Macrophage mobilization and morphology during lens regeneration from the iris epithelium in newts: studies with correlated scanning and transmission electron microscopy

The lens was removed from both eyes of adult newts (Notophthalmus viridescens), and the eyes were fixed in Karnovsky's fixative every 2 days 0-20 days after operation. Anterior half-eyes were prepared by standard procedures for scanning electron microscopy of the surface. Before fixation, the posterior iris surface was cleaned of adhering vitreous mechanically with forceps or by treatment with bovine testicular hyaluronidase or with hyaluronidase and collagenase. Some specimens were cryofractured in buffer or ethanol transverse to the mid-dorsal iris, and the fractured surface viewed with scanning electron microscopy (SEM). Cells with various combinations of ridges, blebs, filopodia, and lamellipodia were observed adhering to the posterior surface of the iris by 6 days after lentectomy. These cells, which exhibited the surface characteristics of macrophages, became more numerous in specimens fixed after longer intervals. Invasion of the iris epithelium was observed in a cryofractured specimen. After observations with SEM, selected specimens were embedded in plastic and sectioned for study with transmission electron microscopy (TEM). The cells on the iris surface had the cytological characteristics of macrophages, and other macrophages were located within the iris epithelium. In specimens fixed 16 or more days after lentectomy, a bulging lens vesicle was regenerating from the dorsal pupillary margin of the iris. Macrophages were absent or few on the surface of this developing lens but remained scattered over the adjoining iris. Roles that might be played by these macrophages during the transdifferentiation of iris epithelium into lens are discussed.     

Macrophage activity in Wolffian lens regeneration

The cell type mainly involved in the phagocytic uptake of melanosomes from iris epithelial cells during Wolffian lens regeneration in the adult newt is identified on the basis of electron and light microscopic evidence as a macrophage of monocytic origin. Appearance of macrophages in iris and ciliary epithelia following lentectomy is a part of leucocytic infiltration of the area, in which granulocytes, mast cells, and other cell types also participate. The general pattern of leucocytic infiltration was studied as a function of time after lentectomy. Infiltration of the iris epithelium by macrophages is reduced when most of the melanosomes have been removed from the cytoplasm of the epithelial cells and finally ceases when depigmentation has been completed. The possibility that an immune mechanism mediated by macrophages is involved in dedifferentiation of iris epithelial cells is discussed.     

Changes in RNA and protein synthesis in the neural retina during lens regeneration in newts

Since neural retina stimulates regeneration of a lens from the dorsal iris in newts, RNA and protein synthesis in the neural retina was investigated during this process. Incorporation of 3H-uridine and 3H-leucine using liquid scintillation counting was employed to compare RNA and protein synthesis in the neural retina from sham-operated control eyes with that in eyes during lens regeneration. An initial increase in 3H-uridine uptake was seen one to three days after lentectomy. This was followed by greater incorporation of 3H-leucine, indicating increased protein synthesis between 5 to 15 days after lens removal. A decrease in 3H-uridine uptake was also seen at 5 to 12 days after lentectomy. After 20 days both the RNA and protein synthesis returned to the normal level. Since the increase in protein synthesis is preceded by an increase in RNA synthesis, the two processes might be related. The results indicate significant changes in the synthesis of macromolecules by the neural retina following lentectomy. These may be indirectly related to the production of the neural retinal factor with stimulates lens differentiation.     

Proliferation of the cells of iris and pigment epithelium of the retina in the early periods after removal of the lens and iris in Spanish newts]

It has been shown by means of autoradiography that following the simultaneous removal of lens and retina in the eyes of adult ribbed newts (Pleurodeles waltlii) the proliferative processes related to the regeneration of retina, rather than lens, are most active at the early stages of eye restoration. During the lens regeneration in the absence of retina, the proliferation of the cells of pars iridica of the dorsal iris zone, a source of lens regeneration, is delayed, possibly due to the increase of the duration of mitotic cycle of these cells.     

Controlling gene loss of function in newts with emphasis on lens regeneration

Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.     

Molecular signatures that correlate with induction of lens regeneration in newts: lessons from proteomic analysis

Background

Amphibians have the remarkable ability to regenerate missing body parts. After complete removal of the eye lens, the dorsal but not the ventral iris will transdifferentiate to regenerate an exact replica of the lost lens. We used reverse-phase nano-liquid chromatography followed by mass spectrometry to detect protein concentrations in dorsal and ventral iris 0, 4, and 8 days post-lentectomy. We performed gene expression comparisons between regeneration and intact timepoints as well as between dorsal and ventral iris.

