Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo.

INTRODUCTION

Genetic studies have shown that three sequential signaling pathways establish the dorsal-ventral pattern of the Drosophila embryo. Each of these pathways generates and transmits spatial information by localizing the activity of an extracellular morphogen, which acts as a ligand for a broadly distributed transmembrane receptor. This review focuses on these ligands, with particular emphasis on the distinct strategy used by each pathway to achieve spatial localization of signaling activity. As a result of these sequential processes, the initial cue for dorsal-ventral asymmetry is progressively refined during the stages of oocyte and embryonic development. Perhaps not surprisingly, an understanding of these processes reveals that Drosophila deploys some of the same molecular mechanisms used by vertebrates for intercellular communication and axial development.

The shape of the Drosophila egg shows dorsal-ventral polarity at the time it is laid. The eggshell is curved on one side, corresponding to the prospective ventral side of the embryo, and flattened on the dorsal side which is marked by a pair of respiratory appendages at the anterior. Soon after cellularization, which occurs about three hours after egg laying, different cell types within the embryo along the dorsal-ventral axis become distinguishable, as determined by changes in cell shape and in the pattern of cell division during gastrulation and germ band elongation. These blastoderm cells eventually give rise to four distinct regions [ILLUSTRATION FOR FIGURE 1 OMITTED]. Cells at the ventral midline become muscle and other mesodermal derivatives, ventrolateral cells give rise to ventral epidermis and the ventral nerve cord, dorsolateral cells give rise to dorsal epidermis, and cells at the dorsal midline form the extraembryonic amnioserosa (14). Mutations that disrupt the embryonic dorsal-ventral pattern fall into one of three classes: maternal effect mutations that perturb the polarity of the embryo and the surrounding eggshell; maternal effect mutations that produce embryonic defects within a wild-type eggshell; and zygotic mutations that affect embryonic pattern within more limited regions of the dorsal-ventral axis. Each group of genes defines one of the three sequential signaling pathways.

The first of these signaling pathways takes place during oogenesis, when the germline-derived oocyte is surrounded by an epithelium of somatically derived follicle cells, which later secrete components of the eggshell. The oocyte produces a dorsalizing signal that is received by neighboring follicle cells, thereby defining the polarity of both the embryo and eggshell (110, 122). The proposed ligand and receptor in this pathway are encoded by the genes gurken and torpedo, respectively. Spatial regulation of the signal is achieved by localizing gurken RNA to the dorsal anterior side of the oocyte.

After fertilization, the second signaling pathway encoded by eleven dorsal group genes and cactus generates a signal on the ventral side of the embryo (14, 131). The proposed ligand and receptor in this pathway are encoded by the genes spatzle and Toll, respectively. The spatially restricted production of the Toll ligand is achieved by a localized proteolytic processing reaction, which is regulated by a cascade originating in the follicle cells. Activation of Toll results in the nuclear translocation of the transcription factor encoded by dorsal. The maternal pathway culminates in a graded ventral-to-dorsal distribution of the Dorsal protein, which controls the transcriptional activation or repression of a set of zygotic genes.

The third signaling pathway defines patterning on the dorsal side of the embryo by using a ligand encoded by decapentaplegic, a member of the TGF[Beta] family (30). Response to this signal is mediated by a heteromeric complex containing a type II receptor encoded by punt and a type I receptor encoded by thick veins or saxophone. A dorsal-to-ventral gradient of decapentaplegic activity apparently arises as a consequence of posttranslational enhancement of decapentaplegic activity on the dorsal side and inhibition on the ventral side.

SIGNALING FROM THE OOCYTE TO SURROUNDING FOLLICLE CELLS

The dorsal-ventral polarity of both the embryo and the eggshell is initiated during oogenesis by a pathway that involves the production of an asymmetric signal from the oocyte. This patterning requires communication within the ovary between the germline-derived oocyte and the surrounding somatically derived follicle cells, which will secrete the eggshell. Mutations in 13 known genes affect the dorsal-ventral pattern of both the eggshell and the embryo (Table 1). Maternal effect mutations in torpedo, gurken, and cornichon lead to a strong ventralization of the eggshell and embryo, indicating that the wild-type gene products are required for the specification of dorsal fates (104a, 120). To determine which cells require these genes, mosaic egg chambers were constructed in which the germline was mutant while the somatic tissue was wild type, or vice versa. This analysis showed that gurken and cornichon are required in the germline (i.e. the oocyte), while torpedo is required in the soma (presumably the follicle cells) (120, 121).

These genetic observations led to a model that the oocyte nucleus, located in the dorsal anterior comer of the oocyte, produces an asymmetric "dorsalizing" signal, possibly encoded by gurken or cornichon [ILLUSTRATION FOR FIGURE 2 OMITTED]. This signal, interpreted by the Torpedo protein, induces the closest neighboring follicle [TABULAR DATA FOR TABLE 1 OMITTED] cells to adopt a dorsal fate; follicle cells located farther away receive little or no signal, and thus enter a ventral fate by default (81, 121). The follicle cells must eventually signal back into the oocyte to establish embryonic polarity, because loss of torpedo activity causes ventralization of the embryonic pattern, in addition to the ventralization of the eggshell.

