The vertebrate central nervous system contains anywhere from 500 to 10,000 functionally distinct subtypes of neurons and glia. Surprisingly little is known about how this amazing diversity of cell types is specified in development. To approach this problem, we have focused on the mouse retina, which contains only seven readily distinguished major cell types, each of which differentiates during well-defined intervals. We have comprehensively profiled gene expression in the mouse retina from the start to the end of neurogenesis using serial analysis of gene expression (SAGE). We determined the cellular expression patterns of over 1000 genes that show dynamic expression during development by SAGE. Projects currently underway or planned include:

1. Functional analysis of candidate regulators of cell specification in retina. We are conducting preliminary functional tests on several dozen transcripts that are selectively expressed in the four main retinal cell types that differentiate postnatally in the mouse – specifically rod photoreceptors, bipolar neurons, amacrine cells and Muller glia. These genes include transcription factors, regulators of signal transduction, and also putative noncoding RNAs. We plan to analyze in detail genes that show effects on cellular development in our initial screens.


2. Regulation of cell-specific transcription in retina. We seek to identify the cascade of transcription factors, and their target sites, that transform a retinal cell from an undifferentiated dividing progenitor cell to a terminally differentiated neuron or glia. We have used bioinformatic approaches to study the putative regulatory regions of genes specifically expressed in photoreceptors, bipolar neurons and Muller glia. We will test the ability of these sequences to regulate cell-specific expression via electroporation and high-throughput transgenesis, and in conjunction aim to identify the transcription factors that bind these sequences.


3. Genomic analysis of hypothalamus development. The mammalian hypothalamus is the central regulator of a broad set of behaviors ranging from the sleep-wake cycle to appetite to the care of offspring, but little is known about the diversity of cell types in the hypothalamus or how these cells are specified in development. We will conduct genomic studies on the developing hypothalamus similar to those we have conducted in the retina – profiling gene expression at various times of development, in various genetic backgrounds and in both male and female animals, and functionally examining genes that show interesting cellular expression patterns. We will also conduct single-cell expression profiling to classify neuronal subtypes in the hypothalamus.

Publications:

Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W. P., Fraioli, R. E., Cho, S.-H., Yung, R., Asch, E., Wong, W. H., and Cepko, C. L. Genomic analysis of mouse retinal development. PLoS Biology, in press.

Cai, L., Huang, H., Blackshaw, S., Liu, J. S., Cepko, C. L., and Wong, W. H. Cluster Analysis of SAGE data: A Poisson Approach. Genome Biology, submitted.

Blackshaw, S., Kuo, W. P., Park, P. J., Tsujikawa, M., Gunnersen, J. M., Scott, H. S., Wee- Boon, M., Tan, S. S., and Cepko, C. L. (2003) MicroSAGE is highly representative and reproducible, but reveals major differences in gene expression between samples obtained from identical tissues. Genome Biology, 4:R17.

Blackshaw, S. and Livesey, F. J. (2002). Applying genomic technologies to neural development. Current Opinion in Neurobiology, 6:110-14.

Browne, S. J., Sullivan, L. S., Blanton, S. H., Cepko, C. L., Blackshaw, S., Birch, D. G., Hughbanks-Wheaton, D., Heckenlively, J. R., and Daiger, S. P. (2002). Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa. Hum. Mol. Genet., 11(5):559-568.

Sharon, D*., Blackshaw, S*. Cepko, C. L., and Dryja, T. P. (2002). Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE). Proc. Natl. Acad. Sci. USA 99:315-20 (* indicates equal contribution by both authors).

Blackshaw, S., Fraioli, R. E., Furukawa, T., and Cepko, C. L. (2001). Comprehensive analysis of photoreceptor gene expression and the identification of candidate retinal disease genes. Cell, 107: 579-89.