Mü ller glia can serve as a source of new neurons after retinal damage in both fish and birds. Investigations of regeneration in the mammalian retina in vitro have provided some evidence that Mü ller glia can proliferate after retinal damage and generate new rods; however, the evidence that this occurs in vivo is not conclusive. We have investigated whether Mü ller glia have the potential to generate neurons in the mouse retina in vivo by eliminating ganglion and amacrine cells with intraocular NMDA injections and stimulating Mü ller glial to re-enter the mitotic cycle by treatment with specific growth factors. The proliferating Mü ller glia dedifferentiate and a subset of these cells differentiated into amacrine cells, as defined by the expression of amacrine cell-specific markers Calretinin, NeuN, Prox1, and GAD67-GFP. These results show for the first time that the mammalian retina has the potential to regenerate inner retinal neurons in vivo.amacrine ͉ Gabaergic neuron ͉ glia I t is well established that the retina of cold-blooded vertebrates regenerates very well after damage (1-3). The avian retina is also capable of limited regeneration of new neurons following neurotoxic damage (4). In both systems, damage to retinal neurons causes Müller glial cells to re-enter the cell cycle, after which they dedifferentiate into retinal progenitors, and ultimately differentiate into neurons. In fish, all types of neurons are regenerated. However, in chicks, only a limited number of different types of inner retinal neurons (amacrine, bipolar, and ganglion cells) are produced; few, if any, photoreceptors are regenerated.In the mammalian retina, by contrast, the proliferative response of Müller glia to injury is even more limited than in chicks. In response to injury in mouse or rat retina, the Müller glia may become reactive and hypertrophy, but few re-enter the mitotic cell cycle. Due to the lack of a spontaneous regenerative response in the mammalian retina, several groups have attempted to stimulate regeneration with intraocular injections of growth factors and/or transcription factors, in vitro or in vivo (5-10). Taken together, the studies of the mammalian retina indicate that Müller glia have a very limited proliferative response to injury, but can be stimulated to re-enter the cell cycle after photoreceptor or inner retinal neuron injury. These studies also reported that some of the progeny of Müller glial mitotic divisions go on to differentiate characteristics of rod photoreceptors. However, these studies failed to detect regenerating inner retinal neurons after damage, unless they were transfected with genes that specifically promote amacrine fate (9). This is puzzling, since in the chick, amacrine cells are the primary neuronal cells that are regenerated after injury. In an attempt to resolve this issue, we have carried out a systematic analysis of the response to injury in the mouse retina, and the effects of growth factor stimulation on Müller glial proliferation. The previous studies have used rats because ...

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.arealization | Auts2 | microarray

The chicken retina is capable of limited regeneration. In response to injury, some Müller glia proliferate and de-differentiate into progenitor cells. However, most of these progenitors fail to differentiate into neurons. The Notch pathway is upregulated during retinal regeneration in both fish and amphibians. Since the Notch signaling pathway maintains cells in a progenitor state during development, we hypothesized that a persistently active Notch pathway might prevent a more successful regeneration in the chick retina. We found that Notch signaling components are upregulated in the proliferating progenitors. We also found that blocking the Notch pathway while Müller glia are de-differentiating into progenitor cells prohibits regeneration; conversely, blocking the Notch pathway after the progenitors have been generated from the Müller glia caused a significant increase in the percentage of new neurons. Thus, Notch signaling appears to play two distinct roles during retinal regeneration. Initially, Notch activity is necessary for the de-differentiation/proliferation of Müller glia, while later it inhibits the differentiation of the newly generated progenitor cells.

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle.

In the developing nervous system, the balance between proliferation and differentiation is critical to generate the appropriate numbers and types of neurons and glia. Notch signaling maintains the progenitor pool throughout this process. While many components of the Notch pathway have been identified, the downstream molecular events leading to neural differentiation are not well understood. We have taken advantage of a small molecule inhibitor, DAPT, to block Notch activity in retinal progenitor cells, and analyzed the resulting molecular and cellular changes over time. DAPT treatment causes a massive, coordinated differentiation of progenitors that produces cell types appropriate for their developmental stage. Transient exposure of retina to DAPT for specific time periods allowed us to define the period of Notch inactivation that is required for a permanent commitment to differentiate. Inactivation of Notch signaling revealed a cascade of proneural bHLH transcription factor gene expression that correlates with stages in progenitor cell differentiation. Microarray/QPCR analysis confirms the changes in Notch signaling components, and reveals new molecular targets for investigating neuronal differentiation. Thus, transient inactivation of Notch signaling synchronizes progenitor cell differentiation, and allows for a systematic analysis of key steps in this process.