Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field.

The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (∼20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4-5 months.

We identified a mechanism whereby retina regeneration in the embryonic chick can be induced by the contribution of stem/ progenitor cells. We show that bone morphogenetic protein (BMP) signaling is sufficient and necessary to induce retina regeneration and that its action can be divided into two phases. By 3 days after postretinectomy (d PR), the BMP pathway directs proliferation and regeneration through the activation of Smad (canonical BMP pathway) and the up-regulation of FGF signaling by the MAPK pathway. By 7d PR, it induces apoptosis by activating p38 (a noncanonical BMP pathway) and down-regulating FGF signaling (by both MAPK and AKT pathways). Apoptosis at this later stage can be prevented, and BMP-induced regeneration can be further induced by inhibition of p38. These results unravel a mechanism for stem/ progenitor cell-mediated retina regeneration, where BMP activation establishes a cross-talk with the FGF pathway and selectively activates the canonical and noncanonical BMP pathways. Retina stem/progenitor cells exist in other species, including humans. Thus, our findings provide insights on how retinal stem cells can be activated for possible regenerative therapies.p38 ͉ FGF B one morphogenetic proteins (BMPs) are secreted signaling proteins that elicit their effect by binding to a heterodimer receptor complex composed of a BMP type I receptor (BMPRIA or BMPRIB) and a BMP type II receptor (BMPRII) (1). The BMP pathway can activate the canonical downstream effector, Smad, or a noncanonical downstream effector, transforming growth factor-␤-activated kinase (TAK1) (1). Several endogenous inhibitors, including noggin, chordin, follistatin, and gremlin, can regulate the ability of BMP to activate these pathways (2).In the developing retina, BMP2, BMP4, and BMP7, as well as the BMP receptors, are expressed in the chick (3) and mouse (4-6) and have been found to play a role in establishing the dorsal/ventral patterning of the retina. They also regulate the differentiation and survival of retinal neurons (7-11). Because of the BMP pathway's importance during retina development and in stem cell biology, we wanted to examine its role in inducing and regulating retina regeneration.A population of retinal stem cells is maintained after retinal development in the anterior margin of the eye in many vertebrates, including humans (12, 13). In most vertebrates, these retinal stem cells remain quiescent and do not respond to injury. However, cells in the anterior margin of the embryonic chick eye respond to injury during a limited time of retina development, providing an opportunity to study the induction process of these stem/progenitor cells (13-15).The embryonic chick has been shown to regenerate a complete retina in Ϸ7 days as long as a retinectomy is performed on or around embryonic day 4 (E4) and a source of FGF is added (14,16,17). In the presence of ectopic FGF2, regeneration takes place by transdifferentiation of the retinal pigmented epithelium (RPE) and the activation of retinal stem/progenitor cells (RS/ R...

Pax-6 is a master regulator of eye development and is expressed in the dorsal and ventral iris during newt lens regeneration. We show that expression of Pax-6 during newt lens regeneration coincides with cell proliferation. By knocking down expression of Pax-6 via treatment with morpholinos, we found that proliferation of iris pigment epithelial cells was dramatically reduced both in vitro and in vivo, and, as a result, lens regeneration was significantly retarded. However, induction of dedifferentiation in the dorsal iris was not inhibited. Pax-6 knockdown early in lens regeneration resulted in inhibition of crystallin expression and retardation of lens fiber induction. Once crystallin expression and differentiation of lens fibers has ensued, however, loss of function of Pax-6 did not affect crystallin expression and lens fiber maintenance, even though the effects on proliferation persisted. These results conclusively show that Pax-6 is associated with distinct early events during lens regeneration, namely control of cell proliferation and subsequent lens fiber differentiation.newt ͉ morpholino ͉ proliferation ͉ crystallin