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Thomas A Reh

Publications and source records attributed to Thomas A Reh.

At least 19 recordsLinked to original sources
PubMed2025

CUT&TIME captures the history of open chromatin in developing neurons.

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

Journal Article↗
PubMed2006

Efficient generation of retinal progenitor cells from human embryonic stem cells.

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 cell-derived 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.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Epidermal growth factor receptor expression regulates proliferation in the postnatal rat retina.

Epidermal growth factor (EGF) is known to promote proliferation of both retinal progenitors and Muller glia in vitro, but several questions remain concerning an in vivo role for this factor. In this study, we investigated whether the EGF receptor (EGFR) is necessary for the maintenance of normal levels of progenitor and Muller glial proliferation in vivo. Here, we show that (1) mice with homozygous deletion of the Egfr gene have reduced proliferation in late stages of retinal histogenesis, (2) EGF is mitogenic for Müller glia in vivo during the first two postnatal weeks in the rodent retina, (3) the effectiveness of EGF as a Müller glial mitogen declines in parallel with the decline in EGFR expression as the retina matures, and (4) following damage to the retina from continuous light exposure, EGFR expression is up-regulated in Müller glia to levels close to those in the neonatal retina, resulting in a renewed mitotic response to EGF. Together with previous results from other studies, these data indicate that the downregulation of a growth factor receptor is one mechanism by which glial cells maintain mitotic quiescence in the mature nervous system.

Animals↗
PubMed2006

Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina.

Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal-ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone beta2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRbeta2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development.

Animals↗
PubMed2006

Pea3 expression is regulated by FGF signaling in developing retina.

FGF signaling has been implicated as an important regulator of retinal development. As a first step in characterizing potential downstream targets of FGF signaling in the retina, we have analyzed expression of Pea3, a member of the Pea3 class of Ets-domain transcription factors, in the developing eye. We find that Pea3 is expressed in the developing retina, and its transcription is regulated by FGF receptor activation. In addition, FGF signaling activates Cath5, a gene necessary for retinal ganglion cell differentiation. These results suggest that FGF signaling via MAPK up-regulates transcription factors that in turn control retinal ganglion cell differentiation.

Animals↗
PubMed2006

Retinal stem cells.

During the embryonic development of the eye, a group of founder cells in the optic vesicle gives rise to multipotent progenitor cells that generate all the neurons and the Müller glia of the mature retina. In most vertebrates, a small group of retinal stem cells persists at the margin of the retina, near the junction with the ciliary epithelium. In fish and amphibians, the retinal stem cells continue to produce progenitors throughout life, adding new retina to the periphery of the existing retina as the eye grows. In birds the new retinal addition is more limited, and it is absent in those mammals that have been analyzed. Nevertheless, cells from the retinal periphery and ciliary body of mammals can be isolated and grown in vitro for extended periods. Methods for the study of both embryonic progenitors and adult retinal stem cells in vitro and in vivo have led to a better understanding of retinal development, allowed for the screening of factors important in retinal growth and differentiation, and enabled the development of methods to direct stem and progenitor cells to specific fates. These methods may ultimately lead to the development of strategies for retinal repair.

Animals↗
PubMed2006

Notch activity is downregulated just prior to retinal ganglion cell differentiation.

The Notch signaling pathway is important at several stages of retinal development including the differentiation of retinal ganglion cells and Muller glia. The downstream effectors of Notch signaling, Hes1 and Hes5, have been shown to be critical in the retina as well. While Notch activity directly regulates Hes1 and Hes5 elsewhere in the nervous system, it has been unclear whether Hes1 and/or Hes5 are directly regulated by Notch activity in the developing retina. Here, we report that both Hes1 and Hes5 are directly regulated by Notch activity during retinal development. Using fluorescence-based Hes1 and Hes5 reporter constructs, we can monitor Notch activity in progenitor cells in the intact retina, and we find that Notch activity is downregulated just prior to retinal ganglion cell differentiation.

Amino Acid Sequence↗
PubMed2005

Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating newt retina.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.

