Role of the Age Factor in Eye Regeneration in the Gastropod Achatina fulica

The dependence of the ability to regenerate the eye on the age of experimental animals was studied in the snail Achatina fulica. The degree of regeneration was estimated by light-microscopic and electrophysiological methods and by analyzing the motor response to visual stimuli. In older age groups, the number of regenerated eye-bearing tentacles decreased, whereas the period of regeneration increased. The regenerated eyes of the snails operated at the age of more than two months remained smaller than normal eyes even after six months. Regeneration of the distal part of the optic nerve was observed, and the regenerated eyes recovered the ability to respond to stimulation by light. In the electroretinogram, the responses of the regenerated eye, compared to the control, were characterized by lower amplitude and longer repolarization and refractory periods. Manifestations of the motor response to visual stimuli in the young snails with regenerating eyes could be regarded as evidence for the recovery of connection between the organ of sight and the central ganglia.     

Electrical responses to amputation of the eye in the mystery snail

Immediately following amputation through the eyestalk of the mystery snail (Pomacea), a persistent ionic current enters the apical amputation surface of the eyestalk stump. The circuit is completed by current driven from undamaged integument of the eyestalk stump and other body regions. The current is relatively steady during the first 10 hours following amputation. Currents subsequently begin a slow decline to base line levels by 60 hours postamputation--a time coincident with wound healing processes. The "battery" driving this ionic current is the internally negative transepidermal potential existing across the snail integument--perhaps the result of a net inward pumping of chloride across the skin. This system is compared to other regeneration models such as the amphibian limb, bone fracture repair, and skin wound healing. We suggest that ionic current may be a control of eye regeneration in the snail.     

Structure and regeneration of the eyes of strombid gastropods

Summary The tips of the eyestalks of three species of strombid gastropods were amputated and the structure of the fully developed eye investigated. The retina contains at least two types of cell: sensory cells bearing long tufts of microvilli with a central cytoplasmic core, and pigment cells with short microvilli.New eyes became visible at the tips of the eyestalk stump 5–16 days after amputation. When the regenerated eyes first appear, they consist of hollow balls of cells with a pigment lined cavity; two types of retinal cells are already distinguishable but their microvilli and cilia are small and sparse. The microvillous tufts and sensory cell contents develop quickly and about 14 days after their first appearance, the eye is a fully formed but miniature organ.     

Inhibition of Lens Regeneration by Nickel Subsulfide in the Japanese Common Newt, Cynops pyrrhogaster

To investigate the effects of nickel subsulfide (Ni₃S₂) on lens regeneration, Ni₃S₂ was administered to the lentectomized newt eye by a single injection into the eye chamber. Lens regeneration was inhibited in the early stage of regeneration with about 60% of the treated eyes after 25 days, while all control eyes had a regenerating lens. The size of the regenerating lens in the treated eyes was the same as in controls. Lens regeneration was inhibited in all treated eyes containing Ni₃S₂ particles within the eye chamber. Inhibition of lens regeneration in the treated eyes was about 80% at 3 months, when all control eyes had already completed lens regeneration. Although the tendency toward pupil disappearance was prominent in the eyes having no regenerating lens, there were also a few eyes which had no regenerating lens and no signs of pupil disappearance. The significance of these results was discussed in relation to the regeneration and carcinogenesis.     

Bromodeoxyuridine labeling reveals a class of satellite-Like cells within the electric organ

When the electric organ (EO) of weakly electric fish is amputated, a blastema forms from which new EO and muscle cells arise. However, the progenitor cells that contribute to the blastema are unknown. We studied regeneration of the electric organ in Sternopygus to answer this question. The EO of this species is composed of electrocyte cells surrounded by peripheral bundles of muscle fibers. Fish were injected with 5′-bromodeoxyuridine (BrdU) 24 h after amputating the terminal portion of the EO. At this time, a population of small cells were labeled in the extracellular matrix between electrocytes and muscle fibers. These cells did not label in control fish injected with saline or in nonamputated BrdU-injected fish. For the first 6 days postamputation, increasing numbers of BrdU-labeled cells appeared at the wound margin. A blastema formed 6 days after amputation and contained numerous BrdU-labeled cells. At 10 days postamputation, clusters of BrdU-positive cells were seen throughout the wound margin and proximal blastema. At 14 days, BrdU-labeled nuclei were present within developing electrocytes. Labeling alternate sections with MF20 antimyosin and AE1 anticytokeratin antibodies confirmed that BrdU-positive multinucleate cells coexpress myosin and cytokeratin epitopes, diagnostic of newly regenerated electrocytes. Electron micrographs reveal that the small cells surrounding muscles and electrocytes are similar; they contain an elongate nucleus, are largely devoid of cytoplasm, and possess few organelles. This morphology and evidence of myogenic potential suggests that these cells are satellite cells. © 1993 John Wiley & Sons, Inc.     

Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO₄-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.     

Two visual systems in one eyestalk: The unusual optic lobe metamorphosis in the stomatopod Alima pacifica

The compound eyes of adult stomatopod crustaceans have two to six ommatidial rows at the equator, called the midband, that are often specialized for color and polarization vision. Beneath the retina, this midband specialization is represented as enlarged optic lobe lamina cartridges and a hernia‐like expansion in the medulla. We studied how the optic lobe transforms from the larvae, which possess typical crustacean larval compound eyes without a specialized midband, through metamorphosis into the adults with the midband in a two midband‐row species Alima pacifica. Using histological staining, immunolabeling, and 3D reconstruction, we show that the last‐stage stomatopod larvae possess double‐retina eyes, in which the developing adult visual system forms adjacent to, but separate from, the larval visual system. Beneath the two retinas, the optic lobe also contains two sets of optic neuropils, comprising of a larval lamina, medulla, and lobula, as well as an adult lamina, medulla, and lobula. The larval eye and all larval optic neuropils degenerate and disappear approximately a week after metamorphosis. In stomatopods, the unique adult visual system and all optic neuropils develop alongside the larval system in the eyestalk of last‐stage larvae, where two visual systems and two independent visual processing pathways coexist. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 3–14, 2018     

Organization and regeneration ability of spontaneous supernumerary eyes in planarians -eye regeneration field and pathway selection by optic nerves-

Planarians can propagate asexually by fission and successive regeneration. During head regeneration, they again form a new pair of eyes, and sometimes supernumerary eyes. The positions of normal and supernumerary eyes and their regeneration abilities are expected to be highly relevant to the question of where and how the field to regenerate eyes is determined. In this study, spontaneously generated supernumerary eyes were classified into various types. In all cases, they were formed in the anterior part of the head. Enucleation of a normal eye elicited regeneration of a new eye; however, enucleation of a supernumerary eye did not. The supernumerary eyes were morphologically and functionally indistinguishable from the normal eyes, revealed by the studies of immunohistology and photophobic response, respectively. From the obtained results, we proposed a model of the eye regeneration field that changes its distribution spatiotemporally during regeneration. Immunohistological studies also showed that the optic nerves from the normal and supernumerary eyes ran independently, which might have implication about the nature of guidance cues for the optic nerves.     

reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins

Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at approximately 35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.     

Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.     

Forelimb regeneration in thyroidectomized adult newts following organ culture and autografting of the thyroid glands

Summary Thyroidectomy and organ culture of adult newt thyroid glands three days prior to forelimb amputation was followed by autografting the glands subcutaneously into the animal's lower jaw region 9, 18 or 25 days postamputation (GC^{9, 18, 25} day series). This was an attempt, utilizing 515 animals, to elucidate further the role of the thyroids in regeneration. Amputated limbs of the thyroidectomized (Thx) and autografted muscle explant (MC = sham) cases underwent stumping or were significantly delayed in their regeneration rate and displayed abnormal morphogenesis compared with control regenerates. In the GC⁹ series newts, regenerates were identical to controls 45 days postamputation. However, regenerates of the GC¹⁸ series cases exhibited delayed and abnormal development at 45 days; but they were not as delayed and had fewer abnormalities than those cases in the Thx and MC groups. Results of the GC²⁵ series newts were similar to those of the Thx group. Within 5 days of autografting the thyroids, epidermal moulting resumed and long-term survival ensued. We conclude that normal limb regeneration in the adult newt is thyroid hormone(s) dependent, specifically the later stages of growth, differentiation and morphogenesis.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.     

Food-attraction conditioning in the snail,Helix pomatia

Adult pulmonate snails (Helix pomatia) were released equidistant between two types of food, carrot and potato, respectively. Naive snails moved in different directions and did not locate either food above chance, although both foods were readily eaten upon direct contact. After a single carrot feeding episode, 75% of the carrot-fed snails moved directly towards the carrot and ate it. Conversely, potato-fed snails located the potato in 67% of the cases. Snails that were fed apple or lettuce behaved like naive animals, with the majority of animals (75% in both cases) locating neither the carrot nor the potato.The ability of snails to locate this particular food after a single feeding episode was maintained for at least 11 days, provided that the snails were not exposed to other foods in the interim. If the animals were fed a different food (but still tested for food-finding ability to the initially conditioned food) their orientation preference decreased gradually over a period of 5 days.Although the snails' orientation is based upon olfactory cues, exposure to food odor alone is not sufficient to enable food-finding; additional feeding related stimuli are necessary.These findings indicate that Helix do not possess a predisposition for the foods tested, and further suggest that processes underlying food-finding and food selection are strongly influenced by learning experiences. The conditioning phenomenon underlying food-finding behavior has been called Food-Attraction Conditioning, and appears to be a crucial link between the ecologies of learning and foraging behaviors. The accessibility of the snail's nervous system should permit neuronal analysis of the mechanisms underlying such a unique and complex learning phenomenon.     

