Cephalopod Lens Research Articles

Membrane structures and functional correlates in the bi-segmented eye lens of the cephalopod.

The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.

Co-option of the limb patterning program in cephalopod eye development

BackgroundAcross the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod.ResultsHere we identify the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. We show radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exd, Meis/hth, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. We assess the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and find the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss.ConclusionThis regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, our study suggests that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures.

Comparative visual acuity of coleoid cephalopods

The pelagic realm of the ocean is characterized by extremely clear water and a lack of surfaces. Adaptations to the visual ecology of this environment include transparency, fluorescence, bioluminescence, and deep red or black pigmentation. While the signals that pelagic organisms send are increasingly well-understood, the optical capabilities of their viewers, especially for predators with camera-like vision such as fish and squid, are almost unknown. Aquatic camera-like vision is characterized by a spherical lens focusing an image on the retina. Here, we measured the resolving power of the lenses of eight species of pelagic cephalopods to obtain an approximation of their visual capabilities. We did this by focusing a standard resolution target through dissected lenses and calculating their modulation transfer functions. The modulation transfer function (MTF) is the single most complete expression of the resolving capabilities of a lens. Since the optical and retinal capabilities of an eye are generally well-matched, we considered our measurements of cephalopod lens MTF to be a good proxy for their visual capabilities in vivo. In general, squid have optical capabilities comparable to other organisms generally assumed to have good vision, such as fish and birds. Surprisingly, the optical capability of the eye of Vampyroteuthis infernalis rivals that of humans.

Homology Modeling of Cephalopod Lens S-Crystallin: A Natural Mutant of Sigma-Class Glutathione Transferase with Diminished Endogenous Activity

The soluble S-crystallin constitutes the major lens protein in cephalopods. The primary amino acid sequence of S-crystallin shows an overall 41% identity with the digestive gland sigma-class glutathione transferase (GST) of cephalopod. However, the lens S-crystallin fails to bind to the S-hexylglutathione affinity column and shows very little GST activity in the nucleophilic aromatic substitution reaction between GSH and 1-chloro-2,4-dinitrobenzene. When compared with other classes of GST, the S-crystallin has an 11-amino acid residues insertion between the conserved α4 and α5 helices. Based on the crystal structure of squid sigma-class GST, a tertiary structure model for the octopus lens S-crystallin is constructed. The modeled S-crystallin structure has an overall topology similar to the squid sigma-class GST, albeit with longer α4 and α5 helical chains, corresponding to the long insertion. This insertion, however, makes the active center region of S-crystallin to be in a more closed conformation than the sigma-class GST. The active center region of S-crystallin is even more shielded and buried after dimerization, which may explain for the failure of S-crystallin to bind to the immobilized-glutathione in affinity chromatography. In the active site region, the electrostatic potential surface calculated from the modeled structure is quite different from that of squid GST. The positively charged environment, which contributes to stabilize the negatively charged Meisenheimer complex, is altered in S-crystallin probably because of mutation of Asn99 in GST to Asp101 in S-crystallin. Furthermore, the important Phe106 in authentic GST is changed to His108 in S-crystallin. Combining the topological differences as revealed by computer graphics and sequence variation at these structurally relevant residues provide strong structural evidences to account for the much decreased GST activity of S-crystallin as compared with the authentic GST of the digestive gland.

Microscopical evaluation of the crystalline lens of the squid ( Loligo opalescens) during embryonic development

The similarity between the cephalopod lens and the teleost (vertebrate) lens can be considered an optical example of convergent evolution. However, the embryology and ultrastructure of the cephalopod lens appear to be different from that of vertebrates, and perhaps unique to the animal kingdom. Using light and scanning electron microscopy, the morphogenesis of the squid (Loligo opalescens) lens is characterized. Results indicate that the posterior lens primordium appears first during development and is derived from cellular processes which extend from a middle group, group 2, of lentigenic (ectodermal) cells. The processes extend from the basal aspect of the lentigenic cells, project down into the optic vesicle during early stages of development, and fuse to form the posterior lens primordium. During later stages, the processes extend from surrounding lentigenic cells and are applied to the stalk of the lens, where they form bud-shaped protrusions. Once applied to the lens, the processes form lens elements that later fuse into plate-like elements evident in later-staged embryo and adult lenses. The anterior lens primordium is derived from an anterior group, group 1, of lentigenic cells, during later stages of development. Lentigenic processes extend from these lentigenic cells and are laid down in a circumferential fashion to form the anterior lens cap. As in the posterior lens, evidence indicates that the anterior lens elements fuse to form plate-like elements. The ultrastructure and morphogenesis of the cephalopod lens is discussed and contrasted with other strategies of lens development.

