Regulation of Gene Expression in Eukaryotes:- Development Is Controlled by Cascades of Regulatory Proteins
For sheer complexity and intricacy of coordination, the patterns of gene regulation that bring about develop ment of a zygote into a multicellular animal or plant have no peer. Development requires transitions in morphology and protein composition that depend on tightly coordinated changes in expression of the genome. More genes are expressed during early development than in any other part of the life cycle. For example, in the sea urchin, an oocyte has about 18,500 different mRNAs, compared with about 6,000 different mRNAs in the cells of a typical differentiated tissue. The mRNAs in the oocyte give rise to a cascade of events that regulate the expression of many genes across both space and time. Several animals have emerged as important model systems for the study of development, because they are easy to maintain in a laboratory and have relatively short generation times. These include nematodes, fruit flies, zebra fish, mice, and the plant Arabidopsis. This dis cussion focuses on the development of fruit flies. Our understanding of the molecular events during develop ment of Drosophila melanogaster is particularly well advanced and can be used to illustrate patterns and principles of general significance. The life cycle of the fruit fly includes complete metamorphosis during its progression from an embryo to an adult (Fig. 1). Among the most important characteristics of the embryo are its polarity (the anterior and posterior parts of the animal are readily distinguished, as are its dorsal and ventral parts) and its metamerism (the embryo body is made up of serially repeating segments, each with characteristic features). During development, these segments become organized into a head, thorax, and abdomen. Each segment of the adult thorax has a different set of appendages. Devel opment of this complex pattern is under genetic control, and a variety of pattern-regulating genes have been discovered that dramatically affect the organization of the body. The Drosophila egg, along with 15 nurse cells, is surrounded by a layer of follicle cells (Fig. 28–35). As the egg cell forms (before fertilization), mRNAs and proteins originating in the nurse and follicle cells are de posited in the egg cell, where some play a critical role in development. Once a fertilized egg is laid, its nucleus divides and the nuclear descendants continue to divide in synchrony every 6 to 10 min. Plasma membranes are not formed around the nuclei, which are distributed within the egg cytoplasm (or syncytium). Between the eighth and eleventh rounds of nuclear division, the nuclei migrate to the outer layer of the egg, forming a monolayer of nuclei surrounding the common yolk-rich cytoplasm; this is the syncytial blastoderm. After a few additional divisions, membrane invaginations surround the nuclei to create a layer of cells that form the cellular blastoderm. At this stage, the mitotic cycles in the various cells lose their synchrony. The developmental fate of the cells is determined by the mRNAs and proteins originally deposited in the egg by the nurse and follicle cells. Proteins that, through changes in local concentration or activity, cause the surrounding tissue to take up a particular shape or structure are sometimes referred to as morphogens; they are the products of pattern regulating genes. As defined by Christiane Nüsslein Volhard, Edward B. Lewis, and Eric F. Wieschaus, three major classes of pattern-regulating genes—maternal, segmentation, and homeotic genes—function in successive stages of development to specify the basic features of the Drosophila embryo’s body. Maternal genes are expressed in the unfertilized egg, and the resulting maternal mRNAs remain dormant until fertilization. These provide most of the proteins needed in very early development, until the cellular blastoderm is formed. Some of the proteins encoded by maternal mRNAs direct the spatial organization of the developing embryo at early stages, establishing its polarity. Segmentation genes, transcribed after fertilization, direct the formation of the proper number of body segments. At least three subclasses of segmentation genes act at successive stages: gap genes divide the devel oping embryo into several broad regions, and pair-rule genes together with segment polarity genes define 14 stripes that become the 14 segments of a normal embryo. Homeotic genes are expressed still later; they specify which organs and appendages will develop in particular body segments. The many regulatory genes in these three classes direct the development of an adult fly, with a head, thorax, and abdomen, with the proper number of segments, and with the correct appendages on each segment. Al though embryogenesis takes about a day to complete, all these genes are activated during the first four hours. Some mRNAs and proteins are present for only a few minutes at specific points during this period. Some of the genescode for transcription factors that affect the expression of other genes in a kind of developmental cascade. Regulation at the level of translation also occurs, and many of the regulatory genes encode translational repressors, most of which bind to the 3UTR of the mRNA . Because many mRNAs are deposited in the egg long before their translation is required, translational repression provides an especially important avenue for regulation in developmental pathways.
