Photopigment gene expression and rhabdom formation in the crayfish (Procambarus clarkii).

This study examines the expression of the photopigment gene in the developing retina of the freshwater crayfish Procambarus clarkii(Crustacea, Malacostraca, Decapoda). Both sense and anti-sense RNA probes were used for in situ hybridization (ISH) of whole embryos collected at various stages during development. A characteristic of retinal development is the formation of screening pigment in the retinular cells of the retinal ommatidia. This pigmentation is seen as a band that begins at the lateral side of the retinal field and progresses medially. At hatching the retina is approximately 50% pigmented. ISH of whole embryos shows that expression of the photopigment gene by the retinular cells correlates with the extent of the screening pigment band in the retina and with the presence of rhabdoms within the ommatidia. Sections taken through embryos after being hybridized indicate that staining is localized in the cytoplasm of the retinular cells and in the axonal region below the basement membrane. No staining reaction was seen in the rhabdoms of older ommatidia. ISH staining was also seen at the anterior midline of the protocerebrum where extraretinal photoreceptors have been reported. The data presented here show a close correlation of opsin expression within the retinular cells of the ommatidia and the formation of the very early rhabdoms, similar to Drosophila. The results will be discussed in relation to recent studies in Drosophila that suggest rhodopsin plays a role in effecting the organization of the terminal web-like cytoskeleton at the base of the developing rhabdom microvilli.

Retinal development in the lobster Homarus americanus. Comparison with compound eyes of insects and other crustaceans.

Pattern formation and ommatidial differentiation were examined in the developing retina of the lobster Homarus americanus using light and electron microscopy. In the lobster the retina differentiates from the surface ectoderm that covers the optic primordia. Initially a single band of proliferation moves across this surface ectoderm. Immediately following this wave of proliferation, rows of ommatidial cell clusters appear. The earliest cell clusters are often seen adjacent to dividing cells of the proliferation band. The changing organization of the first seven rows of ommatidial clusters, visible at the surface of the retina, reveals events in early ommatidial differentiation. A rosette-like cluster of 18 cells forms the first row. Each stage following the rosette clusters occurs in a separate staggered row. Developing ommatidia have a central cluster of retinula cells, whose organization changes at each stage. Four cone cells enclose the retinula cells in each cluster and extend to the surface. In the seventh row, rhabdome formation begins and the retinula cells recede, leaving only cone cells visible at the retinal surface. This change initiates the two-tiered organization of the adult ommatidium. In 70% embryos, asymmetries in the position of the R8 axon around R7 create an equatorial line separating the dorsal and ventral halves of the retina. Possible mechanisms for the formation of these asymmetries are discussed. Postembryonic growth of the retina continues in stage VI juvenile animals along the ventral edge of the retina.

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii.

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8-10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.

Localization of actin in the retina of the crayfish Procambarus clarkii.

The distribution of actin in the retina of the crayfish was investigated at the LM level using FITC-phalloidin. Fluorescent staining was associated with the main rhabdom and eighth cell rhabdom, the zonula adherens junctions between retinula cells, and the basement membrane of the retina. EM and S1 decoration were used to confirm the presence of actin and identify its structural relationships. Phalloidin staining of the rhabdom and S1 decoration of actin filaments in the rhabdom microvilli confirmed earlier findings that actin is a component of the microvillus cytoskeleton in the crayfish. At the zonula adherens junctions, actin filaments, identified by S1 decoration, run longitudinally within the plaque of the junction. At the extreme proximal end of the rhabdom, actin filaments associated with the junctions fill each small area of retinula cell cytoplasm. In the basement membrane, EM and S1 decoration show that basilar cells contain large bundles of actin filaments which are associated with cell-matrix adherens junctions. Foot cells which lie immediately below the rhabdom also contain similar junctions and actin is tentatively identified in these cells. The functional role of actin at these various locations is discussed in relation to retinal organization in the crayfish and other invertebrates.

Changes in the microvillus cytoskeleton during rhabdom formation in the retina of the crayfish Procambarus clarkii.