Growth and optical development of the ocular lens of the squid ( Sepioteuthis lessoniana)

Lens focal properties (spherical aberration), diameter and relative anterior/posterior proportions were measured photographically for Japanese squid ( Sepioteuthis lessoniana) at ages 4–6 weeks, 7–9 weeks and 7–8 months. The measures involved photographing the refractive effects of lenses in a physiological solution, with and without an index matching fluid (polyvinylpyrroloidone solution), on a parallel array of fine helium-neon laser beams. Spherical aberration was determined from measurement of back vertex distance. Similar measurements were made on lenses from the eyes of cuttlefish ( Sepia officinalis). The cephalopod lens develops as hemispheric halves from separate ectodermal sources. The posterior component contributes, on average, about 60% of axial lens diameter in S. lessoniana of all ages and 55% in S. officinalis. However, these proportions vary widely in both species. All lenses of both species show that spherical aberration is neutralized, although small variations in back vertex distance (positive and negative spherical aberration) were measured. Preliminary measures indicate that the refractive index distribution within the cephalopod lens varies in a manner reminiscent of teleost lenses. Squid lenses from animals 7–9 weeks of age were optimally corrected for spherical aberration. Some squid of this age, from a separate tank, showed a high incidence of cataract development. In each case, lens opacification was caused by deterioration of the posterior lens component.

Immunolocalization of S-crystallins in the developing squid (Loligo opalescens) lens.

S-crystallins are the predominant soluble proteins of the squid lens. Of these, S-III crystallin is the major component and S-I and S-II crystallin are the minor lens components. The lens has a posterior and anterior segment, each derived from separate groups of ectodermal cells referred to as lentigenic cells. In the present study, the appearance of S-crystallins during the development of the lens of Loligo opalescens was followed by immuno-cytochemistry. S-crystallins of the lens and lentigenic cells were first observed at day 17 (Arnold stage 27) of embryogenesis. S-crystallins were not confined to a single region, but were present in the middle group (group 2) of lentigenic cells, the posterior lens primordium, and the processes connecting the lentigenic cells and the posterior lens primordium. Two days later (Arnold stage 28), the S-crystallins were also observed in the anterior group (group 1) of lentigenic cells, the anterior lens primordium, and the processes connecting the cells with the anterior lens primordium. Thus, during development, S-crystallins accumulate first in the posterior lens primordium and subsequently in the anterior lens primordium and their respective lentigenic cells and connecting lentigenic processes. Incubated sections of the adult lens and lentigenic cells also show specific immuno-peroxidase staining when compared with controls. This evidence in combination with a recent investigation (West [1993] Ph.D. dissertation), which indicates that the cephalopod lens continues to grow throughout adulthood, suggests that squid lens crystallins are synthesized during adulthood.

Primary structure and lens-specific expression of genes for an intermediate filament protein and a β-tubulin in cephalopods

Intermediate filament (IF) protein and tubulin cDNAs of cephalopod eye lenses were cloned and sequenced. The rod regions of the deduced IF proteins of the squid and octopus were more similar (68% identical) than were head (33% identical) and tail (40% identical) regions. The rod sequences were closer to squid neuronal IF protein (39% identical) than to any other known IF protein. There was only 31% identity between the rod regions, 21–30% identity between the head regions and 23–32% identity between the tail regions of the present IF proteins of cephalopods and other invertebrates. The rod regions of the cephalopod IF proteins contained the 6 heptads characteristic of nuclear lamins, consistent with an evolutionary relationship between IF proteins and lamins. The present octopus α-tubulin was 93% and β-tubulin was 87% identical to the corresponding tubulins of insects and vertebrates. SDS-PAGE and peptide sequencing indicated that the order of abundance of the cephalopod lens cytoskeletal proteins was IF proteins, actin and tubulins. Northern blot hybridization revealed a 4 kb mRNA for the octopus IF protein and 2.9 and 7.3 kb mRNAs for the squid IF protein; the α-tubulin mRNA was about 1.8 kb in the octopus and squid, and the β-tubulin mRNA was about 2.8 kb in the octopus. The α-tubulin mRNA was present in all tissues examined; by contrast, the present β-tubulin and IF protein mRNAs appeared specialized for lens expression.

A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed Ω-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the Ω-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.

Electrical coupling between fibre cells in amphibian and cephalopod lenses.

