Section VIIControl of Proliferation and Entry into the Meiotic Pathway

A. The glp-1 Signaling Pathway

In both the hermaphrodite and male, a signal from the somatic DTC promotes germ-line proliferation and/or inhibits germ-cell entry into meiotic prophase (also see Section IV, Somatic Gonad). Three genes that directly mediate DTC signaling are glp-1 , lag-1 , and lag-2 . Genetic and molecular characterization of these genes indicates that the glp-1 signaling pathway is homologous to the Notch signaling pathway, which functions in cell fate specification in Drosophila (Artavanis-Tsakonas et al. 1995; for the related lin-12 signaling pathway, see Greenwald, this volume; for the role of glp-1 in embryogenesis, see Priess and Schnabel, this volume). Partial loss-of-function mutations in lag-1 and lag-2 and null mutations in glp-1 have essentially the same phenotype as that resulting from DTC ablation: entry of all germ cells into the meiotic pathway. The glp-1 gene, the function of which is required continuously in the germ line (Austin and Kimble 1987), encodes a transmembrane protein related to the receptors Notch and LIN-12 (Austin and Kimble 1989; Yochem and Greenwald 1989). The lag-2 gene encodes a transmembrane protein homologous to Drosophila Delta, the ligand for Notch, and is expressed in the DTC but not the germ line (Henderson et al. 1994; Tax et al. 1994). The lag-1 gene encodes an intracellular protein homologous to Drosophila suppressor of hairless (Su[H]) and the CBF1 (also known as RBPJk) family of mammalian DNA-binding proteins (Christensen et al. 1996).

The GLP-1/Notch family receptors possess cytoplasmic ankyrin repeats that are critical for signaling. Mutations in the ankyrin repeats of GLP-1 disrupt receptor activity (Kodoyianni et al. 1992; Lissemore et al. 1993). Overexpression of the cycloplasmic domain, including the ankyrin repeats, from GLP-1, LIN-12, and Notch leads to constitutive signaling (for review, see Greenwald 1994). In Drosophila tissue culture cells, when Notch binds ligand, Su(H) is displaced from the ankyrin repeats and translocates to the nucleus (Fortini and Artavanis-Tsakonas 1994). glp-1 signaling may be mechanistically similar in that LAG-1 binds to a portion of cytoplasmic domain that includes the ankyrin repeats of GLP-1 (M. Bosenberg and J. Kimble, pers. comm.). It is important to note that the GLP-1 cytoplasmic domain does not simply function to regulate LAG-1 negatively as glp-1 and lag-1 have the same germ-line loss-of-function phenotype. Thus, both act to promote germ-line proliferation (an analogous argument holds for Notch/ Su[H] signaling pathways).

Signaling activity of the glp-1 pathway must be spatially restricted to establish and maintain normal germ-line polarity. LAG-2, expressed by the DTC, is a localized ligand (Henderson et al. 1994). Tethering of LAG-2 via its transmembrane domain is important for normal germ-line polarity as a secreted form of the ligand, driven from the lag-2 promoter, produces ectopic germ-line proliferation (Fitzgerald and Greenwald 1995).

glp-1 expression is also spatially restricted (Crittenden et al. 1994). In young adult hermaphrodites and males, immunohistochemical staining shows that membrane-associated GLP-1 is present in the proliferating germ-cell population (1 to ~20 cell diameters from the DTC). Membrane-associated GLP-1 decreases rapidly in the transition zone and is not detected in the pachytene region. glp-1 RNA is found throughout the hermaphrodite germ line but limited to the more distal region in males. Thus, glp-1 expression appears to be regulated at both translational and transcriptional levels.