Animals↗
PubMed2005

Sonic hedgehog regulates proliferation of the retinal ciliary marginal zone in posthatch chicks.

The ciliary marginal zone (CMZ) has long been known to be a source of postembryonic neuronal production in the retinas of fish and amphibians, and more recently, birds. However, there is little known about the factors that are required for the maintenance of this neural stem cell zone. The cells of the CMZ respond to mitogens such as endothelial growth factor, insulin-like growth factor-1, and insulin, factors that are also mitogenic for embryonic retinal progenitors, suggesting that the continued expression of embryonic mitogenic factors might be required to maintain the postembryonic proliferative potential of the CMZ. To test this hypothesis, we examined the expression and functional role of a critical embryonic retinal progenitor mitogen, Sonic hedgehog (Shh) in the regulation of proliferation of the cells of the CMZ. We have found that Shh is concentrated at the retinal margin of postembryonic chicks. Moreover, we report that intraocular injection of Shh stimulates proliferation of the CMZ cells, whereas cyclopamine, an inhibitor of the Shh pathway, inhibits CMZ proliferation. We conclude that Shh signaling is an important factor in the maintenance of postembryonic retinal neurogenesis.

Animals↗
PubMed2005

Retinoid X receptor (gamma) is necessary to establish the S-opsin gradient in cone photoreceptors of the developing mouse retina.

PURPOSE: The retinoid X receptors (RXRs) are members of the family of ligand-dependent nuclear hormone receptors. One of these genes, RXRgamma, is expressed in highly restricted regions of the developing central nervous system (CNS), including the retina. Although previous studies have localized RXRgamma to developing cone photoreceptors in several species, its function in these cells is unknown. A prior study showed that thyroid hormone receptor beta2 (TRbeta2) is necessary to establish proper cone patterning in mice by activating medium-wavelength (M) cone opsin and suppressing short-wavelength (S) cone opsin. Thyroid hormone receptors often regulate gene transcription as heterodimeric complexes with RXRs. METHODS: To determine whether RXRgamma cooperates with TRbeta2 to regulate cone opsin patterning, the developmental expression of RXRgamma was examined, and cone opsin expression in RXRgamma-null mice was analyzed. RESULTS: RXRgamma was expressed in postmitotic cones and was transiently downregulated at the time of S-opsin onset in both mouse and human cones. RXRgamma-null mice expressed S-opsin in all cones, similar to the TRbeta2-null mice. Unlike TRbeta2-null mice, which did not express M-opsin, RXRgamma-null mice had a normal pattern of M-opsin expression. CONCLUSIONS: RXRgamma is essential (along with TRbeta2) for suppressing S-opsin in all immature cones and in dorsal cones in the mature retina, but it is not necessary for M-opsin regulation. These results demonstrate a critical role for RXRs in regulating cell differentiation in the CNS and highlight a remarkable conservation of opsin regulation from Drosophila to mammals.

Animals↗
PubMed2005

Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGF beta signaling.

The number of proliferating cells in the rodent retina declines dramatically after birth. To determine if extrinsic factors in the retinal micro-environment are responsible for this decline in proliferation, we established cultures of retinal progenitors or Muller glia, and added dissociated retinal neurons from older retinas. The older cells inhibited proliferation of progenitor cells and Muller glia. When these experiments were performed in the presence of TGF(beta)RII-Fc fusion protein, an inhibitor of TGF(beta) signaling, proliferation was restored. This suggests a retina-derived TGF(beta) signal is responsible for the developmental decline in retinal proliferation. TGFbeta receptors I and II are expressed in the retina and are located in nestin-positive progenitors early in development and glast-positive Muller glia later in development. RT-PCR and immunofluorescence data show TGF(beta)2 is the most highly expressed TGF(beta)ligand in the postnatal retina, and it is expressed by inner retinal neurons. Addition of either TGF(beta)1 or TGF(beta)2 to postnatal day 4 retinas significantly inhibited progenitor proliferation, while treatment of explanted postnatal day 6 retinas with TGF(beta) signaling inhibitors resulted in increased proliferation. Last, we tested the effects of TGF(beta) in vivo by injections of TGF(beta) signaling inhibitors: when TGF(beta) signaling is inhibited at postnatal day 5.5, proliferation is increased in the central retina; and when co-injected with EGF at postnatal day 10, TGF(beta)inhibitors stimulate Muller glial proliferation. In sum, these results show that retinal neurons produce a cytostatic TGF(beta) signal that maintains mitotic quiescence in the postnatal rat retina.

