- Email: [email protected]
Apoptosis in early ocular morphogenesis in the mouse
Developmental Brain Research 112 Ž1999. 129–133
Short communication
Apoptosis in early ocular morphogenesis in the mouse Lois K. Laemle a
a,b,)
, Michele Puszkarczuk a , Richard N. Feinberg
a
Department of Anatomy, Cell Biology and Injury Sciences, UMDNJ, New Jersey Medical School, 185 South Orange AÕenue, Newark NJ 07103, USA b Department of Ophthalmology, UMDNJ, New Jersey Medical School, Newark NJ 07103, USA Accepted 13 October 1998
Abstract Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5–E11.5, were embedded in paraffin, sectioned at 5 mm and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling ŽTUNEL. method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Ocular development; Cell death; Apoptosis
The present study has utilized current concepts of apoptosis and contemporary methodology to re-evaluate ocular morphogenesis in utero. The eye is formed from neural ectoderm, surface ectoderm and mesenchyme. Normal ocular development depends upon dynamic interactions between these embryonic tissues, as well as between individual cells within each tissue. These include the death of cells in predictable locations and at precise stages of development by a process known as programmed cell death ŽPCD. w3,6,8,12x. Genetic control over cell differentiation is well-established; however, the concept that cellular death can be under genetic control Žapoptosis. is relatively new w5x. We have used the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling ŽTUNEL. method, which identifies apoptotic cells, to re-evaluate the cellular death described in earlier studies of ocular development in the mouse embryo. This methodology, applied for the first time to embryonic optic primordia, enables us to confirm much of the temporal and spatial pattern of cell
)
Corresponding [email protected]
author.
Fax:
q 1-973-972-7489;
E-mail:
death reported previously in the component tissues of the embryonic eye and to identify the mechanism of cell death as apoptosis. Mammalian ocular development has been studied most extensively in the retina, and primarily during the early postnatal period w1,8,9,15x. Relatively few studies have focused on the early embryonic period. Using hematoxylin- and eosin-stained tissue, Silver and Hughes w14x described a reproducible temporal and spatial pattern of pyknotic nuclei in the developing optic primordia of C56BLr6J mice. They identified this process as ‘necrosis’ or ‘PCD’. However, the distinction between apoptosis and necrosis has never been addressed in this system. Our objectives were twofold: first, to determine the contribution of apoptotic mechanisms to prenatal stages of normal eye formation; second, to provide a foundation for understanding the role of apoptosis in congenital ocular anomalies. We used ZRDCT-N mouse embryos, because they are the genetic controls for congenitally anophthalmic ŽZRDCT-AN. mutants. Thirty-three embryos from 8.5 to 11.5 days of gestation were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and oriented so that the optic primordia were sectioned in either the sagittal, transverse or coronal
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 3 - 9
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L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133
plane. For some embryos, serial sections were stained with hematoxylin; for others, alternate sections were stained with hematoxylin or with the TUNEL method w2x. The TUNEL method is based on the specific binding of terminal deoxynucleotidyl transferase ŽTdT. to 3X OH ends of DNA, resulting in a polydeoxynucleotide polymer w2x. Briefly, sections were deparaffinized and rehydrated to phosphate-buffered saline ŽPBS.. Nuclear DNA was exposed by treatment with proteinase K ŽSigma.. Sections were then incubated with TdT to incorporate biotinylated deoxyuridine at sites of DNA breaks, and the signal was amplified by avidin-peroxidase, enabling conventional histochemical identification by light microscopy. The current study employed the APOTAG kit ŽONCOR Sciences. and used 3,3X-diaminobenzidine tetrahydrochloride ŽDAB, Sigma. as the chromogen. Using this method, apoptotic cells are identified by the brown reaction product in the nuclei and nuclear fragments. Sections were counterstained with light green or methylene blue. Apoptotic cells stained with hematoxylin were identified by their diminished size and their deeply pyknotic and fragmented nuclei. Apoptotic cells were mapped from serial sections