Germ-line polarity is lost in a glp-1(gf) mutant which displays a tumorous phenotype (L. Wilson Berry and T. Schedl, unpubl.). The glp-1(gf) mutant receptor is constitutively active; germ-cell proliferation occurs after elimination of the ligand (double mutant with lag-2[lf]) or its source (DTC ablation). The receptor encoded by the glp-1(gf) allele contains an amino acid substitution at a conserved residue in a region of the extracellular domain adjacent to the transmembrane domain. In glp-1(gf) homozygous hermaphrodites and males, germ cells proliferate continuously. In glp-1(gf)/glp-1(lf) animals, normal germ-line polarity is initially established, but as adults age, the population of proliferating germ cells expands proximally to more than 60 germ-cell diameters from the distal tip. Spatial restriction of glp-1 expression is also lost in glp-1(gf) animals: Ectopically proliferating germ cells contain membrane-associated GLP-1. The correlation between proliferation and expression, both in wild type where glp-1 signaling is limited by the localized ligand and in glp-1(gf) where signaling is independent of ligand, suggests that proliferation (or glp-1 signaling) positively regulates glp-1 expression.

How does DTC signaling influence proliferation up to approximately 20 germ cell diameters away from its cell body (Crittenden et al. 1994)? The DTC sends out cytoplasmic processes (D. Hall, pers. comm.). Visualization of these processes by expression of green fluorescent protein (Chalfie et al. 1994) in the DTC reveals that they extend more than ten germ-cell diameters from the distal tip of adult gonads (Fitzgerald and Greenwald 1995). If such processes contained LAG-2, they might directly signal GLP-1 in germ cells that are some distance from the distal tip. An alternative hypothesis is based on the observation that LAG-2 produced by the DTC is internalized into the germ line and appears to colocalize with the internal punctate GLP-1 (Henderson et al. 1994). The internalized LAG-2–GLP-1 complexes may produce a signal that is propagated locally in the germ-line syncytium.

In principle, the absence of signaling activity by the glp-1 pathway may be sufficient for germ cells to enter the meiotic pathway. Initial entry into meiosis in the L3 may occur solely because proximal germ cells have escaped the influence of the DTC (Kimble and White 1981). It is also possible that somatic gonad cells that neighbor proximal germ cells have an active role in promoting entry into meiotic prophase in L3.

B. Suppressors and Enhancers of glp-1

To identify additional components of the glp-1 signaling pathway and genes that modulate its activity, mutations that suppress (sog genes; Maine and Kimble 1993) or enhance (ego genes; Qiao et al. 1995) the phenotype of glp-1 (ts) mutants have been sought. sog mutants do not bypass the requirement of glp-1 , as they fail to suppress a glp-1 null mutation. Since partial loss-of-function alleles of lag-1 represent one class of ego mutants, this enhancer screen can identify components of the glp-1 signaling pathway. The remaining ego mutants display a heterogeneous mix of sterile phenotypes. Future characterization will determine the null phenotype and the molecular nature of the sog and ego genes.

C. Proximal Proliferation

Mutations in more than ten genes display a proximal proliferation (Pro) phenotype, for example, lin-12(lf) (Seydoux et al. 1990), shv mutants (R. Francis et al., unpubl.), pro(q540) (L. Kadyk and J. Kimble, pers. comm.), let-42(gf) (B. Westlund and T. Schedl, unpubl.), and ego-3 (Qiao et al. 1995). In most cases, it is not yet known whether the mutations affect components of the glp-1 signaling pathway or disrupt meiotic prophase functions (see gld-1 below). In the case of lin-12(lf), inappropriate signaling between the somatic gonad and the germ line is the likely cause of the proximal proliferation phenotype. During the L1 and L2 stages, the gonadal anchor cell (AC) and its precursors contact the germ line. In lin-12(lf) mutants and in the wild type when certain somatic gonad blast cells are ablated, proximal germ cells fail to enter the meiotic prophase, resulting in proximal proliferation (Seydoux et al. 1990). A likely scenario for the lin-12(lf) Pro phenotype is that LAG-2 expressed in the AC and its precursors (for the anchor cell/ventral uterine decision, see Greenwald, this volume) inappropriately activates germ-line GLP-1, a process that is normally blocked or limited by surrounding somatic gonad cells and lin-12 (+) activity.