Generation of transgenic newt Cynops pyrrhogaster for regeneration study

To take advantage of the ample potential for tissue regeneration by the newt, a technique to create transgenic newt was developed. The technique was based on a procedure for producing transgenic Xenopus, but modified to adapt to the different sperm morphology and to overcome the refractoriness of newt eggs to activation by normal cleavage. Sperm was collected from mature testes early in winter, permeabilized with digitonin, but without treatment of egg extract. Efficient egg activation was achieved by coinjection of inositol 1,4,5-trisphosphate (IP3) with DNA-sperm nucleus complex. Transgenic Cynops for EGFP/DsRed2 genes under the control of cytomegalovirus (CMV) enhancer/promoter showed nonmosaic widespread expression of reporter genes in embryos, swimming larvae, and adults after metamorphosis. Transgenic newt carrying EGFP gene under regulation of betaB1-crystallin promoter expressed the transgene uniquely in the lens. During lens regeneration after lens removal, EGFP expression occurred, reflecting the lens regeneration process. The newt transgenesis technique described here is likely to be of wide use in monitoring and manipulating gene expression in the study of molecular mechanisms underlying tissue regeneration.     

Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.     

Lens formation by pigmented epithelial cell reaggregate from dorsal iris implanted into limb blastema in the adult newt

In newt lens regeneration, the dorsal iris has lens forming ability and the ventral iris has no such capability, whereas there is no difference in the morphological criteria. To investigate the real aspects of this characteristic lens regeneration in the newt at the cellular level, a useful model system was constructed by transplanting the dorsal and ventral reaggregate derived from singly dissociated pigmented epithelial cells of the iris into the blastema of the forelimb in the newt. The lens was formed from the dorsal reaggregate with high efficiency, but not from the ventral one. No lens formation was observed in the implantation of the reaggregate into the tissue of the intact limbs. In detailed examination of the process of lens formation from the reaggregate, it was shown that tubular formation was the first step in the rearrangement of cells within the reaggregate. This was followed by depigmentation, vesicle formation with active cell growth, and the final step was lens fiber formation by transdifferentiation of epithelial cells composing the lens vesicle. The process was almost the same as in situ lens regeneration except the reconstitution of the two-layered epithelial structure was embodied as flattened tubular formation in the first step. The present study made it possible for the first time to examine lens forming ability in the reaggregate mixed with dorsal and ventral cells, because the formation of a reaggregate was started from singly dissociated cells of the dorsal and ventral cells of the iris. Mixed reaggregate experiments indicated that the existence of the dorsal cells in a cluster within the reaggregate is important in lens formation, and ventral cells showed an inhibitory effect on the formation. The present study demonstrated that the limb system thus constructed was effective for the analysis of lens formation at the cellular level and made it possible to examine the role of dorsal and ventral cells in lens regeneration.     

Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster

The sperm-binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome-reacted sperm were inseminated. No acrosome-intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome-reacted sperm were revealed to bind to a seat-like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome-reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin-Sepharose. These results indicated that sperm binding was mediated by heparin-like molecules expressed on the surface of acrosome-reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.     

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.     

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.