, some cells were considered as axon-bearing amacrine cells. However, the total number of labeled cells was rather small. At least two morphologically different types terminated in each of the inner plexiform sublayers. Narrow- and wide-field arbors indicated the existence of a diversified network. The colocalization studies revealed that the neurotransmitters and neuropeptides overlapped partially with axonin-1 expression. This indicated that axonin-1-immunoreactive amacrine cells were also functionally diverse.     

Differential expression of neuronal calcium sensor-1 in the developing chick retina

Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) binding protein that has been implicated in the regulation of neurotransmission and synaptogenesis. In this study we investigated the developmental expression and localization of NCS-1 in the chick retina. Single- and double-labeling experiments with three-dimensional reconstruction as well as ultrastructural data of the distribution of NCS-1 suggest that this protein is also involved in axonal process outgrowth. We found an early expression of NCS-1 in ganglion cells and their axons, in amacrine, and in horizontal cells, whereas photoreceptors were immunonegative at embryonic stages. In the early posthatching days we found strong immunostaining for NCS-1 in horizontal cells and their processes in the outer plexiform layer. In contrast, synaptic vesicle protein 2 (SV2) was prominent only in photoreceptor synaptic terminals. Ultrastructural analysis confirmed that NCS-1 was localized postsynaptically in horizontal cell processes, whereas presynaptic terminals were immunonegative. However, at late posthatching days we observed that photoreceptor ribbon synapses (from rods and/or cones) also expressed NCS-1. Thus the results support the notion that NCS-1 is involved in neuronal process outgrowth and is localized in pre- and postsynaptic compartments including mature photoreceptor synapses.     

Differential expression of phospholipase D1 in the developing retina

Expression patterns of phospholipase D1 (PLD1) in the developing rat retina were investigated using immunocytochemistry and Western blot analysis and compared with the expression patterns of glutamine synthetase. PLD1 immunoreactivity appeared first in a few neuroblasts in the middle of the mantle zone of the primitive retina by embryonic (E) day 13. PLD1-immunoreactive primitive ganglion cells were characterized in the ganglion cell layer by E17. Faint immunoreactivity at E17 profiled radially orientated cells and this pattern appeared up to postnatal (P) day 7. In the ganglion cell layer at P3, displaced amacrine cells and ganglion cells were classified. At P5, presumptive horizontal cells and amacrine cells were identified. By P7, a thin outermost layer of newly formed segments of the photoreceptor cells was also PLD1 immunoreactive. PLD1 immunoreactivity at P8 was limited to radial Müller cells and the outer segment layer of the photoreceptor cells, and the expression pattern was conserved to adulthood. Western blot analysis showed relatively high amounts of PLD1 protein at E17 and P3, a decrease at P7, and moderate amounts from P8 onward. Co-expression of PLD1 with glutamine synthetase in the retina appeared first after birth in differentiating neurons and in Müller cells by P8; thereafter the pattern was maintained. The expression pattern of the PLD1 during development of the retina suggests that PLD1 plays important roles in glutamate-associated differentiation of both specific neurons and radial glial cells, and in glutamate-mediated cellular signalling in Müller cells.     

Ultrastructural observations on the expression of axonin-1: implications for the fasciculation of sensory axons during axonal outgrowth into the chick hindlimb.

To help understand how axons interact as they grow into the developing chick hindlimb, we used electron microscopy in conjunction with immunoperoxidase staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axons travel together in bundles, tightly apposed to one another. In contrast, motoneuron axons are more widely spaced, although motoneuron axons situated at the perimeter of sensory axon bundles are found in close contact with neighboring sensory axons. Sensory growth cones and lamellipodia tend to be located centrally within the bundles, with several lamellipodia typically being found stacked together. Strikingly, regions of close axonal apposition are accompanied by axonin-1 expression, suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow together, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axons. Axons situated at the periphery of sensory bundles are typically partly labelled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells. Thus, axonin-1 appears to become redistributed within the membranes of axons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically to axonin-1, appears uniformly distributed on even those axons that would have an asymmetric distribution of axonin-1. Thus, the localization of axonin-1 strongly suggests that it plays an important role in sensory axon fasciculation, but the relative contributions of its interactions with various potential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculation is important in sensory axon guidance, sensory axons may also explore the local environment.     

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.     

Differential expression and cellular localization of doublecortin in the developing rat retina

Doublecortin is 40 kDa microtubule-associated phosphoprotein required for neuronal migration and differentiation in various regions of the developing central nervous system. We have investigated the expression and cellular localization of doublecortin in the developing rat retina using immunocytochemistry and Western blot analysis. The expression of doublecortin was high from embryonic day 18 (E18) until E20 and was low during the postnatal period. The doublecortin immunoreactivity first appeared in a few radially orientated cells in the mantle zone of the primitive retina at E15. From E16 onward, the immunoreactivity appeared in two different regions: the inner part of the retina and middle of the neuroblastic layer. In the inner part, the somata of cells in the ganglion cell layer, in the distal row of the neuroblastic layer and profiles in the inner plexiform layer showed doublecortin immunoreactivity up to postnatal day 1 (P1). Afterwards, the doublecortin immunoreactivity persisted in the inner plexiform layer until P15, although the intensity decreased gradually with the maturation of the retina. In the middle of the neuroblastic layer, doublecortin immunoreactivity appeared in the radially orientated cells. These cells transformed into horizontal cells. The doublecortin immunoreactivity persisted in these cells up to P21. Given these results, doublecortin may play an important role in the migration and differentiation of specific neuronal populations in developmental stages of the rat retina.     

