Single Pigment Cell Research Articles

Laser capture microdissection of pigment cells from Salmonid fish skin to identify differentially expressed genes in an enriched cell population

In order to determine the function of pigment cell types in different pigment pattern of skin, we have applied the laser capture microdissection method (LCM) for isolation of different pigment cell types from skin sections of marble trout (Salmo marmoratus) and brown trout (S. trutta), characterized with labyrinthine and spotted skin pattern, respectively. RNA was isolated from dissected pigment cell populations and used for expression analysis. Expression profiles of candidate genes, potentially involved in pigmentation, in LCM isolated pigment cell population, were more specific compared to differently pigmented skin regions, containing a heterogeneous mixture of cells, including more types of chromatophores. Moreover, they could provide important information about particular pigment cell type function and its contribution to different pigment pattern. Protocol of combined LCM and genomic approach to study single pigment cell populations can be applied in the future, but needs to be further optimized in order to yield RNA of sufficient quantity and quality required for i.e. transcriptomic analyses.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

Urochordate βγ-Crystallin and the Evolutionary Origin of the Vertebrate Eye Lens

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.

Reconstructing the eyes of Urbilateria.

The shared roles of Pax6 and Six homologues in the eye development of various bilaterians suggest that Urbilateria, the common ancestors of all Bilateria, already possessed some simple form of eyes. Here, we re-address the homology of bilaterian cerebral eyes at the level of eye anatomy, of eye-constituting cell types and of phototransductory molecules. The most widespread eye type found in Bilateria are the larval pigment-cup eyes located to the left and right of the apical organ in primary, ciliary larvae of Protostomia and Deuterostomia. They can be as simple as comprising a single pigment cell and a single photoreceptor cell in inverse orientation. Another more elaborate type of cerebral pigment-cup eyes with an everse arrangement of photoreceptor cells is found in adult Protostomia. Both inverse larval and everse adult eyes employ rhabdomeric photoreceptor cells and thus differ from the chordate cerebral eyes with ciliary photoreceptors. This is highly significant because on the molecular level we find that for phototransduction rhabdomeric versus ciliary photoreceptor cells employ divergent rhodopsins and non-orthologous G-proteins, rhodopsin kinases and arrestins. Our comparison supports homology of cerebral eyes in Protostomia; it challenges, however, homology of chordate and non-chordate cerebral eyes that employ photoreceptor cells with non-orthologous phototransductory cascades.

First evidence of mitochondrial lensing in two species of the “Typhloplanoida” (Plathelminthes, Rhabdocoela): phylogenetic implications

Based on electron-microscopical observations the light-sensing organs ofProxenetes deltoides andPtychopera westbladi, representatives of the “Typhloplanoida” Trigonostominae, are described. The photoreceptors in both species belong to the type of rhabdomeric pigment cup ocelli.P. deltoides has a single pigment cell and three sensory cells.P. westbladi possesses eyes made up of a single pigmented cup cell and a single sensory cell. The dioptric apparatus in the eyes ofP. deltoides is formed by three proliferations of the cup cell containing giant mitochondria. InP. westbladi, the elements focalizing incoming light also consist of modified mitochondria which are arranged in the section of the cup cell covering the eye cavity. With regard to the new findings, mitochondrial lensing is hypothesized as an autapomorphy of a monophylum encompassing distinct taxa or all members of the free-living Rhabdocoela; the Neodermata also belong to this monophylum.

Lectin binding pattern in human retinal pigment epithelium

A comparative lectin histochemical study of human retinal pigment epithelium (RPE) was performed to investigate the lectin binding pattern of normal, reactive and proliferating RPE. Normal RPE with attached sensory retina was found to bind the lectins Con A, WGA, PNA and RCA I. Reactive and proliferating RPE in retinal detachment and in photocoagulation scars revealed the same lectin binding pattern although its cellular topography changed. RPE-macrophages showed an additional reaction with SBA. In periretinal membranes of human PVR the typical lectin binding pattern of Con A, WGA, PNA and RCA I was found in pigmented and in a subpopulation of non-pigmented cells, suggesting that these lectin-positive elements were of RPE-origin. Additionally, single pigmented cells positive for SBA were found indicating macrophage differentiation. Thus lectin histochemistry provides a tool for cytochemical identification of RPE and its morphologic variants by revealing a specific combination of sugar-binding sites.

Identification of a low-threshold T-type calcium channel in bovine ciliary epithelial cells.

