
	1999 Summer Scholars Program

Previous: Grace Messah

Next: Brandon Roberts

Notch1 Signaling Requires Intracellular Processing

By Amy Nichols
Mentors: Stacey Huppert, PhD and Raphael Kopan, PhD
Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO

Notch is a transmembrane protein responsible for cell fate decisions during embryonic and post-embryonic development. It functions as an intracellular communication mechanism between neighboring cells by binding to ligands on the cell surface. The expression of Notch on the surface of each cell is variable. When cells interact this variability is emphasized. The expression level of Notch in a cell defines it as either a signaling (low levels) or receiving (high levels) cell. This segregation of cells results in a differentiation process where, for example, signaling cells become neurons and receiving cells become epithelium. The molecular process is still being investigated, but at this time the essential mechanisms are known. Notch is a large protein consisting of 36 epidermal growth factor (EGF) like repeats, six ankyrin repeats, and a CSL binding domain (RAM23). The binding process between Notch and the ligand occurs at the eleventh and twelve EGF-like repeats on the extracellular portion of Notch. When the receptor and ligand bind, it has been proposed that the intracellular domain is cleaved and the resulting peptide acts as an indirect transcription factor via CSL. It is proposed that Notch activates multiple genes by changing the conformation of CSL, a repressor, so that it acts as an activator. This investigation focuses on the necessity of the intracellular cleavage.

To ensure that Notch 1 was necessary for development, a knockout mouse was created that lacked the Notch1 gene. It was found that the null Notch1 embryos died between 9.5 days postcoitum (dpc) and 11.5 dpc. Phenotypically the null embryos appeared small, less developed, lacking defined somites, and had a distended pericardial sack¹. The next question posed was how to mutate Notch1 to illustrate the necessity of intracellular cleavage. A sequencing process was undertaken to locate the cleavage site. This was accomplished by cross referencing the sequence of the cleaved peptide against the sequence of the whole protein. Once the site was located, a variety of point mutations were evaluated to see if processing could be eliminated. A single base change from thymine to guanine in the DNA proved to alter the primary structure of the protein enough to disrupt the cleavage event in cell culture². This mutation was introduced into the mouse genome and through a series of matings of heterozygous mice, mutant/wild type and homozygous mutant embryos were produced¹. The embryos were harvested from 8.5 dpc to 11 dpc. Each embryo was genotyped and stained. The genetic results confirmed the observations that the embryos appearing phenotypically similar to the null, with distended pericardial sacks, were indeed homozygous mutant animals. The heterozygous and the homozygous wild type animals grew larger and were further along in development. By 1 ldpc, homozygous mutant embryos were found to be in the process of resorption.

This work concludes that Notchl processing of the intracellular domain is necessary for viability. The mutants resembled the null in phenotype, but further detailed phenotypical analysis should reveal the true phenotypic similarities. Differences in phenotype may allude to independent functions of CSL. Also, further work needs to investigate if Notchl has signaling cascades that reach the nucleus independently of the cleavage process.

¹ Swiatek et al. (1994) Genes & Development 8: 707-719; Conlon, Reaume, and Rossant (1995) Development 121: 1533-1545.
²Schroeter, Kisslinger, and Kopan (1998) Nature 393: 382-386.

Previous: Grace Messah

Scholars Index

Next: Brandon Roberts

This page was last updated on Fri, Jun 16, 2000 at 2:16:41 PM by Tom Elgin with Userland Frontier.