Determinative roles of FGF and Wnt signals in iris-derived lens regeneration in newt eye

Total regeneration of experimentally excised lens from the dorsal part of the iris-pigmented epithelium of newts has been a key model of tissue regeneration via cells originating from a foreign tissue. Due to the strict spatial restriction of the lens origin in the newt iris, it has often been assumed that only the dorsal iris cells are endowed with an intrinsic potential to give rise to lens tissues. However, our reinvestigation of the process revealed completely different mechanisms underlying lens regeneration and its spatial restriction, comprising the following two steps: (i) Fibroblast growth factor (FGF) 2-dependent proliferation of iris-pigmented epithelium and activation of early lens genes ( Pax6, Sox2, MafB ) over the entire circumference of the iris; and (ii) dorsal iris-restricted activation of the canonical Wnt signals (involving Wnt2b and Frizzeld4) that leads to localized expression of late lens genes ( Prox1, Sox1, β-crystallin ). Injection of FGF2 into normal eyes specifically elicited the second lens development from the dorsal iris, and the administration of recombinant Wnt3a to the cultured iris-pigmented epithelium caused even ventral iris-derived lens development. Thus, it is concluded that the regulation of FGF2 and Wnt signals is a determinative of the iris-derived lens regeneration in the newt eye.     

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.     

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.     

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.     

Normal newt limb regeneration requires matrix metalloproteinase function

Newts regenerate lost limbs through a complex process involving dedifferentiation, migration, proliferation, and redifferentiation of cells proximal to the amputation plane. To identify the genes controlling these cellular events, we performed a differential display analysis between regenerating and nonregenerating limbs from the newt Notophthalmus viridescens. This analysis, coupled with a direct cloning approach, identified a previously unknown Notophthalmus collagenase gene (nCol) and three known matrix metalloproteinase (MMP) genes, MMP3/10a, MMP3/10b, and MMP9, all of which are upregulated within hours of limb amputation. MMP3/10b exhibits the highest and most ubiquitous expression and appears to account for the majority of the proteolytic activity in the limb as measured by gel zymography. By testing purified recombinant MMP proteins against potential substrates, we show that nCol is a true collagenase, MMP9 is a gelatinase, MMP3/10a is a stromelysin, and MMP3/10b has an unusually broad substrate profile, acting both as a stromelysin and noncanonical collagenase. Exposure of regenerating limbs to the synthetic MMP inhibitor GM6001 produces either dwarfed, malformed limb regenerates or limb stumps with distal scars. These data suggest that MMPs are required for normal newt limb regeneration and that MMPs function, in part, to prevent scar formation during the regenerative process.     

FGF signals induce Caprin2 expression in the vertebrate lens

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.     

FGF signals from the nasal pit are necessary for normal facial morphogenesis

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.     

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.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

Mesonephric FGF signaling is associated with the development of sexually indifferent gonadal primordium in chick embryos

The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression.     

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.     

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that EY needs to collaborate with additional factors for eye induction. We examined ectopic eye induction by EY in the wing disc and found that eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both HH and DPP signaling. Although EY in the anterior compartment induced dpp and dac, these were not sufficient for eye induction. Coexpression experiments show that EY needs to collaborate with high level of HH and DPP to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene.     

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.     

Cdx2 initiates histodifferentiation of the midgut endoderm

Null mutation or haploinsufficiency of Cdx2 results in the development of heterotopic lesions with a gastric phenotype in the midgut endoderm. Conversely transgenic expression of Cdx2 in the stomach causes the endoderm to differentiate into intestinal-type mucosa. We demonstrate that the mesoderm adjacent to intestinal heterotopic areas expresses stomach specific Barx1 while the surrounding mesoderm is Barx1 negative. We conclude that the initiation of gut histodifferentiation lies in the endodermal expression of Cdx2 and that endodermal/mesodermal cross-talk involving Barx1 with appropriate feedback loops results in the development of the postnatal gut phenotype.