BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration

Abstract

Lens regeneration in adult newts is a classic example of faithfully regenerating an entire organ via transdifferentiation1-6. After lentectomy, intriguing regulation allows the pigment epithelial cells (PECs) of the dorsal iris, but not the ventral, to dedifferentiate and then differentiate to form a new lens. This regulation might provide clues to the lack of lens regeneration in higher vertebrates. Six-3 and pax-6 known for their ability to induce ectopic lenses during embryogenesis7,8 and members of the BMP pathway, which are regulators of the dorsal/ventral axis establishment in embryos9 were examined for their role in induction of lens regeneration. Here we show that lens regeneration from the ventral iris is possible by inhibiting the BMP pathway or by transfecting ventral iris cells with six-3 and concomitant treatment with retinoic acid. In intact irises six-3 is expressed higher in the ventral iris. During regeneration, however, only levels in the dorsal iris are significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3/RA or BMP inhibitors. Therefore, transcriptional regulation associated with competency for lens regeneration, aims to increase levels over established thresholds and not to merely render a regulatory gene as dorsal-specific. Lack of induction in the axolotl, a salamander incapable of lens regeneration seems to be associated with repression of six-3 expression.

Signaling during lens regeneration

“…Contrary to its early role in embryonic lens specification and induction, Pax-6 appears to play a role during the later stages of regeneration. One line of evidence that supports this notion is that overexpression of Pax-6 in newt ventral iris PECs did not induce transdifferentiation of the ventral iris into lens [32]. Further evidence pointing to a role of Pax-6 in later events of lens regeneration such as proliferation of both the dorsal and ventral iris PECs and regulation of crystallin synthesis has been shown through the use of morpholino technology.…”

“…Perhaps it is the increase in Six-3 expression above the normal threshold levels for both the dorsal and ventral iris that is the key to inducing lens regeneration as opposed to the relative amount of Six-3. Treatment of ventral iris PECs with Six-3/RA causes the ventral iris tissue to adopt an expression profile similar to that seen in the dorsal iris during lens regeneration [32]. In relation to the BMP pathway, we have shown that treatment of newt ventral iris tissue with chordin increases the expression of Six-3 and Pax-6 [32].…”

“…However, during regeneration only the dorsal iris shows a significant increase in Six-3 expression. In addition, treatment of ventral iris PECs with Six-3/ retinoic acid induced transdifferentiation of the lens regeneration incompetent ventral iris [32]. Importantly, Six-3 alone did not cause transdifferentiation of ventral iris PECs but required the presence of retinoic acid (RA).…”

“…However, we have recently shown that BMPs play important functional roles in lens regeneration. We reported that lens regeneration can be induced from the regeneration incompetent ventral iris with two separate BMP inhibition treatments [32]. In this study newt iris explants (dorsal and ventral) were treated with recombinant mouse chordin protein or a truncated, signaling incompetent form of human BMPR-IA to inhibit BMP signaling and then re-implanted into a host newt to observe any effect on lens regeneration.…”

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt

“…They achieve this by the process of transdifferentiation of terminally differentiated cells at the site of the injury. During this process one cell type changes to another, by dedifferentiating [3][4][5]. It is conceivable that such an event may involve global changes in gene expression, implicating a large number of genes [6].…”