FIGURE 1 Life cycle of the fruit fly Drosophila melanogaster. Drosophila undergoes a complete metamorphosis, which means that the adult insect is radically different in form from its immature stages, a transformation that requires extensive alterations during development. By the late embryonic stage, segments have formed, each containing specialized structures from which the various appendages and other features of the adult fly will develop.
Maternal Genes Some maternal genes are expressed within the nurse and follicle cells, and some in the egg itself. Within the unfertilized Drosophila egg, the mater Nalgene products establish two axes—anterior-posterior and dorsal-ventral—and thus define which regions of the radially symmetric egg will develop into the head and abdomen and the top and bottom of the adult fly. A key event in very early development is establishment of mRNA and protein gradients along the body axes. Some maternal mRNAs have protein products that diffuse through the cytoplasm to create an asymmetric distribution in the egg. Different cells in the cellular blastoderm therefore inherit different amounts of these proteins, setting the cells on different developmental paths. The products of the maternal mRNAs include transcriptional activators or repressors as well as translational repressors, all regulating the expression of other pattern regulating genes. The resulting specific patterns and sequences of gene expression therefore differ between cell lineages, ultimately orchestrating the development of each adult structure.
FIGURE 2 Early development in Drosophila. During develop ment of the egg, maternal mRNAs (including the bicoid and nanos gene transcripts, discussed in the text) and proteins are deposited in the developing oocyte (unfertilized egg cell) by nurse cells and follicle cells. After fertilization, the two nuclei of the fertilized egg divide in synchrony within the common cytoplasm (syncytium), then migrate to the periphery. Membrane invaginations surround the nuclei to create a monolayer of cells at the periphery; this is the cellular blastoderm stage. During the early nuclear divisions, several nuclei at the far posterior become pole cells, which later become the germ-line cells.
The anterior-posterior axis in Drosophila is defined at least in part by the products of the bicoid and nanos genes. The bicoid gene product is a major anterior morphogen, and the nanos gene product is a major posterior morphogen. The mRNA from the bicoid gene is synthesized by nurse cells and deposited in the unfertilized egg near its anterior pole. Nüsslein-Volhard found that this mRNA is translated soon after fertilization, and the Bicoid protein diffuses through
the cell to create, by the seventh nuclear division, a concentration gradient radiating out from the anterior pole (Fig. 3a). The Bicoid protein is a transcription factor that activates the expression of a number of seg mentation genes; the protein contains a homeodomain (p. 1090). Bicoid is also a translational repressor that in activates certain mRNAs. The amounts of Bicoid protein in various parts of the embryo affect the subsequent ex pression of a number of other genes in a threshold dependent manner. Genes are transcriptionally activated or translationally repressed only where the Bicoid protein concentration exceeds the threshold. Changes in the shape of the Bicoid concentration gradient have dramatic effects on the body pattern. Lack of Bicoid protein results in development of an embryo with two abdomens but neither head nor thorax (Fig. 3b); however, embryos without Bicoid will develop normally if an adequate amount of bicoid mRNA is injected into the egg at the ap propriate end. The nanos gene has an analogous role, but its mRNA is deposited at the posterior end of the egg and the anterior-posterior protein gradient peaks at the posterior pole. The Nanos protein is a translational repressor.
FIGURE 3 Distribution of a maternal gene product in a Drosophila egg. (a) Micrograph of an immunologically stained egg, showing distribution of the bicoid (bcd) gene product. The graph measures stain intensity. This distribution is essential for normal develop ment of the anterior structures of the animal. (b) If the bcd gene is not expressed by the mother (bcd /bcd mutant) and thus no bicoid mRNA is deposited in the egg, the resulting embryo has two posteriors (and soon dies).
A broader look at the effects of maternal genes reveals the outline of a developmental circuit. In addition to the bicoid and nanos mRNAs, which are deposited in the egg asymmetrically, a number of other maternal mRNAs are deposited uniformly throughout the egg cytoplasm. Three of these mRNAs encode the Pumilio, Hunchback, and Caudal proteins, all affected by nanos and bicoid (Fig. 4). Caudal and Pumilio are in volved in development of the posterior end of the fly. Caudal is a transcriptional activator with a homeodomain; Pumilio is a translational repressor. Hunch back protein plays an important role in the development of the anterior end and is also a transcriptional regulator of a variety of genes, in some cases a positive regulator, in other cases negative. Bicoid suppresses trans lation of caudal in the anterior and also acts as a transcriptional activator of hunchback in the cellular blastoderm. Because hunchback is expressed both from maternal mRNAs and from genes in the developing egg, it is considered both a maternal and a segmentation gene. The result of the activities of Bicoid is an increased concentration of Hunchback at the anterior end of the egg. The Nanos and Pumilio proteins act as translational repressors of hunchback, suppressing synthesis of its protein near the posterior end of the egg. Pumilio does not function in the absence of the Nanos protein, and the gradient of Nanos expression confines the activity of both proteins to the posterior region. Translational repression of the hunchback gene leads to degradation of hunchback mRNA near the posterior end. However, lack of Bicoid protein in the posterior leads to expression of caudal. In this way, the Hunchback and Caudal proteins become asymmetrically distributed in the egg.
