Delta signaling from the germ line controls the proliferation and differentiation of the somatic follicle cells during Drosophila oogenesis

By H. Johnston. 2001

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The Notch family of receptors play a central role in an evolutionarily conserved signaling pathway that regulates numerous cell fate decisions in many organisms (Artavanis-Tsakonas et al. 1999). Notch encodes a large transmembrane receptor for the ligands Delta (Dl), Serrate (Ser), and related orthologs in other species (collectively called DSL), which are also transmembrane proteins with large extracellular domains, and signaling therefore requires direct cell-cell contact. On ligand binding, Notch receptor undergoes a presenilin-dependent proteolytic cleavage to release the intracellular domain from the membrane (De Strooper et al. 1999; Struhl and Greenwald 1999; Ye et al. 1999). This domain can then translocate to the nucleus, where it acts as a transcriptional activator in association with Suppressor of Hairless (Su[H]; Bailey and Posakony 1995; Lecourtois and Schweisguth 1995). The best characterized function of Notch in Drosophila is in lateral inhibition during the specification of the neuroblasts and sensory organ precursors of the nervous system (Lehmann et al. 1983; Campos-Ortega and Knust 1990). In this process, one cell in a cluster of equipotent precursor cells expresses higher levels of a Notch ligand, such as Delta, and activates the Notch pathway in the neighboring cells to turn on the expression of the E(spl) complex genes (Bailey and Posakony 1995; Lecourtois and Schweisguth 1995). As these transcription factors inhibit proneural gene expression, this prevents the cells from adopting a neural fate (Oellers et al. 1994). Mutants in this pathway therefore result in a neurogenic phenotype in which too many cells adopt a neural fate at the expense of epidermis. Although Notch signaling usually regulates cell fate determination, in a few cases it has been shown to have other effects on other aspects of cell behavior. For example, the Caenorhabditis elegans Notch homolog, GLP-1, regulates the decision between mitosis and meiosis in the germ line, whereas mouse Notch homologs control neurite outgrowth in differentiated cortical neurons (Berry et al. 1997; Kimble and Simpson 1997; Sestan et al. 1999). One place where Notch signaling plays an essential, but ill-defined, role is in the process of axis formation in Drosophila, which takes place during the development of the oocyte in the ovary (Ruohola et al. 1991). Oogenesis begins at the anterior of the germarium, when a germ line stem cell divides asymmetrically to give rise to another stem cell and a cystoblast, which then undergoes four mitoses with incomplete cytokinesis to generate a cyst of 16 interconnected germ cells (Spradling 1993). As the germ line cyst moves down the germarium, it becomes surrounded by somatically derived follicle cells, which form an epithelium around the cyst, and a stalk of six to eight cells that separates it from the preceding cyst. The first important function of Notch signaling is in migration of the follicle cells, as adjacent egg chambers are often fused in flies that are mutant for a temperature-sensitive allele of Notch under nonpermissive conditions, and this gives rise to giant compound egg chambers that contain multiple germ line cysts in a single follicular epithelium (Ruohola et al. 1991; Xu et al. 1992; Goode et al. 1996a). This Notch-dependent encapsulation of the cyst by the follicle cells is required for the initial polarization of the anterior-posterior (A-P) axis. One of the 16 germ line cells is selected to become the oocyte in region 2b and then moves to the posterior of the cyst to generate the first A-P polarity, and this cell rearrangement is driven by DE-cadherin–dependent adhesion between the oocyte and the follicle cells that migrate around the posterior (Godt and Tepass 1998; Gonzalez-Reyes and St. Johnston 1998). Clonal analysis has revealed that the follicle cells are already divided into two populations in the germarium. One lineage gives rise to the stalk cells and the polar cells, which are a pair of follicle cells at each end of the egg chamber that connect to the stalk, and both cell types never divide once the egg chamber has formed (Tworoger et al. 1999). The rest of the follicle cells derive from a separate lineage and form the epithelium around the cyst (Margolis and Spradling 1995). These cells, which we will refer to as epithelial follicle cells, go through five further rounds of mitosis before switching to the endocycle during stages 7 through 9, in which they become polyploid by cycling through S phases without intervening cell divisions (Calvi et al. 1998). It is unclear exactly when and where the Notch pathway is required for the migration of the follicle cells around the