MOLECULAR ANALYSIS OF NEURAL CELL DEVELOPMENT

Project: Research project

Description

The long term objectives of this project are to understand at the molecular
level some of the genetic and epigenetic factors responsible for cell
differentiation and pattern formation in the mammalian Central Nervous
System. With this knowledge it will be possible to understand more fully a
wide range of developmental disorders and degenerative diseases that affect
the nervous system. Much of this proposal utilizes the retina as a model for CNS development.
Previous work from this laboratory has defined the developmental appearance
and some aspects of the cellular regulation of a wide array of retinal
molecules. One of the major aims of the current proposal is to further
characterize nuclear proteins essential for the transcriptional regulation
of one photoreceptor molecule, the visual pigment protein opsin. Gel
retardation and DNase I protection assays will be used to identify tissue-
specific binding proteins and their binding sites. Transient transfection
assays using the opsin promoter and beta-galactosidase or luciferase
reporter genes will be used to determine functionally important sites.
Developmental studies will test the hypothesis that a change in level or
activity of some of these proteins is important for the onset of opsin
transcription. This analysis will be extended to other photoreceptor proteins whose
appearance follows a very similar time course to that of opsin. In this
project we will focus on the cGMP-gated ion channel of outer segments that
is primarily responsible for the light dependent changes in membrane
conductance. This analysis will determine whether promoter regions of this
gene bind any proteins that interact with the opsin promoter to provide
information about common regulatory mechanisms during neural development. To study the role of diffusible factors in CNS development, known factors
and second messengers will be tested for their effects in culture on cell
proliferation, cell differentiation and specific transcription of defined
retinal genes. The presence of a number of embryonic growth factors will
be measured in developing retina using PCR amplification with primers
chosen from published sequences. A combination of these approaches will
allow testing of the hypothesis that commitment to become a neuronal cell
type and the onset of differentiation are regulated by changes in growth
factor levels or responses. Monoclonal antibodies will be prepared against partially purified nuclear
proteins. These antibodies will allow the testing of hypotheses concerning
the generation of cell type specificity of transcription by either unique
combinations of ubiquitous nuclear proteins or cell-type specific nuclear
proteins. Together, the specific aims in this proposal represent a focused effort to
define and understand the cellular and molecular processes governing the
formation of neural cell types and transcription of cell-type specific
genes of known function.
StatusFinished
Effective start/end date7/1/832/28/99

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $208,219.00
  • National Institutes of Health
  • National Institutes of Health: $219,721.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

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Opsins
Retina
Nuclear Proteins
Cell Communication
Genes
Epigenomics
Proteins
Cell Differentiation
Protein Binding
Monoclonal Antibodies
Growth Cones
Retinal Pigments
Carrier Proteins
Deoxyribonuclease I
Second Messenger Systems
beta-Galactosidase
Central Nervous System
Photoreceptor Cells
Ion Channels
Genetic Promoter Regions