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In vitro Studies on Differentiation of the Optic Stalk in the Chick Embryo
Differentiation
Differentiation 16, 189-191 (1980)
(3 Springer-Vcrlag I080
In vitro Studies on Differentiation of the Optic Stalk in the Chick Embryo ROBERT J. ULSHAFER’ and A N D a CLAVERT Institut d’Embryologie, Strasbourg, France
An orderly pattern of cell death accompanies growth of retinal ganglion cell axons through the optic stalk of the chick embryo. In order to determineifthe cell death process in this adage is preprogrammed at earlier stagesor if other factors play a role, we cultured optic stalk primordia at a stage prior to retinal differentiation, either alone or in the presence of head or limb bud mesenchyme. When optic stalk was cultured alone, many cells differentiated into neurons. However, when mesenchymecells of either head or limb bud origin were combined with the stalk, the stalk cells either degenerated, were unrecognizable in the mesenchymemass, or retained their epithelialarrangement and became pigmented. Mesenchyme and/or neural crest which normally migrate around the stalk at the same time that ganglion cell axons penetrate this structure may therefore be involved in some aspect of the cell death process. Since many optic stalk cells in vitro differentiateinto neurons, these cells may represent the population of cellswhich in situ would normally die. Introduction The optic stalk-optic cup preparation offers an interesting model system for studies on growth and directed migration of retinal axons (and neurons, in general), cell death during embryogenesis, and gliogenesis. A gradient of dying cells exists with time in the optic stalk of the chick embryo during migration of retinal fibers ill. Large intercellular spaces in the mouse optic stalk, possibly resulting from autolysis or phagocytosis of dead cells, have been shown to contain axonal growth cones of retinal ganglion cells [2,31 and therefore alignment of spaces may provide channels in the neuroepithelium through which axons grow. Optic cup ablation at stages prior to differentiation of retinal ganglion cells does not prevent degeneration of the stalk so the axons themselves probably do not induce the cells to die 111. Under those conditions, the stalk completely degenerated and gliogenesis did not occur. Cell death in the stalk therefore appears to be determined 1 Present address and address for reprint requests: Department of Ophthalmology, Baylor College of Medicine, Texas Medical Center, Houston, Texas, 77030 USA
prior to and independently of fiber penetration but arrest of cell death and subsequent gliogenesis may require the presence of ingrowing optic fibers. Cell death may therefore be preprogrammed at the time of primary neural induction. Alternately, mesenchyme and neural crest which migrate rostrally around the stalk prior to penetrationof ganglioncell axons may be causal agents in the cell death process. The purpose of the present study was to determine which of these two phenomena, if either, plays the primary role in initiation of cell death in the stalk. To test this, we analysedthe growth and differentiation of the stalk in culture with and without the presence of mesenchyme or ectomesenchyme.
Methods Eggs of white Leghorn chickens were incubated for 2-3 days and embryos removed at Hamburger and Hamilton (41 stage 18-19 (prior to merentiation and penetration of the optic stalk by retinal ganglion cell axons). Under a dissecting microscope the intact anterior neural tube was removed and the following structures were isolated: optic stalk, optic cup, optic stalk combined with optic cup and surrounding ectomesenchyme. Optic stalk was also isolated and combined with
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R. J. Ulshafer and A. Clavert: Optic Stalk Differentiation in vitro
mesenchyme from the limb bud. Tissues were cultured on a semi-solid coagulum consisting of 2 parts Hank's salt solution, 1 part fetal calf serum, 1 part cock plasma, and 6 parts chick embryonic extract in a humidified incubator at 37" C in an atmosphereof 5% CO, in air 151. Tissue explants were transferred to fresh media at 48 h intervals until the 8th day when they were fxed in Bouin's solution, dehydrated, and embedded in paraffin. Serial sections were cut at 8 pm and alternate sectionswere stained by either the Bodian technique, which is specific for fine nerve fibers [61, or Harris hematoxylin and eosin.
