In order to determine the role that six-3
play in the induction of transdifferentiation of the ventral iris, ventral iris cells were transfected in the presence or absence or retinoic acid (RA) with the appropriate constructs and examined for induction by utilizing an in vitro
transplantation system that reproduces the conditions seen in vivo10–12
. Retinoids have been shown to affect regeneration and to determine morphogenesis and differentiation of several tissues including the eye and limb13–16
. In addition, dorsal or ventral iris explants were treated with soluble BMP-4, BMP-7, chordin and a soluble competitor for BMPR-1A.
Following transfection and implantation of aggregated PECs, scores of eyes were examined (see Supplementary Information). As a rule, untransfected dorsal PEC aggregates transdifferentiate to lens while the ventral ones do not. Under the conditions outlined in Methods short term culturing of cells does not interfere with the potential for lens transdifferentiation. Dorsal aggregates produced a lens in over 83% of the cases (10/12), while the ventral ones, as expected, did not (0/11) (). It has been shown before, through beta galactosidase staining, that the lens is indeed derived from the aggregate12
. Dorsal aggregates transfected with the constructs with RA treatment also transdifferentiated to lens (not shown). However, with ventral PECs, only one particular protocol, transfection of PECs with six-3
in the presence of retinoic acid, led to the induction of lens transdifferentiation (). This induction occurred at a comparable rate (3/4; 75%) to that seen in the dorsal aggregates. Neither treatment with retinoic acid alone nor transfection of ventral PEC cultures with six-
3 alone was able to induce transdifferentiation. In the BMP series we found that inhibition of the pathway by either the BMPR-1A competitor or chordin resulted in the induction of a lens from the ventral explants (3/15 and 1/8 respectively) (). The incidence of induction was low (17%), however, we regard this as highly significant in light of the failure of the untreated ventral explants to differentiate to lens (0/27; 0% induction). This is in agreement with the established role of BMPs in maintaining ventral identity during embryogenesis and the fact that inhibition of BMPs binding to receptors results in dorsalization9
. Interestingly, treatment of the dorsal iris explants with BMP-7, and to a lesser degree BMP-4, significantly inhibited their ability to transdifferentiate to lens (1/12; 8.3% and 5/12; 41.6% respectively). Such results clearly indicate that BMPs maintain the ventral identity and inhibition of the pathway dorsalizes the ventral iris allowing transdifferentiation.
Lens induction from ventral PECs
In order to further probe the mechanism of induction, we decided to undertake a detailed gene expression profiling of six-3 and BMPR-1A during lens regeneration and during the experimental treatments that lead to the induction of lens regeneration from the ventral iris. Pax-6 expression was also assessed because of its known association with six-3. We selected to work with samples of iris isolated 2, 4 and 8 days post lentectomy. During this time, dedifferentiation events that lead to regeneration from the dorsal iris have been initiated. Moreover, at later stages the vesicle starts expressing crystallins and differentiating to lens. Since these genes are also expressed in the differentiating lens their induction-related expression might be ‘contaminated.’ Several interesting points emerged from the expression patterns, which somewhat were very surprising and call for a revision of our view of the mechanism of lens regeneration. First, both dorsal and ventral iris showed expression of all three genes. When the data were analyzed to compare between the dorsal and ventral iris we found that the three genes were expressed higher in the intact ventral iris. This pattern was maintained by day 8 but with a lesser relative fold change (). When the data, however, were analyzed in a different way to compare expression in the 2, 4 and 8-day dorsal iris with the intact dorsal iris and the 2, 4 and 8-day ventral iris with the intact ventral iris, to correlate expression with the process of regeneration, an interesting pattern emerged: The levels of six-3 were elevated in the dorsal iris only and seem comparable at this time to the ventral ones. BMPR-1A and pax-6 were also slightly up-regulated (). Up-regulation of six-3 in the dorsal iris started at day 4 (), while for pax-6 and BMPR-1A at day 8 (). In other words, increase of six-3 levels seems to be important during the dedifferentiation process in the dorsal iris. Since regeneration occurs only from the dorsal iris and since the ventral iris also expresses these genes, our data suggest that gene regulation associated with the competency for lens regeneration aims to increase levels over a particular threshold and not simply rendering a regulatory gene as dorsal-specific. Such a pattern for six-3 is clearly shown when the expression of the different time points is also presented in comparison to intact dorsal iris in one cluster ().
Expression during lens regeneration and induction
Treatment of ventral iris cells with six-3/RA, which resulted in induction of transdifferentiation, showed a similar pattern of up-regulation of six-3, pax-6, and BMPR-1A when compared to the untransfected ventral cells (). Treatment of the cells with RA alone or transfection of six-3 alone, which failed to induce irises to differentiate to lens, did not show such a pattern (not shown). Similarly, treatment of ventral iris explants with chordin, which also resulted in induction, invoked marked up-regulation of six-3 and pax-6, and to a lesser degree of BMPR-1A, in the treated ventral irises, as compared with the increase in the untreated irises (). BMPR-1A transcriptional regulation might not be that important for the induction. Interestingly the rate of increase (as relative fold change) in the treated ventral irises is comparable to the increase in the regenerating 8-day dorsal iris. In other words the treated ventral irises that were able to transdifferentiate to lens adopted a gene expression profile (especially for six-3) that is seen only in the dorsal iris during dedifferentiation and regeneration. This in turn indicates that when the ventral irises are coaxed to mimic patterns of regulatory events seen in the dorsal iris they are able to be “dorsalized” and therefore transdifferentiate into lens.