Changes in the microvillus cytoskeleton during the formation of the light-receptive rhabdom in the crayfish retina were examined at four structurally distinct stages. The cytoskeleton of microvilli in early rhabdoms is composed of a regularly packed bundle of 12-25 actin filaments. The polarity of S1 decorated filaments indicates that the plus end of the actin filaments is located at the microvillus tip. The hexagonal packing of filaments within the bundle, their spacing, and the presence of cross-striations along the bundle in longitudinal sections indicate the filaments are held together by cross-linking proteins. Electron microscopic observations and data from three-dimensional reconstructions of individual microvilli indicate that the filaments arise from a concentration of dense material at the tip of the microvillus and extend into the cytoplasm as a rootlet. Over the four developmental stages examined there is an increase in the number of microvilli forming the rhabdomeres and a 50% decrease in the mean cross-sectional area of individual microvilli. During this same period the number of actin filaments forming the microvillus cytoskeleton also decreases. Following this decline, microvilli of late stage rhabdoms, which are structurally similar to adults, contain only two to four filaments. These changes are discussed in relation to the three phases of growth described for stereocilia and brush border microvilli.

Effect of rigid gas permeable contact lens wetting solutions on the rabbit corneal epithelium.

This investigation was designed to compare the effects of three rigid gas permeable (RGP) contact lens solutions on the rabbit corneal epithelium. Boston Advance Conditioning Solution, Boston Conditioning Solution, and Allergan Wet-N-Soak, which are preserved with 0.0015% polyaminopropyl biguanide, 0.006% chlorhexidine gluconate, and 0.003% benzalkonium chloride, respectively, were evaluated by scanning (SEM) and transmission electron microscopy (TEM). Our results show that Boston Advance Conditioning Solution is significantly more toxic to the corneal epithelium than either Boston Conditioning Solution or Allergan Wet-N-Soak Plus. This is presumably due to the presence and concentration of the preservative, 0.0015% polyaminopropyl biguanide. Although this study was conducted using rabbits, the results raise clinical concerns for human RGP contact lens wearers.

The diurnal pattern of protein and photopigment synthesis in the retina of the crayfish, Procambarus clarkii.

The interrelationship between the diurnal cycle of membrane loss and synthesis of new rhabdom components remains a key element in forming a complete picture of the turnover of photopigment-containing membrane in the crayfish photoreceptor cell. In order to examine this aspect of the turnover process, the diurnal pattern of photopigment synthesis was examined using an in vitro incubation system for incorporation of 3H-leucine into photoreceptor protein. The incorporation of 3H-leucine into total protein and photopigment specifically was measured in photoreceptors isolated from incubated retinas. The results indicate that for both total protein and photopigment there is no significant variation in the rate of synthesis during the 12-12 light-dark cycle. These data combined with earlier data on diurnal membrane loss from the rhabdom suggest that light-stimulated rhabdom membrane loss is superimposed on a diurnally constant level of synthesis and assembly of new rhabdom constituents.

Regional morphological variations within the crayfish eye.

The existence of structural asymmetries has been quantitatively demonstrated in the crayfish compound eye. Variations in the size of the rhabdomes and corneal facets, as well as the size and extent of the accessory reflecting pigment cells, have been found. It was determined that the mean rhabdome diameter within a 70 degrees arc in the dorsal quadrant of the retina is 11-19% smaller than the mean rhabdome diameter in the remaining areas of the eye. Also, the extent of the accessory reflecting pigment cells is diminished over an area corresponding generally to the dorsal region of smaller rhabdomes. Corneal facet size and shape vary over the surface of the cornea, with smaller facets occurring in the dorsal region. Both the mean rhabdome diameter and the mean corneal facet area for whole eyes increases linearly in animals ranging in size from 3.9-12 cm. The estimated number of corneal facets, and therefore the number of rhabdomes, increases from an average of 4700 in the 3-6.9 cm size range to about 6000 in 7-12 cm animals. These data indicate that structural asymmetries and various size-related parameters exist in the crayfish eye and should be considered in any quantitative analysis of this structure.

Development of the crayfish retina: a light and electron microscopic study.

The development of the crayfish retina was examined in embryos and first, second and third instars with both and light and electron microscope. Light microscopic observations indicate that differentiation begins at the posterior portion of the optic disc and progresses in an anterior direction. Development of screening pigment, dioptric elements, and rhabdoms all parallel this posterior to anterior gradient in the retina. Tracer studies in early embryos reveal that the retina is separated from the proximal neuropil regions by a distinct vascular space. This observation suggests that the source of new cells for the retina may not be the more proximal cell proliferation zone as previously indicated. It is proposed that mitotic activity within the retina and/or differentiation of cells from the anterior surface layer of the eye may be sources for addition of new cells to the retina. Proto-ommatidial clusters of seven retinula cells occur very early at the posterior region of the embryonic retina. Initially the receptor cells extend throughout the entire thickness of the retina, but later they withdraw from beneath the cornea to occupy only the proximal portion of the retina. Microvilli of the rhabdom arise from the centrally opposed membranes of the retinula cells in each cell cluster. Each new microvillus contains a core of fine filaments which extend out into the cytoplasm at its base. As development of the microvilli continues, the core filaments appear to be lost or altered, but the cytoplasmic bundles at the base of the microvilli persist.

