The union of egg and sperm initiates complex developmental processes that ultimately produce a new multicellular animal. However, the roles played by egg and sperm in embryonic development are by no means equivalent. Without an egg, neither sperm nor any other cell can even begin the complex processes that lead to an embryo and ultimately to an adult. All eggs contain a molecular blueprint to guide the assembly of the embryo's complex structures and sequester stores of cellular constituents for use by the growing embryo. In recent years, my laboratory has analyzed some of the earliest steps in egg construction, a fascinating period that appears to be similar in a wide range of organisms.
Model Stem Cells
Drosophila eggs arise from germline stem cells (GSCs) that reside at the tip of each ovariole. Using genetic screens and by gene profiling of purified GSCs, my group has identified and studied genes that are required for GSC function, as well as others that control the onset of egg development. Our research revealed that GSCs are regulated by the specialized cellular microenvironment created by nearby somatic cell types, i.e., a "niche." In conjunction with research from several other labs, this work has led to a detailed molecular model of GSC maintenance and differentiation. We found that the niche cannot reprogram introduced foreign cells but can stimulate them to divide. However, we did discover conditions under which young germ cells that are just beginning to differentiate can revert to a fully functional stem cell fate. We are extending these studies to other types of stem cells in the ovary: escort stem cells (ESCs) and follicle cell stem cells (SSCs). ESCs resemble the somatic cyst progenitor stem cells of the testis, indicating that the male and female stem cell niches are much more similar than previously believed. The escort cells require JAK/STAT signaling to maintain the normal cellular architecture surrounding the GSCs and to prevent GSC loss, providing new insight into how all the cellular constituents of the niche are coordinated.
A favorable new system for studying stem cells has been identified through our discovery that the Drosophila adult intestine is maintained by hundreds of pluripotent intestinal stem cells (ISCs). ISCs divide to produce an enteroblast that either differentiates into an absorptive cell that lines the gut or into an enteroendocrine cell. We found that these cell fate decisions and the rate of cell proliferation downstream from the ISC are controlled by Notch signaling, much as they are in vertebrates. Interestingly, ISCs do not reside next to a stromal cell analogous to the cap cell, suggesting that they are controlled by a different type of niche. This work suggests that ISCs and their basic program of differentiation have been conserved in evolution from before the divergence of vertebrates and invertebrates.
Eggs Develop Within Polarized Germ Cell Cysts
Following the stem cell stage, nascent Drosophila female germ cells undergo four special divisions that result in the formation of a germ cell cyst. These divisions are rapid, synchronous, and leave all 16 cells interconnected by special intercellular bridges known as ring canals. In Drosophila, just one cell within each cyst differentiates as an egg, while the other 15 function as nurse cells, providing materials to the developing oocyte by transporting them through the ring canals. By studying the corresponding stages of mouse oogenesis, which take place in fetal ovaries, our group discovered that a similar process of cyst formation occurs in this species as well. In the mouse, the cysts break down at about the time of birth, when approximately one in three germ cells becomes surrounded by somatic cells to form follicles, while the others die. Our proposal, based on our studies of Drosophila and mouse cysts, is that a critically important process occurs during this stage—the selective removal of damaged molecules and organelles. As a result of this, the cytoplasm of new oocytes would be freed of the molecular damage arising during the previous generation and become capable of supporting another complete life cycle.
A key aspect of germline cysts is their asymmetry. Individual cyst cells divide unequally and acquire different amounts of an unusual germ cell–specific organelle known as the fusome. Our studies of both flies and mice show that many germ cell components reorganize during the cyst stage. Drosophila, mitochondria, Golgi vesicles, centrosomes, and segments of endoplasmic reticulum move along the fusome, through ring canals, and aggregate into "clouds" that eventually coalesce into a large Balbiani body in newly formed follicular oocytes. Several Drosophila RNAs destined for embryonic germ cells associate with the Balbiani body, supporting a connection between Balbiani body constituents and germ cells. We suspect that the organized movements of these key constituents of cell cytoplasm are part of the mechanisms that supply embryonic germ cells with functional organelles, including mitochondria with undamaged genomes. Less-functional or damaged constituents may fail to move or may be actively directed into germ cells that are designated for recycling, explaining the evolutionary conservation of germline cysts, Balbiani bodies, and germ cell death. We also found a Balbiani body in mouse oocytes within young cysts, despite the fact that no maternal components associated with germ cell specification have so far been identified in mammals.
New Tools for Analyzing Complex Tissues
We are engaged in two large-scale projects to generate Drosophila strains containing single-transposon insertions that facilitate genetic analysis. As part of the Berkeley Drosophila Genome Project (BDGP), we have helped generate, characterize, and make publicly available P-element insertion strains that are being widely used to study Drosophila gene function. So far about 6,000 different genes have been disrupted, corresponding to 45 percent of the estimated 13,800 Drosophila genes.
Genomic analysis of gene function in multicellular organisms currently has great difficulty dealing with the complex anatomy of animal tissues and the rapid spatial and temporal changes in gene expression that are commonplace during development and in adults interacting with realistic environments. Surmounting this complexity is an important general problem that also is a major limiting factor in our work to understand the Drosophila ovary. Consequently, postdoctoral fellow Michael Buszczak initiated a project to generate lines containing single, random P-element insertions that each fuse an endogenous gene product to green fluorescent protein (GFP). In most cases, the fusion protein expressed by such flies accurately reproduces the developmental expression and subcellular localization of the endogenous gene product. Using an automated embryo sorter, we have selected from more than 35 million embryos >7,500 lines expressing GFP. We have mapped the genomic location of their insertions in collaboration with BDGP, and identified about 1,000 lines that fuse distinct gene products. This has generated a collection of sufficient breadth and quality for the rapid study of many aspects of cellular and tissue physiology by examining the dynamic patterns of GFP expression within appropriate lines.