in representative embryos of each age, using Neurolucida, a computerized image analysis system from Microbrightfield, and 3-D reconstructions were made. From E8.5 to E9 ŽFig. 1a,d., the optic primordia appear as evaginations of uniform thickness from the neural tube. The walls of the optic stalk and vesicle are comprised of a pseudostratified epithelium that has two to three levels of nuclei. The optic vesicles are separated from the surface epithelium by a distinct layer of mesenchyme and are closely invested by a prominent capillary network ŽFig. 1d.. At E9, the optic stalk has narrowed, mesenchyme between the neural epithelium and surface ectoderm is reduced to scattered cells, and capillaries may or may not be evident between the optic vesicle and surface ectoderm. Mitotic figures are common in the deeper layers of the optic vesicle, in the mesenchyme and surface ectoderm. Contact occurs between neural epithelium in the ventral portion of the optic vesicle and surface ectoderm. Between E9 and E9.5, the region of contact between the optic vesicle and surface ectoderm expands. Although mesenchymal cells are usually absent from the region of contact, cells are occasionally trapped between the two tissue layers ŽFig. 1e.. A well-formed vascular supply surrounds the optic stalk and margins of the optic vesicle, but stops at the region of contact. Between E9.5 and E10, the surface epithelium thickens to form the lens placode ŽFig. 1b.. The neural epithelium of the ventral wall of the optic vesicle doubles in thickness; the epithelium of the distal wall triples and the epithelium of the dorsal wall remains unchanged from E8.5. Between E10 and E10.5, the lens placode invaginates to form the lens pit, and the optic vesicle invaginates to form the optic cup ŽFig. 1b,e.. At E10.5, the ventral region of the pigment epithelium is composed of a single layer of cells, while dorsally it is still
pseudostratified. Formation of the lens vesicle is completed between E10.5 and E11 ŽFig. 1c,f.. The hyaloid vessels are present at E11, and the pigment epithelium becomes uniform in thickness. Between E11 and E11.5, the lens detaches from the surface ectoderm. Apoptotic cells were first observed at E9 ŽFig. 2a.. From E9 to E9.5, hematoxylin staining revealed pyknotic cells that were scattered throughout the wall of the optic vesicle and optic stalk, and in the lens epithelium. In adjacent sections, cells in these locations stained positively with the TUNEL method ŽFig. 2a,d.. Mesenchymal cell death in the region between the optic vesicle and the lens epithelium was observed in fewer than 20% of primordia examined. Although scarce, these cells when present, could be identified in both hematoxylin and TUNEL-stained sections. Between E 9.5 and E10, apoptosis in the optic vesicle became more localized, with apoptotic cells disappearing from the dorsal region by E10. From E10 to 10.25 ŽFig. 1b,e, Fig. 2b,e., apoptotic cells were concentrated in the ventral and distal walls of the optic cup, where invagination was beginning, at the margins of the lens pit and in the dorsal region of the lens placode. At E10.5, increased numbers of apoptotic cells were observed in the optic cup, particularly in the ventral wall, and in the lens epithelium. Apoptotic cells were also present in the central region of the pigment epithelium. At E11 ŽFig. 1c,f, Fig. 2c,f., apoptotic loci were observed in the proximal optic cup, retinal pigment epithelium, and optic stalk. The temporal and spatial distribution of apoptosis in the lens placode, optic vesicle and optic cup were consistent with localized regions of cell necrosis described previously by Silver and Hughes in the C57B1r6J mouse w14x and the rat w13x. Since mesenchymal cell death was observed infrequently in our tissue, we could not confirm this process as a major determinant of normal ocular development. In this regard, our observations support the findings of Harch et al. w4x rather than those of Silver and Hughes w14x. In our preparations, inflammation was absent and pyknotic cells were scattered as opposed to being clustered. These morphological characteristics are consistent with death by an apoptotic mechanism rather than by necrosis w7x. Labeling of cells with the TUNEL method provides further evidence that cells are dying by apoptosis rather than necrosis w2x. Jacobson et al. w6x suggest five reasons for elimination of cells by PCD during development. It is likely that at least four play a significant role in ocular development. These include control of cell numbers, elimination of abnormal cells, production of differentiated cells without organelles, and sculpting of structures. It has been shown that both intrinsic properties and environmental factors regulate differentiation in the vertebrate eye w1,9–11,15,16x. Diffusible molecules such as growth factors which have been identified at early stages of mammalian ocular development include fibroblast growth factors ŽFGFs., insulinlike growth factors ŽIGFs., epidermal growth factors ŽEGFs., platelet-derived growth factors ŽPDGFs., nerve
