Dynamic Development

CONTENTS

Main Page Dynamic Development

The Foundations of Developmental Biology

Gametogenesis

From Sperm and Egg to Embryo

Genetic Regulation of Development

Organizing the Multicellular Embryo

Generating Cell Diversity


Dynamic Development at a Glance


Learning Resources

Research Resources

The Developmental Biology Journal Club

Developmental Biology Tutorial

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.

Overview

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)

  • Maternal co-ordinate genes- Anterior or posterior end of egg is missing. bicoid mutants lack anterior head structures, nanos mutants have no abdomen.
  • Gap genes- There are large "gaps" in the embryo- many adjacent segments are missing. Krüppel mutants are missing T1-A5, hunchback mutants are missing head segments and knirps mutants lack posterior segments.
  • Pair-rule genes- Have only half the number of parasegments- they are actually missing every other parasegment. fushi-tarazu mutants lack odd-numbered parasegments and even-skipped mutants lack even-numbered parasegments.
  • Segment polarity genes- Within each segment either the anterior or posterior half is duplicated. gooseberry and engrailed mutants have the posterior part of each segment replaced by the anterior part of the adjacent segment.

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)

  • Maternal co-ordinate genes- bicoid mRNA is localized at the anterior end of the embryo, nanos mRNAs are at the posterior end. First expressed during oogenesis.
  • Gap genes- Krüppel mRNA is in parasegments 4-6, hunchback mRNA is in the anterior half of the embryo and knirps mRNA is in the posterior end. First expressed at syncytial blastoderm.
  • Pair-rule genes- form seven stripes of transcription around each embryo. First expressed at syncytial blastoderm.
  • Segment polarity genes- form fourteen stripes of transcription around each embryo. First expressed at cellular blastoderm.

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 determine whether another is to see if the expression pattern of one gene in the mutant 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):

  • Localized irradiation of the anterior poles of Smittia embryos resulted in the loss of head structures and the formation of embryos which had two abdomens in mirror image symmetry.
  • Embryo ligation experiments showed that the formation of structures in the middle of the embryo depended on substances localized in the anterior and posterior poles of the embryo.
  • Cytoplasmic leakage, and cytoplasmic transplantation experiments showed that there were substances at the anterior and posterior ends of the embryo that were responsible for organizing the global pattern of the embryo.

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 affecting the dorsal ventral pattern were also identified but they will not be discussed any further in this course. 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 resulted in smaller deletions at both ends of the embryo. There is also a maternal system control the development of the dorsal-ventral axis. (The dorsal ventral and terminal groups will not be discussed further.)

Anterior group

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 fairly a 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 it's 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.)

Posterior group

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. This 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 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.


Summary

  • segmentation is controlled by localized maternal determinants in the anterior and posterior polar cytoplasm
  • genetic screens identified several genes including bicoid (anterior determinant) and nanos (posterior determinant)
  • bicoid mRNA located at the anterior pole, translated and the protein diffuses to form a concentration gradient
  • acting as a transcription factor, bicoid influences fate (gene expression) in a concentration dependent manner
  • acting as a RNA binding protein, bicoid inhibits the translation of caudal mRNA in the anterior part of the embryo resulting in an anterior to posterior gradient of caudal homeodomain protein.
  • nanos mRNA is localized to the posterior pole
  • nanos protein diffuses throughout the posterior part of the embryo and blocks the translation of maternal hunchback mRNA. In the absence of nanos, the maternal hunchback mRNA is translated and blocks the activation of the zygotic gap gene knirps.
  • nanos does not bind the 3'UTR of hunchback directly but rather acts a spatially localized co-factor for another posterior group gene, pumillio


Learning Objectives

  • How would you determine if one gene in the segmentation hierarchy regulates the expression of another?
  • In the case of a transcription factor, what methods could you use to see if the interaction was indirect?
  • What methods would you use to determine whether a RNA binding protein acted directly or indirectly?
  • Outline the role of bicoid in the regulation of hunchback and caudal.
  • Explain how transgenes with containing different elements of the hunchback promoter fused to the lacZ coding regions were used to show that bicoid could regulate the transcription of genes in a concentration dependent manner.
  • Outline the interactions between nanos, pumillio, and maternal hunchback and how they affect the regulation of the gap gene knirps. (N.B. This is a good example of direct and indirect interactions)


Digging Deeper

Links to Related Material

(See Christianne Nüsslein-Volhard and Drosophila Embryogenesis in Zygote; optional.)

FLY MORPH-O-GENESIS


Reviews* and References

Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology. Third edition. Saunders College Pub. Philadelphia.

Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA.

Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill. New York.

Murata, Y., and Wharton, R. P. (1995). Binding of pumillio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell 80: 747-56.

*Nüsslein-Volhard, C., Frohnhofer, H. G., and Lehmann, R. (1987). Determination of anteroposterior polarity in Drosophila. Science 238: 1675-81.

Nüsslein-Volhard, C., and Wieschaus, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature 287: 795-801.

*Rivera-Pomar, R., and Jäckle, H. (1996). From gradients to stripes in Drosophila embryogenesis: filling in the gaps. Trends Genet 12: 478-83.

Shostak, S. 1991. Embryology. An Introduction to Developmental Biology. HarperCollins. New York.

*St. Johnston, D., and Nüsslein-Volhard, C. (1992). The origin of pattern and polarity in the Drosophila embryo. Cell 68: 201-219.

Wharton, R. P., and Struhl, G. (1991). RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos. Cell 67: 955-67
.

Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London.


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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Monday, August 4, 1998