The Molecular Biology Of Djrosophila Embryogenesis

In the remaining sections of this chapter we focus on the early embryonic development of the fruit fly, Drosophila melanogcister. The molecular details of how development is regulated are better understood in this system than in any other animal embryo. The various mechanisms of cell communication discussed in the first half of this chapter, and those of gene regulation discussed in the previous chapters, are brought together in this example*

Localized determinants and cell signaling pathways are both used to establish positional information that result in gradients of regulatory proteins that pattern the anterior-posterior (bead-tailÍ Rod dorsal-ventral (back-belly) body axes. These regulatory proteins—activators and repressors — control die expression of genes whose products define different regions of the embryo. A recurring theme is the use of complex regulatory DNAs—particularly complex enhancers-—to bring transcriptional activators and repressors to genes where they function in a combinatorial manner to produce sharp on/off patterns of gene expression.

An Overview of Drosophiia Embryogenesis

Life begins for the fruit fly as it does for humans: adult males inseminate females. A single sperm cell enters a mature egg, and the haploid sperm and egg nuclei fuse to form a diploid, "zygotic" nucleus. This nucleus undergoes a series of nearly synchronous divisions within the central regions of the egg. Because there are no plasma membranes separating the nuclei, the embryo now becomes what is called a syncitium—that is, a single cell with multiple nuclei. With the next series of divisions, the nuclei begin to migrate toward the cortex or periphery of the egg. Once located in the cortex, the nuclei undergo another three divisions leading to the formation of a monolayer of approximately 6,000 nuclei surrounding the central yolk. During a 1-hour period, from 2 to 3 hours after fertilization, cell membranes form between adjacent nuclei.

Before the formation of cell membranes, the nuclei are totipotent or uncommitted; they have not yet taken on an identity and can si ill give rise to any cell type, jus* after celiularization, however, nuclei have become irreversibly "determined" to differentiate into specific tissues in the adulf fly. This process is described in Box 18-4, Overview of Drosophila Development. The molecular mechanisms responsible for this dramatic process of determination are described in the remaining sections of tliis chapter.

A Morphogen Gradient Controls Dorsal-Ventral Patterning of the Drosophila Embryo

The dorsal-ventral patterning of the early Drosophila embryo is controlled by a regulatory protein called Dorsal, which is initially distributed throughout the cytoplasm of the unfertilized egg. After fertilization, and after the nuclei reach the cortex of the embryo, the Dorsal protein enters nuclei in ventral and lateral regions but remains in the cytoplasm in dorsal regions (Figure 18-13). The formation of this Dorsal gradient in nuclei across the embryo is very similar, in

Toll receptors nuclei at periphery

Toll receptors nuclei at periphery

Dorsal protein released to nuclei

nucleus

Pelle Û Tube cytoplasm ^

Toll receptor perivitelline space extracellular ventral signal (Spätzle fragment)

Toll receptor perivitelline space extracellular ventral signal (Spätzle fragment)

FIGURE 18-13 Spätzle-Toll and Dorsal gradient, (a) The circles represent cross sections through early Dtosophila embryos. The Toll receptor is uniformly distributed throughout the plasma membrane of the precelluiar embryo. The Spätzle signaling molecule is distributed in a gradient with peak levels in the ventralmost regions As a result, more Toll recep tors are activated in ventral regions than in lateral and dorsal regions. This gradient in "loll signaling creates a broad Dorsal nuclear gradient (b) Details the Toll signaling cascade Active tion of the Toll receptor leads to the activation of the Pelle kinase in the cytoplasm. Pelle either directly or indirectly phosphorylates the Cactus protein, which binds and inhibits the Uorsal protein. Phosphorylation of Cactus causes its degradation, so that Dorsal is released from the cytoplasm into nudet.

principle, to the formation of the Gli activator gradient within ventral cells of the vertebrate neural tube.

Regulated nuclear transport of the Dorsal protein is controlled by a cell signaling molecule called Spätzle. This signal is distributed in a ventral-to-dorsal gradient within the extracellular matrix present between the plasma membrane of the unfertilized egg and the outer egg shell. After fertilization, Spätzle hinds to the cell surface Toll receptor. Depending on the concentration of Spätzle, and thus the degree of receptor occupancy in a given region of the syncitial embry o, Toll is activated to a greater or lesser extent. There is peak activation of Toll receptors in ventral regions—where the Spätzle concentration is highest—and progressively lower activation in more lateral regions. Toll signaling causes the degradation of a cytoplasmic inhibitor, Cactus, and the release of Dorsal from the cytoplasm into nuclei. This leads to the formation of a corresponding Dorsal nuclear gradient in the ventral half of the early embryo. Nuclei located in the ventral regions of the embryo contain peak levels of the Dorsal protein, wrhile those nuclei located in lateral legions contain lower levels of the protein.

