Regeneration of tissue occurs by a variety of means. In many invertebrates, regeneration of whole tissues occurs by a determination and differentiation of quasi-embryonic population of cells. Examples of this are the differentiation of interstitial cells and neoblasts in Hydra and Planaria (Goss1969). In vertebrates, specialized populations of cells are also involved in the regeneration of many organs and tissues; for example, satellite cells which give rise to mammalian muscles, and chondrogenic blastemic cells that give rise to cartilage (Carlson, 1981). However, examples of organisms with the capacity for replacement of external sensory organs are extremely limited. Many amphibians have the ability to replace tails, limbs, a complete eye lens and a large portion of the retina from remaining pigmented epithelium. However, none are able to replace the loss of an entire eye (Ross, 1964).

Eakin and Ferlatte (1973) found that the Garden Snail (Helix aspersa) had the ability to regenerate a complete functional eye 32 days postamputation. The process of regeneration occurred through the mid-eye stalk and began by an invagination of integumentary epithelium at the apex of the eyestalk stump to produce a shallow cleft or "eye cup." Differentiation of all components of the eye occurred by transdetermination of these epithelial cells.

The eye of the Garden Snail is at the tip of the cephalic eyestalk and has following principle features: Transparent cornea which is continuous with the capsule of the eye; a large, secreted lens (about 150um in diameter); and a retina composed of four strata; villous, pigmented, somatic and neural (Brandergbuger, 1967.)

The principle component of the villous layer of the retina is the microvilli that extend from the dome-shaped distal end of the receptor cells to the undersurface of the lens. The villi touch the surface of the lens, except the short lateral ones. The tip of those that reach the lens appear bent, possibly due to hydrostatic pressure in the lens. Spaces between the microvilli and the lens are filled with a finely granular humor. It is not whether the microvilli are the light-sensitive organelles, but the information available on their structure, physiology, and biochemistry strongly supports that they contain a photopigment (Branderbuger and Eakin, 1967.)

The pigmented layer is composed primary of support cells containing many black granules concentrated distally , and their nuclei lie in the somatic layer of the retina. The supportive cells lie laterally to the two types of sensory cells in the pigmented layer of the retina, and all are bounded together by zonula adhers junctions near their distal ends (Eakin and Brandenburger, 1975.) The neural layer is composed of relatively scarce (about 12 per eye), and are located in the basal half of the retina (somatic and neural layer). Neurites from the photosensory cells and those from the ganglion cells pass into the neural layer of the retina, concentrate at or near the optic axis, and enter the neural swelling at the head of the optic nerves. The capsule of the eye contains glial and muscular cells that embedded in a collagenous matrix (Ferlatte and Eakin, 1973).

Eye regeneration in snails shares interesting similarities to the well-studies regeneration of amphibian limbs, that is dependent on an intact nerve supply (Eakin and Ferlatte, 1973) and the eye regenerates in the presence of applying a steady ionic current to the eyestalk ( Bever and Borgens, 1987). However, it is not known that whether light is a directional stimulus on the organization of the stratified layer of cells of the regenerating eye, and the role of this directional cue on the regeneration process forms the major goal of this paper.

Twenty garden snails were caught on the campus of C.S.U Long Beach. The snail shells were numbered with nail polish, and they were divided into two groups; ten snails were put under the light, ten were put in the dark. The eyes of these snails were cut through the eyestalk by a very sharp knife, and these eyes used to study the structure of normal eye. The snails were fed with pieces of lecttuce, chalk and maintained at moisture environment. Both groups of the snails were allowed to regenerate their eye in 25 days.

Transmission electron microscope(TEM) and light microscope preparation:

The eyes were fixed immediately with 2% glutaraldehyde(pH=6.7) in Milliionigs phosphate buffer for one hour. Following the primary fixation, the eyes were rinsed in 3 time ten minutes changes of phosphate buffer . They were then secondarily fixed in 1% osmium tetroxide in phosphate buffer for one hour following by rinsing 3 time ten minutes changes of buffer. Osmium tetroxide worked as a secondary fixative of tissue structure reacting primarily with lipid moieties. This oxidized heavy metal added density and contrast to the biological tissues(Ressel, Bozzika 1992). The eyes were dehydrated with a cold graded ethanol series, and following by for fifteen minutes changes of 100% ethanol, two fifteen minutes changes of propylene oxide, and then infiltrated with Spur resin epoxy for period of sixteen hour. The purpose of fixation was to protect tissues against disruption during embedding and sectioning and exposing to the electron beam. The eye were orientated laterally in flat embedding molds and cured in a vacuum oven set at 65c.

