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Constance L Cepko

Publications and source records attributed to Constance L Cepko.

31 records · Page 2Linked to original sources
PubMed2003

Electroporation and RNA interference in the rodent retina in vivo and in vitro.

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

Animals↗
PubMed2003

MicroSAGE is highly representative and reproducible but reveals major differences in gene expression among samples obtained from similar tissues.

BACKGROUND: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate. RESULTS: We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals. CONCLUSIONS: MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

3T3 Cells↗
PubMed2003

Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina.

Retinal progenitor cells regulate their proliferation during development so that the correct number of each cell type is made at the appropriate time. We found that the homeodomain protein Prox1 regulates the exit of progenitor cells from the cell cycle in the embryonic mouse retina. Cells lacking Prox1 are less likely to stop dividing, and ectopic expression of Prox1 forces progenitor cells to exit the cell cycle. During retinogenesis, Prox1 can be detected in differentiating horizontal, bipolar and AII amacrine cells. Horizontal cells are absent in retinae of Prox1-/- mice and misexpression of Prox1 in postnatal progenitor cells promotes horizontal-cell formation. Thus, Prox1 activity is both necessary and sufficient for progenitor-cell proliferation and cell-fate determination in the vertebrate retina.

Animals↗
PubMed2003

Thyroid hormone and retinal development: an emerging field.

Thyroid hormone appears to play a critical, yet not fully understood, role in the development of the neuroretina. This review focuses on recent experiments in the rodent, chicken, and amphibian, with an emphasis on how the hormone and its receptor isoforms influence retinal cell proliferation and cell fate decisions. The initial results are fueling the next generation of experiments in the retina, which promise to provide insights into the mechanisms of thyroid hormone action in a wide variety of developing neural tissue.

Amphibians↗
PubMed2003

Irx4-mediated regulation of Slit1 expression contributes to the definition of early axonal paths inside the retina.

Although multiple axon guidance cues have been discovered in recent years, little is known about the mechanism by which the spatiotemporal expression patterns of the axon guidance cues are regulated in vertebrates. We report that a homeobox gene Irx4 is expressed in a pattern similar to that of Slit1 in the chicken retina. Overexpression of Irx4 led to specific downregulation of Slit1 expression, whereas inhibition of Irx4 activity by a dominant negative mutant led to induction of Slit1 expression, indicating that Irx4 is a crucial regulator of Slit1 expression in the retina. In addition, by examining axonal behavior in the retinas with overexpression of Irx4 and using several in vivo assays to test the effect of Slit1, we found that Slit1 acts positively to guide the retinal axons inside the optic fiber layer (OFL). We further show that the regulation of Slit1 expression by Irx4 is important for providing intermediate targets for retinal axons during their growth within the retina.

Animals↗
PubMed2002

The dorsal-ventral axis of the neural retina is divided into multiple domains of restricted gene expression which exhibit features of lineage compartments.

The neural retina is a complex sensory structure designed to receive, integrate, and transmit visual information. An important aspect of retinal development is the establishment of pattern along the dorsal-ventral (D-V) and anterior-posterior (A-P) axes. The recent identification and functional characterization of a dorsal-specific and a ventral-specific transcription factor suggested that the D-V axis is divided into two domains. This study characterizes the expression patterns of these and other D-V markers, and establishes that the retina is subdivided into at least four domains of gene expression along this axis. The composition and spatial relation of these expression domains alters our model of D-V patterning, suggesting more complexity in the way that the retina is patterned than was previously recognized. As domains of gene expression within developing tissues sometimes comprise compartments whose borders are not crossed by clonally related cells, we performed a retroviral lineage study. A strong preference for cells to remain in their original domain of gene expression was observed, suggesting that these borders comprise developmental compartments.

Animals↗
PubMed2002

Ena/VASP proteins regulate cortical neuronal positioning.

Development of the multilayered cerebral cortex involves extensive regulated migration of neurons arising from the deeper germinative layers of the mammalian brain. The anatomy and formation of the cortical layers has been well characterized; however, the underlying molecular mechanisms that control the migration and the final positioning of neurons within the cortex remain poorly understood. Here, we report evidence for a key role of Ena/VASP proteins, a protein family implicated in the spatial control of actin assembly and previously shown to negatively regulate fibroblast cell speeds, in cortical development. Ena/VASP proteins are highly expressed in the developing cortical plate in cells bordering Reelin-expressing Cajal-Retzius cells and in the intermediate zone through which newly born cells migrate. Inhibition of Ena/VASP function through retroviral injections in utero led to aberrant placement of early-born pyramidal neurons in the superficial layers of both the embryonic and the postnatal cortex in a cell-autonomous fashion. The abnormally placed pyramidal neurons exhibited grossly normal morphology and polarity. Our results are consistent with a model in which Ena/VASP proteins function in vivo to control the position of neurons in the mouse neocortex.

