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Neuroectoderm of the Early Embryonic Rot Eye Scanning Electron Microscopy Dennis E. Morse and Patricia S. McCann A more accurate description of the changes that occur in the neuroectodermal portion of the developing eye is possible if the surface ectoderm and its underlying mesectoderm are dissected away prior to scanning electron microscopic analysis. A clean preparation of the basal surface of the neuroectoderm with its basal lamina can be prepared by this method. The primitive eyes form during day 11 as lateral diverticula from the forebrain in the rat embryo. These optic vesicles initially have a broad attachment to the diencephalon. By day 12, a true optic stalk connects the optic vesicle to the brain. As the vesicle approaches the surface ectoderm, it involutes to form the optic cup. During day 13, the cup deepens and creates a prominent rim on all but its ventral side. This cleft in the ventral portion of the optic cup is known as the optic fissure. Three portions of the neuroectodermal eye are apparent at this stage: the optic cup, optic stalk, and a short narrow region that joins these two—the collum. The optic fissure extends into the collum but ends abruptly at the junction of the collum with the stalk. The fissure closes on day 14. Its only remnants at this time are a shallow groove in the optic cup and a small patent portion in the collum that permits passage of the intraocular vessels. Invest Ophthalmol Vis Sci 25:899-907, 1984 The developing vertebrate eye serves as the model system for studies of such diverse morphogenetic problems as induction,1 neuron development,2 cell death, 34 melanogenesis,5 and extracellular matrix production by epithelia.6'7 Formation of both the optic cup and fissure involves all of these processes. Of the two, the optic cup has received more of the attention in published works. The optic fissure is a prominent, albeit transitory, groove on the ventral aspect of the optic vesicle in all but the most primitive vertebrates.8 In mammals, this groove typically is described to extend the entire length of the optic stalk, which connects the optic vesicle to the forebrain. The purpose of the fissure is twofold. First, it creates a channel whereby the intraocular vessels can enter the optic cup. Second, while the retinal neurons do not actually enter the fissure, it is established that these neurons do not successfully extend their processes to the diencephalon in the absence of the optic fissure.29"12 In each case, the formation of the fissure provides direct access between the cup and stalk. 213 Within a short period of time after its formation, the optic fissure is obliterated by the fusion of its two lips. Fusion begins near the midpoint of the fissure and proceeds in either direction. 814 Electron microscopic study of the fissure is limited to that of Geeraets.15 Her observations are based on thin sections from the golden hamster eye. Presently, there is no published report that uses scanning electron microscopy to study the formation of the optic fissure. By microdissection, we are able to remove the surface ectoderm and subjacent mesectoderm to expose the neuroectoderm, which gives rise to the optic vesicle, stalk, cup and fissure. This method avoids the treatment with enzymes and provides the entire structure for study rather than relying on random fractures or razor cuts through the head region to expose the deeperlying neuroectodermal portion of the embryonic eye. This paper describes the scanning electron microscopy of normal rat optic fissure formation. Materials and Methods Female Sprague-Dawley rats (approximately 200 g) were caged overnight with a male rat of the same strain. The presence of sperm in a vaginal smear performed the following morning was used as an indication of pregnancy. The day of the positive smear was termed day 0. A pregnant female rat was placed in a separate cage and maintained until the desired stage of gestation. Ages used in this study were 11-, 12-, 12.5-, 13-, 13.5-, and 14-days gestation. All animals used in this study were handled in accordance with the ARVO Resolution on the Use of Animals in Research. From the Department of Anatomy, Medical College of Ohio, Toledo, Ohio. Submitted for publication: November 9, 1983. Reprint requests: Dennis E. Morse, PhD, Department of Anatomy, Medical College of Ohio, C.S. 10008, Toledo, OH 43699. 