The lenses of vertebrate and cephalopod eyes differ ontogenetically and in other respects. The vertebrate lens, derived from a single cell type, consists mainly of long fibre cells continuously produced by division and elongation of columnar epithelial cells near the lens equator. Almost 50% of the fibre cell surface consists of junctional complexes and the internal resistance, from point to point within the lens, is low compared with the surface membrane resistance. Thus the vertebrate lens is expected to behave as a well coupled syncytial system. The cephalopod lens, however, is formed by the fusion of two distinct cell types; the anterior segment has the same ontogenetic origin as the cornea but the posterior segment shares a common origin with the retina, and the plane of contact of the two cell types can be seen in light-microscope sections. Most of the lens is composed of long fibre cells similar in appearance to those found in the vertebrate lens, and membrane junctional regions between adjacent fibres have also been tentatively identified. We now describe electrophysiological investigations of cellular communications in the cephalopod lens, which show marked differences in the intercellular electrical coupling within the vertebrate (amphibian) and cephalopod lens.

Isofocusing and immunological investigations on cephalopod lens proteins

Soluble lens proteins from Octopus vulgaris, Sepia officinalis, and Loligo vulgaris were analyzed by thin-layer isoelectric focusing and compared by various immunochemical methods using antibodies directed against total soluble lens protein antigens from the said three species. The results show close similarity between Sepia and Loligo lens proteins. Antibodies made against Sepia and Loligo lens proteins did not react against Octopus total lens soluble protein antigens. Our results suggest a common ancestry for Sepia and Loligo which is different from that of Octopus.

A comparison of ion concentrations, potentials and conductances of amphibian, bovine and cephalopod lenses

1. The concentrations of sodium, potassium and chloride in frog and bovine lenses showed a normal intracellular ion distribution with the sum of the internal cations approximately equal to the external sum. In the cephalopod lens, however, the sum inside was much lower than that outside.2. The membrane potentials of frog, Sepiola and bovine lenses were -63, -63 and -23 mV respectively. A comparison of the electrical data with the Nernst potentials predicted from ion concentration data indicated that sodium and chloride ions as well as potassium contributed to the membrane potential in frog and bovine. In contrast, the membrane and Nernst potentials for potassium were equal in Sepiola.3. Substituting potassium for sodium in the external medium depolarized lens potentials in all three species. Estimates of the relative permeabilities of sodium, potassium and chloride were obtained by fitting the Goldman-Hodgkin-Katz equation to the potential data.4. The potassium permeability was determined directly by (42)K efflux measurements and values of 2.99, 9.83 and 3.13 (x (-8) m sec(-1)) were obtained for frog, Sepiola and bovine lenses respectively.5. The effect of raising external potassium on the efflux rate constant was determined and there was reasonable agreement between experiment and theory (Kimizuka-Koketsu) in frog and bovine lenses, but the Sepiola data indicated that the potassium permeability decreased by a factor of 2.6 when the external potassium was raised from 10 to 120 mM-K+.6. The measured specific conductances, obtained using two internal micro-electrodes, were 7.7, 15.9 and 9.9 (Sm(-2)) for frog, cephalopod and bovine lenses respectively. These data compare with computed values (Kimizuka-Koketsu theory) of 7.5, 14.1 and 17.2 (Sm(-2)).7. The effect of increasing external potassium on the conductance was also tested and there was good agreement between experiment and theory (assuming constant permeabilities) only in the amphibian lens. However, when the cephalopod data were corrected assuming a 2.6-fold decrease in P(K) for a twelvefold increase in potassium, then there was excellent agreement between experiment and theory.8. The bovine measured conductances were much lower than the theoretical values throughout the range of external potassium concentrations and several explanations were proposed to account for the discrepancies.

Cephalopod lens proteins

Cephalopod lens proteins have been studied by electrophoresis, immunological methods and ultracentrifugal sedimentation. The most important result is the systematic order difference between the decapods and octopods, as revealed by their electrophoretic eye lens patterns and their corresponding serological cross-reactions. In their sedimentation patterns, however, they show great similarity.

Fine structure of the development of the cephalopod lens

The development of the cephalopod lens involves migration and fusion of lentigenic processes which form a lens primordium which projects into the optic vesicle. The lens material is elaborated in the lens primordium and eventually the cytoplasm of the lentigenic processes is obliterated. Apparently the lens material forms from the content of Golgi vesicles which migrate from the cell body into the lens primordium. The role of the microtubules in this process is discussed.