Genetic Control of Segmentation in Drosophila The Maternal Legacy
by Dr. William Brook Department of Medical Biochemistry University of Calgary
The fertilized egg of Drosophila melanogaster gives rise to a segmented fully-differentiated maggot over the course of a 24 hour embryonic period. The genetic, molecular, and embryological analyses of embryonic segmentation in Drosophila have led to a detailed description of how maternally deposited spatial determinants coordinate zygotic gene expression during embryogensis. The resulting description is the most complete to date of the genetic control of a developmental process in a metazoan. The reason we know so much about this process in Drosophila is because of the availability of sophisticated genetic approaches.
The genetic control of segmentation involves a cascade of gene regulation occurring largely before the onset of the cellular blastoderm stage (~2.5 to 3 hours of development). The cascade (Fig. 1) begins with the diffusion of spatially localized maternal factors, the products of the coordinate genes (i.e. bicoid, nanos, caudal, etc.), from the anterior and posterior poles of the embryo. These control the spatial patterns of transcription of the gap genes (i.e. hunchback. Krüppel, knirps, etc.). The gap genes are amongst the earliest expressed zygotic genes and they encode transcription factors. The gap genes are expressed overlapping territories along the anterior to posterior axis of the fly embryo. These genes act to sub-divide the embryo into broad domains (anterior, middle, posterior). Each domain encompasses the progenitors of several contiguous segments. The gap genes regulate each other and the next set of genes in the hierarchy, the pair-rule genes (even-skipped, hairy, fushi-tarazu, etc.). Pair-rule genes are expressed in 7 stripes of cells corresponding to every other segment. Pair-rule genes encode transcription factors that establish the expression of the segment polarity genes (wingless, hedgehog, engrailed, etc.), many of which are expressed in 14 segmentally repeated stripes. Unlike the other classes of segmentation genes, the segment polarity genes include regulatory proteins other than transcription factors (i.e. secreted signaling molecules, receptors, kinases, etc.) and they mediate interactions between cells. The end result of the hierarchy is a series of segments that have identical repeated segment polarity gene expression patterns. The final group of genes are the homeotic genes which control the character (i.e. head, thorax, abdomen) of each segment. We will discuss these later.
Figure 1. Hierarchy of Gene Control of Segmentation in Drosophila
How was all this worked out?
1) Mutations in the segmentation genes gave us the first clue that the genes might function in a hierachy because the different classes of genes affectd progressively smaller regions of the embryo.
(See Browder et al., 1991, Fig. 14.18; Gilbert, 1997, Fig. 14.18; Kalthoff, Fig. 21.6; Wolpert, 1998, Fig. 5.3)
2)in situ RNA and protein expression patterns of these genes correlated well with the mutant phenotypes AND told us when the genes were first expressed.
(See Browder et al., 1991, Figs. 14.19 and 14.20; Gilbert, 1997, Fig. 14.19; Kalthoff, Fig. 21.20; Wolpert et al., Fig. 5.4)
3) Genetic experiments were used to determine whether these genes interact. The first step in determining whether one gene regulates the expression of another is to see if the expression pattern of one gene is affected by mutation of another (Fig. 2). For instance, if gene A is important for determining the temporal and spatial expression of gene B, then in an a/a mutant, the expression of the B mRNA will be altered either in its timing (it might not be expressed at all) or in its pattern (the region of expression will change). Remember, genetic tests do not indicate whether an interaction is direct or indirect.
Figure 2. Genetic tests to determine whether genes interact with one-another.
4) Molecular experiments determined the structure of these genes. Many (but not all) are transcription factors, meaning that they bind to DNA and cause transcription of a gene. Sometimes these genes regulate not only the transcription of other genes, but also of themselves and sometimes rather than increasing the expression level of a gene, they decrease it.
(See Browder et al. 1991, Fig. 14.26; Gilbert, 1997, Fig. 14.25; Kalthoff, Fig. 21.22; Wolpert et al., Fig. 5.18)
Embryological Evidence Suggests the Existence of Morphogen Gradients in Insect Embryos
During the first three hours of Drosophila development, the fertilized egg undergoes several rounds of nuclear division in the absence of cell division. At the time of cellularization there is no morphological evidence for segmentation or differentiation but cells at that stage are determined with respect to the segment they will form in the adult and in the larval fly. What controls this determination? Results from experimental manipulations of insect embryos in the 1960's and 1970's (localized irradiation, embryo ligations, cytoplasmic leakage, and cytoplasmic transplantation experiments) suggested that there were determinants localized in the cytoplasm at the anterior and posterior poles of the eggs which had long range effects on the fate of the entire embryo. Some of this evidence included (these data are summarized in Nüsslein-Volhard et al., 1987):
One idea was that these determinants could diffuse throughout the embryo forming a concentration gradient and that they could specify different fates at different concentration thresholds. This idea of "concentration dependent morphogens" was very controversial but as you will see there is mounting evidence that these ideas are in fact quite close to the truth. But what were the determinants?
