Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards.

BACKGROUND: Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma. RESULTS: Combining histology, optics, mass spectrometry, and UV and Raman spectroscopy, we found that the extensive variation in color patterns within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural color by constructive interference of light with guanine nanocrystals. More specifically, we show that 1) the hue of the vivid dorsolateral skin is modulated both by variation in geometry of structural, highly ordered narrowband reflectors, and by the presence of yellow pigments, and 2) that the reflectivity of the white belly and of dorsolateral pigmentary red marks, is increased by underlying structural disorganized broadband reflectors. Most importantly, these interactions require precise colocalization of yellow and red chromatophores with different types of iridophores, characterized by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH or redox state of pigments provide yet another source of color variation in squamates. CONCLUSIONS: Precisely colocalized interacting pigmentary and structural elements generate extensive variation in lizard color patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of the size, shape, and orientation of nanocrystals, and the superposition of specific chromatophore types. This study opens up new perspectives on Phelsuma lizards as models in evolutionary developmental biology.

The "benzothiazine" chromophore of pheomelanins: a reassessment.

The characteristic absorption and photochemical properties of pheomelanins are generally attributed to "benzothiazine" structural units derived biogenetically from 5-S-cysteinyldopa. This notion, however, conveys little or no information about the structural chromophores responsible for the photoreactivity of pheomelanins. At pH 7.4, natural and synthetic pheomelanins show a defined maximum around 305 nm, which is not affected by reductive treatment with sodium borohydride, and a monotonic decrease in the absorption in the range 350-550 nm. These features are not compatible with a significant proportion of structural units related to 2H-1,4-benzothiazine and 2H-1,4-benzothiazine-3-carboxylic acid, the early borohydride-reducible pheomelanin precursors featuring absorption maxima above 340 nm. Rather, these features would better accommodate a contribution by the nonreducible 3-oxo-3,4-dihydrobenzothiazine (lambdamax 299 nm) and benzothiazole (lambdamax 303 nm) structural motifs, which are generated in the later stages of pheomelanogenesis in vitro. This conclusion is supported by a detailed liquid chromatography/UV and mass spectrometry monitoring of the species formed in the oxidative conversion of 5-S-cysteinyldopa to pheomelanin, and would point to a critical reassessment of the commonly reported "benzothiazine" chromophore in terms of more specific and substantiated structural units, like those formed during the later stages of pheomelanin synthesis in vitro.

Fluorescence microscopy of microtubules in cultured cells.

Cytoplasmic microtubules are noncovalent polymers of the protein tubulin. In the cells, the main function of microtubules is to provide tracks for organelle transport. Two experimental approaches based on fluorescence microscopy are commonly used to examine organization of microtubules in mammalian tissue culture cells. The first experimental approach involves indirect immunofluorescence staining of chemically fixed cells with tubulin antibody. Fluorescence microscopy of immunostained specimens allows the examination of the distribution of microtubules in the cytoplasm at the moment of fixation. The second experimental approach involves introduction of tubulin subunits covalently labeled with a fluorochrome into the cytoplasm of living cells. Time-lapse fluorescence microscopy of cells containing labeled tubulin subunits allows to examine changes in the spatial organization of microtubules in the cytoplasm and also to directly observe their behavior. In this chapter, we describe preparation of samples for fluorescence microscopy of microtubules.

Tracking melanosomes inside a cell to study molecular motors and their interaction.

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (approximately 2 nm) and temporal (approximately 1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.

Antioxidant defenses and DNA damage induced by UV-A and UV-B radiation in the crab Chasmagnathus granulata (Decapoda, Brachyura).

The photoprotector role of pigment dispersion in the melanophores of the crab, Chasmagnathus granulata, against DNA and oxidative damages caused by UV-A and UV-B was investigated. Intact and eyestalkless crabs were used. In eyestalkless crabs, the dorsal epidermis of the cephalothorax (dispersed melanophores) and the epidermis of pereiopods (aggregated melanophores) were analyzed. Intact crabs showed only dispersed melanophores in the two epidermis. Antioxidant enzymes activity and lipoperoxidation content were analyzed after UV-A (2.5 J/cm2) or UV-B (8.6 J/cm2) irradiation. DNA damage was analyzed by single cell electrophoresis (comet) assay, after exposure to UV-B (8.6 J/cm2). UV-A radiation increased the glutatione-S-transferase activity in the pereiopods epidermis of eyestalkless crabs (P<0.05). UV-B radiation induced DNA damage in the dorsal epidermis of eyestalkless crabs (P<0.05). In pereiopod epidermis of eyestalkless crabs, there was no significant difference between control and UV-B-exposed crabs. In the pereiopods epidermis of eyestalkless, the control group showed higher scores of DNA damage and approximately 50% of cellular viability. Because in eyestalkless and irradiated crabs the cellular viability was approximately 5%, it was not possible to observe nuclei for determination of DNA damage. The findings show that melanophores can play a role in the defense against harmful effects of a momentary exposure to UV radiation.