Diurnal changes of lysosome-related bodies in the crayfish photoreceptor cells.

The effect of illumination on the degradation of microvillar membrane in the invertebrate photoreceptor cell has been correlated with the appearance in the cytoplasm of certain distinct lysosome-related bodies. Three types of organelles were distinguished in the retinula cell cytoplasm of the crayfish, multivesicular bodies (MVB), both large (.20-1.50 micron) and small (1.49-0.30 micron), combination bodies (CB), and lamellar bodies (LB). Under diurnal lighting conditions significant temporal differences were found in the appearance of these three classes of organelles in the retinula cell. Small MVB are present at a consistent level throughout most of the diurnal cycle but show peak numbers at 30 min after light onset and again after 6 h of dark adaptation. Large MVB increase significantly 1 h after light onset and remain elevated through 4 h in the light. After 4 h the large MVB decline gradually for the remaining light period. Combination bodies and LB do not begin to increase until 1 h after light onset and are at peak levels between 4 and 6 h into the light period. The minimum rhabdome diameter coincides with the peak levels of large MVB, CB, and LB. These data support support the hypothesis that light causes microvillar membrane breakdown, resulting in the initial production of MVB which in turn undergo degradation to form CB and finally LB. This primary degradative response appears to be completed within the first 8 h of the light period.

The distribution of 3H-leucine labeled protein in the retinula cells of the crayfish retina.

The synthesis and distribution of 3H-leucine labeled protein was studied under conditions of diurnal lighting in the retinula cells of the crayfish retina with both light and electron microscopic autoradiography. Times ranging from two minutes to seven days after an intracardiac injection were analyzed. Quantification of the electron microscopic autoradiograms revealed that labeling of the cytoplasm was greater than the rhabdome at 2, 5, and 30 minutes and reached a peak at 12 hours. The rhabdome showed increasing activity after 5, 30, and 60 minutes, also reaching a peak at 12 hours. Radioactive label in cytoplasmic multivesicular bodies was higher than activity measured in either the total cytoplasm or rhabdome at all times except two minutes. Two temporally different microvillar labeling patterns were seen under diurnal lighting conditions. (1) Microvilli forming the slightly enlarged distal tip of the rhabdome retained their radioactivity at 1, 3, and 7 days, when labeling of the rest of the rhabdome microvilli was decreasing. (2) In the remainder of the microvilli, labeling at 1 and 12 hours appeared as a gradient which declined toward the proximal end of the rhabdome. This gradient subsequently reversed itself, showing heavier proximal labeling at three days. In a second experiment, labeling patterns in light and dark adapted rhabdomes were compared. In the dark, a distinct gradient of activity was observed with radioactivity concentrated distally and declining toward the proximal end of the rhabdome. A more even distribution of label was present in the light adapted eye, but a slight distal-proximal gradient was still present. The dark adapted rhabdomes had more radioactivity per unit area than those exposed to light.

Neurofilament and glycogen changes during cold acclimation in the trochlear nucleus of lizards (Sceloporus undulatus).

In lizards (Sceloporus undulatus), long term (13 or 19 weeks) acclimation to an environment of 6 degrees C produces a striking increase in the argyrophilic neurofibrillar network in most large perikarya of the trochlear nucleus. In electron micrographs the cells contain numerous bundles of 10-30 regularly-spaced 90 A neurofilaments. In the cells from warm acclimated animals, a plexus of neurofibrils is seen by light microscopy. The electron micrographs show scattered neurofilaments and fewer, thinner bundles than in the cold. Within the cell bodies of the cold animals, glycogen particles are organized in regional accumulations from which other organelles are excluded except for the bundles of neurofilaments which are distributed throughout the cytoplasm. The aggregations of rough endoplasmic reticulum (RER) are also penetrated by the neurofilament bundles. The increased neurofilamentous network in the cold is not accompanied by obvious changes in the amount or distribution of RER or of microtubules which are present in limited numbers in both conditions. The dendrites of trochlear cells and axon terminals within the nucleus also show a cold induced increase in neurofilaments, as well as in the distinctive accumulations of glycogen particles.