Genetic screens for maternal-effect mutations: coordinate genes
Genetics provided the way to find the determinants. A few maternal and zygotic mutations that affected the pattern of segmentation had been identified and this inspired Christiane Nusslein-Volhard, Eric Wieschaus and colleagues in the late 1970's, to begin large scale systematic genetic screens for maternal effect and zygotic lethal mutations (Nüsslein-Volhard et al., 1987) affecting the pattern of differentiation of the embryonic epidermis. Screening for mutations that affect segmentation was a simple but very powerful idea. Their screen was quite exhaustive and identified well over 100 mutations affecting various aspects of embryonic development. Amongst the maternal effect lethal mutations, three groups of genes were found that had similar effects on anterior posterior pattern. Mutations of the anterior group (including the gene bicoid) result in the deletion of anterior structures, mutations of the posterior group (including the genes nanos and oskar) result in the deletion of posterior abdominal segments and terminal group mutations result in smaller deletions at both ends of the embryo. There is also a maternal system that controls the development of the dorsal-ventral axis. (The dorsal ventral and terminal groups will not be discussed further.)
bicoid: Weak alleles of bicoid resemble the effects of anterior cytoplasmic leakage experiments. Stronger alleles result in the loss of most anterior structures and occasional pattern duplications. bicoid mutant embryos can be rescued by anterior injection of wild-type cytoplasm or bcd+ mRNA produced in vitro. The bicoid mRNA is localized to the anterior end of the embryo and translated forming an A/P protein gradient. bicoid is a homeodomain protein which can act as a transcription factor, which means that it is regulating the transcription of a gene. What genes does bicoid regulate? One of the targets of bicoid is the gap gene called hunchback. bicoid activates hunchback by binding to specific DNA sequences (binding sites) at the 5'-end of the gene. hunchback becomes transcribed in the anterior part of the embryo but, unlike bicoid, its expression stops in a fairly discrete line. How is this accomplished? Binding sites function in an all or nothing fashion. Therefore, within a gradient of bicoid protein is a point where the levels of bicoid are not high enough to activate hunchback. It is called a threshold. Nusslein-Vollhard's group showed this by altering the number of copies of the bicoid gene and showing that this altered the posterior limit of expression of hunchback. They also used a reporter gene to show that if only low affinity binding sites are present, the reporter will need a high concentration of bicoid to be activated (therefore its expression domain is smaller). However, if it has high affinity binding sites, it will be activated with a low concentration of bicoid (it's expression domain extends towards the posterior). bicoid regulation of hunchback is an example of how the affinity of DNA binding might turn a gradient of spatial information into a sharp threshold of spatial information (see Figure 7 in St. Johnston and Nüsslein-Volhard, 1992). As well as acting as a transcription factor, bicoid can bind to specific RNA sequences. This second activity is important in the production of a gradient of another homeodomain protein called caudal. The caudal message is uniformly distributed in the oocyte cytoplasm but the protein is distributed as a gradient opposite to that of bicoid. caudal protein is uniformly translated in bicoid mutant embryos, suggesting that bicoid affects the levels of caudal translation. It turns out that bicoid can bind to specific sequences in the 3'UTR of the caudal message and inhibit its translation. Thus where bicoid levels are high, caudal levels are low and vice versa. caudal may act as a transcriptional activator of posterior gap genes such as knirps and giant.. (See Rivera-Pomar and Jäckle, 1996 for a review of how bicoid affects caudal translation.) (Note: caudal is an example of a gene that was NOT identified in genetic screens. It was first identified based on its sequence similarity to other homeodomain genes. It is important to keep in mind that not all important genes can be picked up by genetic means.)
nanos/pumillio The nanos mRNA is localized to the posterior end of the egg and nanos protein forms a P/A gradient. It also prevents translation of a specific maternal mRNA . This is where it gets complicated, because hunchback , the gene whose zygotic transcription is stimulated by bicoid, has both a maternal and a zygotic phase. It is the translation of the maternal hunchback mRNA that is repressed by nanos(Wharton and Struhl, 1991). In nanos mutant embryos, maternal hunchback mRNA is uniformly translated, while in wild-type embryos, the mRNA is not translated in the posterior half of the embryo. Unlike bicoid, which directly binds to the 3'UTR of the caudal mRNA, nanos does not bind hunchback mRNA. Rather, it somehow is required for the binding of another member of the posterior group, pumillio, to the hunchback 3'UTR(Murata and Wharton, 1995). The pumillio protein is expressed throughout the cytoplasm of the early embryo and nanos provides positional specificity for its binding to the hunchback 3'UTR. When maternal hunchback is translated throughout the whole embryo in nanos mutants, it represses another gap gene, knirps, resulting in the deletion of most of the abdomen of the embryo. These phenotypes of nanos and knirps are almost identical and that suggests that the only function of nanos is to repress maternal hunchback in the posterior part of the embryo to allow the expression of knirps. In both cases of translational control (bicoid inhibition of caudal translation and nanos/pumillio inhibition of maternal hunchback) it was found that the repression worked though cis-acting sequences in the 3' UTRs of the mRNAs. The two most important pieces of supporting evidence were 1) showing that uniformly transcribed transgenes would be translated in the hunchback or caudal pattern if they were fused to the appropriate 3'UTR and 2) demonstrating that bicoid and pumillio could bind the 3'UTR RNA in vitro. Next, the zygotic genome takes over responsibility for patterning the embryo. As we shall discuss, transcription from the zygotic genome depends upon input from proteins encoded by the coordinate genes.
Links to Related Material
(See Christianne Nüsslein-Volhard and Drosophila Embryogenesis in Zygote; optional.)
Reviews* and References
Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology.
Third edition. Saunders College Pub. Philadelphia.
Murata, Y., and Wharton, R. P. (1995). Binding of pumillio to
maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell
Shostak, S. 1991. Embryology. An Introduction to Developmental Biology.
HarperCollins. New York.
Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London.
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Last revised Monday, August 4, 1998