These expression patterns for six-3 pose the following question: Are there subpopulations in the dorsal or ventral iris that might account for these differences? To answer this question we used immunostaining to assess the distribution of six-3 expressing cells. We stained serial sections along the nasal-temporal axis that spanned the whole iris (with distinct dorsal and ventral portions) and we counted the positive cells. Six-3 positive cells were found throughout the examined 8-day dorsal and ventral irises without apparent differences in their distribution (). These results show that six-3 up-regulation is not attributed to expression in more cells. When the aggregates (or explants) transdifferentiated to lens nearly all cells participated, agruing against six-3 expressing subpopulations as well (). Since expression of six-3 and BMPR-1A in lens regeneration has not been reported before we also examined their expression throughout the process. In we show expression in dorsal and ventral iris at early stages (before vesicle formation) and in during later stages (in vesicle or regenerating lens). Ventral iris of the later stages is also positive (not shown). The presence of six-3 in dorsal and ventral iris is consistent with the QPCR data, immunostaining, however, is not quantitative.
Expression of six-3 and BMPR-1A during regeneration
The next question is whether the “newt treatments” are unique to the newt or if they can induce lens transdifferentiation in irises from other vertebrates. To answer this question we used another salamander, the axolotl, which possesses the ability to regenerate limbs or tail, but not the lens. None of the treatments induced transdifferentiation, indicating that the treatments are most likely newt-specific. However, it might be too premature to preclude the participation of six-3 and BMP inhibition in lens regeneration in other species. In the newt the lens is regenerated from the dorsal iris and in the premetamorphic frog from the cornea, two strategies that differ from the embryonic induction of lens development as well. This might argue against absolute conservation of the inductive mechanisms. To receive some insight that could explain the axolotl data we examined gene expression in intact irises, in irises 8 days after lentectomy and in treated irises. In contrast to what was observed in the newt, the expression profiles in the axolotl differ considerably in both intact and 8-day irises (). The intact and 8-day ventral irises do not show higher levels of expression over the dorsal ones. Moreover, six-3 was severely down regulated in the irises 8-days after lentectomy when compared with the intact irises. Pax-6 and BMPR-1A were slightly upregulated in both dorsal and ventral irises (). This expression pattern is diametrically opposite to what we observed in the newt and indicates that a negative regulation of six-3 as well as regulation in establishing thresholds might result in inability for lens regeneration in the axolotl. So, why did the treatments fail to induce lens transdifferentiation? We believe because repression of six-3 persists even after the treatments. Indeed, treatment with chordin, which mediated six-3 and pax-6 up-regulation in the induced newt ventral irises failed to do so in the axolotl (compare with ). Several explanations can account for the axolotl results. First, six-3 repression could be regulated more tightly than in the newt possibly by an inhibitor of its transcription and function. Second, the axolotl PECs do not respond to these treatments equally and they may require optimized conditions. Third, the mechanism of induction of lens regeneration in different vertebrates follows unique pathways.
It is rather interesting that pax-6
was unable to induce transdifferentiation of the ventral iris, even though it was shown to be up-regulated in the chordin-treated ventral irises. However, in pax-6
transfected cells six-3
was not upregulated (not shown) and this might be the reason why pax-6
was not able to induce transdifferentiation. Based on other results from our laboratories we now believe that pax-6 is rather involved in later events of lens regeneration, such as the proliferation of PECs in both the dorsal and ventral iris and control of crystallin synthesis17
. The fact that RA was also necessary for the induction most likely indicates that other factor(s) regulated by RA are involved, a synergism that has been shown in other studies as well16, 18,19
. Interestingly, both inhibitors of the BMP pathway and the six-3/pax-6
loop are part of a network identified during induction of eye development20
. The up-regulation of six-3
in chordin treated iris explants () suggests that the BMP signaling is upstream of the six-3/pax-6
Previous work by us and others has shown that other important regulators of lens differentiation, such as FGFs, Sox2, MafB
and members of the hedgehog pathway are expressed in both dorsal and ventral iris21,22
. This goes against the commonly held belief that regulatory genes involved in lens regeneration should be dorsal-specific. However, the detailed quantitative studies reported here suggest novel regulatory events involved in the induction of lens regeneration. Collectively, our data presented here demonstrate that induction of lens regeneration can be achieved in non-competent adult tissues. This is important because ectopic lens formation has never been shown in adults and opens new avenues in the field of vertebrate lens regeneration.