899 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 900 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / August 1984 Vol. 25 Fig. 1. The neuroectoderm gives rise to two lateral diverticula on day 11. These early optic vesicles (OV) have a broad attachment to the forebrain at this stage. The midline neural groove is seen in its final stages of closure in this anterior view of the head region. The surface ectoderm and mesectoderm have been removed. A few surface ectodermal cells are adherent in the neural groove region (arrows). Bar = 100 j*m. The rats were injected intraperitoneally (IP) with sodium pentobarbitol just prior to surgery. After the rat was fully under the effect of the anesthesia, a laparotomy was performed. The uterine horns were exposed, excised, and placed immediately in cold (4°C) 2.5% glutaraldehyde-2% paraformaldehyde buffered with 0.2 N cacodylate. The final solution pH was 7.4. Embryos were dissected free and then placed in fixative at least overnight. Following initial fixation, the embryos were rinsed in 0.2 N cacodylate buffer, postfixed in 2% osmium tetroxide in 0.144 N cacodylate buffer and then rinsed with 0.144 N cacodylate buffer. Specimens were dehydrated in an ascending series of ethanols and critical point dried with liquid carbon dioxide as the transitional fluid. Tissue was mounted on aluminum stubs using silver conductive paste and sputtercoated with gold-palladium. Exposure of the study area was accomplished by partial dissection of the specimen while in the initial fixative. In most cases, the surface ectoderm was stripped off and various amounts of the mesectoderm were teased away at this stage of preparation. At the 11-12.5 days gestation stages, the lens placode was still a part of the surface ectoderm and, thus, was removed with that layer. The lens separated from the surface ectoderm at later stages. In some of these specimens, the lens and other contents of the optic cup and fissure were dissected out. After the specimens were dried and mounted, additional mesectoderm was either picked, brushed or blown away to adequately expose the neuroectoderm. Results The neural portion of the developing visual system is evident morphologically on day 11 of gestation in Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 No. 8 NEUROECTODERM OF THE EMBRYONIC EYE / Morse and McCann 901 Fig. 2. A posterior view of the left portion of the specimen in Figure 1 shows a well-delineated optic vesicle (OV) and the early indication of the narrower optic stalk (OS). It is common for mesectodermal elements to be more adherent to the optic vesicles than to other portions of the neuroectoderm at this stage. Bar = 100 nm. the rat embryo. Mechanical removal of surface ectoderm and its underlying mesectoderm at this stage reveals that a pair of lateral diverticula are budding from the primitive forebrain. These optic vesicles initially have a broad attachment to the brain, but very soon acquire a narrowed connection termed the optic stalk (Figs. 1, 2). Cells of the mesectoderm over the optic vesicles and neuropore adhere to the neuroectoderm. On day 12, two important processes occur. The optic stalk lengthens considerably so that the optic vesicle comes to lie farther from the brain and closer to the surface ectoderm. Synchronously, the optic vesicle begins its characteristic involution to create the optic cup (Figs. 3, 4). A shallow furrow forms on the ventral surface of the optic cup. The furrow, the presumptive optic fissure, is bounded on either side by folds of neuroectoderm and filled by mesectodermal tissue. In the living state, the optic cup is occupied by the forming lens and mesectoderm. The lens vesicle develops from the surface ectoderm at this time. It is still connected to the surface ectoderm by the lens stalk and thus is most often removed with that layer. The smooth texture of the neural portion of the eye at these early stages is most likely caused by the basal lamina which covers the exposed (basal) aspect of cells of the neuroectoderm. Several changes are noted by the end of day 13. The optic stalk lengthens farther. A slightly narrower neck region connecting the stalk to the optic cup is evident (Fig. 5). We refer to this region as the collum. The lens vesicle separates from the surface ectoderm and typically remains in the optic cup when the surface ectoderm is removed. Mesectoderm occupies the remainder of the cup and also fills and camouflages the optic fissure (Figs. 5, 6). Removal of the lens and much Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 902 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1984 Fig. 3. The surface ectoderm and mesectoderm are removed from the optic cup (OC) and ventral aspect of the stalk (OS) in this specimen from a 12-day embryo. The optic fissure is present on the ventral portion of the cup. The fissure is wide and shallow with the lips of the cup which form its border (asterisks) far apart. Bar = 100 Atm. of the mesectoderm reveals the fissure as a deep slit in the area of the optic cup (Figs. 7-9). As it extends to the collum from the optic cup, the fissure becomes more shallow and ends abruptly at the junction of the collum with the optic stalk (Figs. 7, 8). The opposing lips of the fissure establish contact during day 13 (Fig. 9). On day 14, the optic fissure closes completely. All that remains of the fissure at this time is a shallow furrow on the ventral aspect of the eyeball (Fig. 10). Intraocular vessels enter the optic cup through a small persistent portion of the optic fissure in the collum (Figs. 10, II). Discussion Analysis of sectioned material for descriptive purposes always introduces the difficulty of relating the Vol. 25 Fig. 4. A lateral view of the optic cup (OC) in a 12-day embryo demonstrates the early optic fissure (OF) in the ventral wall of the cup. The lens stalk is still attached to the surface ectoderm at this stage and is removed with that layer. Mesectodermal tissues also have been removed except at the outermost rim of the cup (arrows). The basal lamina of the neuroectodermal cells of the cup give the surface a homogeneous appearance. Bar = 100 /im. information found in a given section to the entire organ or tissue. Factors contributing to interpretive problems in sectioned material include specimen orientation relative to the cutting edge, section thickness, and staining characteristics. In many instances, correlative scanning electron microscopy helps minimize some of these difficulties by presenting the three-dimensional morphology. This paper utilizes scanning electron microscopy to study the optic fissure of the rat embryo. Two contributions to the knowledge of visual system development are presented. One is technical in nature and the other clarifies a major discrepancy in the literature regarding the extent of the optic fissure. Previously published studies of the neuroectodermal portion of the developing eye, particularly at the electron microscopic level, concentrate predominantly on Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 No. 8 NEUROECTODERM OF THE EMDRYONIC EYE / Morse and McConn Fig. 5. During day 13, the optic stalk (OS) continues to elongate. The optic fissure is obscured by mesectodermal tissues (M) in this preparation. The fissure is more of a slit now as the opposing lips, which form its margins (asterisks), come closer together. The lens vesicle (L) has separated from the surface ectoderm and now typically remains in the optic cup when surface ectoderm is removed. Bar = 100 fim. the optic cup and its derivatives. The optic fissure is mentioned in passing or not at all. The dense collection of mesectodermal tissue surrounding the optic cup and stalk has forced the scanning electron microscopist to resort to random razor cuts or fractures through this organ16 or to "view" the structures based on features reflected by the surface ectoderm.17 The present paper describes a technique to remove tissues that cover the neuroectodermal portion of the forming eye. Microdissection permits a clean preparation ofjust the intact optic cup and stalk and obviously allows a much more accurate description of these structures as they change with development. This technique has been applied to descriptive studies of other developing systems with equal success.18 It may provide a mechanism for the analysis of the eye using specific labels and markers for cellular and matrical components such as those 900 Fig. 6. Mesectodermal cells (M) remain in the optic fissure (OF) during the time of closure on day 13. Bar = 5 fim. recently used by Hilfer and Yang7 and Yang and Hilfer.16 Most modern as well as classic textbooks of embryology and ophthalmology describe the optic fissure to extend not only through the optic cup but along the ventral surface of the optic stalk. The degree to which the fissure is affiliated with the stalk varies as much among authors as species studied. On the one hand, the opticfissureis said to extend the entire length of the stalk to reach