Cognin distribution during differentiation of embryonic chick retinal cells in vitro

Differentiation of individual retina neurons is closely linked to development of retina function. This differentiation may be intrinsic to the cell or determined by the position of the cell within the developing tissue. Retina cognin, a cell-cell recognition protein, which may itself mediate positiondependent cell interactions in vivo exhibits a characteristic change in distribution during embryonic chick development. Cognin is progressively lost from the outer retina in a manner which appears position-dependent. ¹⁰ We asked if this change in cognin distribution was actually position-dependent or intrinsic to the retina cells. Neural retina cells from 8-day-old chick embryos were cultured in vitro. Continued differentiation of the cultured cells was demonstrated by neurite outgrowth and characteristic increases in choline acetyltransferase and glutamic acid decarboxylase activity. In such cultures, the characteristic developmentally related disappearance of retina cognin occurred as in vivo. This indicated that this aspect of retina neuronal differentiation was independent of position within the tissue and likely intrinsic to individual cells after 8 days of embryonic development.     

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.     

The cell recognition molecule, cognin, mediates choline acetyltransferase activity in embryonic chick retina.

Cell signaling and cell-cell interactions play an important role in neuronal differentiation in the embryonic CNS. Previous work (Hausman, R.E., Vivek Sagar, G.D. and Shah, B.H., Dev. Brain Res., 59 (1991) 31-37) had shown that cholinergic differentiation in the embryonic chick retina depends on insulin and neuron-neuron interactions. Here, we pursued the molecular nature of that dependence on cell interactions. The embryonic chick retina is known to contain several cell adhesion or recognition molecules. We asked if retina cognin, a 50 kDa cell surface-associated protein, played a role in controlling cholinergic differentiation in the developing chick retina. As previously, cholinergic differentiation was measured by two markers: choline acetyltransferase (ChAT) activity and high-affinity choline uptake. We used polyclonal antibody to cognin to determine if blocking cognin-mediated cell interactions would affect the normal embryonic increases in these cholinergic markers. We demonstrated a 40% inhibition of the normal developmental appearance of ChAT activity in retina neuronal cultures from early development, but no effect in cultures from more differentiated retina. The inhibition was selective for retina, since it was not seen in neural tissues like cerebrum and cerebellum that also express ChAT. In contrast to the effect of insulin, choline uptake was not affected by treatment with cognin antibody. Antibodies to two other cell recognition molecules present in the retina (Ng-CAM and N-cadherin) did not block the normal developmental appearance of ChAT. These results suggest that cognin-mediated interactions play a unique role in the control of one aspect of cholinergic differentiation in the developing chick retina.     

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.     

Target Preference of Embryonic Retina Cells and Retinal Cell Lines is Cell-autonomous, Position-specific, Early Determined and Heritable

Retinal ganglion cells (RGCs) form the topographic connection between retina and optic tectum in the developing avian embryo. In vitro , neurons with the morphological traits and marker expression of RGCs were found both in single-cell cultures from embryonic day (E) 6 chick retina and in retinal cell lines derived from E3.5 quail retina. Rapid and substantial differentiation of RGC-like cells could be induced in the lines by addition of fibroblast growth factor aFGF or bFGF. RGC-like cells were examined with respect to their target discrimination properties as single cells in the stripe carpet assay. In this assay system, alternating stripes of membrane vesicles prepared from the anterior and posterior tectum are offered to growing axonal processes as a substrate. Temporal RGC-like cells, both primary cells prepared from the temporal retina and immortalized cells of those retinal lines derived from the temporal retina, avoid stripes of membrane vesicles from posterior tectum; they prefer to grow on membrane vesicles from the anterior tectum, which is their in vivo target. Nasal RGC-like cells did not exhibit a target preference, in accordance with previous findings. Together the experiments show that target preference of RGCs is a cell-autonomous and heritable mechanism that is determined early and is position-dependent.     