L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133 Fig. 1. Markers of ocular development in the sighted ZRDCT-N mouse. Morphology of the optic primordium at E9 Ža,d., E10–E10.5 Žb,e. and E11 Žc,f.. Ža. E9 is characterized by formation of the optic vesicle Žov.. The optic vesicle is separated from the surface ectoderm by a thin layer of mesenchyme Ž=115.. Žb. E10 is characterized by thickening of the surface ectoderm to form the lens placode, invagination of the optic vesicle to form to optic cup, and initial invagination of the lens placode Ž=150.. Žc. At E11, formation of the lens vesicle is complete Ž=150.. Žd. Higher magnification of an E9 optic vesicle shows the closely associated vascular network which surrounds the optic stalk and vesicle Ž).. A thin layer of mesenchyme Žwhite arrows. can be seen between the optic vesicle and the surface ectoderm Ž=400.. Že. Optic primordium at E10.5. Invagination of the lens placode and optic vesicle proceeds normally with mesenchymal cells Žarrows. present between the lens pit and optic cup Ž=400.. Žf. Developing eye at E11, showing a locus of apoptotic cells in the presumptive retina. Note that there are multiple apoptotic bodies, of which four are indicated by arrowheads. The eye shown in this figure is contralateral to the eye in Žc. Ž=300..
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L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133
Fig. 2. Spatial and temporal distribution of apoptotic cells. Ža–c. Show 3-D reconstructions of optic primordia at E9 Ža., E10 Žb. and E11 Žc.. Žd–f. Show apoptotic cells stained by the TUNEL method Žarrowheads.. In the reconstruction, each dot represents an apoptotic cell. Each mapped tissue section is represented by a different color. Thus, all red dots represent apoptotic cells mapped from a single tissue section, blue from a different section, etc. At E9 Ža,d., apoptosis is sparse. Apoptotic cells are scattered throughout the optic vesicle and to a lesser extent in the lens epithelium. By E10 Žb,e., apoptosis is more localized to the ventral and distal regions of the optic cup and the dorsal lens placode, while disappearing from the dorsal optic cup. At E11 Žc,f., apoptosis has localized primarily to the central region of the presumptive retina and adjacent retinal pigment epithelium. Magnifications: Žd. =280; Že. =330; Žf. =560.
growth factors ŽNGFs., transforming growth factors ŽTGFs. and mesodermal growth factors ŽMGFs. w11,16x. Cell death could result from deficient or excessive production of these or other factors required for differentiation, production of these factors at inappropriate times in development, or receipt of opposing signals. Since extracellular matrix molecules selectively bind and store growth factors w16x, delivery of growth factors or other survival signals to the developing optic primordium can be regulated by the composition of the extracellular matrix. Furthermore, the availability of the necessary hormones and growth factors may be determined by the number, position, and permeability of periocular blood vessels. In summary, we have described the spatial and temporal patterns of cell death in optic primordia of the ZRDCT-N mouse embryo between E8.5 and E11.5. Use of the TUNEL method and current criteria for analysis of our data has allowed us to conclude that this cellular death takes place by apoptosis. Our results provide the groundwork for
evaluating the role of apoptosis in abnormal ocular development, and for future studies of the role of growth factors, extracellular matrix molecules, and vascular supply in normal and abnormal ocular morphogenesis.
Acknowledgements This work was supported by a grant from the UMDNJ Foundation and a grant to the Department of Ophthalmology from Research to Prevent Blindness. The authors would like to thank Ms. Erminia Cafasso and Ms. Ana Cuevas for their technical assistance.
References w1x D. Altshuler, C. Cepko, A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro, Development 114 Ž1992. 947–957.
