SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cellderived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.photoreceptors ͉ eye development ͉ neurogenesis T he neural retina is subject to a number of degenerative conditions, including retinitis pigmentosa, age-related macular degeneration, and glaucoma. Although there are a number of sources of progenitors for regeneration in nonmammalian vertebrates, these are greatly reduced or absent in the adult mammalian retina (1). By contrast, recent reports show that retinal progenitor cells can be derived from mouse ES cells (2, 3), and may provide an alternative to adult derived retinal stem cells. In other regions of the central nervous system, the transplantation of neurons derived from embryonic stem cells has led to some promising results. Dopaminergic neurons derived from mouse, monkey, and human embryonic stem cells have been shown to integrate into the brain after transplantation and partially restore function in animal models of Parkinson's disease (4-8). Oligodendrocytes derived from ES cells can repair some of the damage caused by spinal cord trauma (9) as well as in mouse models of spinal demyelination (10-12). ResultsWe have developed methods for deriving retinal neurons from human embryonic stem (hES) cells. The current molecular genetic model of vertebrate embryogenesis (13) suggests that there are several sequential induction steps. Forebrain development requires that both BMP and Wnt signaling are antagonized (14)(15)(16)(17)(18)(19). Although the specific molecular signals required for eye field specification are not completely defined in any model system, insulin-like growth factor-1 (IGF-1) mRNA injections into Xenopus embryos specifically promote eye induction (20). Therefore, to direct the ES cells to an anterior neural fate, we treated embryoid bodies with a combination of noggin (a potent endogenous inhibitor of the BMP pathway) and Dickkopf-1 (dkk1; a secreted antagonist of the Wnt͞␤-catenin signaling pathway (14, 21)) and IGF-1. The embryoid bodies were cultured for 3 days in the three factors (Fig. 1A) and t...

The retina of warm-blooded vertebrates is believed to be incapable of neural regeneration. Here we provide evidence that the retina of postnatal chickens has the potential to generate new neurons. In response to acute damage, numerous Müller glia re-entered the cell cycle, and shortly thereafter, expressed CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. These progenitor-like cells transiently expressed neurofilament. Newly formed cells became distributed throughout the inner and outer nuclear layers of the retina, and remained for at least three weeks after damage. Some of these newly formed cells differentiated into retinal neurons, a few formed Müller glia, and most remained undifferentiated, with continued expression of Pax6 and Chx10. These cells continued to proliferate when grown in culture, with some differentiating into retinal neurons or Müller glia. We propose that, in response to damage, Müller glia in the retina are a potential source of neural regeneration.

Summary Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina; photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem (ES) cells could in principle provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ES cells. We now show that retinal cells derived from human ES cells will migrate into mouse retinas following intra-ocular injection, settle into the appropriate layers and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx -/- mice (a model of Leber's Congenital Amaurosis), the hES cell derived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hES cells can, in principle, be used for photoreceptor replacement therapies.

Color vision is facilitated by distinct populations of cone photoreceptors in the retina. In rodents, cones expressing different opsin photopigments are sensitive to middle (M, 'green') and short (S, 'blue') wavelengths, and are differentially distributed across the retina. The mechanisms that control which opsin is expressed in a particular cone are poorly understood, but previous in vitro studies implicated thyroid hormone in cone differentiation. Thyroid hormone receptor beta 2 (TR beta 2) is a ligand-activated transcription factor that is expressed in the outer nuclear layer of the embryonic retina. Here we delete Thrb (encoding Tr beta 2) in mice, causing the selective loss of M-cones and a concomitant increase in S-opsin immunoreactive cones. Moreover, the gradient of cone distribution is disturbed, with S-cones becoming widespread across the retina. The results indicate that cone photoreceptors throughout the retina have the potential to follow a default S-cone pathway and reveal an essential role for Tr beta 2 in the commitment to an M-cone identity. Our findings raise the possibility that Thrb mutations may be associated with human cone disorders.

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons1–6. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage1,7 in zebrafish and is necessary for regeneration8. Although Ascl1 is not expressed in mammalian MG after injury9, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro10 and in vivo after NMDA (N-methyl-D-aspartate) damage in young mice11. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression11. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC–seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.

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 ...

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.