Role of nitric oxide in photoreceptor survival in embryonic chick retinal cell culture

The presence of nitric oxide synthase (NOS) in chick retina during development has allowed us to study the role of nitric oxide (NO) during retinal differentiation in dissociated chick retinal cell culture from embryonic day 6. We have demonstrated the presence of nicotinamide adenine dinucleotide phosphate diaphorase staining in these cultures after 3 days in vitro (Div), with a maximal intensity after 8 Div, corresponding to embryonic day 14. Immunohistochemistry studies confirmed the presence of the two isoforms of NOS, NOS-I and -III, in dissociated retinal cell cultures at 8 Div. Addition of NG-monomethyl-L-arginine, a NOS inhibitor, to retinal cell cultures prevented NO production but did not modify the appearance and the survival of ganglion and amacrine cells. However, immunohistochemical analysis with distinct markers for photoreceptor cells (rods and cones) showed that inhibition of endogenous NOS in retinal cell cultures prevented the developmental decrease of rod number between 5 and 8 Div, thus supporting the hypothesis that NO may be involved in the cell death of rods during the development of the retina.     

The development distribution of vimentin in the chick retina

The developmental distribution of vimentin was localized in chick retina using light and electromicroscopic immunohistochemical techniques. From embryonic day 4 (E4) to E8 vimentin was present in cells throughout the retina. By E10 vimentin immunoreactivity was restricted to Müller cells. In tissue culture of chick neural retina vimentin was found to be restricted to flat cells that are thought by others to be derived from Müller cells. Neurons in culture did not contain vimentin.     

Induction of glutamine synthetase in embryonic neural retina: its suppression by the gliatoxic agent alpha-aminoadipic acid

Competence for cortisol-mediated induction of glutamine synthetase (GS) is a differentiation marker of embryonic neural retina. Earlier work has indicated that the induction and accumulation of GS is localized in the Müller glia cells. This localization was presently confirmed by the finding that the gliatoxin D,L-alpha-amino-adipic acid (AAA) reduces responsiveness to GS induction by 60--90% due to preferential damage to Müller cells. The tests were performed on organ cultures of retina tissue from chick embryos, and on retina cell aggregates in which there is tissue reconstruction. The presence of GS-inducible Müller cells was monitored by immuno-staining of tissue sections with anti-GS antiserum. Reduction of GS inducibility due to pretreatment with AAA resulted in virtual absence of cells that immunostained for GS. The preferential toxicity of AAA for Müller cells was also demonstrated by cell viability tests; it was further corroborated by the finding that treatment with AAA greatly reduced the level of carbonic anhydrase activity, another enzyme localized predominantly in Müller cells, but did not affect gamma-aminobutyric acid transaminase and choline acetyl transferase, neuronal enzymes. Susceptibility of Müller cells to AAA was found to increase with embryonic development of the retina. We suggest that acquisition of susceptibility for AAA represents another differentiation marker of embryonic Müller cells.     

Hair cell differentiation in the developing chick cochlea and in embryonic cochlear organ culture.

We have defined a method for growing chick embryonic cochleae in organ culture that preserves many aspects of hair cell differentiation. Cochlear ducts were isolated from embryonic day 8 chicks, placed in organ culture, and incubated for 48 hours (to a point equivalent to embryonic day 10). The cultured ducts were then fixed and processed for scanning electron microscopy. As controls, cochlear ducts at embryonic days 8 and 10 were dissected and immediately fixed and processed for scanning electron microscopy. We chose this period to culture cochleae because at the corresponding time in vivo hair cells undergo a dynamic phase of differentiation. During this time, the number of stereocilia in the stereociliary bundle increases, and two to three rows of stereocilia nearest the kinocilium elongate, initiating the staircase pattern of the bundle. Also, the orientation of many hair cells shifts from nonpolarized at embryonic day 8 to polarized toward the inferior edge of the basilar papilla at embryonic day 10. Many of these aspects of hair cell differentiation proceed normally in organ culture. The appropriate distal-to-proximal gradients of hair cell density, apical surface area, and stereociliary number are preserved. Elongation of the 1-2 stereociliary rows next to the kinocilium continues, and more stereociliary bundles are oriented toward the inferior edge in cultured cochleae than in embryonic day 8 chicks. It appears that cochlear organ culture can serve as an effective method with which to study how hair cell differentiation is regulated.     