L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133 w2x Y. Gavrieli, Y. Sherman, S.A. Ben-Sasson, Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell Biol. 119 Ž1992. 493–501. w3x A. Glucksman, Cell death in normal vertebrate ontogeny, Cambridge Philos. Soc. Biol. Rev. 26 Ž1951. 59–86. w4x C. Harch, H.B. Chase, N.I. Gonslaves, Studies on an anophthalmic strain of mice, Dev. Biol. 63 Ž1978. 352–357. w5x H. Horvitz, H. Ellis, P. Sternberg, Programmed cell death in nematode development, Neuroscience Comment 1 Ž1982. 56–65. w6x M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 Ž1997. 347–354. w7x J.F.R. Kerr, A.H. Wyllie, A.R. Curie, Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics, Br. J. Cancer 26 Ž1972. 239–257. w8x R.A. Lang, Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege, Cell Death Diff. 4 Ž1997. 12–20. w9x T. Matsuo, N. Osumi-Yamashita, S. Noji, H. Ohuchi, E. Koyama, F. Myokai, N. Matsuo, S. Taniguchi, H.L. Doi, S. Iseki, Y. Ninomiya, N. Fujiowara, T. Watanabe, K. Eto, A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells, Nat. Genet. 3 Ž1993. 299–304.
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w10x D. Papermaster, Apoptosis of the mammalian retina and lens, Cell Death Diff. 4 Ž1997. 21–28. w11x L.W. Reneker, D.W. Silversides, K. Patel, P.A. Overbeek, TGF-a can act as a chemoattractant to perioptic mesenchymal cells in developing mouse eyes, Development 121 Ž1995. 1669–1680. w12x J.W. Saunders Jr., Death in embryonic systems, Science 154 Ž1996. 604–612. w13x J. Silver, F.W. Hughes, The role of cell death during morphogenesis of the mammalian eye, J. Morphol. 140 Ž1973. 159–170. w14x J. Silver, F.W. Hughes, The relationship between morphogenetic cell death and the development of congenital anophthalmia, J. Comp. Neurol. 157 Ž1974. 281–302. w15x K. Tomita, M. Ishibashi, K. Nakahara, S.-L. Ang, S. Nakanishi, F. Guillemot, R. Kageyama, Mammalian hairy and enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis, Neuron 16 Ž1996. 723–734. w16x B.J. Tripathi, R.C. Tripathi, A.M. Livingston, N.S.C. Borisuth, Role of growth factors in development of the eye, Am. J. Anat. 192 Ž1991. 442–471.
Short communication
Apoptosis in early ocular morphogenesis in the mouse Lois K. Laemle a
a,b,)
, Michele Puszkarczuk a , Richard N. Feinberg
a
Department of Anatomy, Cell Biology and Injury Sciences, UMDNJ, New Jersey Medical School, 185 South Orange AÕenue, Newark NJ 07103, USA b Department of Ophthalmology, UMDNJ, New Jersey Medical School, Newark NJ 07103, USA Accepted 13 October 1998
Abstract Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5–E11.5, were embedded in paraffin, sectioned at 5 mm and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling ŽTUNEL. method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Ocular development; Cell death; Apoptosis
The present study has utilized current concepts of apoptosis and contemporary methodology to re-evaluate ocular morphogenesis in utero. The eye is formed from neural ectoderm, surface ectoderm and mesenchyme. Normal ocular development depends upon dynamic interactions between these embryonic tissues, as well as between individual cells within each tissue. These include the death of cells in predictable locations and at precise stages of development by a process known as programmed cell death ŽPCD. w3,6,8,12x. Genetic control over cell differentiation is well-established; however, the concept that cellular death can be under genetic control Žapoptosis. is relatively new w5x. We have used the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling ŽTUNEL. method, which identifies apoptotic cells, to re-evaluate the cellular death described in earlier studies of ocular development in the mouse embryo. This methodology, applied for the first time to embryonic optic primordia, enables us to confirm much of the temporal and spatial pattern of cell
)
Corresponding [email protected]
author.