Melanophore sublineage-specific requirement for zebrafish touchtone during neural crest development.

The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.

Touchtone promotes survival of embryonic melanophores in zebrafish.

An outstanding problem in the study of vertebrate development is the identification of the genes that direct neural crest precursor cells to adopt and maintain specific differentiated cell fates. In an effort to identify such genes, we have carried out a mutagenesis screen in zebrafish and isolated mutants that lack neural crest-derived melanophores. In this manuscript we describe the phenotype of one such mutant, touchtone(b722) (tct), and the map position of the gene it defines. Analysis of expression of dopachrome tautomerase (dct) and microphthalmia (mitfa) suggests that melanophore precursors are specified normally in homozygous tct mutants. However, differentiated melanophores are pale, small, and about half of them have disappeared by 48 h of development, apparently by cell death. We show that melanophores require Tct function cell autonomously. Signals from the receptor tyrosine kinase receptor C-kit are essential for survival of melanophores in zebrafish and mammals. However, differences in the phenotypes of tct and c-kit homozygous mutants, and an absence of interaction between c-kit and tct heterozygotes, suggest that Tct functions independently of the C-kit pathway. Other neural crest derivatives, including other pigment cell types, appear normal in tct mutants. Interestingly, tct mutant embryos undergo a temporary period of near complete paralyzis during the second day of development, although markers of axons of motor and sensory neurons look normal in this period. A fraction of tct(b722) mutants survive to adulthood, but mutant adults are small, indicating a role for Tct in post-larval growth. The tct gene maps to a small interval near a telomere of chromosome 18. Thus, we have identified a zebrafish gene that when mutated produces semi-viable offspring and that may serve as a model of human diseases that have both pigmentation and neurological symptoms.

Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Height changes associated with pigment aggregation in Xenopus laevis melanophores.

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgCl2-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

Design, synthesis, and biological evaluation of non-peptidic ligands at the Xenopus laevis skin-melanocortin receptor.

Taking the tripeptide D-Trp-Arg-Leu-NH(2) as a lead for a Xenopus laevis skin-melanocortin (MC) receptor antagonist, thirteen non-peptidic compounds were synthesized and biologically evaluated at Xenopus laevis melanophores. Six competitive antagonists (shown by Schild analysis) and one partial agonist were identified with moderate activity (IC(50): 5-10 microM). Tryptophanamides with aliphatic side chains were inactive whereas basic residues restored activity. Introducing an imidazole residue yielded partial agonist activity (EC50: 32 microM). Interestingly, constraining the inactive S-tryptophan-isoamylamide to a beta-carboline ring yielded an MC receptor antagonist (42). The specificity for MC receptors was tested at various G-protein coupled receptors. In conclusion, the synthesis of non-peptidic MC receptor antagonists is described which may serve as lead compounds for further studies.

Dopamine and melanin-concentrating hormone neurons are distinct populations in the rat rostromedial zona incerta.

Zona incerta (ZI) is a controversial diencephalic area with a variety of cytoarchitectonic subdivisions, neurotransmitters and related functions. Medial ZI synthesizes dopamine (A13 group) and tyrosine hydroxylase (TH, a catecholamine synthesizing enzyme), which has been considered a neurochemical marker for this region. The rostromedial ZI also expresses melanin-concentrating hormone (MCH), but it is not known whether dopamine and MCH are colocalized. By using double label immunohistochemistry we analyzed the distribution of TH and MCH in the rat ZI. We found that MCH and TH neurons are intermingled but are not colocalized.

Induction and characterization of mutations at the b locus of the medaka, Oryzias latipes.

