Evolutionary developmental studies of cyclostomes and the origin of the vertebrate neck

Because they lack some gnathostome-specific traits, cyclostomes have often been regarded as representing an intermediate state linking non-vertebrate chordates and gnathostomes. To understand the evolutionary origins of the jaw and paired fins, lamprey embryos and larvae have been used as comparative models. The lack of the jaw–neck region is a conspicuous feature specific to cyclostomes; however, the absence of these features has been largely neglected both in evolutionary developmental studies and in the field of classical comparative embryology. This review seeks to develop a possible evolutionary scenario of the vertebrate neck muscles by taking the cucullaris (trapezius) muscle as the focus. By combining the comparative embryology of lampreys and gnathostomes, and considering the molecular-level developmental mechanism of skeletal muscle differentiation, this review argues that the establishment of the vertebrate neck deserves to be called an evolutionary novelty based on the remodeling of mesenchymal components between the cranium and the shoulder girdle, which involves both mesodermal and neural crest cell lineages.     

A new cephalopod with soft parts from the Upper Carboniferous Francis Creek Shale of Illinois, USA

A ten armed fossil from the Pennsylvanian Francis Creek Shale and belonging to the famous Mazon Creek biota is described as a cephalopod. Apart from a superficial resemblance to the coleoids, there is little basis for an assessment of the animal's affinities. A mineralized Bask shaped structure preserved in calcite and seen in both part and counterpart in fragmentary form may represent the fossilized remains of an internal organ such as the stomach or ink sac A small, dark circular spot positioned anterior to the appendages could be the animal's eye although mere is no internal structure visible. The extremely rare occurrence of Palaeozoic cephalopods with soft parts has so far been Ended to the Francis Creek Shale and the Devonian Hunsrückschiefer of Germany. The Mazon Creek biota includes many fossils which show a remarkable standard of soft part preservation which in some cases includes the fossilization of eye spots and stomach traces. Previously deserted Mazon Creek cephalopods have included the tentacular impression of a squid complete with arm hooks, and the radulae and shells of both coleoids and nautiloids. The specimen described here is the first from the Palaeozoic to show any hint of organ preservation.     

Analysis of adhesion proteins from neural eye tissues of eight-day-old chick embryos

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epitheliumin vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10⁻¹² M solutions.     

Evaluation of the role of the retinal G protein-coupled receptor (RGR) in the vertebrate retina in vivo

The retinal G protein-coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein-coupled receptors. RGR may play a role as a photoisomerase in the production of 11-cis-retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11-cis-retinol dehydrogenase (RDH5), is to regenerate 11-cis-retinal under light conditions and RDH5 is expected to function in the light-independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr-/- single and rdh5-/-rgr-/- double knockout mice under a number of light conditions. The most striking phenotype of rgr-/- mice after a single flash of light includes light-dependent formation of 9-cis- and 13-cis-retinoid isomers. These isomers are not formed in wild-type mice because either all-trans-retinal is bound to RGR and protected from isomerization to 9-cis- or 13-cis-retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all-trans-retinyl esters and an attenuated recovery of 11-cis-retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic-like photoisomerization-driven production of 11-cis-retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod-dominated mice.     

Fossilized eggs from the Pennsylvanian of Illinois

Nine siderite concretions from the Middle Pennsylvanian, Francis Creek Shale (Carbondale Formation, Desmoinesian Series, West‐phalian C‐D), in the Mazon Creek area, Will‐Kankakee Counties, Illinois, U.S.A. preserve clusters of impressions of small eggs. Differential staining of the matrix suggests that the eggs were originally spawn within gelatinous strings containing 1 or 2 rows of eggs. Unfortunately, these egg impressions lack the diagnostic features needed to identify the zoological taxon (taxa) from which they originated.     

Culture of ratretinal pigment epithelium

Summary A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been cultured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be useful in studying the defect(s) associated with this mutation. This work was supported by grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, and the George Gund Foundation, Cleveland, Ohio. Breeding pairs of RCS-p⁺ rats were provided through National Institutes of Health Research Contract EY-3-2133 to Dr. R. L. Sidman.     

Decay of vertebrate characters in hagfish and lamprey (Cyclostomata) and the implications for the vertebrate fossil record

The timing and sequence of events underlying the origin and early evolution of vertebrates remains poorly understood. The palaeontological evidence should shed light on these issues, but difficulties in interpretation of the non-biomineralized fossil record make this problematic. Here we present an experimental analysis of decay of vertebrate characters based on the extant jawless vertebrates (Lampetra and Myxine). This provides a framework for the interpretation of the anatomy of soft-bodied fossil vertebrates and putative cyclostomes, and a context for reading the fossil record of non-biomineralized vertebrate characters. Decay results in transformation and non-random loss of characters. In both lamprey and hagfish, different types of cartilage decay at different rates, resulting in taphonomic bias towards loss of 'soft' cartilages containing vertebrate-specific Col2α1 extracellular matrix proteins; phylogenetically informative soft-tissue characters decay before more plesiomorphic characters. As such, synapomorphic decay bias, previously recognized in early chordates, is more pervasive, and needs to be taken into account when interpreting the anatomy of any non-biomineralized fossil vertebrate, such as Haikouichthys, Mayomyzon and Hardistiella.     

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.     