Localization of gurken RNA Defines Dorsal-Ventral Asymmetry

The molecular identities of gurken and torpedo provided support for the proposed signaling pathway from the oocyte to the follicle cells. The gurken gene is expressed in the oocyte and encodes a signaling molecule. The open reading frame corresponds to a 33-kd TGF[Alpha]-like protein containing a putative signal peptide at the amino-terminus and a transmembrane domain near the carboxy-terminus of the molecule (90). The torpedo gene, which is expressed in the follicle cells, encodes the Drosophila epidermal growth factor (EGF) receptor (99, 116). By analogy to mammalian TGF[Alpha], which binds and activates the EGF receptor (24), a form of the Gurken protein could be the ligand for Torpedo.

The localization of the gurken transcript appears to be crucial for defining the spatial asymmetry of the egg chamber along both the anterior-posterior and dorsal-ventral axes at two different stages in oogenesis. In young egg chambers (stages 1-7), gurken RNA is localized to the posterior margin of the oocyte. This pattern becomes more diffuse by stage 8, with gurken RNA accumulating along the anterior margin. This change in RNA localization coincides with the attachment of microtubules to the anterior margin, which occurs during the reorganization of the cytoskeleton (22, 141). Soon thereafter, the oocyte nucleus moves anteriorly from its central position and to the prospective dorsal side of the oocyte (80). The displacement of the nucleus towards one side may arise stochastically and is the earliest visible dorsal-ventral asymmetry in the egg chamber (121). At late stage 8 and during stage 9, the gurken RNA becomes spatially restricted to the dorsal anterior side of the oocyte nucleus.

In the first signaling event requiring the gurken-torpedo pathway, the anterior-posterior polarity of the oocyte is defined. During the early stages of oogenesis, the gurken RNA localized to the presumptive posterior margin is required to induce neighboring polar follicle cells to adopt a posterior fate (41a, 104a). During stages 6-7, these follicle cells then generate a signal back to the oocyte (41), in a process that requires Notch and Delta in the follicle cells (108) and the activity of protein kinase A in the oocyte (75a). This signaling results in the establishment of a polarized microtubule network extending from the anterior to the posterior pole (21) that directs localization of the maternal transcripts bicoid and oskar to opposite poles of the oocyte (27, 130, 131) and thereby defines anterior-posterior asymmetry. In the absence of gurken activity, the polar follicle cells at both ends of the oocyte follow the default anterior pathway, resulting in the formation of a symmetric microtubule cytoskeleton. The oocyte develops two anterior poles, with the accumulation of bicoid RNA at each pole and oskar RNA in the center (41a, 104a).

The dorsal-ventral polarity of the oocyte is established by the gurken-torpedo pathway in a second signaling event at stage 9, when the gurken transcript becomes localized to the dorsal anterior corner of the oocyte nucleus. Soon thereafter, Gurken protein accumulates in the cytoplasm and the plasma membrane on the dorsal side of the oocyte (104a). In contrast to the spot-like localization of gurken RNA, Gurken protein assumes a more elongated stripe-like distribution along the anterior-posterior axis (104a).

Production of the active signal requires the activities of cornichon and brainiac in the germline, in addition to the gurken gene (6, 42). cornichon is not only necessary for generating the dorsalizing signal, but also acts in the earlier signaling event that specifies anterior-posterior polarity of the egg chamber (41a, 104a). cornichon encodes a hydrophobic protein that could be involved in the membrane localization or proper activation of the Gurken protein (104a).

Laser ablation of the nucleus produces a ventralized phenotype, providing functional evidence that the nucleus (or a closely associated structure) acts as the source of the dorsalizing signal (87). Mutations in all of the genes that act upstream of gurken in the genetic pathway affect the localization of gurken RNA to the nucleus [ILLUSTRATION FOR FIGURE 2 OMITTED]. Maternal effect mutations in fs(1) K10, spire, cappuccino, or squid cause an apparent dorsalization of the egg chamber and the resulting embryo (66, 81, 150). In these mutant egg chambers, the gurken transcript is normally localized to the posterior margin of the oocyte in the early stages of oogenesis. However, instead of being sharply restricted to the region around the dorsal side of the oocyte nucleus, the gurken transcript accumulates along the anterior margin of the oocyte from early stage 9 through later stages of oogenesis (90). A maternal effect allele of orb (20, 76) produces a complex embryonic phenotype that is also caused by the mislocalization of gurken RNA (105).

The products encoded by both the K10 and squid genes accumulate in the oocyte nucleus (16, 66), and may regulate the production of an activity required for the localization of gurken RNA. Eggs laid by K10 and squid females were initially described as being dorsalized (66, 149, 150). However, a careful examination of the mutant embryonic phenotypes provided the surprising finding that although these embryos are dorsalized at their anterior ends, they are ventralized at their posterior ends (105). In fact, the dorsal-ventral axis in these embryos appears to be rotated nearly 90 [degrees], so that cells at the anterior end are now directed to a dorsal fate, while cells at the posterior end adopt a ventral fate. As the anterior-posterior axis remains unaffected with respect to eggshell polarity in these mutant embryos, the dorsal-ventral axis is nearly parallel to the anterior-posterior axis. Thus, proper gurken RNA localization is required to establish the orthogonal orientation of the anterior-posterior and dorsal-ventral axes (105) (see below).

Eggs laid by cappuccino and…