The activation of some Dorsal target genes requires peak levels of the Dorsal protein, whereas others can be activated by intermediate and low levels, respectively. In this way, the Dorsal gradient specifies three major thresholds of gene expression across the dorsal-ventral

Box 18-4 Overview of Drosophila Development

After the sperm and egg haploid nuclei fuse, the diploid, zygotic nucleus undergoes a series of ten rapid and nearly synchronous cleavages Within the central yolky regions of the egg. Large microtubule arrays emanating horn the centnolcs oi the dividing nuclei help direct the nudei from central regtons toward the periphery of the egg (Box 18-1 Figure 1). After eight cleavages, the 256 zygotic nuclei begin to migrate to the periphery. During this migration they undergo two more cleavages (Box Figure 1, nuclear cleavage cycle 9). Most but not all, of the resulting approximately 1,000 nuclei enter the cortical regions of the egg (Box 18-4 Figure 1, Nudeat cleavage cyde 10). The others ("vitellophages") remain tn central regions where they play a somewhat obscure role in development.

Once the rnajonty of the nudei reach the cortex at about 90 minutes Following fertilization, they fast dCquire competence to transcribe Pol II genes. Thus, as in many other organisms such as Xenopus, there seems to be a "mid-blastula transition," whereby early blastomeres (or nudei) are transcriptionally silent dunng rapid periods of mitosis. While causality is undear, it does seem that DNA undergoing intense bursts of replication cannot simultaneously sustain transcription. These and other observations have led to the suggestion that there is competition between the large macromoleailar complexes promoting replication and transcription. Because transcriptional competence is only achieved when the nuclei reach the cortex, it has been suggested that peripheral regions aintain localized determinants. However, recent gene expression studies have stripped much of the mystery from the cortex, for example, the segmentation gene, hunchback, is uniformly transcribed in all of the nuclei present in the anterior half of the early embryo. This expression encompasses both the peripheral nudes that have entered cortical regions, as well as the vitellophages that remain in the yolk.

After the nudei reach die carter they undergo another three rounds of cleavage (for a total of 13 divisions after fertilization), leading to the dense packing of about 6,000 columnar-shaped nudei enclosing the central yolk (Box 18-4 Figure I, Nuclear cleavage cyde 14), Technically, the eiTibryo is still a synatium, although histochemical staining of early embryos with antibodies against cytoskeletal proteins indicate 3 highly structured mesh-work surrounding each nudeus. Dunng a 1-hour period, from 2 to 3 hours after fertilization, the embryo undergoes a dramatic celWanzation process, whereby cell membranes are formed between adjacent nudei (Box 18-4 Figure J, Nuclear deavage cyde 14) By 3 hours after fertilization, the embryo has been trans-

Fusion Pronuclei

BOX 18-4 FIGURE 1 Drosophila embryogenesis. Drosophiiia embryos are oriented with tie future head pointed up. The numbers refer to the number of nudoar deavages. Nuclei are stained white within the embryos. For example, stage 1 contains the single zygotic nucleus resulting from the fusion of the sperm and egg pronuclei. Stage 2 contains 2 nudei arising from the first d'vtston of the zygotic neudeus. At stage 10 there are approximately bOO nudei and most are arranged in a single layer at the cortext (periphery of the embryo). At Nudear deavage cycle 14 there are Over 6,000 nude densely packed in a monolayer in the cortex Cellularization occurs during this stage. (Source: Courtesy of W. fcaker and C. Shubiger.)

BOX 18-4 FIGURE 1 Drosophila embryogenesis. Drosophiiia embryos are oriented with tie future head pointed up. The numbers refer to the number of nudoar deavages. Nuclei are stained white within the embryos. For example, stage 1 contains the single zygotic nucleus resulting from the fusion of the sperm and egg pronuclei. Stage 2 contains 2 nudei arising from the first d'vtston of the zygotic neudeus. At stage 10 there are approximately bOO nudei and most are arranged in a single layer at the cortext (periphery of the embryo). At Nudear deavage cycle 14 there are Over 6,000 nude densely packed in a monolayer in the cortex Cellularization occurs during this stage. (Source: Courtesy of W. fcaker and C. Shubiger.)

farmed into a cellular blastoderm, comparable to the "hollow ball of cells" that characterize the btastulae of most other embryos.