with mesenchyme, either from the head (i.e., ectomesenchyme which includes neural crest) or limb bud, cells in the stalk either degenerated, were unidentifiable in the mesenchyme mass, or retained an epithelial arrangement and became pigmented (Fig. lc). A similar phenomenon occurs in vivo when the optic cup is removed but its stalk and surrounding mesenchyme are left intact [ 11 or when choroid fissure is prevented from forming due to the presence of antimitotic drugs during a critical period, which thus causes the absence of optic nerve [71. Tissue culture experiments on early morphogenesisof the amphibian eye have shown that mesenchyme is necessary for differentiationof pigment epithelium(P. E.) and normal cupping of the optic primordium [81. Sinceour experiments were performed at much later stages (i.e., following invagination of the optic vesicle), a certain pop ulation of cells had already been determined to develop into pigment epithelium, and for this reason we identified pigmented cells in all optic cup preparations. We noted that in optic cups cultured without mesenchyme,P. E. cells were always arranged in an aggregate, frequently in the matrix between neural retina and lens, when present. Op tic anlage, cultured with ectomesenchyme, however, usually developed a normal arrangement of cells: a single
Results and Discussion
The culturing conditionssupported normal differentiation of retinal cell types in optic cup preparations as discerned by morphologic examination: by eight days nuclear and plexiform layers had formed, axons were present, and usually grew together in a well-defined fiber layer. In those cases where stalk was cultured alone, many neurons differentiated. The tissue mass appeared to have organised mantle and marginal layers (Fig. la), although a ventricle or neurocoel was never seen. Fibers in both layers occasionally formed small fascicles which coursed together for several hundred micra before dissipating through the tissue (Fig. 1b). When the stalk was cultured
Flg. 1. Optic stalks cultured alone (a, b) or with limb bud mesenchyme (c) for dght days (Bodian stain). a Cells of the optic stalk have largely differentiated into neurons. The tissue appears to have formed mantle and marginal layers, similar to cells in the embryonic neural tube. b High power magnification of a. Many fibers are arranged in a large fascicle. c Optic stalk cultured with limb bud mesenchyme did not differentiateinto neurons. In fact, in this specimen the stalk retained its primitive neuroepithelial arrangement of cells and many of its cells contain pigment granules. The mesenchyme (Me) immediately around the stalk remnant (0s) is much darker than the rest of the mesodermal mass
R. J. Ulshafer and A. Clavert: Optic Stalk Differentiation in vitro
layer of P.E. outside and apposed to the neural retina. Thus, mesenchyme appears to play a sequential role throughout morphogenesis of the P. E., originally as an inductive agent and later in stabilization of the cellular arrangement in this tissue. Our system differs from those anlagen which are known to be preprogrammed to die, such as in morphogenesis of the knee joint 191 or in the posterior necrotic zone (PNZ) of the chick limb bud. For example,when the chick PNZ was excised at stage 2 1 or beyond and cultured in a manner similar to that in OUT experiments, a large number of necrotic cells and phagocytes accumulated in the cultures which resembled in number and appearance those observed in situ at stage 24. In that experiment, when prospective PNZs were cultured in the presence of wing mesoderm, necrosis did not occur unless the two tissues were separatedby at least 1-2 mm and contact did not occur between the two tissues [lo]. A genetically programmed "death clock" therefore does not seem to be responsible for cell death in the optic stalk of the chick unless external agents in the immediate environment are responsible for turning it on. Cell death which is induced or determined at the time of primary neural induction similarlydoes not appear to be the causal agent since post-induction explants remain viable unless cultured in the presence of mesenchyme. Mesenchyme has been suggested to be involved in suppressing neural cell proliferation in the developing neural tube [ 111 and in two cases of genetically eyeless animals: the eyeless axolotl [ 121 and the anophtalmic mouse [131. Mesenchyme and neural crest which migrate around the stalk may similarly be involved in some aspect of cell death processes in the normal optic stalk. Since many optic stalk cells in culture differentiate into neurons, these cells may represent the population of cells which in situ would normally die. Acknowledgements: The authors would like to thank Drs. J. V. Ruch and V. Karcher-Durcicfor their technical advise on the tissue culture
191 studies. This work was supported in part by Grant ATP.68.78-100 from INSERM. Dr. Ulshafer was sponsored by the NSF/CNRS Exchange of Scientist's Program.