Fax:
q 1-973-972-7489;
E-mail:
death reported previously in the component tissues of the embryonic eye and to identify the mechanism of cell death as apoptosis. Mammalian ocular development has been studied most extensively in the retina, and primarily during the early postnatal period w1,8,9,15x. Relatively few studies have focused on the early embryonic period. Using hematoxylin- and eosin-stained tissue, Silver and Hughes w14x described a reproducible temporal and spatial pattern of pyknotic nuclei in the developing optic primordia of C56BLr6J mice. They identified this process as ‘necrosis’ or ‘PCD’. However, the distinction between apoptosis and necrosis has never been addressed in this system. Our objectives were twofold: first, to determine the contribution of apoptotic mechanisms to prenatal stages of normal eye formation; second, to provide a foundation for understanding the role of apoptosis in congenital ocular anomalies. We used ZRDCT-N mouse embryos, because they are the genetic controls for congenitally anophthalmic ŽZRDCT-AN. mutants. Thirty-three embryos from 8.5 to 11.5 days of gestation were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and oriented so that the optic primordia were sectioned in either the sagittal, transverse or coronal
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 3 - 9
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L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133
plane. For some embryos, serial sections were stained with hematoxylin; for others, alternate sections were stained with hematoxylin or with the TUNEL method w2x. The TUNEL method is based on the specific binding of terminal deoxynucleotidyl transferase ŽTdT. to 3X OH ends of DNA, resulting in a polydeoxynucleotide polymer w2x. Briefly, sections were deparaffinized and rehydrated to phosphate-buffered saline ŽPBS.. Nuclear DNA was exposed by treatment with proteinase K ŽSigma.. Sections were then incubated with TdT to incorporate biotinylated deoxyuridine at sites of DNA breaks, and the signal was amplified by avidin-peroxidase, enabling conventional histochemical identification by light microscopy. The current study employed the APOTAG kit ŽONCOR Sciences. and used 3,3X-diaminobenzidine tetrahydrochloride ŽDAB, Sigma. as the chromogen. Using this method, apoptotic cells are identified by the brown reaction product in the nuclei and nuclear fragments. Sections were counterstained with light green or methylene blue. Apoptotic cells stained with hematoxylin were identified by their diminished size and their deeply pyknotic and fragmented nuclei. Apoptotic cells were mapped from serial sections in representative embryos of each age, using Neurolucida, a computerized image analysis system from Microbrightfield, and 3-D reconstructions were made. From E8.5 to E9 ŽFig. 1a,d., the optic primordia appear as evaginations of uniform thickness from the neural tube. The walls of the optic stalk and vesicle are comprised of a pseudostratified epithelium that has two to three levels of nuclei. The optic vesicles are separated from the surface epithelium by a distinct layer of mesenchyme and are closely invested by a prominent capillary network ŽFig. 1d.. At E9, the optic stalk has narrowed, mesenchyme between the neural epithelium and surface ectoderm is reduced to scattered cells, and capillaries may or may not be evident between the optic vesicle and surface ectoderm. Mitotic figures are common in the deeper layers of the optic vesicle, in the mesenchyme and surface ectoderm. Contact occurs between neural epithelium in the ventral portion of the optic vesicle and surface ectoderm. Between E9 and E9.5, the region of contact between the optic vesicle and surface ectoderm expands. Although mesenchymal cells are usually absent from the region of contact, cells are occasionally trapped between the two tissue layers ŽFig. 1e.. A well-formed vascular supply surrounds the optic stalk and margins of the optic vesicle, but stops at the region of contact. Between E9.5 and E10, the surface epithelium thickens to form the lens placode ŽFig. 1b.. The neural epithelium of the ventral wall of the optic vesicle doubles in thickness; the epithelium of the distal wall triples and the epithelium of the dorsal wall remains unchanged from E8.5. Between E10 and E10.5, the lens placode invaginates to form the lens pit, and the optic vesicle invaginates to form the optic cup ŽFig. 1b,e.. At E10.5, the ventral region of the pigment epithelium is composed of a single layer of cells, while dorsally it is still
pseudostratified. Formation of the lens vesicle is completed between E10.5 and E11 ŽFig. 1c,f.. The hyaloid vessels are present at E11, and the pigment epithelium becomes uniform in thickness. Between E11 and E11.5, the lens detaches from the surface ectoderm. Apoptotic cells were first observed at E9 ŽFig. 2a.. From E9 to E9.5, hematoxylin staining revealed pyknotic cells that were scattered throughout the wall of the optic vesicle and optic stalk, and in the lens epithelium. In adjacent sections, cells in these locations stained positively with the TUNEL method ŽFig. 2a,d.. Mesenchymal cell death in the region between the optic vesicle and the lens epithelium was observed in fewer than 20% of primordia examined. Although scarce, these cells when present, could be identified in both hematoxylin and TUNEL-stained sections. Between E 9.5 and E10, apoptosis in the optic vesicle became more localized, with apoptotic cells disappearing from the dorsal region by E10. From E10 to 10.25 ŽFig. 1b,e, Fig. 2b,e., apoptotic cells were concentrated in the ventral and distal walls of the optic cup, where invagination was beginning, at the margins of the lens pit and in the dorsal region of the lens placode. At E10.5, increased numbers of apoptotic cells were observed in the optic cup, particularly in the ventral wall, and in the lens epithelium. Apoptotic cells were also present in the central region of the pigment epithelium. At E11 ŽFig. 1c,f, Fig. 2c,f., apoptotic loci were observed in the proximal optic cup, retinal pigment epithelium, and optic stalk. The temporal and spatial distribution of apoptosis in the lens placode, optic vesicle and optic cup were consistent with localized regions of cell necrosis described previously by Silver and Hughes in the C57B1r6J mouse w14x and the rat w13x. Since mesenchymal cell death was observed infrequently in our tissue, we could not confirm this process as a major determinant of normal ocular development. In this regard, our observations support the findings of Harch et al. w4x rather than those of Silver and Hughes w14x. In our preparations, inflammation was absent and pyknotic cells were scattered as opposed to being clustered. These morphological characteristics are consistent with death by an apoptotic mechanism rather than by necrosis w7x. Labeling of cells with the TUNEL method provides further evidence that cells are dying by apoptosis rather than necrosis w2x. Jacobson et al. w6x suggest five reasons for elimination of cells by PCD during development. It is likely that at least four play a significant role in ocular development. These include control of cell numbers, elimination of abnormal cells, production of differentiated cells without organelles, and sculpting of structures. It has been shown that both intrinsic properties and environmental factors regulate differentiation in the vertebrate eye w1,9–11,15,16x. Diffusible molecules such as growth factors which have been identified at early stages of mammalian ocular development include fibroblast growth factors ŽFGFs., insulinlike growth factors ŽIGFs., epidermal growth factors ŽEGFs., platelet-derived growth factors ŽPDGFs., nerve
L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133 Fig. 1. Markers of ocular development in the sighted ZRDCT-N mouse. Morphology of the optic primordium at E9 Ža,d., E10–E10.5 Žb,e. and E11 Žc,f.. Ža. E9 is characterized by formation of the optic vesicle Žov.. The optic vesicle is separated from the surface ectoderm by a thin layer of mesenchyme Ž=115.. Žb. E10 is characterized by thickening of the surface ectoderm to form the lens placode, invagination of the optic vesicle to form to optic cup, and initial invagination of the lens placode Ž=150.. Žc. At E11, formation of the lens vesicle is complete Ž=150.. Žd. Higher magnification of an E9 optic vesicle shows the closely associated vascular network which surrounds the optic stalk and vesicle Ž).. A thin layer of mesenchyme Žwhite arrows. can be seen between the optic vesicle and the surface ectoderm Ž=400.. Že. Optic primordium at E10.5. Invagination of the lens placode and optic vesicle proceeds normally with mesenchymal cells Žarrows. present between the lens pit and optic cup Ž=400.. Žf. Developing eye at E11, showing a locus of apoptotic cells in the presumptive retina. Note that there are multiple apoptotic bodies, of which four are indicated by arrowheads. The eye shown in this figure is contralateral to the eye in Žc. Ž=300..
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Fig. 2. Spatial and temporal distribution of apoptotic cells. Ža–c. Show 3-D reconstructions of optic primordia at E9 Ža., E10 Žb. and E11 Žc.. Žd–f. Show apoptotic cells stained by the TUNEL method Žarrowheads.. In the reconstruction, each dot represents an apoptotic cell. Each mapped tissue section is represented by a different color. Thus, all red dots represent apoptotic cells mapped from a single tissue section, blue from a different section, etc. At E9 Ža,d., apoptosis is sparse. Apoptotic cells are scattered throughout the optic vesicle and to a lesser extent in the lens epithelium. By E10 Žb,e., apoptosis is more localized to the ventral and distal regions of the optic cup and the dorsal lens placode, while disappearing from the dorsal optic cup. At E11 Žc,f., apoptosis has localized primarily to the central region of the presumptive retina and adjacent retinal pigment epithelium. Magnifications: Žd. =280; Že. =330; Žf. =560.