Animals↗
PubMed2005

TWIST is expressed in human gliomas and promotes invasion.

TWIST, a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, has been shown to promote tumor cell metastasis and to enhance survival in response to cytotoxic stress. Our analysis of rat C6 glioma cell-derived cDNA revealed TWIST expression, suggesting that the gene may play a role in the genesis and physiology of primary brain tumors. To further delineate a possible oncogenic role for TWIST in the central nervous system (CNS), we analyzed TWIST expression in human gliomas and normal brain by using reverse transcription polymerase chain reaction, Northern blot analysis, in situ hybridization, and immunohistochemistry. TWIST expression was detected in the large majority of human glioma-derived cell lines and human gliomas examined. Levels of TWIST mRNA were associated with the highest grade gliomas, and increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis and subsequent malignant progression. TWIST was also expressed in embryonic and fetal human brain, and in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.

Animals↗
PubMed2004

Different aspects of gliosis in retinal Muller glia can be induced by CNTF, insulin, and FGF2 in the absence of damage.

PURPOSE: In response to acute damage, Muller glia in the retina have been shown to dramatically alter their expression of filamentous proteins. Since damaged retinal cells are known to produce growth factors such as insulin-like growth factor (IGF), ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF), the altered expression of filaments in Muller glia in response to retinal damage may be induced by some of these factors. The purpose of this study was to assay whether growth factors influence the expression of filamentous proteins in Muller glia in the intact retinas of postnatal chickens. METHODS: We assayed for changes in expression levels of IGF-I, IGF-II, CNTF, FGF1, and FGF2 in N-methyl-D-aspartate(NMDA) damaged retinas by using quantitative PCR. In undamaged retinas, we assayed whether intraocular injections of insulin, CNTF, or FGF2 influenced glial expression of glial fibrillary acidic protein (GFAP), neurofilament, RA4, vimentin and beta3 tubulin by using immunocytochemistry on frozen sections. RESULTS: We demonstrated that levels of mRNA for IGF-II, FGF1, FGF2, and CNTF were increased in the postnatal chicken retina in response to neurotoxic damage. This was coincident with increased glial expression of GFAP and filamentous neuronal proteins. The combination of insulin and FGF2 caused postmitotic Muller glia to transiently increase their expression of vimentin and putative neuron specific filamentous proteins such as neurofilament, beta3 tubulin and RA4. By comparison, insulin or FGF2 alone had minor effects on glial expression of cytoskeletal proteins. Although neurofilament expression was not induced by CNTF, this growth factor stimulated Muller glia to express GFAP. CONCLUSIONS: We conclude that the phenotype of postmitotic Muller glia is plastic and can be regulated by retinal damage, and these damage induced changes in phenotype can be induced by exogenous growth factors in the absence of damage.

Animals↗
PubMed2004

Leber congenital amaurosis linked to AIPL1: a mouse model reveals destabilization of cGMP phosphodiesterase.

Leber congenital amaurosis (LCA4) has been linked to mutations in the photoreceptor-specific gene Aryl hydrocarbon interacting protein like 1 (Aipl1). To investigate the essential role of AIPL1 in retina, we generated a mouse model of LCA by inactivating the Aipl1 gene. In Aipl1(-/-) retinas, the outer nuclear layer develops normally, but rods and cones then quickly degenerate. Aipl1(-/-) mice have highly disorganized, short, fragmented photoreceptor outer segments and lack both rod and cone electroretinogram responses. Recent biochemical evidence indicates that AIPL1 can enhance protein farnesylation. Our study reveals that rod cGMP phosphodiesterase, a farnesylated protein, is absent and cGMP levels are elevated in AIPL1(-/-) retinas before the onset of degeneration. Our findings demonstrate that AIPL1 enhances the stability of phosphodiesterase and is essential for photoreceptor viability.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Persistent progenitors at the retinal margin of ptc+/- mice.

