Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

The fine structure of limb tissues of the adult newt, Diemictylus viridescens

The limb tissues of the adult newt investigated for their fine structure include epidermis, subcutaneous glands, dermis, striated muscle, peripheral nerves and blood vessels. This survey complements and extends previous observations, emphasizing intercellular junctions, and the ubiquitous “glycocalyx” (= polysaccharide-protein lamella, around cells and adjacent to epithelia). Our survey touches on the characteristic tonofilaments, intercellular desmosomes and basal hemidesmosomes of the epidermis. The subcutaneous glands consist of secretory cells with a granular product, and myoepithelial cells; intercellular desmosomes are present. The adepidermal reticulum of collagen fibrils reveals periodic regions of intersecting fibrils ( = nodules), and fibril continuity with the underlying dermis: a striking feature is the adipose tissue closely applied to the adepidermal reticulum. The limb striated muscle displays typical banded myofibrils, and a triad system with centrotubules in the I-band close to the Z-band: terminal sacs of sarcoplasmic reticulum complete the triad system. A particularly prominent glycocalyx is applied to the surface of the sarcolemma. The peripheral nerves of the limb possess connective tissue sheaths with prominent vesiculation of the cell membranes, and an occasional intercellular desmosomal junction. Blood vessels typically have endothelial cells with prominently vesiculated plasma membranes. This investigation serves as the basis for recognizing the fine structure of tissue responses to trauma, their repair, and regeneration.     

Cellular cooperation in hapten-carrier responses in the newt, Triturus viridescens

Heterologous erythrocyte responses in the three hematopoietic organs, spleen, liver and kidney, were studied in the American Common Newt, Triturus (Diemictylus) viridescens. These responses were measured by immunocytoadherence. Horse (HRBC) and sheep (SRBC) red cells were used as immunogens. Background and response values to HRBC were consistent in all three organs. Activity levels and their kinetics were greater in spleen than in liver or kidney. Anti-SRBC activity levels proved to be too variable and as a consequence HRBC's were used in subsequent studies. Secondary responses in the newt showed modest increases in the spleen, a decrease in liver activity levels and a proportionately large (5×) increase within kidney. While splenectomy prior to immunization did not effect primary response activity in liver and kidney, it caused a three-fold enhancement of the usual loss of activity in secondary responses within liver. Kidney secondary response levels were unaffected by prior splenectomy. Spleen-liver lymphoid traffic may be involved in anamnestic responses in the newt.The main question had to do with whether one could demonstrate cellular cooperation in immune responses in so primitive a vertebrate. Chicken (CRBC) and toad (TRBC) red cells were used as carriers for the hapten trinitrophenol (TNP). Pre-immunization with the carrier immunogen led to the development of rosette forming cells (RFC) specific for the hapten in spleen, liver, and kidney after challenge with the TNP on the same carrier. Injection of the TNP carrier alone or pre-immunization with a different carrier caused no TNP-specific RFC's to be generated. The anti-TNP responses were assayed with HRBC and TNP-HRBC. Prior treatment with TNP-HSA (human serum albumin), before TNP-HRBC assay, blocked the RFC with TNP-HRBC and helped to establish the specificity of the hapten-specific RFC's. These kinds of immune responses in the newt require at least two cooperating cellular populations. Cellular cooperation may have been an early phylogenetic feature of immune responses.     

Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens).

Two non-glial phagocytes were found to participate along with ependymoglial cells in Wallerian degeneration of the severed optic nerve of the newt (Triturus viridescens). The first type of non-glial cell (polymorphonuclear phagocyte) was positively identified as a neutrophil and participates in the early stages of degeneration. Cells of this type make a brief appearance, reaching a peak by the second postoperative day (2 p.o.d.), and quickly diminish until few can be found by 4 p.o.d. Neutrophils invade the degenerating optic nerve from surrounding connective tissue spaces, most likely, through channels which penetrate the nerve parenchyma. The second type of non-glial cell is an invading mononuclear phagocyte which exhibits characteristics of microglial cells reported in other vertebrate species. Such cells appear in the nerve much later than the neutrophils and towards the end of Wallerian degeneration (6-10 p.o.d.). Their mode of entry and exit appears to be the same as that reported for neutrophils. The neutrophils and microglial-like, mononuclear phagocytes may serve to supplement the histolytic action of the ependymoglial cells, picking up scattered fragments of degenerating myelin and axons.     

A cytochemical study of myeloid bodies in the retinal pigment epithelium of the newt Notophthalmus viridescens

Summary It has been suggested (Yorke and Dickson 1984) that myeloid bodies (MBs) in the retinal pigment epithelium (RPE) of the newt, Notophthalmus viridescens, may represent areas of endoplasmic reticulum where lipids, such as 11-cis retinal derived from phagocytized outer segment tips, accumulate prior to esterification. Experiments in which an artificial ester substrate was added during in-vitro incubations have shown that esterase activity is represented in all areas of the newt RPE endoplasmic reticulum, including sites adjacent to all MBs. In related tests in which the localization of enzyme activity was restricted to areas of the cell where there had been accumulations of naturally-occurring (endogenous) esters, the products of ester hydrolysis were restricted to profiles of endoplasmic reticulum associated with lipid droplets, and with the interior of about 20% of those MBs that appeared completely circular in sections. This enzyme activity was not associated with other MB configurations. Results from endogenous-ester hydrolysis were identical to those obtained after staining with ZIO. This ZIO-reactivity was not affected by pre-incubation with agents that blocked or protected sulphydryl groups, and ZIO-reactive sites associated with MBs did not form complexes with digitonin. These observations suggest that MBs are a site of lipid-ester formation, but that they do not represent unique intracellular areas for this activity.     