The b locus is one of the most familiar pigmentation loci in the medaka, but its biochemical function is still unknown. Here we report induction of new mutations at the b locus by radiation and ENU. We also characterized all these mutations and previously isolated spontaneous ones on the phenotypic basis. Unexpectively, all the 18 induced mutations reduced melanin contents in both eyes and skin correlatively, although degree of reduction was varied from mutations to mutations. Moreover, presumed null mutants (bs8, bg8, bc2, bd3, bd6, bg13, bg19, bg24) had slightly melanized (dark red) eyes. These results suggest that the b-locus product plays an important but not a critical role in melanogenesis. The spontaneous mutants were divided into two types: one (bdl2, bdl3, and bp) had similarities with the induced mutants in that they had slightly colored eyes and skin, the other (bv, B', bd, bdl1, and b) exhibited normally black eyes but lightly colored skin. The present study supports our recent results (Fukamachi et al., 2001) that mutational changes were found in the coding region of the b gene in some of the mutants which reduced both eyes and skin melanogenesis, while the mutational change for the b allele could not be found there. We speculate that the bv, B', bd, bdl1, and b alleles might arise by the mutations in the regulatory region for skin melanogenesis.

Identification of 3,4-didehydroretinal isomers in the Xenopus tadpole tail fin containing photosensitive melanophores.

It is well characterized that melanophores in the tail fin of Xenopus laevis tadpoles are directly photosensitive. In order to better understand the mechanism underlying this direct photosensitivity, we performed a retinal analysis of the tail fins and eyes of Xenopus tadpoles at stages 51-56 using high performance liquid chromatography (HPLC). Following the extraction of retinoids by the formaldehyde method, a fraction containing retinal and/or 3,4-didehydroretinal isomers from the first HPLC analysis were collected. These isomers were then reduced by sodium borohydride to convert retinal and/or 3,4-didehydroretinal isomers into the corresponding retinol isomers to prepare for a second HPLC analysis. Peaks of 11-cis and all-trans 3,4-didehydroretinol were detected in the eyes and tail fins containing melanophores, but they were not detected in the tail fins without melanophores. The amounts of 11-cis and all-trans 3,4-didehydroretinol were 27.5 and 5.7 fmol/fin, respectively, and the total quantity of 3,4-didehydroretinal was calculated at approximately 5 x 10(6) molecules/melanophore. These results strongly suggest the presence of 11-cis and all-trans 3,4-didehydroretinal in melanophores of the tadpole tail fin, which probably function as the chromophore of photoreceptive molecules.

Biosensing of opioids using frog melanophores.

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.

The electric charge of pigment granules in pigment cells.

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be -1.5 x 10(-16) and -1.7 x 10(-16) C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.

Melanin-concentrating hormone system in the brain of the lungfish Protopterus annectens.

The neurochemical anatomy of the lungfish brain is of particular interest, because many features in these animals might be representative of the common ancestor of land vertebrates. In the present study, we have investigated the localization and biochemical characteristics of melanin-concentrating hormone (MCH)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. The most prominent group of MCH-immunoreactive cell bodies was found in the dorsal hypothalamus. Additional groups of MCH-immunoreactive perikarya were detected in the telencephalon within the medial and dorsal pallium, the medial subpallium, and the ventral part of the lateral subpallium. Brightly immunofluorescent nerve fibers were seen in the anterior olfactory nucleus, the ventral part of the medial pallium, the medial subpallium, and the anterior preoptic area. In the diencephalon, the hypothalamus and the medial region of the dorsal thalamus exhibited a dense accumulation of fibers. MCH-immunoreactive fibers were also found in the tectum and the tegmentum of the mesencephalon and within the reticular formation of the rhombencephalon. In the pituitary, several small groups of cells of the intermediate lobe showed a bright fluorescence. Reversed-phase high-performance liquid chromatography (HPLC) analysis of diencephalon and pituitary extracts resolved a major MCH-immunoreactive peak that coeluted with synthetic salmon MCH. The distribution of MCH in the brain of P. annectens suggests that, in lungfishes, this peptide may exert neuromodulator or neurotransmitter functions. The presence of MCH-like immunoreactivity in the intermediate lobe of the pituitary indicates that, in dipnoans, MCH may also act as a typical pituitary hormone.

Melanopsin: An opsin in melanophores, brain, and eye.

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of Xenopus laevis. Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin's function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.

The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells.

Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.