Novel organization of microtubules in cultured central nervous system neurons: formation of hairpin loops at ends of maturing neurites

Using the high voltage electron microscope, we have examined cultured embryonic neurons in order to understand better the organization of microtubules in developing neurites. We found that, in embryonic chick retina neurons, microtubules were abundant in the ends of neurites and showed an unusual pattern of organization. Most striking was the presence of microtubule loops; after entering the flattened region of a growth cone, microtubules frequently made tight 180 degrees turns. Occasionally these looping microtubules re-entered the neurite and returned in the direction of the cell body. Positive identification of the loop structures as microtubules was made by specific immunocytochemical labeling. Quantitative analysis showed that more than half of the retina neurons that were dissociated on embryonic day 8 and kept in culture for 4 to 6 days (E8C4 and E8C6) contained at least one microtubule that made a 180 degrees turn at flat regions along or at the tips of neurites. The area within the loops typically contained larger membranous organelles, whereas only small vesicles were seen outside the loops. Fine filaments were seen to interconnect the loops at various places, suggesting the possibility that they played a role in maintaining the shape of microtubule loops. Examination of other neurons showed that tight microtubule loops were prominent in chick spinal cord neurons, but they were rarely seen in neurons of the sympathetic ganglia or dorsal root ganglia or in NG108-15 cloned cells. Developmentally, no loops were observed in E8C1 retina neurons, but retina neurons dissociated from older embryos (12 days) did show loops after 1 day in culture; these data suggest that microtubule loops may be abundant around embryonic day 12 to 13 in the chick retina. The possible significance of this unusual microtubule organization to the control of neurite growth and bidirectional transport is discussed.     

Apoptotic cell death of proliferating neuroepithelial cells in the embryonic retina is prevented by insulin

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT-mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor-deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl-3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S-phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl-3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.     

Changes in patterns and synthesis of proteins in embryonic neural retina studied by 2-dimensional gel electrophoresis

Changes in protein patterns during early differentiation of embryonic neural retina (chick) were studied by 2-dimensional gel electrophoresis. The procedures employed here made it possible to visualize the overall population of proteins present in the tissue at a given time and, on the same gel to distinguish labeled from unlabeled proteins. 2-Dimensional gels were stained by a highly sensitive silver stain to visualize, map and quantitate proteins (and polypeptides) resolved by electrophoresis; the same gels were then autoradiographed in order to differentiate between actively synthesized and pre-existing proteins at each development stage. The effectiveness of this combinative analysis was first verified by identifying and localizing glutamine synthetase, an inducible enzyme marker of retina differentiation. Next, protein patterns in retina tissue at 2 embryonic ages were compared. Of the large number of spots visualized by the above methods approximately 10% showed distinct qualitative-quantitative developmental changes; these were grouped into 7 classes representative of major modes of alteration of protein patterns during cell differentiation.     

Expression of functional N-methyl-D-aspartate receptors during development of chick embryo retina cells: in vitro versus in vivo studies