or nearly reach the lateral wall of the forebrain in man,81319"22 chick,23 pig,24 or all vertebrates.25 Others report that the fissure extends only onto the distal portion of the stalk in man,14-26 chick,27 and rat and mouse.2-4 A substantial number of publications are nonspecific in their descriptions of the optic fissure relative to the optic stalk. Most simply state that the fissure extends along the ventral surface of the stalk.1015-28'32 Care must be taken in defining the parts of the neuroectodermal portion of the eye. It is the ventral portion of the optic cup that is retarded in its "growth," which Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 904 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Augusr 1984 Vol. 25 Fig. 7. Three neuroectodermal regions of the eye can be recognized at 13 days gestation: optic cup (OC); collum (C); optic stalk (OS). Some of the mesectoderm has not been removed from the proximal portion of the optic fissure (OF). The fissure extends through the cup and collum portions only. No evidence of the fissure is seen on the optic stalk. The lens has been removed from the optic cup. Bar = 100 nm. Fig. 8. The eye from a 13-day embryo with surface ectoderm, lens, and mesectoderm removed demonstrates that the optic fissure (OF) is restricted to the optic cup (OC) and collum (C). No evidence of the fissure is found on the optic stalk (OS). Bar = 100 jjm. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 No. 8 NEUROECTODERM OF THE EMBRYONIC EYE / Morse and McConn 905 Fig. 9. The surface ectoderm, mesectoderm, and lens have been removed from this 13-day specimen, which is viewed from the lateral aspect. The optic cup (OC) is deep and lips of the optic fissure (OF) appear to be in contact at some points on the ventral portion of the cup. Bar = 100 pm. leads to the formation of the optic fissure. If one observes only the ventral aspect at these stages, the initial tendency is to classify the region that contains the proximal portion of thefissureas optic stalk. However, careful analysis of the entire optic cup/optic stalk transition region reveals that the great majority of the fissure is associated with the cup. The, narrow segment, which connects the optic cup and stalk, has been recognized only recently. Its formation is quite likely due to a focus of cell death since this region corresponds to the constricted and necrotic zone (necrotic area 6) in the C57BL-6J mouse embryo.9 We have termed this region the collum for two reasons. First, this portion of the neuroectodermal outgrowth from the diencephalon possesses the characteristics implied by this term, ie, a constricted portion of any organ or structure that connects two parts. Second, this is the only portion of the neuroectoderm outside of the optic cup that contains optic fissure. This distinguishes it from the optic stalk. With this classification of the parts of the early neuroectodermal eye, some of the apparent disparity in the literature regarding the optic fissure is explained. This is especially true for those authors who describe a fissure only on the distal stalk.2<4>14-26-27 it is likely these authors were referring to the collum. The possibility of variation in the optic fissure location due to species differences must be considered. This potential is less credible, however, when it is realized that major discrepancies exist for descriptions of the same species by different laboratories. Planes of section may account for some of the disagreement. The direction of the fissure in the collum is very oblique. Thus, the fissure is a shallow groove in the proximal neck and a deep cleft in the distal portion. This produces a furrow through which vasoformative tissues that give rise to the intraocular vessels enter the developing eyeball without crossing the rim of the optic cup and without piercing epithelial layers. As indicated by Mann13 and Silver and Robb,2 the same effect is created for the axons of retinal neurons that leave the optic cup to form the optic nerve and establish connections in the brain. The mechanism by which the central canal of the optic stalk is occluded is unknown. It is implied in most works that the infolding of the stalk to create the opticfissurecontributes to the narrowing and eventual loss of this communication between the cavity of the Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 906 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1984 Fig. 10. The optic fissure is closed by day 