Thick sections and thin sections for light microscope and transmission electron microscope were prepared by using glass knives on a Porter-Blum MT-2 ultramicrotome. The thick sections were stained with freshly filtered 0.5% toluidine bllue in 1% borax for light microscope, and thin sections were double stained with Lead Citrate a Uranyl Acetate (Ressel, Bozzole, 1992). Transmission electron micrograph were taken using JEOL 1200 EX II transmission electron microscope operated at 80kv.

Scanning electron microscope preparation:

The eyes were frozen in liquid nitrogen after they were fixed through primary fixation and dehydrated through series of alcohol. They were cut through the optic axis of the eyes while they were immersing in the liquid nitrogen. The specimen was dried by critical point drying. This technique was successful because, at the critical point, the specimen was totally immersed in a dense vapor phase devoid of the damaging liquid/air interface one wished to avoid (Ressel, Bozzika, 1992). The eyes were attached to a metalic stub, and then coated with gold-palladium by a sputter coating. The metal coating prevented the buildup of high voltage charges on the specimen by conducting the charge to ground (Ressel, Bozzola, 1992). Moreover, the metal coating served as a good sources of second electron. The eyes were observed under The Scanning Electron Microscope that was operated at 25kv.

Anatomy of the intact eye

The eye of Garden Snail is located at the anterior tip of the cephalic eyestalk (Fig.1). The tissuses which allow light to enter the eye are situated at the apex of the eyestalk, surrounded by eyestalk epithelum, with an acellular lens beneath (Fig.2). The spherical lens is surrounded by the retina. The retina has four layer as observe under the light microscope: an inner villus layer is about 4 um in length, a prominent pigment layer which contains spherical black pigments about 0.3um in diameter, a somatic layer and a neural layer. The neural components project through the optic nerve (Fig.3). Thus, the general anatomy of the control eyes of this Garden Snail follows the general description of ealier studies (Eakin and Brandenburger, 1967; Ferlatte and Eankin, 1975).

Figure1: The SEM photo of the eyestalk (longitudinal section). (x42)

Figure2: The SEM photo of the eye (longitudinal section). (x50)

Figure3: Light micrograph of a thick section (longitudinal section) of a normal eye. (CO) cornea; (L) lens; (NF) nerve fiber (NS) neural swelling; (1) villous; (2) pigmented; (3) somatic; (4) neural.

Ultrastructure of regenerating eyes

Figure 4: SEM photo of microvilli of the retina (X42). (MV) microvilli, (L) lens

Although cellular regeneration is observed 25 days postamputation in the animals maintain in the presence of light and absence of light, the regeneration is incomplete in both case.

Transmission electron microscopy of the regenerating eyes of animals expose to light reveals two types of cells in the retina: sensory and pigmented cells. These sensory cells are microvilli (mv), about 3um in length and 0.09um in diameter, extending from the outer surfaces of the receptor cells and touch the surface of the lens (Fig.5). The microvilli appears to be twisted (Fig.7 and Fig.8) which implying that the regenerating process is incomplete.

For the group of snail expose to dark, the transmission electron micrographs of regenerating eye show that microvilli and pigment granules are regenerated next to the capsule layer of the eye (Fig.10).

Numerous membrane-bound spheroid pigment granules are regenerated abundant in the supportive cells of the retina in both group of snails. However, they are lack of uniformity in appearance, and were still in the process of differentiation (Fig.9, Fig.12). The supportive cells are connected to one another and to sensory cells via long stretch of septate desmosomes (Fig.6).

In the studies of Eakin and Brandenburger (1967), they pointed out that the sources of lens material in Garden Snail derived from both retinal and corneal cells. Secretory vesicles that contained lens material original formed in the Gogi apparatus of these cells. The exuded secretion subsequently aggregated into small bodies which fused to establish the primordial lens that continued to grow by accretion. For the group of snails in the absence of light, the lens regenerated incomplete. However, there are areas that contain very dense small granules (Fig.13) that similar, and are presumed to that of lens material as described by Eakin and Brandenburger (1967).

Figure 5: TEM of 25 days of the Helix aspersa's regenerating eye showing the basic structure of a normal eye (X12). (MV) microvilli (PG) pigment granules. Scale bar: 167microns.