The influence of the nerve on lower jaw regeneration in the adult newt, Triturus viridescens

The present investigation was undertaken in an attempt to determine the role played by the nerve in the regeneration of the lower jaw of the adult newt, Triturus viridescens. The results indicated that the number of nerve fibers normally available at the amputation surface was very low compared with that of the newt forelimb. Furthermore, denervation of the lower jaw reduced the number of nerve fibers available to an extremely low level and maintained the number at a low level for up to four weeks without intervening redenervations. The regenerative events in the denervated and amputated lower jaws were indistinguishable histologically from those in amputated jaws having normal innervation. This presented an apparent exception to the general rule that regeneration of external body parts is dependent on the nerve. Several possible explanations are proposed by which this apparent exception might be explained. The process following amputation might be an exaggerated form of wound healing and tissue regeneration which can occur in the absence of nerves. The tissues of the lower jaw might be more sensitive to the influence of those nerve fibers present. The nerve fibers themselves might be qualitatively different and thus exert a greater influence on the tissues.     

Lamellar to tubular conformational changes in the endoplasmic reticulum of the retinal pigment epithelium of the newt,Notophthalmus viridescens

Summary The retinal pigment epithelium (RPE) of the newt (Notophthalmus viridescens) was examined ultrastructurally under both in-vivo and in-vitro conditions. Five distinct conformations of smooth endoplasmic reticulum (SER), two lamellar and three tubular, were observed. The two lamellar conformations included myeloid bodies, which have previously been described (Yorke and Dickson 1984), and fenestrated SER. The latter appeared as layers of flattened or curved cisternae which were penetrated by fenestrations. Fenestrated SER became indistinguishable from the highly branched and convoluted random-tubular SER through the formation of an intermediate configuration (tubular sheets). The remaining tubular SER conformations appeared to arise from random-tubular SER through a progressive reduction in branching and a straightening of individual tubules. Fascicular SER was represented by the hexagonal organization of straight, unbranched tubules into bundles (fascicles). Spiral SER consisted of a similar hexagonal arrangement, but the unbranched tubules spiralled about one another. Neighbouring tubules in areas of spiral SER were also joined together by pairs of electrondense bars. Although lamellar (especially myeloid bodies) and random-tubular configurations of the SER were common features in vivo, fascicular and spiral SER were primarily conformations encountered in vitro. Conditions favouring bilayer lipid phases also appear to facilitate the formation of both myeloid bodies and fascicular SER. These conditions included increased duration of incubation, low (<20° C) incubation temperatures, and Ca²⁺-free incubations with EGTA. Random-tubular SER was most prevalent in media supplemented with fetal calf serum and also after warmer (30° C) incubation temperatures. We speculate that the different conformations of SER observed in the newt RPE may be due, in part, to lipid phase transitions within the membranes of this organelle. However, the specific formation of fascicular and spiral SER may also involve some additional factor, possibly a protein.Supported by grant # MT-5039 from the Medical Research Council of Canada to DHD     

The fine structure of the adepidermal reticulum in the basal membrane of the skin of the newt,Triturus

The fine structure of the tapetum of the cat eye has been investigated by electron microscopy. The tapetum is made up of modified choroidal cells, seen as polygonal plates grouped around penetrating blood vessels which terminate in the anastomosing capillary network of the choriocapillaris. The tapetal cells are rectangular in cross-section, set in regular brick-like rows, and attain a depth of some thirty-five cell layers in the central region. This number is gradually reduced peripherally, and is replaced at the margin of the tapetum by normal choroidal tissue. The individual cells are packed with long slender rods 0.1 micro by 4 to 5 micro. The rods are packed in groups and with their long axes oriented roughly parallel to the plane of the retinal surface. Each cell contains several such groups. Cells at the periphery or in the outer layers of the tapetum are frequently seen to contain both tapetal rods and melanin granules, the latter typical of the choroidal melanocytes. Also melanocyte granules may have intermediate shapes. These observations plus the similar density of the two inclusions lead to the belief that the tapetal rods may be melanin derivatives. A fibrous connective tissue layer lies between the tapetum and the retina. The subretinal capillary network, the choriocapillaris, rests on this layer and is covered by the basement membrane of the retinal epithelium. The cytoplasm of the retinal epithelium exhibits marked absorptive modifications where it comes in contact with the vessels of the choriocapillaris. This fibrous layer and the basement membrane of the retinal epithelium apparently comprise the structural elements of Bruch's membrane.