Insm1 is a transcriptional repressor associated with neuroendocrine tumors23
, small cell lung cancer24
, and the terminal cell divisions associated with neurogenesis25–28
. Although Insm1 plays an important role during nervous system development, surprisingly little is known about Insm1’s action in the adult nervous system. Furthermore, the mechanisms controlling insm1
gene induction and underlying Insm1’s action on progenitor differentiation remain poorly understood.
Zebrafish harbor 2 insm1
genes referred to as insm1a
. In the adult retina, insm1a
is normally expressed in the neurogenic ciliary marginal zone, but can also be found in the central retina of fish with photoreceptor degeneration30
. Our studies suggest that Insm1a is a multifaceted transcriptional repressor that plays an essential role in the formation, expansion and differentiation of MG-derived progenitors during retina regeneration. These studies highlight how a single transcription factor can assume diverse roles at different stages of regeneration and point to the cellular environment as a critical factor in determining Insm1a function. Furthermore, our studies revealed signaling mechanisms underlying injury-dependent insm1a
gene induction and also identified mechanisms by which Insm1a acts to control MG reprograming, along with the proliferation and differentiation of MG-derived progenitors. These results may have important implications for stimulating retina regeneration in mammals and for preventing uncontrolled MG proliferation in diseased and damaged human retinas. Furthermore our studies suggest mechanisms underlying Insm1
gene function and action in mammals.
Although Insm1 is best known as a transcriptional repressor that is associated with terminal cell division and neuronal differentiation, we uncovered a number of novel roles for Insm1a during retina regeneration. First, Insm1a links ascl1a
gene induction with dkk
gene repression, which we previously showed was necessary for MG dedifferentiation and retina regeneration5
. Second, using a novel protocol of delayed MO electroporation and gene knockdown, we found that Insm1a regulates the zone of injury-responsive MG flanking the injury site. This later function of Insm1a was only observed if MG were allowed to initially dedifferentiate and generate progenitors, suggesting that knockdown of Insm1a in these progenitors stimulated reprogramming of neighboring MG so they could generate additional progenitors. A possible mechanism underlying this effect was suggested by our finding that Insm1a controls hb-egfa
gene expression, whose product was recently shown to stimulate MG reprogramming and progenitor formation in the uninjured retina16
Investigation of the mechanisms initiating injury-dependent Insm1a induction identified Ascl1a, a gene product that is a nodal point for a number of signaling cascades during retina regeneration4,5,31,32
. We previously showed that ascl1a
gene expression is regulated by HB-EGF and is among the earliest gene inductions following retinal injury16
. Here we show Ascl1a is necessary for injury-dependent insm1a
induction. Thus, together these studies identify an HB-EGF/Ascl1a/Insm1a/dkk
signaling cascade as a critical signaling mechanism underlying the formation of injury-induced MG-derived progenitors. Interestingly, Ascl1 and Insm1 induction are also associated with Dkk
repression in certain human lung cancers24,33
and pancreatic endocrine tumors34,35
, perhaps suggesting a conserved signaling pathway.
In addition to regulating dkk
gene expression, Insm1a also feeds back to inhibit hb-egfa
promoter activity. This kind of feedback is often associated with oscillations36,37
and appears to underlie the biphasic pattern of insm1a
gene expression during retina regeneration and also helps restrict injury-responsive MG to the injury site.
Insm1a knockdown at 4 dpi not only expanded the zone of MG reprogrammed to produce progenitors, but also dramatically increased progenitor proliferation at the expense of differentiation. This action is consistent with Insm1’s role in mammals where it is associated with cell cycle exit. Insm1 appears to stimulate cell cycle exit by sequestering cyclin D1 with its proline-rich cyclin D1 binding domain38
. However, this domain is missing in zebrafish Insm1a (Supplementary Fig. S7
), suggesting another mechanism of action. Remarkably, we found that Insm1a not only suppressed a gene expression program that drives cell proliferation, but also relieved repression of p57kip2
, a cyclin kinase inhibitor that along with p27kip1
drives cell cycle exit during mouse retina development20,39
. In the mouse retina, reduced expression of p27kip1
is associated with MG proliferation and reactive gliosis40
. Interestingly, our transcriptome analysis of MG and MG-derived progenitors revealed that p27kip1
is constitutively expressed in these two populations. Whether this constitutive expression in the injured zebrafish retina helps prevent a gliotic response and promotes a regenerative one is not known.
In summary, our data suggest Insm1a plays at least 3 important roles during retina regeneration (). First, it contributes to MG reprogramming and the generation of progenitors by inhibiting Dkk expression and releasing the Wnt/β-Catenin pathway from inhibition. Second, it helps sculpt the zone of injury-responsive MG by regulating hb-egfa gene expression. Third, it contributes to the cessation of retina regeneration by stimulating cell differentiation via the suppression of genetic programs driving cell proliferation. Finally, our studies revealed mechanisms by which Insm1a mediates these effects and the signaling pathways underlying insm1a gene regulation in the injured retina. These studies place Insm1a among the key factors underlying retina regeneration and provide novel insight into signaling pathways that may help shift the response of MG in the injured mammalian retina from reactive gliosis towards retinal repair.