growth factors ŽNGFs., transforming growth factors ŽTGFs. and mesodermal growth factors ŽMGFs. w11,16x. Cell death could result from deficient or excessive production of these or other factors required for differentiation, production of these factors at inappropriate times in development, or receipt of opposing signals. Since extracellular matrix molecules selectively bind and store growth factors w16x, delivery of growth factors or other survival signals to the developing optic primordium can be regulated by the composition of the extracellular matrix. Furthermore, the availability of the necessary hormones and growth factors may be determined by the number, position, and permeability of periocular blood vessels. In summary, we have described the spatial and temporal patterns of cell death in optic primordia of the ZRDCT-N mouse embryo between E8.5 and E11.5. Use of the TUNEL method and current criteria for analysis of our data has allowed us to conclude that this cellular death takes place by apoptosis. Our results provide the groundwork for
evaluating the role of apoptosis in abnormal ocular development, and for future studies of the role of growth factors, extracellular matrix molecules, and vascular supply in normal and abnormal ocular morphogenesis.
Acknowledgements This work was supported by a grant from the UMDNJ Foundation and a grant to the Department of Ophthalmology from Research to Prevent Blindness. The authors would like to thank Ms. Erminia Cafasso and Ms. Ana Cuevas for their technical assistance.
References w1x D. Altshuler, C. Cepko, A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro, Development 114 Ž1992. 947–957.
L.K. Laemle et al.r DeÕelopmental Brain Research 112 (1999) 129–133 w2x Y. Gavrieli, Y. Sherman, S.A. Ben-Sasson, Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell Biol. 119 Ž1992. 493–501. w3x A. Glucksman, Cell death in normal vertebrate ontogeny, Cambridge Philos. Soc. Biol. Rev. 26 Ž1951. 59–86. w4x C. Harch, H.B. Chase, N.I. Gonslaves, Studies on an anophthalmic strain of mice, Dev. Biol. 63 Ž1978. 352–357. w5x H. Horvitz, H. Ellis, P. Sternberg, Programmed cell death in nematode development, Neuroscience Comment 1 Ž1982. 56–65. w6x M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 Ž1997. 347–354. w7x J.F.R. Kerr, A.H. Wyllie, A.R. Curie, Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics, Br. J. Cancer 26 Ž1972. 239–257. w8x R.A. Lang, Apoptosis in mammalian eye development: lens morphogenesis, vascular regression and immune privilege, Cell Death Diff. 4 Ž1997. 12–20. w9x T. Matsuo, N. Osumi-Yamashita, S. Noji, H. Ohuchi, E. Koyama, F. Myokai, N. Matsuo, S. Taniguchi, H.L. Doi, S. Iseki, Y. Ninomiya, N. Fujiowara, T. Watanabe, K. Eto, A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells, Nat. Genet. 3 Ž1993. 299–304.
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w10x D. Papermaster, Apoptosis of the mammalian retina and lens, Cell Death Diff. 4 Ž1997. 21–28. w11x L.W. Reneker, D.W. Silversides, K. Patel, P.A. Overbeek, TGF-a can act as a chemoattractant to perioptic mesenchymal cells in developing mouse eyes, Development 121 Ž1995. 1669–1680. w12x J.W. Saunders Jr., Death in embryonic systems, Science 154 Ž1996. 604–612. w13x J. Silver, F.W. Hughes, The role of cell death during morphogenesis of the mammalian eye, J. Morphol. 140 Ž1973. 159–170. w14x J. Silver, F.W. Hughes, The relationship between morphogenetic cell death and the development of congenital anophthalmia, J. Comp. Neurol. 157 Ž1974. 281–302. w15x K. Tomita, M. Ishibashi, K. Nakahara, S.-L. Ang, S. Nakanishi, F. Guillemot, R. Kageyama, Mammalian hairy and enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis, Neuron 16 Ž1996. 723–734. w16x B.J. Tripathi, R.C. Tripathi, A.M. Livingston, N.S.C. Borisuth, Role of growth factors in development of the eye, Am. J. Anat. 192 Ž1991. 442–471.