The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the NMDA receptors was followed by monitoring the changes in the intracellular free calcium concentration ([Ca2+](i)), in response to NMDA or to L-glutamate. The expression of the subunits NMDAR1, NMDAR2A/B and NMDAR2C in the retinospheroids and in chick retinas were determined by Western blot analyses. The changes in [Ca2+](i) in response to 400 microM NMDA increased from 5 h in vitro to 3 days in vitro (DIV) and remained constant until 14 DIV, whereas the [Ca2+](i) response to 500 microM L-glutamate increased from 5 h in vitro to 3 DIV and decreased slightly until 14 DIV. In the retinospheroids, the expression of the NMDAR1 and NMDAR2A/B subunits increased from 5 h in vitro until 14 DIV, whereas the NMDAR2C subunit increased from 5 h in vitro until 10 DIV and remained constant until 14 DIV. In the retinas, the expression of NMDAR1 increased from embryonic day 8 (E8) until E15, decreased until E18, and increased again until day 22 (post-hatched 1, PH1). The NMDAR2A/B increased from E8 until E18 and decreased slightly until PH1, whereas the NMDAR2C subunit increased from E8 until E15, remained constant until E18, and increased again until PH1. The results suggest that NMDA receptors are expressed and functionally active at early embryonic stages in the retina and in retinospheroids, before synapse formation (E12). However, the calcium responses to NMDA were relatively constant from 3 DIV until 14 DIV, showing no correlation with the increase in the expression of the studied NMDA receptor subunit during the same period. Also, the patterns of NMDA receptor subunits expressed in chick embryo retina cells cultured in vitro and in retina cells developing in vivo were similar.     

An in vitro model of proliferation and differentiation of the chick retina: coaggregates of retinal and pigment epithelial cells

Pigment epithelial (PE) cells exert a pronounced organizing effect when added to embryonic day (E) 5-6 chick retinal cells in a reaggregation system such that after a period of 14-21 d of culture, the main layers of an intact E10-E14 retina are reconstructed (Vollmer et al., 1984). In the present study we investigated the time course of the formation of retina-like structures in retinal-pigment epithelial aggregates, in particular, the fate of the PE cells and their influence on processes of differentiation within the aggregates. PE cells first form a core in the center of the aggregates and migrate to the periphery at later stages. The PE core affects the organization of proliferation and differentiation, phenomena that were monitored using 3H-thymidine autoradiography and AChE histochemistry, respectively. A double-staining procedure combining both techniques on the same section is described. Soon after aggregation, a matrix zone and a zone of differentiated cells are formed. At later steps, proliferation becomes gradually restricted to a narrow band within the aggregates comparable to the in vivo situation. The spatiotemporal pattern of withdrawal from mitosis resembles that of the in vivo retina. Proliferation in aggregates is sustained over a longer period with PE, as compared with aggregates formed by retinal cells alone. AChE staining in aggregates in the presence of PE shows a layered appearance, while there is only crude sorting-out of labeled and unlabeled cells in aggregates composed of retinal cells only. The basis of PE cell action as well as the relevance of this in vitro system for understanding normal eye development are briefly discussed.     

Transient muscarinic calcium mobilisation in transdifferentiating as in reaggregating embryonic chick retinae

Two independent in vitro regeneration systems of the embryonic chick retina (E4-5) were used to study the mobilisation of intracellular calcium by the neurotransmitters acetylcholine (ACh) and glutamate, as measured by Fura-2 fluorescence changes. Retinal pigment epithelium (RPE) explants under the influence of basic fibroblast growth factor transdifferentiate into a retina-like tissue with normal laminar organisation, while rosetted spheres reaggregated from fully dispersed cells of the embryonic retina will achieve only an inferior tissue organisation, characterised by regions of an inverted retina [Layer et al., Neuroreport 12 (2001) A39-46]. ACh induced a pronounced Ca(2+) response in young explants, and a similar but less pronounced response in reaggregates; this response decreased almost entirely after 1 week in culture. In contrast, a Ca(2+) response to glutamate became detectable later, continuously increasing during this period. The response to ACh was strictly mediated by muscarinic ACh receptors (mAChRs), since it was inhibited by preincubation with atropine, but not tubocurarine; correspondingly, it was mimicked by muscarine, but not nicotine. Studies with mAChR blockers, preferentially acting on the m1-, m2-, or m3-receptor subtypes, suggested that the muscarine-induced Ca(2+) response is mediated by m1- and/or m3-type mAChRs, but not by the m2-type. These results show that (i) similar to the in vivo retina, in both a transdifferentiating and a reaggregating system an early muscarinic Ca(2+) response is active, which (ii) roughly parallels periods of cell proliferation, and (iii)-as evidenced by the reaggregates-does not depend on any tissue pre-organisation. In contrast, a response to glutamate becomes prominent only when tissue differentiation commences. This is the first demonstration of a physiological response in regenerating chick retinas, supporting their validity as models both of retinal development and regeneration.     