The hedgehog signaling pathway is a key regulator of neural development, affecting both proliferation and differentiation of neural progenitors. Sonic hedgehog (Shh) is a mitogenic factor for retinal progenitors in vitro. To determine whether this signaling system is important in vivo for regulating retinal progenitor proliferation, we analyzed mice with a single functional allele of the Shh receptor patched (ptc). We found that ptc+/- mice had increased numbers of neural progenitors at every stage of retinal development that we examined. In addition, these mice had persistent progenitors at the retinal margin for up to 3 months of age, reminiscent of the ciliary marginal zone of lower vertebrates. To test whether the progenitors at the retinal margin of ptc+/- mice could be induced to regenerate retinal neurons in response to damage, we bred ptc+/- mice onto a retinal degeneration background (pro23his rhodopsin transgenic) and labeled newly generated cells with combined immunohistochemistry for bromodeoxyuridine and retinal neuron and photoreceptor-specific markers. We found newly generated neurons and photoreceptors at the retinal margin in ptc+/-;pro23his mice. We propose that the Shh pathway may act as a regulator of both prenatal and postnatal retinal growth.

Animals↗
PubMed2004

NeuroD induces the expression of visinin and calretinin by proliferating cells derived from toxin-damaged chicken retina.

Müller glia have been shown to be a potential source of neural regeneration in the avian retina. In response to acute damage Müller glia de-differentiate, proliferate, express transcription factors found in embryonic retinal progenitors, and some of the progeny differentiate into neurons and glia (Fischer and Reh [2001a] Nat. Neurosci. 4:247-252). However, most of the cells produced by proliferating Müller cells appear to remain undifferentiated. The purpose of this study was to test whether the neurogenic gene NeuroD can promote the differentiation of proliferating cells derived from the postnatal chick retina. We used recombinant avian retroviruses to transfect green fluorescent protein (GFP) or NeuroD. The majority of cells transfected with GFP remained undifferentiated, with a few cells differentiating into calretinin-immunoreactive neurons. Many cells transfected with the NeuroD-virus expressed calretinin, neurofilament, or visinin, while most cells remained undifferentiated. The number of calretinin-expressing cells that were generated was increased approximately 20-fold with forced expression of NeuroD. In addition, we found that cells transfected with NeuroD never expressed glutamine synthetase, a marker of mature Müller glia, suggesting that NeuroD suppresses glial differentiation. We conclude that NeuroD stimulates cells from the toxin-damaged chicken retina to acquire some neuronal phenotypes. We propose that most of these cells were derived from Müller glia.

Animals↗
PubMed2004

Identification of ciliary epithelial-specific genes using subtractive libraries and cDNA arrays in the avian eye.

The ciliary epithelium of the ciliary body is derived from the anterior rim of the developing optic cup. Several recent studies have found that developmental abnormalities in this tissue can underlie congenital glaucoma. However, there is little known about the development of the ciliary epithelium. To better understand the developmental events responsible for the specification of this domain of the optic cup, we used a subtractive library, differential screening approach along with the construction of cDNA arrays to identify genes expressed in the ciliary epithelium of the chicken. We identified many genes specifically expressed in the ciliary epithelium, including a number that had been described previously as enriched in the ciliary epithelium of other species. By analyzing the expression of these genes during eye development, we were able to correlate the onset of ciliary epithelial gene expression with a reduction in mitotic activity in this region. We propose that the mechanisms that regulate the expression of ciliary epithelial genes are linked to the reduction in proliferation that results in the epithelial monolayer in this region.

Animals↗