References 1. Ulshafer R, Clavert A (1979) Cell death and optic fiber penetration in the optic stalk of the chick. J Morphol 162:67 2. Silver J, Robb RM (1979) Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia. Develop Biol 68: 175 3. Oberdorfer MD, Silver J (1979) Cell death and neuroepithelial extracellularspaces in the mammalian embryoniceye. J Cell Biol 83:604 4. Hamburger V, Hamilton HL (195 1) A series of normal stages in development of the chick embryo. J Morphol 88:49 5. Ruch JV (1967) Action du phosphate de axamethazone surla differenciationde bronches souchesembryonnairesin vivoet in vitro. CR Soc Biol (Paris) 161: 1339 6. Bodian D (1936) A new method for stainingnerve fibers and nerve endings in mounted paraflii sections. Anat Rec 65:89 7. Clavert A, Gabriel-Robez 0 (1974) Anomalies oculaires determinks par le chloraminophkne chez la souris. CR SOCBiol (Pans) 168: 11 15 8. Lopashov GV (1963) Developmental mechanisms of vertebrate eye rudiments. Macmillan, New York 9. Fell HB, Canti R G (1934) Experiments on the development in vitro of the avian knee joint. Proc Roy Soc Lond [Biol] 116:316 10. Fallon JF, Saunders JW (1968) In vitro analysis of the control of cell death in a zone of prospective necrosis from the chick wing bud. Develop Biol 18:553 11. Takaya H, Watanabe T (1961) Differential proliferation of the ependyma in the developing neural tube of amphibian embryo. Embryologia 6: 169 12. Ulshafer R, Hibbard E (1979) An SEM and TEM study of suppression of eye development in eyeless mutant axolotls. Anat Embryo1 (Berl) 156:29 13. Silver J, Hughs F W (1974) The relationship between morphogenetic cell death and the development of congenitalanophtalmia J Comp Neurol 157:281 Received September 1979/Accepted February 1980
Differentiation 16, 189-191 (1980)
(3 Springer-Vcrlag I080
In vitro Studies on Differentiation of the Optic Stalk in the Chick Embryo ROBERT J. ULSHAFER’ and A N D a CLAVERT Institut d’Embryologie, Strasbourg, France
An orderly pattern of cell death accompanies growth of retinal ganglion cell axons through the optic stalk of the chick embryo. In order to determineifthe cell death process in this adage is preprogrammed at earlier stagesor if other factors play a role, we cultured optic stalk primordia at a stage prior to retinal differentiation, either alone or in the presence of head or limb bud mesenchyme. When optic stalk was cultured alone, many cells differentiated into neurons. However, when mesenchymecells of either head or limb bud origin were combined with the stalk, the stalk cells either degenerated, were unrecognizable in the mesenchymemass, or retained their epithelialarrangement and became pigmented. Mesenchyme and/or neural crest which normally migrate around the stalk at the same time that ganglion cell axons penetrate this structure may therefore be involved in some aspect of the cell death process. Since many optic stalk cells in vitro differentiateinto neurons, these cells may represent the population of cellswhich in situ would normally die. Introduction The optic stalk-optic cup preparation offers an interesting model system for studies on growth and directed migration of retinal axons (and neurons, in general), cell death during embryogenesis, and gliogenesis. A gradient of dying cells exists with time in the optic stalk of the chick embryo during migration of retinal fibers ill. Large intercellular spaces in the mouse optic stalk, possibly resulting from autolysis or phagocytosis of dead cells, have been shown to contain axonal growth cones of retinal ganglion cells [2,31 and therefore alignment of spaces may provide channels in the neuroepithelium through which axons grow. Optic cup ablation at stages prior to differentiation of retinal ganglion cells does not prevent degeneration of the stalk so the axons themselves probably do not induce the cells to die 111. Under those conditions, the stalk completely degenerated and gliogenesis did not occur. Cell death in the stalk therefore appears to be determined 1 Present address and address for reprint requests: Department of Ophthalmology, Baylor College of Medicine, Texas Medical Center, Houston, Texas, 77030 USA
prior to and independently of fiber penetration but arrest of cell death and subsequent gliogenesis may require the presence of ingrowing optic fibers. Cell death may therefore be preprogrammed at the time of primary neural induction. Alternately, mesenchyme and neural crest which migrate rostrally around the stalk prior to penetrationof ganglioncell axons may be causal agents in the cell death process. The purpose of the present study was to determine which of these two phenomena, if either, plays the primary role in initiation of cell death in the stalk. To test this, we analysedthe growth and differentiation of the stalk in culture with and without the presence of mesenchyme or ectomesenchyme.