Results

Our analysis revealed gene expression patterns associated with the ability of the dorsal iris for transdifferentiation and lens regeneration. Proteins regulating gene expression and various metabolic processes were enriched in regeneration timepoints. Proteins involved in extracellular matrix, gene expression, and DNA-associated functions like DNA repair formed a regeneration-related protein network and were all up-regulated in the dorsal iris. In addition, we investigated protein concentrations in cultured dorsal (transdifferentiation-competent) and ventral (transdifferentiation-incompetent) iris pigmented epithelial (IPE) cells. Our comparative analysis revealed that the ability of dorsal IPE cells to keep memory of their tissue of origin and transdifferentiation is associated with the expression of proteins that specify the dorso-ventral axis of the eye as well as with proteins found highly expressed in regeneration timepoints, especially 8 days post-lentectomy.

Conclusions

The study deepens our understanding in the mechanism of regeneration by providing protein networks and pathways that participate in the process.

     

BDNF increases the phagocytic activity in cultured iris pigment epithelial cells

To investigate the effect of brain derived neurotrophic factor (BDNF) on the phagocytic activity in iris pigment epithelial (IPE) cells, purified porcine photoreceptor outer segments (POS) were applied to cultured IPE cells for three hours. To measure phagocytic activities, bound and total POS were differentially stained using a double immunofluorescence staining method. BDNF increased the binding of POS in IPE cells in a dose-dependent manner. Ingestion of POS, however, was not affected throughout the concentrations used in this study. To investigate the signal transduction pathways of BDNF, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and MAPK/ERK kinase (MEK) inhibitor, PD98059, were used for this study. LY294002 had no effect on the binding and ingestion of POS in BDNF-treated IPE cells. On the other hand, PD98059 completely inhibited the increase of POS binding in BDNF-treated cells and also decreased the ingestion of POS. These results indicate that increased POS binding activity by BDNF and the decreased ingestion of POS were mediated through the MAPK pathway.     

Neurogenesis during caudal spinal cord regeneration in adult newts

 After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube. Received: 30 November 1998 / Accepted: 22 December 1998     

Cellular analysis on localization of lens forming potency in the newt iris epithelium

The localization of a lens forming potency in the iris epithelium was studied by autoradiographic analysis of the distribution of ³H-thymidine labelled cells to be participated in lens regeneration in newts. DNA synthesis started from the dorsal portion of the iris epithelium around 4 days after lentectomy. 5 days after lentectomy, a large number of labelled cells were mostly found in the dorsal sector, showing strong contrast to the ventral and lateral sectors of iris, which contained a few labelled cells. The labelled index (the number of labelled cells/the number of cells in the definite pigmented area of the iris epithelium) of the dorsal sector attained the highest value, 29.7 ± 2.35, on day 7 after lentectomy, and dropped temporarily. This was followed by the second peak on day 12. The dorso-ventral ratio of the labelled index reached to the highest value, 6.87 ± 0.67, on day 5. This ratio decreased rapidly after the completion of a lens rudiment, and it became about 1. In “chase” experiments by diluting the radio-isotope with excess cold thymidine, it was obviously shown that most of the cells labelled with the radio-isotope and distributed in the dorsal marginal iris 5 days after lentectomy participated in the formation of a lens regenerate during the period of chasing. From these results, the following conclusion was drawn. The iris epithelium consists of at least 2 different cell populations; one is capable of transformation into lens cells and is distributed mostly in the dorsal portion of the iris epithelium, while the other has no potency for transformation and is able to grow to compensate a loss of the dorsal marginal cells which transformed into lens cells during the process of lens regeneration.     

Tissue factor expression in newt iris coincides with thrombin activation and lens regeneration

Lens regeneration in adult salamanders occurs at the pupillary margin of the mid-dorsal iris where pigmented epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. It is not understood how the injury caused by removal of the lens (lentectomy) in one location is linked to initiating the response in a different spatial location (dorsal iris) and to this particular sector. We propose that the blood provides a link between the localised coagulation and signal transduction pathways that lead to regeneration. A transmembrane protein (tissue factor) is expressed in a striking patch-like domain in the dorsal iris of the newt that localises coagulation specifically to this location, but is not expressed in the axolotl, a related species that does not show thrombin activation after lentectomy and cannot regenerate its lens. Our hypothesis is that tissue factor expression localises the initiation of regeneration through the activation of thrombin and the recruitment of blood cells, leading to local growth factor release. This is the first example of gene expression in a patch of cells that prefigures the location of a regenerative response, and links the immune system with the initiation of a regenerative program.     