One of the most compelling aspects of classical embryology is the intnnsic beauty of the material, The early embryos of most marine organisms, such as ascidians, are visually stunning. Unfortunately, the Drosopbila embryo ts rather ugly; its salvation has been the unprecedented visualization of gene expression patterns. The differential gene activity that has been so graphically visualized in the early embryo using a variety of molecular and histochemical tools is not simply a manifestation of cell fate specification. Rather, some of the first genes to be visualized encode regulatory proteins that actually dictate eel! fate Thus, the molecular studies have literally illuminated the mysterious process of cell fate specification and determination.

When frie nuclei enter the cortex of the egg they are totipotent and can form any adult cell type. The location of each nucleus, however, now determines its fate. The 30 or so nudei that migrate into posterior regions of the cortex encounter localized protein determinants, such as Oskar, which program these naive nuclei to form the germ cells (Box 18-4 Figure 2). Among the putative determinants contained in the polar plasm are large nudeoprotem complexes, called polar granules. The posterior nuclei bud off from the main body of the embryo along with the polar granules, and the resulting pole cells differentiate into either sperm or eggs, depending on the sex of the embryo. The microinjection of polar plasm into abnormal locations, such as central and anterior regions, results in the differentiation of supernumerary pole cells.

Cortical nuclei that do not enter the polar plasm are destined to form the somatic tissues. Again, these nudei are totipotent and can form any adult cell type. However, within a very bnef period, perhaps as little as 30 minutes, each nudeus is rapidly programmed (or specified) to follow a particular pathway of differentiation. This specification process occurs during the period of ceilularization, although there is no reason to believe that the deposition of cell membranes between neighboring nudei is critical for determining cell fate. Dilfeient nuclei exhibit distinct patterns of gene transcription prior to the completion of cell formation. By 3 hours after fertilization, each cell possesses a fixed positional identity, so that those located in anterior regions of the embryo will form head structures in the adult fly, whereas cells kxrated in posterior regions will form abdominal structures.

©Y\ fertilized egg pole granules

Unfertilized Egg With Nucleus

nuclei migrate 'jW to periphery, cetl boundaries stari to form pole cells

nuclei migrate 'jW to periphery, cetl boundaries stari to form pole cells

BOX 18-4 FIGURE 2 Development of germ ceils. Poler granules located in the posterior cytopiesm of the unfertilized egg contain germ cell determinants, and the Nanos rnRNA, which is important for the development of the abdominal segments, Nudei (centre! dots) begin to migrate to the periphery. Those that enter posterior regions sequester the polar granules and form the pole cells, which form the germ ceils. The remaining cells (somatic cells) form aU of the other (issues in the adult fly (Source: Adapted from Schneiderman HA 1976- Insect development. In Symposia of the Rcyal Entomological Society of London 8: 3-34. (ed, PA Lawrence). Copyright © 1976. Reprinted by permission of Blackwell Science.)

Box 18-4 (Continued)

A variety oi genetic and experimental studies have shown that cell fate specification is controlled by localized maternal determinants that are deposited into the egg during oogenesis. The first evidence for such determinants came from ligation experiments, in which a hair was tied around the middle of Drcsophila embryos. If (his separation between the anterior and posterior halves occurred early, during syndtial blastoderm stages, then central regions of the embryo failed to form thoracic structures such as wings and halteres (Box 18-4 figure 3a). However, if tiie ligation was done later, after celUaruaton, then these sftuc-tures were properly formed (Box 18-4 Figure 3b). These and related experiments suggested that one or more critical determinants diffused into posterior regions from the anterior pole and that this determinants) could be trapped in anterior regions by separating the halves of early embryos with a hair.