Inhibition of beta1-integrin expression reduces clone size during early retinogenesis.

We have used an antisense retrovirus strategy to test the role of beta1-integrin in the early period of clone development in the embryonic chick neural retina. Analyses of clone size and dispersion demonstrated a marked effect of reducing the levels of expressed beta1-integrin on this critical phase of retina cell and tissue development.     

Development of tyrosine hydroxylase-like immunoreactive structures in the chick retina: three-dimensional analysis.

This study was designed to investigate the developmental profile of tyrosine hydroxylase-like immunoreactive structures in the chick retina in both frozen sections and wholemount preparations. In frozen sections, cells with tyrosine hydroxylase-like immunoreactivity were first detected in 10 to 15 cell rows from the innermost part of the inner nuclear layer on embryonic or incubation day 11. They were seen in the inner cell rows of the inner nuclear layer during later periods; by embryonic day 18, the immunoreactive cells were located 1 to 3 cell rows outward from the innermost part of the inner nuclear layer where mature immunoreactive cells mainly exist. The immunoreactive cells began to give rise to processes on embryonic day 13. The processes (possibly dendrites) gradually increased in number and intensity in sublayers 1 and 4 of the inner plexiform layer during prenatal life. Several days after hatching, an abrupt increase in immunoreactive processes was noted in sublayer 1 but not in sublayer 4. On the sixth postnatal day, retinal neural elements immunoreactive for tyrosine hydroxylase seemed to exhibit a distribution pattern similar to that of the adult chick. In wholemount retinas, immunoreactive cells were initially detected at the earliest stage of embryonic day 12 in a small circle termed "starting area" occupying the ventral part of the temporal retinal field. The closer to the "starting area," the earlier the retinal area began to express many immunoreactive cells. Thus tyrosine hydroxylase cell density in individual retinal areas, as represented by cell number per square millimeter, peaked in different developmental periods varying from embryonic day 12 to day 14. At this stage, immunoreactive cells were arranged irregularly in the retina. Thereafter, the cell density as well as total cell number gradually declined and reached a plateau around embryonic day 20 when tyrosine hydroxylase-like immunoreactive cells, like those in the mature retina, showed an even distribution throughout the retina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout

We have analyzed the patterns of cell proliferation and cell death in the retina and optic tectum of the brown trout (Salmo trutta fario) throughout embryonic and postembryonic stages. Cell proliferation was detected by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), and apoptosis by means of the TUNEL method. Haematoxylin and DAPI staining were also used to demonstrate apoptotic cells. Photoreceptor cell differentiation was assessed by immunohistochemistry with antibodies against opsins. Throughout embryonic development, PCNA-immunoreactive (PCNA-ir) cells become progressively restricted to the peripheral growth zone of the retina, which appears to be the principal source of new retinal cells from late embryos to adults. However, some PCNA-ir cells are observed secondarily in the differentiated retina, first in the inner nuclear layer of 15-mm alevins and later in the outer nuclear layer of 16-mm alevins, after differentiation of the first rods in the central retina, as demonstrated with opsin immunocytochemistry. Our observations also support the view that the PCNA-ir cells observed secondarily in the INL of the central retina of alevins are photoreceptor precursors. The number and distribution of apoptotic cells in the retina and optic tectum of the trout change throughout development, allowing distinction of several waves of apoptosis. Cell death is detected in proliferating areas at early stages, then in postmitotic or differentiating areas, and later concurring temporal and spatially with the establishment of visual circuits, thus indicating a relationship between apoptosis and proliferation, differentiation and synaptogenesis.