Animals↗
PubMed2002

Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (adRP) is a heterogeneous set of progressive retinopathies caused by several distinct genes. One locus, the RP10 form of adRP, maps to human chromosome 7q31.1 and may account for 5-10% of adRP cases among Americans and Europeans. We identified two American families with the RP10 form of adRP by linkage mapping and used these families to reduce the linkage interval to 3.45 Mb between the flanking markers D7S686 and RP-STR8. Sequence and transcript analysis identified 54 independent genes within this region, at least 10 of which are retinal-expressed and thus candidates for the RP10 gene. A screen of retinal transcripts comparing retinas from normal mice to retinas from crx-/crx- knockout mice (with poorly differentiated photoreceptors) demonstrated a 6-fold reduction in one candidate, inosine monophosphate dehydrogenase 1 (IMPDH1; EC 1.1.1.205). Since many of the genes known to cause retinitis pigmentosa are under CRX control in photoreceptors, IMPDH1 became a high-priority candidate for mutation screening. DNA sequencing of affected individuals from the two American RP10 families revealed a GAC-->AAC transition in codon 226 substituting an asparagine for an aspartic acid in both families. The identical mutation was also found in a British RP10 family. The Asp226Asn missense mutation is present in all affected individuals tested and absent from unaffected controls. The aspartic acid at codon 226 is conserved in all IMPDH genes, in all species examined, including bacteria, suggesting that this mutation is highly deleterious. Subsequent screening of probands from 60 other adRP families revealed an additional family with this mutation, confirming its association with retinitis pigmentosa and the relatively high frequency of this mutation. Another IMPDH1 substitution, Val268Ile, was also observed in this cohort of patients but not in controls. IMPDH1 is a ubiquitously expressed enzyme, functioning as a homotetramer, which catalyzed the rate-limiting step in de novo synthesis of guanine nucleotides. As such, it plays an important role in cyclic nucleoside metabolism within photoreceptors. Several classes of drugs are known to affect IMPDH isoenzymes, including nucleotide and NAD analogs, suggesting that small-molecule therapy may be available, one day, for RP10 patients.

Amino Acid Motifs↗
PubMed2002

The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells.

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.

5' Untranslated Regions↗
PubMed2002

Sox-9 and cDachsund-2 expression in the developing chick telencephalon.

The expression patterns of the transcription factor, Sox-9, and of the nuclear factor, cDachsund-2, were examined in the developing chick telencephalon. Both genes were expressed in the ventricular zone and in the subventricular zone of the telencephalon during the period of neurogenesis. Whereas Sox-9 was not expressed in postmitotic tissues, cDachsund-2 was specifically expressed in the neostriatum and in subdivisions of the hyperstriatum embryonically and in posthatch chicks.

Animals↗
PubMed2002

The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation.

The paired type homeodomain gene, Rax, was previously identified as a key molecule in early eye formation in mice and humans. We report the expression patterns of two Rax family members from chicken, Rax and RaxL, and on the function of RaxL in photoreceptor development. Both Rax and RaxL are expressed in early retinal progenitor cells, with Rax being expressed at a significantly higher level than RaxL. At the time that photoreceptors begin to form, RaxL appears at a relatively high level in a subset of cells within the zone of proliferating progenitor cells. Subsequently, it is expressed in cells migrating to the photoreceptor layer, where it is highly expressed during the initial, but not late, stages of photoreceptor differentiation. To test the function of RaxL, a putative dominant-negative allele of RaxL comprising a fusion of the engrailed repressor domain and a region of RaxL (EnRaxLDeltaC) was introduced in vivo into the early chick eye using a retroviral vector. EnRaxLDeltaC, but not the dominant negative Rax (EnRaxDeltaC), caused a significant reduction in expression of early markers of photoreceptor cells. Examination of the transactivation activity of RaxL on a reporter construct bearing a canonical photoreceptor-specific enhancer element showed that RaxL exhibited significant activation activity, and that this activity was severely diminished in the presence of EnRaxLDeltaC. The effect on photoreceptor gene expression in vivo was specific in that other cell types were unaffected, as was general proliferation in the retina. The reduction in numbers of cells expressing photoreceptor markers was probably due to decreased survival of developing photoreceptor cells, as there was increased apoptosis among cells of the retina expressing dominant-negative RaxL. We propose that RaxL plays a role in the initiation of differentiation, and also possibly commitment, of photoreceptor cells in the chicken retina.

Amino Acid Sequence↗
PubMed2001

Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE).

We used the serial analysis of gene expression (SAGE) technique to catalogue and measure the relative levels of expression of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium (RPE) from one or both of two humans, aged 88 and 44 years. The cone photoreceptor contribution to all transcription in the retina was found to be similar in the macula versus the retinal periphery, whereas the rod contribution was greater in the periphery versus the macula. Genes encoding structural proteins for axons were found to be expressed at higher levels in the macula versus the retinal periphery, probably reflecting the large proportion of ganglion cells in the central retina. In comparison with the younger eye, the peripheral retina of the older eye had a substantially higher proportion of mRNAs from genes encoding proteins involved in iron metabolism or protection against oxidative damage and a substantially lower proportion of mRNAs from genes encoding proteins involved in rod phototransduction. These differences may reflect the difference in age between the two donors or merely interindividual variation. The RPE library had numerous previously unencountered tags, suggesting that this cell type has a large, idiosyncratic repertoire of expressed genes. Comparison of these libraries with 100 reported nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a retinal disease and 53 are RPE-specific genes. We expect that these libraries will serve as a resource for understanding the relative expression levels of genes in the retina and the RPE and for identifying additional disease genes.

Adult↗