Methods Eggs of white Leghorn chickens were incubated for 2-3 days and embryos removed at Hamburger and Hamilton (41 stage 18-19 (prior to merentiation and penetration of the optic stalk by retinal ganglion cell axons). Under a dissecting microscope the intact anterior neural tube was removed and the following structures were isolated: optic stalk, optic cup, optic stalk combined with optic cup and surrounding ectomesenchyme. Optic stalk was also isolated and combined with
0301-468l/80/OO 16/0189/8 01.OO
190
R. J. Ulshafer and A. Clavert: Optic Stalk Differentiation in vitro
mesenchyme from the limb bud. Tissues were cultured on a semi-solid coagulum consisting of 2 parts Hank's salt solution, 1 part fetal calf serum, 1 part cock plasma, and 6 parts chick embryonic extract in a humidified incubator at 37" C in an atmosphereof 5% CO, in air 151. Tissue explants were transferred to fresh media at 48 h intervals until the 8th day when they were fxed in Bouin's solution, dehydrated, and embedded in paraffin. Serial sections were cut at 8 pm and alternate sectionswere stained by either the Bodian technique, which is specific for fine nerve fibers [61, or Harris hematoxylin and eosin.
with mesenchyme, either from the head (i.e., ectomesenchyme which includes neural crest) or limb bud, cells in the stalk either degenerated, were unidentifiable in the mesenchyme mass, or retained an epithelial arrangement and became pigmented (Fig. lc). A similar phenomenon occurs in vivo when the optic cup is removed but its stalk and surrounding mesenchyme are left intact [ 11 or when choroid fissure is prevented from forming due to the presence of antimitotic drugs during a critical period, which thus causes the absence of optic nerve [71. Tissue culture experiments on early morphogenesisof the amphibian eye have shown that mesenchyme is necessary for differentiationof pigment epithelium(P. E.) and normal cupping of the optic primordium [81. Sinceour experiments were performed at much later stages (i.e., following invagination of the optic vesicle), a certain pop ulation of cells had already been determined to develop into pigment epithelium, and for this reason we identified pigmented cells in all optic cup preparations. We noted that in optic cups cultured without mesenchyme,P. E. cells were always arranged in an aggregate, frequently in the matrix between neural retina and lens, when present. Op tic anlage, cultured with ectomesenchyme, however, usually developed a normal arrangement of cells: a single
Results and Discussion
The culturing conditionssupported normal differentiation of retinal cell types in optic cup preparations as discerned by morphologic examination: by eight days nuclear and plexiform layers had formed, axons were present, and usually grew together in a well-defined fiber layer. In those cases where stalk was cultured alone, many neurons differentiated. The tissue mass appeared to have organised mantle and marginal layers (Fig. la), although a ventricle or neurocoel was never seen. Fibers in both layers occasionally formed small fascicles which coursed together for several hundred micra before dissipating through the tissue (Fig. 1b). When the stalk was cultured
Flg. 1. Optic stalks cultured alone (a, b) or with limb bud mesenchyme (c) for dght days (Bodian stain). a Cells of the optic stalk have largely differentiated into neurons. The tissue appears to have formed mantle and marginal layers, similar to cells in the embryonic neural tube. b High power magnification of a. Many fibers are arranged in a large fascicle. c Optic stalk cultured with limb bud mesenchyme did not differentiateinto neurons. In fact, in this specimen the stalk retained its primitive neuroepithelial arrangement of cells and many of its cells contain pigment granules. The mesenchyme (Me) immediately around the stalk remnant (0s) is much darker than the rest of the mesodermal mass
R. J. Ulshafer and A. Clavert: Optic Stalk Differentiation in vitro