14. The remnant of the fissure is a shallow groove (arrowheads) on the ventral aspect of the optic cup. The optic stalk (OS) is fractured. An intraocular vessel (arrow) enters the optic cup through a persistent portion of the optic fissure in the collum. This area is illustrated in Figure 11. Bar = 100 jim. Fig. 11. Higher magnification of the area indicated by the arrow in Figure 10 demonstrates a cross-sectional fracture of an intraocular vessel as it traverses a small patent portion of the optic fissure to enter the optic cup. This patency is in the collum. Bar = 10 ^m. optic vesicle and primitive ventricular system of the brain. Evidence from the rat embryo in this paper shows that is not the case. A reevaluation of the components of the optic stalk and their fate and contribution to eye morphogenesis is needed. 6. Key words: optic cup, optic fissure, scanning electron microscopy, rat embryo, neuroectoderm 8. References 9. 1. Spemann H: The development of the vertebrate eye as an example of a composite organ. In Embryonic Development and Induction. New York, Hafner Publishing Company, 1967, pp. 40-76. 2. Silver J and Robb RM: Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia. Dev Biol 68:175, 1979. 3. Glucksmann A: Cell deaths in normal vertebrate ontogeny. Biol Rev 26:59, 1951. 4. Silver J and Hughes AFW: The role of cell death during morphogenesis of the mammalian eye. J Morphol 140:159, 1973. 5. Mund ML and Rodrigues MM: Embryology of the human retinal Vol. 25 7. 10. 11. 12. 13. pigment epithelium. In The Retinal Pigment Epithelium, Zinn KM and Marmor MF, editors. Cambridge, Harvard University Press, 1979, pp. 45-52. Hay ED and Revel J-P: Fine Structure of the Developing Avian Cornea. New York, S Karger, 1969, pp. 4-116. Hilfer SR and Yang J-JW: Accumulation of CPC-precipitable material at apical cell surfaces during formation of the optic cup. Anat Rec 197:423, 1980. Duke-Elder S and Cook C: Normal and abnormal development. In System of Ophthalmology, Duke-Elder S, editor. St. Louis, CV Mosby, 1963, pp. 29-48. Silver J and Hughes AFW: The relationship between morphogenetic cell death and the development of congenital anophthalmia. J Comp Neurol 157:281, 1974. Jackson CG: Prenatal development of the eye in the golden hamster. Am J Anat 146:303, 1976. Jackson CG: Prenatal development of the microphthalmic eye in the golden hamster. J Morphol 167:65, 1981. Ulshafer RJ and Clavert A: Cell death and optic fiber penetration in the optic stalk of the chick. J Morphol 162:67, 1979. Mann IC: A general outline of the development of the optic vesicle and the associated mesoderm. In The Development of the Human Eye. Cambridge, University Press, 1928, pp. 14-45. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017 No. 8 NEUROECTODERM OF THE EMDRYONIC EYE / Morse ond McConn 14. O'Rahilly R: The early development of the eye in staged human embryos. Contrib Embryol 38:1, 1966. 15. Geeraets R: An electron microscopic study of the closure of the optic fissure in the golden hamster. Am J Anat 145:411, 1976. 16. Yang J-JW and Hilfer SR: The effect of inhibitors of glycoconjugate synthesis on optic cup formation in the chick embryo. DevBiol 92:41, 1982. 17. Kaufman M: Cephalic neurulation and optic vesicle formation in the early mouse embryo. Am J Anat 155:425, 1979. 18. 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Walls GL: The genesis of the vertebrate eye. In The Vertebrate Eye. Bloomfield Hills, MI, The Cranbrook Press, 1942, pp. 104109. 26. Woollam DHM: Embryology. In Modern Ophthalmology, Sorsby A, editor. Philadelphia, JB Lippincott, 1972, pp. 171182. 27. Romanoff AL: The organs of special sense. In The Avian Embryo. New York, Macmillan, 1960, pp. 381-384. 28. Keibal F: The development of the sense organs. In Human Embryology, Keibal F and Mall F, editors. Philadelphia, JB Lippincott, 1912, pp. 218-230. 29. Last RJ: Development. In Eugene Wolff's Anatomy of the Eye and Orbit. Philadelphia, WB Saunders, 1968, pp. 419-429. 30. Hendrickx AG, Bollert JA, and Houston ML: Description of stages XV, XVI, XVII and XVIII. In The Embryology of the Baboon, Hendrickx AG, editor. Chicago, The University of Chicago Press, 1971, pp. 103-125. 31. Trevor-Roper P: Embryology. In The Eye and Its Disorders. Oxford, Blackwell Scientific Publications, 1974, pp. 76-79. 32. Kuwabara T: Development of the optic nerve of the rat. Invest Ophthalmol 14:732, 1975. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933348/ on 06/17/2017