Duplicated Pax6 genes in Glomeris marginata (Myriapoda: Diplopoda), an arthropod with simple lateral eyes

Composite (facetted) eyes comprised by several units, termed ommatidia, are an ancestral feature in the arthropods. Some arthropods, however, do not possess composite eyes, obviously by secondary reduction. Reductions on the level of conserved eye developmental genes are one possibility to reduce the visual system. The genes of the Pax6 family have been shown to be key regulators of visual system development in a wide variety of animals. Reduction of Pax6 expression may therefore be expected in a species with reduced eyes. Here I have investigated the myriapod Glomeris marginata that displays very simple eyes. Glomeris, however, possesses two Pax6 genes that, based on their sequence, are similar to Drosophila eyeless (ey) and twin of eyeless (toy), respectively. Both genes are highly expressed in the optic lobes and the ventral nerve cord of developing embryos. Furthermore, homologs of other high-ranking eye developmental genes like hedgehog, decapentaplegic, dachshund, and homothorax are expressed in the optic lobes. This indicates that eye reduction in Glomeris is not realized at the level of the Pax6 genes or other genes on the upper levels of the eye development network. I suggest instead that the simple eyes of Glomeris are the product of changes at a much lower level in the network, probably at the level of genes directly regulating ommatidia development or ommatidia number and arrangement.     

Basic fibroblast growth factor induces retinal regeneration in vivo

In the present study, we have investigated the effect of basic fibroblast growth factor (bFGF) on retinal regeneration in the stage 22-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.     

Bioengineered human growth hormone supports limb regeneration in the hypophysectomized newt Notophthalmus viridescens

It is well documented that growth hormone (GH) replacement therapy will restore normal limb regeneration to hypophysectomized adult newts. However, it is also known that the GH preparations used in previous reports were contaminated by other pituitary hormones shown to support regeneration when administered free of GH. The recent availability of bioengineered human GH was studied for its ability to restore the regenerative capacity to hypophysectomized newts. Five days posthypophysectomy adult newts were subject to forelimb amputation distal to the elbow. Animals were divided into three groups (n greater than 20). Each received one of three GH preparations: pituitary-derived bovine GH, pituitary-derived human GH, or bioengineered human GH. GH was administered via intraperitoneal injection (0.029 IU/50 microliters) on alternate days for either the first 5 days (total of 3 injections) or for 35 days (total of 18 injections). Pituitary-intact and hypophysectomized control newts were subjected to forelimb amputation and injected with hormone diluent. All newts that received GH demonstrated normal limb regeneration to the early digitiform stage by 35 days postamputation. None of the hypophysectomized control newts showed any evidence of regeneration. We conclude that GH alone can restore the ability to undergo normal limb regeneration to hypophysectomized newts.     

3H-thymidine incorporation into the macrophages in the process of eye regeneration in adult tritons]

Changes in the number of labelled macrophages in the regenerating eye cavity of the adult common newts were studied within 2 to 12 days following the injection of 3H-thymidine and removal of retina and lens from the eyes. A small number of labelled macrophages was found in the eye cavity (0,2--5.9%) at all regeneration stages under study upon the pulse 3H-thymidine incorporation. Their number rapidly increased and attained by the end of observation (12 days) 73%. These results suggest that mainly mature non-dividing forms of macrophages which completed the mitotic cycle S-phase in the places of their active reproduction migrate in the eye cavity. The sharp increase of the number of labelled macrophages in the eye cavity is determined by the migration of those macrophages the precursors of which were labelled as a result of both the pulse 3H-thymidine incorporation and reutilization of the labelled precursors of DNA synthesis. New portions of labelled macrophages migrate in the eye cavity within 2 to 4 days.     

Principles of regeneration revealed by the planarian eye

One approach to elucidating the principles of regeneration is to investigate mechanisms that regenerate a target organ. Planarian eyes are discrete, visible structures that are dispensable for viability, making them powerful for studying the logic of regeneration. Fate specification in eye regeneration occurs in stem cells (neoblasts), generating eye progenitors. Eye progenitor production is not responsive to the presence or absence of the eye, with regeneration explained by constant progenitor production in the appropriate positional environment. Eye progenitors display coarse spatial specification. A combination of eye-extrinsic cues and self-organization with differentiated eye cells dictate where migratory eye progenitors target. Finally, guidepost-like cells influence regenerating axons to facilitate the restoration of eye circuitry. These findings from the eye as a case study present a model that explains how regeneration can occur.