Pax-6 and Prox 1 expression during lens regeneration from Cynops iris and Xenopus cornea: evidence for a genetic program common to embryonic lens development

Lens regeneration from non-lens ocular tissues has been well documented in amphibians, from the dorsal iris in the newt and from the outer cornea in Xenopus. To understand the early molecular events which govern lens regeneration, we examined the expression of two early marker genes of normal lens development, Pax-6 and Prox 1. In both Cynops (newt) iris and Xenopus cornea, Pax-6 is expressed soon after lentectomy in a region broader than that giving rise to the regenerating lens, indicative of an important role for Pax-6 in determination of the regeneration potential. Then Prox 1 expression begins within the Pax-6-expressing tissue, and these Prox 1-expressing cells give rise to the regenerating lens. This sequence of events also takes place in the lens placode of the embryo, indicating that the presence of the same genetic program operates in both embryonic lens development and lens regeneration, at least partly. In the Cynops iris, Pax-6 expression occurs initially in the entire marginal region of the iris after lentectomy but then becomes restricted to the dorsal region. Further studies are expected to elucidate the mechanism of this long-standing problem of the dorsal-restriction of lens regeneration from the newt iris.     

Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb

Dorsal iris from the eyes of adult Notophthalmus viridescens was transplanted into the blastema of regenerating limbs, subcutaneously in the limb or shoulder region, into the dorsal fin of larval newts and into the hindbrain of larval Ambystoma maculatum. The iris implants into the blastema regenerated lens vesicles or lenses with fibers in 40–75% of the cases. Multiple lenses were found in a few instances. No lenses developed from iris implants into the dorsal fin. Twenty percent of subcutaneous implants of iris formed lenses or lens vesicles, but lens regeneration from implants into the brain occurred only rarely. Denervation of the limb at the time of iris transplantation into the blastema greatly reduced the number of lenses regenerated. Studies on nerve fiber distribution in dorsal fin, subcutaneous areas, and denervated and innervated regenerating limbs, using the Bodian method, showed a general correlation between density of nerve fibers in the implant site and the incidence of lens regeneration from iris implants into that site. These results provide some evidence for a trophic action of nerve fibers on lens regeneration from the iris.     

Macrophage procoagulant-inducing factor. In vivo properties and chemotactic activity for phagocytic cells

Murine macrophage procoagulant-inducing factor (MPIF) is a lymphokine with chemical properties distinct from a number of well-characterized cytokines. MPIF induces procoagulant activity on the surface of macrophages and thus may play a central role in the expression of cell-mediated immunity. Highly enriched MPIF-alpha and -beta, separated by virtue of their basic isoelectric point and affinity for heparin, induced local induration and fibrin deposition and cellular infiltration similar to that observed in delayed type hypersensitivity reactions, when injected intradermally. Margination with of polymorphonuclear leukocytes (PMN) along the endothelium as well as increased PMN infiltration was evident after 4 h. In contrast to other inflammatory mediators (e.g., C5a, IL-1) reactivity was sustained, with greater numbers of mononuclear cells apparent 24 h after skin testing. Changes in the dermis were evident 4 h after MPIF injection with increased numbers of cells near areas where spaces in the collagen bundles had formed. Dermal thickening was evident after 24 h and collagen fiber structure was disrupted. Extravascular fibrinogen/fibrin was most prominent 24 h after testing. LPS, which induces macrophage procoagulant activity in vitro, did not induce the histopathologic changes evident with MPIF. MPIF was chemotactic for PMN and macrophages in vitro. Chemotactic activity was heat-labile and not due to C5a. Migration was dependent on a concentration gradient, as determined by checkerboard analysis, indicating that MPIF promoted chemotaxis rather than chemokinesis. Experiments reported here suggest that MPIF is an important mediator of fibrin deposition and cellular infiltration characteristic of cell-mediated immune response.     

Intracellular levels of adenosine 3':5'-cyclic monophosphate in the dorsal iris of the adult newt, during lens regeneration

The intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of cAMP. After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of cAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.     

Melanin granule metabolism in the iris and retinal pigment epithelium in adult tritions after completion of eye regeneration

It was concluded that the newly synthesized melanin granules were replaced in the pigmented tissues of the newt eye on the basis of redistribution of the cells of pigment epithelium of retina and iris labelled by 3H-DOPA 2.5 and 6.5 months after the isotope injection. The replacement of melanin granules and, correspondingly, melanin synthesis proceed more actively in the peripheral zones of the pigment epithelium of retina. The depigmentation of cells preceding the melanin synthesis appears to be realized with the participation of macrophages.