Segmentation Genes Gap genes, pair-rule genes, and segment polarity genes, three subclasses of segmentation genes in Drosophila, are activated at successive
stages of embryonic development. Expression of the gap genes is generally regulated by the products of one or more maternal genes. At least some of the gap genes encode transcription factors that affect the expression of other segmentation or (later) homeotic genes. One well-characterized segmentation gene is fushi tarazu (ftz), of the pair-rule subclass. When ftz is deleted, the embryo develops 7 segments instead of the normal 14, each segment twice the normal width. The Fushi-tarazu protein (Ftz) is a transcriptional activator with a homeodomain. The mRNAs and proteins derived from the normal ftz gene accumulate in a striking pat tern of seven stripes that encircle the posterior two thirds of the embryo (Fig. 5). The stripes demarcate the positions of segments that develop later; these segments are eliminated if ftz function is lost. The Ftz protein and a few similar regulatory proteins directly or indirectly regulate the expression of vast numbers of genes in the continuing developmental cascade.
FIGURE 4 Regulatory circuits of the anterior-posterior axis in a Drosophila egg. The bicoid and nanos mRNAs are localized near the anterior and posterior poles, respectively. The caudal, hunchback, and pumilio mRNAs are distributed throughout the egg cytoplasm. The gradients of Bicoid (Bcd) and Nanos proteins lead to accumulation of Hunchback protein in the anterior and Caudal protein in the posterior of the egg. Because Pumilio protein requires Nanos protein for its activity as a translational repressor of hunchback, it functions only at the posterior end.
FIGURE 5 Distribution of the fushi tarazu (ftz) gene product in early Drosophila embryos. (a) In the normal embryo, the gene product can be detected in seven bands around the circumference of the embryo (shown schematically). These bands (b) appear as dark spots (generated by a radioactive label) in a cross-sectional autoradiograph and (c) demarcate the anterior margins of the segments in the late em bryo (marked in red).
FIGURE 7 Effects of mutations in homeotic genes in Drosophila. (a) Normal head. (b) Homeotic mutant (antennapedia) in which antennae are replaced by legs. (c) Normal body structure. (d) Homeotic mutant (bithorax) in which a segment has developed incorrectly to produce an extra set of wings.
Homeotic Genes Loss of homeotic genes by mutation or deletion causes the appearance of a normal appendage or body structure at an inappropriate body position. An important example is the ultrabithorax (ubx) gene. When Ubx function is lost, the first abdominal segment develops incorrectly, having the structure of the third thoracic segment. Other known homeotic mutations cause the formation of an extra set of wings, or two legs at the position in the head where the antennae are nor mally found (Fig. 7). The homeotic genes often span long regions of DNA. The ubx gene, for example, is 77,000 bp long. More than 73,000 bp of this gene are in introns, one of which is more than 50,000 bp long. Transcription of the ubx gene takes nearly an hour. The delay this imposes on ubx gene expression is believed to be a timing mechanism involved in the temporal regulation of subsequent steps in development. The Ubx protein is yet another transcriptional activator with a homeodomain . Many of the principles of development outlined above apply to eukaryotes from nematodes to humans. Some of the regulatory proteins themselves are con served. For example, the products of the homeobox containing genes HOX 1.1 in mouse and antennapedia in fruit fly differ in only one amino acid residue. Of course, although the molecular regulatory mechanisms may be similar, many of the ultimate developmental events are not conserved (humans do not have wings or antennae). The discovery of structural determinants with identifiable molecular functions is the first step in understanding the molecular events underlying devel opment. As more genes and their protein products are discovered, the biochemical side of this vast puzzle will be elucidated in increasingly rich detail.