The characteristics of an inward current in single pigmented cells from the bovine ciliary body were examined using the whole cell recording technique. The inward current appeared to activate at potentials 30 mV positive to a holding potential of -80 mV, although activation studies revealed that from more negative holding potentials the current activated at about -75 mV, peaked at around -10 mV, and reversed at a potential of +40 mV. The current was small, peak value -72 +/- 24 pA (n = 13), and exhibited rapid activation and slower inactivation kinetics, both processes being voltage sensitive. Inactivation occurred at holding potentials more positive than -120 mV and was complete at -50 mV with a half-maximal membrane potential of -88 mV. The overlap of the inactivation and activation curves between -75 and -50 mV means that, in the steady state, entry of calcium will be greatest in this range and calcium will thus enter the cell at the resting membrane potential. The current was resistant to tetrodotoxin and nifedipine. It was partially blocked by Cd2+ and almost completely blocked by Ni2+. It is concluded that this current is a low-threshold T-type calcium current.

Cell counts and maps in the larval central nervous system of the ascidian ciona intestinalis (L.)

Although the ascidian tadpole larva harbors a prospectively valuable prototype of the chordate nervous system, with extensively characterized neural plate cell lineages, the simple cellular composition of the resultant central nervous system (CNS) is not documented in detail. The average total number of cells in the larval CNS of Ciona intestinalis is 335 (range +/- 4, n = 3), 65 or 66 of which reside in the nerve cord of the tail. The estimates were made by tracing and counting the number of nuclei in serial semithin (1 micron) sections cut longitudinally through three larvae, fixed no later than 2 hours after hatching. Within a single fourth larva, L4, 266 cells constituted the CNS in the trunk region of the larva, 45 of which occurred within the visceral ganglion, 215 in the sensory vesicle, and 6 in the neck between the two. Each cell was assigned to one of thirteen categories. Most (182, roughly 68%) are classified as ependymal, a specialized non-neural cell peculiar to embryonic and larval chordates, from their position lining the cavities of the neural tube's elaborations or from clear similarities in the cytological appearance to those that do. Five cells are accessory cells of the sensory structures: three lens cells and a pigment-cup cell in the ocellus, and a single pigment cell in the otolith. Of the remaining 79 cells, 36 are sensory, 17 receptors in the ocellus and 19 presumed hydrostatic pressure receptors; these lie on the right and left sides of the sensory vesicle, respectively. Eighteen of the visceral ganglion cells have been tentatively classified as neurons, as have the remaining 25 cells which form two clusters in the posterior region of the sensory vesicle.

The ultrastructure of the sense organs of some turbellaria rhabdocoela. I. The eyes of Polycystis naegelii K�lliker (Eukalyptorhynchia Polycystididae)

Each pigment-cup eye of Polycystis naegelii consists of two retinal clubs and a single pigmented cell. The latter is divided into two cavities by a septum. Under bright illumination the photoreceptor process appears as a disk containing membranous laminar whorls; under faint illumination the latter are replaced by numerous straight, closely packed, microvilli. This morphological variation is correlated with the intensity of the photoreceptor's exposure to light. The lenticular structures described by previous light microscopists have not been observed.

Ultrastructure of the extracutaneous pigment cells in the plaice (Pleuronectes platessa, L., Teleostei)

The extracutaneous pigment cell system of the plaice (Pleuronectes platessa L.) was examined by light and electron microscopy in selected regions, including two cutaneous regions for comparison. The extracutaneous pigmentation consists of guanocytes and melanocytes with differing distributions within the body. The eyeless side lacks melanocytes. The pigment cells are differentiated as very flat elements with long processes. They display an affinity for loose connective tissue at boundary layers such as the peritoneal epithelium, organ capsules or blood vessels, to which they are parallelly arranged at a very constant distance. In some locations guanocytes are intimately associated with melanocytes forming "reduced chromatophore units". Extracutaneous pigment cells are poor in mitochondria, endoplasmic reticulum, microfilaments, caveolae intracellulares, ribosomes and glycogen granules, all of which are more abundant in cutaneous pigment cells and pigment cells of the eye. In extracutaneous guanocytes the crystals are loosely arranged parallel to the cell surface, in cutaneous guanocytes perpendicular. Cells with rod-like vesicular cisternae are described as "guanoblasts". No single pigment cell was found exhibiting different types of pigment granules. The varying colors of extracutaneous pigmentation arise from varying combinations of guanocytes and melanocytes in addition to the color of the tissue itself.

Ultrastructure of the eyes of Arctonoë vittata grube (polychaeta, polynoidae)

The photoreceptors of Arctonoë vittata are rhabdomeric. The retina is formed of pigment and receptor cells. From the distal part of the receptor cell arises a receptoral process that gives rise to lateral microvilli. The microvilli fill the space between the pigment cup and the lens. The receptor cell body and the proximal part of the receptoral process contain melanin in the form of pigment bodies. The pigment cells form a septum 1 μm in diameter, which passes through the rhabdomeric region and then forms a crystalline lens element. The lens consists of a number of such crystalline elements each secreted by a single pigment cell. The pigment bodies of the pigment cells also contain melanin. A marked change in the shape of the lens and an enlargement of the pupil in the eyes of dark-kept animals suggests that the eyes are functional photoreceptors.