layer of P.E. outside and apposed to the neural retina. Thus, mesenchyme appears to play a sequential role throughout morphogenesis of the P. E., originally as an inductive agent and later in stabilization of the cellular arrangement in this tissue. Our system differs from those anlagen which are known to be preprogrammed to die, such as in morphogenesis of the knee joint 191 or in the posterior necrotic zone (PNZ) of the chick limb bud. For example,when the chick PNZ was excised at stage 2 1 or beyond and cultured in a manner similar to that in OUT experiments, a large number of necrotic cells and phagocytes accumulated in the cultures which resembled in number and appearance those observed in situ at stage 24. In that experiment, when prospective PNZs were cultured in the presence of wing mesoderm, necrosis did not occur unless the two tissues were separatedby at least 1-2 mm and contact did not occur between the two tissues [lo]. A genetically programmed "death clock" therefore does not seem to be responsible for cell death in the optic stalk of the chick unless external agents in the immediate environment are responsible for turning it on. Cell death which is induced or determined at the time of primary neural induction similarlydoes not appear to be the causal agent since post-induction explants remain viable unless cultured in the presence of mesenchyme. Mesenchyme has been suggested to be involved in suppressing neural cell proliferation in the developing neural tube [ 111 and in two cases of genetically eyeless animals: the eyeless axolotl [ 121 and the anophtalmic mouse [131. Mesenchyme and neural crest which migrate around the stalk may similarly be involved in some aspect of cell death processes in the normal optic stalk. Since many optic stalk cells in culture differentiate into neurons, these cells may represent the population of cells which in situ would normally die. Acknowledgements: The authors would like to thank Drs. J. V. Ruch and V. Karcher-Durcicfor their technical advise on the tissue culture
191 studies. This work was supported in part by Grant ATP.68.78-100 from INSERM. Dr. Ulshafer was sponsored by the NSF/CNRS Exchange of Scientist's Program.
References 1. Ulshafer R, Clavert A (1979) Cell death and optic fiber penetration in the optic stalk of the chick. J Morphol 162:67 2. Silver J, Robb RM (1979) Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia. Develop Biol 68: 175 3. Oberdorfer MD, Silver J (1979) Cell death and neuroepithelial extracellularspaces in the mammalian embryoniceye. J Cell Biol 83:604 4. Hamburger V, Hamilton HL (195 1) A series of normal stages in development of the chick embryo. J Morphol 88:49 5. Ruch JV (1967) Action du phosphate de axamethazone surla differenciationde bronches souchesembryonnairesin vivoet in vitro. CR Soc Biol (Paris) 161: 1339 6. Bodian D (1936) A new method for stainingnerve fibers and nerve endings in mounted paraflii sections. Anat Rec 65:89 7. Clavert A, Gabriel-Robez 0 (1974) Anomalies oculaires determinks par le chloraminophkne chez la souris. CR SOCBiol (Pans) 168: 11 15 8. Lopashov GV (1963) Developmental mechanisms of vertebrate eye rudiments. Macmillan, New York 9. Fell HB, Canti R G (1934) Experiments on the development in vitro of the avian knee joint. Proc Roy Soc Lond [Biol] 116:316 10. Fallon JF, Saunders JW (1968) In vitro analysis of the control of cell death in a zone of prospective necrosis from the chick wing bud. Develop Biol 18:553 11. Takaya H, Watanabe T (1961) Differential proliferation of the ependyma in the developing neural tube of amphibian embryo. Embryologia 6: 169 12. Ulshafer R, Hibbard E (1979) An SEM and TEM study of suppression of eye development in eyeless mutant axolotls. Anat Embryo1 (Berl) 156:29 13. Silver J, Hughs F W (1974) The relationship between morphogenetic cell death and the development of congenitalanophtalmia J Comp Neurol 157:281 Received September 1979/Accepted February 1980