Systematic genetic screens by Enc Wieschaus and Chnstiane Nusslein-Vollhard identified approximately 30 "segmentation genes" thai control the early patterning of the Drosophila embryo. This involved the examination of thousands of dead embryos. At the midpoint of embryogencsis, the ventral skin, or epidermis, secretes a cutide that contains many fine hairs, or dentidcs. Each body segment of the embryo contains a characteristic pattern of denticles Three different dasses of segmentatii m genes were identified on the basts of causing specific disnjptions in the denticle patterns of dead embryos. Mutations in the so-callcd "gap* genes cause the deletion of several adjacent segments (Box 18-4 Figure 4). For example, mutations in the gap gene knirps cause the loss of the second through seventh abdominal segments (normal embryos possess eight such segments). Mutations in the "pair-rule" genes cause the loss of alternating segments. For example, mutations in the even-skipped (eve) gene cause the loss of the even-numbered abdominal segments. Finally, mutations in segment polarity genes do not alter the normal number of segments, but instead, cause patterning defects within every segment. Fui example, normal segments contain denudes in one region, but ate naked in the other. In certain segment polarity mutants, such as hedgehog, both regions of every segment contain denttdes.

Knirps Mutation

BOX 18-4 FIGURE 3 Ligation experiment When a hair is used to separate the anterior and posterior halves of early embryos, then determinants emanating from the anterior pole fail to enter posterior regions. As a result, the embryos develop into abnormal flies that lack thoracic structures. In contrast, when the hair separates older embryos (series on the right), then the determinant already entered posterior legions and a normal thorax forms.

BOX 18-4 FIGURE 3 Ligation experiment When a hair is used to separate the anterior and posterior halves of early embryos, then determinants emanating from the anterior pole fail to enter posterior regions. As a result, the embryos develop into abnormal flies that lack thoracic structures. In contrast, when the hair separates older embryos (series on the right), then the determinant already entered posterior legions and a normal thorax forms.

knirps

Denticle Pattern Drosophila

BOX 18-4 FIGURE 4 Darkf ield images of nonnat and mutant circles, (a) The pattern of denticle hairs in this normal embryo ate slightly different among the different body segments (labeled T1 through A8 in the image), (b) The Knirps mutant (having a mutation in the gap gene knirps), shown here, fads the second through seventh abdominal segments. (Source: Nusslein-Vfolhard C and Wieschaus E. 1980. Mutations affecting segment number and polarity in Drosophila. Nature 267 795-801 Images courtesy of Eric Wieschaus, Princeton University.)

axis of embryos undergoing cellularization about two hours after fertilization. These thresholds initiate ihe differentiation of three distinct tissues: mesoderm, ventral neurogenic ectoderm, and dorsal neurogenic ectoderm (Figure 18-14). Each of these tissues goes on to form distinctive cell types in the adult fly. The mesoderm forms flight muscles and internal organs, such as the fat body, which is analogous to our liver. The ventral and dorsal neurogenic ectoderm form distinct neurons iii the ventral nerve cord.

We now consider the regulation of three different targefgenes that are activated by high, intermediate, and low levels of the Dorsal protein — twist, rhomboid, and sog. The highest levels of the Dorsal gradient — that is, in nuclei with the highest levels of Dorsal protein — activate the expression of the twist gene in the ventrahnost 18 cells that form the mesoderm (figure 16-14). The tivist genu is not activated in lateral regions, the neurogenic ectoderm, where there are intermediate and low levels of the Dorsal protein. The reason for this is that the twist regulatory UNA contains two low-affinity Dorsal binding sites (Figure 18-14). Therefore, peak levels of the Dorsal gradient are required (or the efficient occupancy of these sites; the lower levels of Dorsal protein present in lateral regions are insufficient to bind and activate the transcription of the twi'si gene.

sog on

FIGURE 18-14 Three thresholds and three types of regulatory DNAs. The twist 5' regulatory DMA contains ¡wo tow affinity Dorsal bmdtng sites thai are occupied only by peak levels of the Dorsal gradient. As a result, twist expression is restricted to ventral nuclei TTre rhomboid 5' enhancer contains a cluster of Dorsal binding sites. Only one of these sites represents an optimal, high affinity Dorsal recognition sequence. This mixture of high and low affrnity sites allows both high and intermediate levels of the Dorsal gradient to activate rhombo/d expression in ventral-lateral regions. Finally, the 5og intronic enhancer contains four evenly-spaced optimal Dorsal finding sites. These allow high, intermediate, and low levels of the Dorsal gradient to activate sog expression throughout lateral regions.

4 optimal Doreai binding sites

4 optimal Doreai binding sites

rhomboid

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