Figure 6: TEM of 25 days regenerating eyes which show microvilli and suupportive cells. (X40,000). Scale bar: 100 nm.

Figure 7: TEM of microvilli that were still in the process of differentiation, (X2500) (see arrow) (MV) microvilli; (H) holes in the tissue which was the result of incomplete fixation. Scale bar:1.0um

Figure 8: TEM of microvilli that were still in the process of differentiation. Scale bar:150nm.

Figure 9: TEM of pigment granules that sill in the process development. See arrow;(m) mitochondria; (PG) pigment granules (X12,000). Scale bar:1microns.

Figure 10: TEM of 25 days regenerating eye of snails in hte dark. (X3000). Scale bar: 0.7 microns. (MV) microvilli in cross section; (PG) pigment granules; (CA) capsule, (C) collagen.

Figure 11: TEM of 25 days regenerating eye of snails exposed to dark (X20,000). Scale bar: 30nm.

Figure 12: TEM of pigment granules in regenerating eye's retina of snails exposed to dark which were still in the process of differentiation. (X25,000). Scale bar: 35nm

Figure 13: TEM of microvilli and dense tissues that simillar to that lens material. (MV) microvilli; (DT) dense tissues.(X30,000). Scale bar: 0.9nm.

Electron micrographs of regenerating retina of snails in the presence of light revealed all components typical of a normal retina; lens, microvilli, supportive cells associated with pigment granules. The narrow, long regenerating microvilli extended from the dome-shaped distal end of the receptor cells to the undersurface of the lens. Numerous spheroid pigment granules regenerated on the distal halves of the supportive cells and making an inner pigmented zone of the retina. The orientation of these regenerating microvilli and pigment granules was similar to the result of studies of Eakin and Ferlatte (1973) on normal eye regeneration in Garden Snail.

However, comparision of the orientation of the regenerating cells in the retina of snails maintained in the absence of light differed from those exposed to light. Thus, the microvilli and pigment granules from animals kept in the absence of light regenerated toward the collagenous capsule layer of the eye instead of being orientated towards the lens as those in the presence of light (Fig.10). This difference implied that light acted as a directional cue on the orientation of regenerating cells in the retina of Garden Snail.

Eakin and Ferlatte (1973) concluded that the gastropod eyes regenerated in responding to a stimulus provided by the cut end fo the optic nerve. Although this nerve was degenerating, its presence was essential to the formation of the new eye. It appeared that the axon of the regenerating eye grew down the path way of the optic nerve to make contact with the cerebral ganglion. They concluded that a regenerating eye would not become functional until the new nerve had become joined to the brain. In the current study, it was not known that whether the regenerating eye of Garden Snail in 25 days period became functional because the limited scope of this project did not permit an assessment of the role of nerve cells in the regenerating process.

Moreover, it was not possible to assess the effects of light on the quantity of microvilli and pigment granules between two group of snails due to problems of incomplete infiltration; there were holes in tissues.

In conclusion, this study revealed that light appeared to be a strong directional cue in determining the orientation and reorganization of regenerating cells of the retina of the Garden Snail. The differentiation of retinal cells tended to develop a normal regenerating eye in the presence of light and abnormal in the absence of light. With further studies of Garden Snail, more differences may be find between the ultrastructure of regenerating eye of snails in the presence of light and the absence of light. At this point, I hope that further studies with wider range of time sample would need to be done to study the effects of darkness on the differentiation and orientation of the retinal cells of Garden Snail, to accesses more usefulness information about this species.

Calson, B.M. (1981) The regeneration of transplantation of entire skeletal muscles in mamals. In: machanism of Growth Control. R.O. Bedker, ed. C.C. Thomas, Springfield, Illinois, pp.27-53.

Eakin, R.M., and J.L. Brandenburger (1967) Differentiation in the eye of a pulmonate snail Helix aspersa. J. Ultrastruct. Res., 18:391-421.

Eakin, R.M., and J.L. Brandenburger (1975) Understanding a snail's pace. Am. Zool. 15:851-863.

Eakin, R.M., and Ferlatte (1973) Studies on eye regeneration in snail, Helix aspersa . J. Exp. Zool., 184:81-96.

Gos, R. J. (1969) Principles of regeneration. Academic Press, New York.

Ross, S.M. (1964) Regeneration. In: Physiology of the Amphibia. J.A. Moore, ed. Academic Press, New York, pp. 545-622.