Tissue interaction between the retinal pigment epithelium and the choroid triggers retinal regeneration of the newt Cynops pyrrhogaster

Complete retinal regeneration in adult animals occurs only in certain urodele amphibians, in which the retinal pigmented epithelial cells (RPE) undergo transdifferentiation to produce all cell types constituting the neural retina. A similar mechanism also appears to be involved in retinal regeneration in the embryonic stage of some other species, but the nature of this mechanism has not yet been elucidated. The organ culture model of retinal regeneration is a useful experimental system and we previously reported RPE transdifferentiation of the newt under this condition. Here, we show that cultured RPE cells proliferate and differentiate into neurons when cultured with the choroid attached to the RPE, but they did not exhibit any morphological changes when cultured alone following removal of the choroid. This finding indicates that the tissue interactions between the RPE and the choroid are essential for the former to proliferate. This tissue interaction appears to be mediated by diffusible factors, because the choroid could affect RPE cells even when the two tissues were separated by a membrane filter. RPE transdifferentiation under the organotypic culture condition was abolished by a MEK (ERK kinase) inhibitor, U0126, but was partially suppressed by an FGF receptor inhibitor, SU5402, suggesting that FGF signaling pathway has a central role in the transdifferentiation. While IGF-1 alone had no effect on isolated RPE, combination of FGF-2 and IGF-1 stimulated RPE cell transdifferentiation similar to the results obtained in organ-cultured RPE and choroid. RT-PCR revealed that gene expression of both FGF-2 and IGF-1 is up-regulated following removal of the retina. Thus, we show for the first time that the choroid plays an essential role in newt retinal regeneration, opening a new avenue for understanding the molecular mechanisms underlying retinal regeneration.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Not lost in translation: Sensing the loss and filling the gap during regeneration

Every organism responds to injuries by reparative processes in order to adapt to the altered conditions. The quality of the adjustment in terms of morphological and functional recapitulation of the original status varies among species. One task is to understand the concepts by which animals with outstanding regenerative capabilities sense what and how much is missing, and how they translate that information to the appropriate responses. These concepts may integrate various kinds of regenerative phenomena although the specific molecular and cellular mechanisms that execute these processes are divergent and depend on the type of the injury. The use of a variety of lesion paradigms could uncover common principles that link injury to successful regeneration. In addition they could indicate means how to further translate this knowledge to the practice of regenerative medicine. We exemplify this possibility by outlining some critical features of dopaminergic neurogenesis in the midbrain of adult salamanders, and the implications for Parkinson's disease.     

Retinoic acid-dependent attraction of adult spinal cord axons towards regenerating newt limb blastemas in vitro

Adult urodele amphibians possess the unique ability to regenerate amputated limbs and to re-innervate these regenerating structures; however, the factors involved in mediating this re-innervation are largely unknown. Here, we investigated the role of retinoic acid (RA) and one of its receptors, RARbeta, in the reciprocal neurotropic interactions between regenerating limb blastemas and spinal cord explants from the adult newt Notophthalmus viridescens. First, we showed that retinoic acid induced directed axonal outgrowth from cultured spinal cord tissue. This RA-induced outgrowth was significantly reduced when spinal cord explants were pre-treated with either the synthetic RAR pan antagonist, LE540, or the specific RARbeta antagonist, LE135. The role of RARbeta was also investigated using co-cultured regenerating limb blastemas and spinal cord explants. Blastemas induced significantly more axonal outgrowth from the near side of co-cultured explants, than from the far side (when cultured less than 1 mm apart). This blastema-induced directed outgrowth from co-cultured spinal cord explants was also abolished in the presence of the RARbeta antagonist, LE135. These data strongly suggest that endogenous retinoic acid is one of the tropic factors produced by the blastema and that it may be capable of guiding re-innervating axons to their targets. Moreover, this interaction is likely mediated by the retinoic acid beta nuclear receptor.     

Lymphangiogenesis promotes lens destruction and subsequent lens regeneration in the newt eyeball, and both processes can be accelerated by transplantation of dendritic cells

We examined whether lymphangiogenesis is essential for the process of lens destruction and subsequent remodeling in the newt eye. Lens regeneration was induced by pricking the lens once with a needle through the cornea. The results showed that the formation of the vacuoles which was mediated by lysosomes occurred in the original lens on 8 days after pricking, and histolysis of the lens was induced 24 h later. At that time, new lymphatic vessels appeared in the normally avascular cornea. Immunofluorescence studies revealed the expression of VEGF receptor not only on the cells in the central cornea but also on those in the dorsal iris. Moreover, dendritic cells (DCs) migrated from the peripheral to the central regions in the cornea to engulf the remains of the lens. Next, to determine the extent to which the DCs are important for lens regeneration, we transplanted the DCs that had engulfed the remains of the lens into the eyeball of the normal animals. Interestingly, lens regeneration began in the dorsal iris of eyeballs into which the DCs were transplanted and also in those in which no DCs were transplanted. However, surgical removal of the spleen of the recipient animals prior to transplantation resulted in both a failure of both the VEGFR expression in the dorsal iris and a failure of the novel regeneration.     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.     

Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.     

SEM observations on polar body elimination and changes of the oocyte surface during egg maturation in the newt Triturus alpestris (Amphibia,Urodela)

Summary We have studied the surface of the animal half of ovulated newt eggs recovered from different portions of the oviduct. The germinative area, about 40 m diameter, is localized in the region of the whitish polar spot, about 450 m diameter. The structural changes in the germinative area are connected with the formation and extrusion of the first and second polar bodies. Of the two types of oviductal eggs observed, those covered with microvilli (type 1) were found only in the ostial portions of the oviduct, whilst those covered with microfolds (type 11) were found more distally. The structural difference between these two types may be related to the known reduction in surface area of the cell membrane during oocyte maturation. Offprint requests to: W. Kilarski     

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 mum in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.     

北京鸭视网膜节细胞的大小、密度和分布

采用Nissl染色法、视神经溃变法和神经元逆行追踪标记辣根过氧化物酶(HRP)法,研究了北京鸭视网膜节细胞(RGCs)的大小和密度及其分布的变化。北京鸭RGCs形态多样,有圆形、椭圆形和多角形等,RGCs总数为1.3×106个(P0),RGCs平均密度为5370个/mm2(P0),在视网膜中央有一个偏向鼻侧的高密度区即中央高密度区(8860个/mm2),由中央区至周边部,细胞密度逐渐降低,颞侧周边部最低(3440个/mm2)。不同区域RGCs大小差异显著,中央区以小细胞为主(62.2±23.3μm2,P0),而周边部RGCs逐渐增大,颞侧周边部最大(P0:133.7±75.7μm2;P8:152.9±55.9μm2)。由此可见,伴随RGCs大小由中央区至周边部的递增而细胞密度呈现递减的变化,这种变化趋势在颞侧周边部最明显。与此同时,随日龄增长,RGCs总数和密度均递减而细胞大小递增。dACs是位于视网膜节细胞层的小神经元,细胞大小为23.7±4.0μm2     

Changes in retinal neuronal populations in the DBA/2J mouse

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, -aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.Jung-Il Moon and In-Beom Kim contributed equally to this work.This work was supported by a Korea Research Foundation Grant (FP 0005) and by BK 21 in Korea.     

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

On the stability of DNA in photoreceptor cells of rat retina

Summary DNA turnover in post-mitotic photoreceptor cells of F344 rat retina was investigated. Developing retinas of newborn rats were labelled by multiple injections of (methyl-³H)thymidine. One eye was removed on day 60 and embedded in paraffin. The groups of rats were killed 180, 365, 540 or 730 days later and the second eye was removed. Autoradiographic studies on pairs of eyes showed no detectable DNA turnover in photoreceptor cells up to the end of the experiment (near median life-span, 50% survival age). The DNA of these photoreceptor cells is not replaced through the life span of the animals; the results thus suggest that it is very stable and possibly protected in a specific manner.     

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.     

Influence of indomethacin on lens regeneration in the newt notophthalmus viridescens

Summary Following lentectomy newts were injected with indomethacin in a variety of carrier solutions at doses ranging from 1.2–120 mg/kg body weight every other day for 15–17 days. The results show that injection of this drug according to the regimen used has no significant effect on regeneration of the lens. The data suggest, but do not prove, that prostaglandins may not play a major role in the early phases of lens regeneration in the newt.     

Freeze-fracture study on the whorls of rough endoplasmic reticulum in the exocrine pancreatic cells of the Japanese newt and African clawed toad

Summary The whorls of rough endoplasmic reticulum (rER) in the exocrine pancreatic cells of starved newts and clawed toads were examined by a freeze-fracture technique. The whorl appeared to be roughly ovoidal in shape and composed of tightly packed, narrow cisternae arranged like the layers of an onion. The clusters of interdigitating projections of the cisternal membranes were located at several places on the whorl. Some of these projections extended to the vesicular rER around the whorl. The fenestra-like, raised or hollowed craters were seldom seen on the fractured membrane faces of the whorls in the exocrine pancreatic cells of the starved newts.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.