James Priess - US grants

myogenesis; muscle cells; developmental genetics; Caenorhabditis elegans; nucleic acid sequence; early embryonic stage; cell cell interaction; gene expression; in situ hybridization; nonmammalian vertebrate embryology;

We want to understand how embryonic cells become committed to particular pathways of differentiation. Molecular and genetic analysis of development in the fruit fly Drosophila and the nematode Caenorhabditis elegans already has resulted in the identification of several key genes involved in determining cell fate. The vertebrate homologs of many of these genes have now been identified, and mutations in some of these homologous genes are associated with tumor formation. Analysis of the function of these genes in model systems like flies and nematodes is likely to provide a better understanding of how the homologous genes function in vertebrates, and possibly suggest therapeutic approaches to correcting functional defects. Although substantial progress has been made in understanding the very early events in the Drosophila embryo, almost nothing is known about the molecules that control early embryogenesis in C. elegans. Because the early development of these two organisms is very different, analysis of embryogenesis in C. elegans should provide a broader understanding of animal development. We have used a genetic approach to identify a small set of genes in C. elegans that appear to play critical roles in determining the fate of early embryonic cells. These genes are named skn-1; skn-2; pie-1; mex-1 and mex-3. The general aim of the proposed work is the molecular analysis of each of these genes and the localization of their gene products in the embryo. skn-1 encodes a protein with a sequence similarity to the DNA-binding domain of a set of transcription factors called BZIP proteins. However in overall structure the skn-1 protein does not resemble BZIP family members and appears to be a novel protein. Experiments are proposed to determine if skn-1 can bind specific DNA sequences, and to identify genes whose products interact with skn-1 to control cell fate. Genetic analysis of pie-1 and skn-2 mutants suggests the products of these genes may interact. Genetic experiments are proposed to identify alleles of pie-1 that will be useful for molecular analysis, as well as other genes that may interact with pie-1. pie-1 and skn-2 mutants lack a particular cell type that mex-3 mutants overproduce. Analysis of mex-3 mutants should provide a better insight into how pie-1 and skn-2 function, and experiments are proposed to complete a phenotypic characterization of the mex-3 embryonic defect.

We are interested in the molecular mechanisms by which embryonic cells stop dividing and assume particular differentiated fates, and are studying this problem in the nematode C. elegans. In normal development both of the first two embryonic cells, called the AB blastomere and the PI blastomere, produce muscles of the pharynx. Although PI produces these cell types autonomously, AB is induced to produce pharyngeal muscles. We want to understand the molecular basis for this inductive event. Several maternally-expressed genes, including glp-1, aph-1, aph-2, pop-1, and mex- 3, are essential for normal pharyngeal development. The glp-1 gene encodes a homolog of the C. elegans gene lin-12 and the Drosophila gene Notch, a family of proteins that now appear to be key regulators of cell-cell interactions in a wide variety of animals. Genes homologous to glp-1 recently have been identified in humans, and aberrant forms of these molecules have been implicated in certain malignancies. Very little is understood about how cell-cell interactions determine cell fate, however the early inductive interaction in the C. elegans embryo is almost ideal for such a study. First, there are only 4 blastomeres in the embryo when the inductive interaction begins. Second, it is possible to dissociate and rejoin wild type or mutant blastomeres at this stage. Finally, the genetics available in C. elegans provides an extremely powerful tool for uncovering genes involved in the inductive interaction. Some of the genes that determine the fate of the inducing cell already have been identified, and one of these (the skn-1 gene) appears to encode a novel, nuclear localized transcription factor. We propose a phenotypic/molecular analysis of the aph-1, aph-2, pop-1, and mex-3 genes which were identified by genetic screens during the previous years of this grant. Knowing the molecular nature and cellular locations of the corresponding gene products should provide a great deal of insight into the nature of cell-cell interactions.

PROJECT SUMMARY/ABSTRACT One of the fundamental steps in the formation of most organs is the assembly of cells into epithelial layers; simple epithelial cells share a common polarity, are linked by apical junctions and share a basal basement membrane. Epithelial precursors often arise in different parts of the embryo and must aggregate together, often sorting from other types of cells, to form a primordium. Cells within the primordium must then undergo changes in position or shape to create organ morphology. Some remodeling-specific behaviors of normal epithelia cells, such as the loss of adhesion, are reminiscent of cancer cells, the vast majority of which originate from epithelial cells. We want to understand how epithelial cells sort and rearrange, and are studying this in the digestive tract of the nematode C. elegans. C. elegans provides a simple genetic system for investigating basic problems of cell biology, and has had major impact on our understanding of diverse processes such as apoptosis or RNA-mediated gene inhibition. The digestive tract consists primarily of two epithelial tubes, the pharynx and the intestine; we will use the pharyngeal cells to study how precursor cells sort from other cell types, and use the intestine to study how epithelial cells rearrange during organogenesis. Although the pharyngeal precursors cells normally arise from contiguous cells, we demonstrated that they have sorting potential when mixed with other cell types. We want to study the cell biology of sorting in this model, and identify genes required for sorting. Cells at both ends of the intestinal tube undergo a rotation during morphogenesis; we believe this rotation functions to align the lumen throughout the length of the tube. We discovered that the anterior intestinal cells are guided to their new positions by an UNC-6/netrin signaling pathway. This pathway is best understood for its function in nervous system development, but several recent studies have demonstrated that it is also important for the development of non-neural tissues. We want to determine how this pathway functions to reposition the anterior cells, and to determine how cells at the posterior end rotate. Finally, we want to use the powerful genetics of C. elegans to identify genes required for both kinds of epithelial remodeling.

DESCRIPTION (provided by applicant): Programmed cell death or apoptosis is a fundamental, highly conserved pathway in animal development, and plays critical roles in normal health, as well as in aging and disease. Apoptotic cells must shut themselves down while leaving surrounding cells and tissues intact, and part of this process involves changes in the cytoskeleton of the apoptotic cells. Studies in the genetic model system C. elegans identified many of the key molecules that regulate and execute apoptosis, and this grant uses C. elegans as a system to understand the apoptotic regulation of the microtubule cytoskeleton. The study focuses on adult gonads, where about 50% of germ cells normally undergo apoptosis. The gonad is an example of a syncytial tissue, where individual cells maintain small cytoplasmic connections with neighboring cells. In a syncytium, the apoptotic removal of nuclei and cytoplasm must be tightly controlled to prevent death of the whole. The connections between cells present the possibility that part or all of the apoptotic cytoplasm might be transferred intact to the syncytium, rather than simply being degraded and recycled as metabolites. This grant examines how the normal microtubule cytoskeleton is dismantled during germ cell apoptosis, and how the new cytoskeleton is organized. It addresses the fate of cytoplasmc components of the apoptotic cell, and whether the new MT cytoskeleton contributes to the transfer of cytoplasm. Through either transfer or digestion, the apoptotic cell ultimately shrinks its cytoplasm, dismantles its cytoskeleton entirely, and is absorbed by other cells. This study identifies a role for the conserved kinase PAR-1 in the final steps of apoptotic remodeling, and addresses possible steps that might be regulated by PAR-1 activity.