cysts, but expression of a constitutively active form of Notch results in the formation of extremely long stalks that can contain stalk, polar, or undifferentiated cells (Larkin et al. 1996). Thus, the early function of Notch may be to induce the formation of the stalk by controlling the determination or behavior of the stalk and polar cell populations. Later in oogenesis, the Notch signaling pathway is required a second time, for the polarization of the A-P axis of the oocyte itself (Ruohola et al. 1991). After the epithelial follicle cells stop dividing at stage 6, they segregate into two subpopulations: the main-body follicle cells in the middle of the egg chamber and ∼200 terminal follicle cells at each pole (Gonzalez-Reyes and St. Johnston 1998). The oocyte, which is now at the posterior, then signals through the Gurken/EGFR pathway to induce the adjacent terminal follicle cells to adopt a posterior rather than an anterior fate (Gonzalez-Reyes and St. Johnston 1994, 1995; Roth et al. 1995). At around stage 7, these posterior follicle cells signal back to the germ line to polarize the microtubule cytoskeleton along the A-P axis of the oocyte, and this defines the A-P axis of the embryo by directing the localization of bicoid mRNA to the anterior of the oocyte and the localization of oskar mRNA and Staufen protein to the posterior (Theurkauf et al. 1992; Clark et al. 1994; Lane and Kalderon 1994; Pokrywka and Stephenson 1995). In addition, this microtubule reorganization triggers the migration of the oocyte nucleus and gurken mRNA from the posterior of the oocyte to the dorsal anterior corner, where Gurken signals for a second time to specify the dorsal-ventral axis of the future embryo (Koch and Spitzer 1983; Neuman-Silberberg and Schupbach 1993; Gonzalez-Reyes et al. 1995; Roth et al. 1995). It is not clear where the Notch pathway acts in the process of axis formation, but both Notchts and Deltats mutations disrupt the polarization of the oocyte at stage 7 (Ruohola et al. 1991). At restrictive temperatures, mutant egg chambers develop a mirror-symmetric A-P axis, in which bicoid mRNA localizes to both poles of the oocyte; oskar mRNA and Staufen protein are transported to the centre; and the oocyte nucleus often fails to migrate from the posterior pole. Although the phenotypes of Notchts and Deltats provided the first evidence that the follicle cells signal to polarize the oocyte, the Notch pathway cannot be directly involved in the signaling itself because Notch germ line clones form a normal A-P axis, indicating that the receptor is required in the follicle cell layer and not in the germ line (Ruohola et al. 1991). Furthermore, Delta germ line clones have also been reported to have no effect on axis formation, and several studies have supported the conclusion that Delta is required in the follicle cells as well (Ruohola et al. 1991; Bender et al. 1993; Larkin et al. 1999). Thus, Delta and Notch must act upstream of the signaling to the oocyte and presumably play a role in the patterning of the follicle cells. In wild-type ovaries, Fasciclin III (FasIII) expression becomes restricted to the two polar follicle cells at each end of the egg chamber by stage 4 of oogenesis, but Notchts egg chambers contain many more FasIII positive cells at the posterior pole (Ruohola et al. 1991; Goode et al. 1996a). This has led to the suggestion that the defects in A-P axis formation may be a consequence of forming too many posterior polar cells at the expense of the stalk cells (Tworoger et al. 1999). However, these egg chambers also show defects in the specification and patterning of the anterior and posterior terminal populations of epithelial follicle cells (Gonzalez-Reyes and St. Johnston 1998; Larkin et al. 1999). Thus, an alternative model is that Notch signaling is required for the formation of the terminal follicle cells, which, unlike the main-body cells, are competent to respond to Gurken by becoming posterior. Despite the large amount of work on the Notch pathway in oogenesis, the role of Delta/Notch signaling in patterning the follicle cells is still obscure, largely because these studies have only examined females that are mutant for Notchts. Because this allele still retains some activity at restrictive temperatures, the phenotype caused by the complete loss of Notch function is unknown, and these experiments have provided no information on where and when Notch signaling occurs in the follicle cell layer. Here we resolve these questions by generating homozygous mutant clones of null alleles of Notch, Delta, and other components in this pathway. Our results rule out the existing models for the function of Notch in oogenesis and reveal instead that this pathway mediates a novel interaction between the germ line and the soma.

Subfields: Genetics, Molecular Biology

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