Dopaminergic cell differentiation in the developing chick retina

The distribution and morphology of dopaminergic (DA) neurons in the chick retina was studied in the course of development. Fluorescent DA cells were first detected on the 13th day of incubation. They were always found in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL). On the 14th day, DA cells were found in the innermost row of the INL. Subsequently their processes extended not only bilaterally along the IPL-INL junction but also vertically into the IPL. As a result, three fiber layers were formed as laminae 1, 3 and 5 in the IPL. In the newly-hatched chick retina, a number of growth cone-like fluorescent structures with fine spikes were seen at the IPL-INL junction, indicating that DA fibers were still growing and elongating at least at hatching. On the 4th postnatal day, the ramification of dendritic processes was very prominent and they often showed a spiral configuration.     

Biosynthesis of gangliosides in the developing chick embryo retina

Chick embryo retinas were cultured with [3H]glucosamine on each day between days 6 and 12 of development. The total labeling of gangliosides decreased from day 6 to day 12. The decrease was mostly due to the decrease in the labeling of disialosyllactosylceramide (GD3), which diminished to less than 2%, while the labeling of the disialoganglioside GD1a decreased to about 30%. Labeling of endogenous gangliosides by incubating retina homogenates with CMP-[3H]N-acetylneuraminic acid, in addition to showing a decrease in the labeling of GD3, showed a twofold increase in the labeling of GD1a. The changes in the pattern of labeling correlated with the decrease of both the incorporation of [3H]thymidine into DNA and the activity of CMP-NeuAc:GM3 sialosyltransferase and with the increase of both the activities of UDP-Ga1NAc:GM3 N-acetylgalactosaminyltransferase and choline acetyltransferase. The results suggest that the shift in the pattern of labeling of gangliosides occurs in association with the transition from the proliferative to the nonproliferative state of neuronal cells. Retinas at each age cultured with [3H]glucosamine for 22 hr maintain the radioactivity in gangliosides for at least 4 additional days in culture. This indicates that within this developmental interval there is not a period characterized by having a high turnover rate of gangliosides.     

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.     

The environment of axonal migration in the developing chick retina: a scanning electron microscopic (SEM) study.

We have made a SEM study of the basal intercellular spaces of the retina in chick embryos of different developmental stages. Since this is the environment where optic axons grow, the structural characteristics of this region might play some role in the orientation of axonal migration towards the choroid fissure. The basal region of undifferentiated retinas is formed by the vitreal expansions of neuroepithelial cells. In pre-axonal stages, the intercellular spaces between these expansions do not show any preferential orientation towards the fissure. The growth cones of ganglion cell axons appear in an apicobasal direction and turn towards the fissure immediately beneath the vitreal surface. Fasciculation is an early event during development and, in the more advanced stages, the vitreal expansions from retinal cells are placed in rows following the same orientation as the axon bundles. These observations are discussed in relationship to current hypotheses on axonal migration and orientation.     

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs, for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly. © 1994 Wiley-Liss, Inc.     

Differential ontogenesis of D1 and D2 dopaminergic receptors in the chick embryo retina

The differentiation of D1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our results reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10(-4) M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D1 receptors that does not desensitize significantly during retina development.     

Heterogeneity of muscarinic cholinergic receptors in the developing chick embryo retina

Heterogeneity of muscarinic cholinergic receptors was investigated in chick embryo retina throughout development and in chicks immediately after hatching. The presence of a homogeneous receptor population was evidenced by antagonist binding. The affinity of antagonists increased up to day 14 of incubation, when synaptogenesis occurs. After this stage, it remained substantially unchanged. The number of receptors increased in embryos until hatching. On the contrary, agonists, such as acetylcholine and carbachol, bound to two (high- and low-affinity) binding sites. Through development, the affinity of both significantly increased until day 14, further substantiating the hypothesis of a maturation of the receptor pattern which precedes synapse formation. Muscarinic cholinergic binding seems to identify 3 critical steps in retinal neuronal development. The first is between 7 and 9 days of incubation, the second when synaptogenesis occurs and the third after initiation of function.     

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.     

The effect of kainic acid on the developing chick retina

Kainic acid (KA) was given in ovo to 6-day-old chick embryos. Examination of the retina after treatment indicated an immediate damage of cell in the ganglion layer. One week later, numerous cells died in the middle inner portion of the nuclear layer, apparently by the lack of an adequate number of targets in the ganglion cell layer.     

Differential expression of acetylcholinesterase molecular forms in neural retina and retinal pigmented epithelium during chick development

Neural retina (NR) and retinal pigmented epithelium (RPE) were used as a model for studying acetylcholinesterase (AChE) expression in neuronal and non-neuronal tissue during development. In chick embryo retina, increasing AChE activity appeared from day 7 to hatching, first in NR and then in RPE. NR contained 3 main AChE forms, 11.3S, 6.5S and 4.5S, resolved by sucrose density gradient centrifugation. An additional 19S form was exclusively detected in RPE whatever the extraction procedure followed. During differentiation, the proportions of AChE molecular form changed until they reached a steady state characteristics of mature neural retina, whereas in RPE, patterns of AChE molecular forms did not change significantly during development. Thus, 19S AChE appeared to be characteristic of non-neuronal retinal tissue. The expression of the AChE molecular forms in NR and RPE, and particularly the 19S form, was independent of the presence of the lens during retinal differentiation.