Site of conversion of endogenous all-trans-retinoids to 11-cis-retinoids in the bovine eye

By use of a new high-resolution high-pressure liquid chromatographic method for the separation of isomeric forms of retinol, retinal, retinyl ester and retinal oxime, various retinoids were analyzed in separated retinal pigment epithelial tissue or neural retinal tissue from fresh bleached bovine eyes after incubation in the dark at either 30 or 4°C for 90 min. 11-cis-Retinoids significantly increased during incubation at 30°C, relative to those at 4°C, in the retinal pigment epithelium, but not in the retina. The major forms of vitamin A in incubated retinal pigment epithelium and neural retina were retinyl esters (70%) and all-trans-retinol (69%), respectively. Thus, in keeping with observations on the isomerization of radioactive retinol in homogenates of eye tissues, the retinal pigment epithelium seems to be the primary site of 11-cis-retinoid formation from endogenous all-trans-retinoids in the bovine eye.     

Heat shock gene-expression in HSP-70 and HSF1 gene-transfected human epidermoid A-431 cells

Thermotolerant cells display attenuated heat shock protein 70 kD (HSP-70) gene expression and signal transduction such as intracellular Ca2+ concentration and inositol trisphosphate in response to sublethal heat. To further investigate the regulation of heat shock gene expression, we developed constructs containing human HSP-70 and HSF1 genes and transfected human epidermoid A-431 cells. These cells were chosen because skin cells are especially vulnerable to heat shock and other environmental stressors. We report that A431 cells can be successfully transfected with HSP-70 and HSF1 genes as shown by the elevated levels of respective message and protein. Overexpression of HSP-70 in cells transfected with HSP-70 gene led to a down-regulation of the HSF1 gene expression. Interestingly, transfection of cells with the HSF1 gene was not associated with increased expression of HSP-70. Exposure of HSF1 gene-transfected cells to heat resulted in a transient but significant increase in HSP-70 gene expression as compared to that found in vector-transfected cells, which was completely inhibited by treatment with staurosporine. In conclusion, we have demonstrated successful transfection of human A-431 cells with HSF1 and HSP-70 genes, where the regulation of their expression can be studied. (Mol Cell Biochem 167: 145-152, 1997)     

Neuronal stem/progenitor cells in the vertebrate eye

We acquire information from the outside world through our eyes which contain the retina, the photosensitive component of the central nervous system. Once the adult mammalian retina is damaged, the retinal neuronal death causes a severe loss of visual function. It has been believed that the adult mammalian retina had no regenerative capacity. However, the identification of neuronal progenitor cells in the retina sheds some light on cellular therapies for damaged retinal regeneration. In this review, we highlight three potential stem/progenitor cells in the eye, the ciliary body epithelium cells, the iris pigmented epithelium cells, and Müller glia. In order to make them prime candidates for the possible treatment of retinal diseases, it is important to understand their basic characters. In addition, we discuss the key signaling molecules that function extracellularly and determine whether neuronal progenitors remain quiescent, proliferate, or differentiate. Finally, we introduce a secreted protein, Tsukushi, which is a possible candidate as a niche molecule for retinal stem/progenitor cells.     

Changes in the Redox State in the Retina and Brain During the Onset of Diabetes in Rats

Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio.Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD⁺/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state     

Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

Retinal long-chain PUFAs (LC-PUFAs, C₁₂-C₂₂) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, C_n>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology.     

Zinc and Energy Requirements in Induction of Oxidative Stress to Retinal Pigmented Epithelial Cells

In age-related macular degeneration (AMD), retinal pigmented epithelium (RPE) cells are believed to be detrimentally affected. It is thought that zinc may play a part in this process. In the past, therefore, zinc supplementation has been suggested as a treatment for AMD. Experimental data shown here confound this view by indicating that whereas low amounts of zinc do protect RPE cells in culture from stress-induced effects, greater amounts of zinc have the opposite influence. These effects are partly dependent upon the health status of the cells. Experimental data presented herein also show that zinc-induced death of RPE cells can, however, be attenuated by compounds such as antioxidants (-tocopherol, trolox, and metipranolol), or cellular energy substrates (pyruvate and oxaloacetate). It is therefore concluded that a combination of zinc and antioxidants or energy substrates rather that zinc alone should provide a safer and more effective way to treat a disease such as AMD.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

Muscarinic receptors binding in retinal pigment epithelium during rat development

[³H]Quinuclidinyl benzylate (³H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of³H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of³H-QNB binding showed two high affinity sites to RPE, with K_B=2.6nM and 45 nM. Specific³H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of³H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.     

Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching

The zonula adherens (ZA) in adult chicken retinal pigment epithelium was examined with cryo-electron microscopic methods. Deep-etching of the cross-fractured ZA showed globules in the intercellular space. These globules apparently correspond to the electron-dense structure seen in thin sections. Deep-etching of obliquely fractured ZA further revealed rod-like structures extending from the extracellular surface into the intercellular space. These rods (mean approximately 9 nm thick, approximately 20 nm long) were straight and sometimes divided into two or three segments. The rods typically canted at approximately 60 degrees with respect to the plasma membrane, and they were often connected to the intercellular globules at their distal ends. When the rods are compared with the isolated cadherins reported previously, it is suggested that a combination of a rod and a globule may represent an extracellular part of cadherin. Membrane particles were observed on the P-face of the ZA plasma membrane, and their distribution density was approximately seven times that of the rods. The freeze-etching also revealed a characteristic particle complex on the ZA cytoplasmic surface, which may represent the cytosolic proteins linking cadherins to actin bundles.     

Glucose Metabolism in Rat Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO₂ production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO₂ yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO₂ production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress.