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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.
We have seen that the segments of the insect larva are all variations on the same basic theme, with segmentation genes defining the basic repetitive module and homeotic selector genes giving each segment its individual character. The same applies to the major appendages of the adult insect body—legs, wings, antennae, mouthparts and external genitalia: they too are variations on a common basic theme. At a finer level of detail, we encounter the same wonderful simplification: the appendages—and many other parts of the body—consist of substructures that are themselves variations on a small number of basic evolutionarily conserved themes.
In this section we follow the course of development in Drosophila through to its end, narrowing our focus at each step to examine one example of the many related structures that are developing in parallel. As we go along, we shall point out parallels with vertebrate structures that develop similarly, using not only the same general strategies, but many of the same specific molecular mechanims. But to avoid interrupting the narrative later, we must first briefly explain some key experimental methods, required to cope with a special problem that arises when we try to discover how genes control the later stages of development.
As emphasized earlier, the same gene may be used repeatedly in many different situations—in different regions of the body, and at different times. Often, loss-of-function mutations disrupt early development so severely that the embryo or larva dies, depriving us of the opportunity to see how the mutation would affect later processes.
One way around this problem is to study conditional mutations. If we have, for example, a temperature-sensitive mutation in the gene of interest, we can maintain the animal during early development at a low temperature, where the gene product functions normally, and then disable the gene product whenever we please by raising the temperature to discover the late functions.
Other methods involve actually modifying the DNA in subsets of cells at late stages of development—a sort of genetic surgery on individual cells that allows mutant groups of cells of a specified genotype to be generated at a chosen time in development. This remarkable feat can be achieved by induced somatic recombination. A current version of this technique uses transgenic flies that have been bred to contain two types of yeast-derived genetic elements: the FLP site-specific recombinase gene, and the FLP Recombinase Target (FRT) sequence. Typically, the animal is homozygous for an insertion of the FRT sequence close to the centromere on a chosen chromosome arm, while a construct consisting of the FLP gene under a heat-shock promoter is inserted elsewhere in the genome. If such a transgenic embryo or larva is given a heat shock (that is, exposed to a high temperature for a few minutes), expression of FLP is induced, and this enzyme catalyzes crossing-over and recombination between the maternal and paternal chromosomes at the FRT site. If the heat shock is adjusted to be sufficiently mild, this event will occur in only one or a few cells, scattered at random. As explained in Figure 21-48, if the animal is also heterozygous for a gene of interest in the crossed-over chromosomal region, the process can result in a pair of daughter cells that are homozygous, the one receiving two copies of the maternal allele of the gene, the other receiving two copies of the paternal allele. Each of these daughter cells will normally grow and divide to give clonal patches of homozygous progeny.
Creation of mutant cells by induced somatic recombination. The diagrams follow the fate of a single pair of homologous chromosomes, one from the father (shaded), the other from the mother (unshaded). These chromosomes have an FRT element (green) inserted (more...)
The occurrence of the cross-over can be detected if the animal is chosen to be also heterozygous for a mutation in a marker gene—a pigmentation gene, for example—that lies on the same chromosome arm as the gene of interest and so undergoes crossing over in company with it. In this way clearly marked homozygous mutant clones of cells can be created to order. Either FLP and FRT, or the analogous Cre and Lox pair of recombination elements, can also be used in other configurations to switch expression of a gene on or off (see Figure 5-82). With these techniques, one can discover what happens, for example, when cells are caused to produce a particular signaling molecule at an abnormal site, or are deprived of a particular receptor.
Instead of using a heat-shock promoter to drive expression of the FLP recombinase, one can use a copy of the regulatory sequence of a gene in the fly's normal genome that is expressed at some interesting time and place. The recombination event will then be triggered, and mutant cells created, at just the sites where that gene is normally expressed. A variant of this technique uses transcriptional regulation machinery borrowed from yeast, rather than genetic recombination machinery, to switch expression of a chosen fly gene reversibly on or off according to the normal pattern of expression of some other chosen fly gene (Figure 21-49).
The GAL4/UAS technique for controlled gene misexpression in Drosophila. The method allows one to drive expression of a chosen gene G at the places and times where some other Drosophila gene H is normally expressed. (A) A transgenic animal is created, (more...)
By switching gene functions off or on at specific times and places in these ways, developmental biologists can set about deciphering the system of genetically specified signals and responses that control the patterning of any organ of the body.
The external structures of the adult fly are formed largely from rudiments called imaginal discs—groups of cells that are set aside, apparently undifferentiated, in each segment of the larva. The discs are pouches of epithelium, shaped like crumpled and flattened balloons, and continuous with the epidermis (the surface layer) of the larva. There are 19 of them, arranged as 9 pairs on either side of the larva plus 1 disc in the midline (Figure 21-50). They grow and develop their internal pattern as the larva grows, until finally, at metamorphosis, they evert (turn inside out), extend, and differentiate overtly to form the epidermal layer of the adult. The eyes and antennae develop from one pair of discs, the wings and part of the thorax from another, the first pair of legs from another, and so on.
The imaginal discs in the Drosophila larva and the adult structures they give rise to. (After J.W. Fristrom et al., in Problems in Biology: RNA in Development [E.W. Hanley, ed.], p. 382. Salt Lake City: University of Utah Press, 1969.)
The cells of one imaginal disc look like those of another, but grafting experiments show that they are in fact already regionally determined and nonequivalent. If one imaginal disc is transplanted into the position of another in the larva and the larva is then left to go through metamorphosis, the grafted disc is found to differentiate autonomously into the structure appropriate to its origin: a wing disc will give wing structures, a haltere disc, haltere structures, regardless of its new site. This shows that the imaginal disc cells are governed by a memory of their original position. By a more complex serial grafting procedure that lets the imaginal disc cells proliferate for an extended period before differentiating, it can be shown that this cell memory is stably heritable (with rare lapses) through an indefinitely large number of cell generations.
The homeotic selector genes are essential components of the memory mechanism. If, at any stage in the long period leading up to differentiation at metamorphosis, both copies of a homeotic selector gene are eliminated by induced somatic recombination from a clone of imaginal disc cells that would normally express that gene, those cells will differentiate into incorrect structures, as though they belonged to a different segment of the body. These and other observations indicate that each cell's memory of positional information depends on the continued activity of the homeotic selector genes. This memory, furthermore, is expressed in a cell-autonomous fashion—each cell appears to maintain its state individually, depending on its own history and genome.
We must now examine how an appendage develops its internal pattern. We shall take the insect wing as our example.
The process begins with the early patterning mechanisms we have already discussed. The anteroposterior and dorsoventral systems of signals in the early embryo in effect mark out an orthogonal grid in the blastoderm, in the form of dorsoventral, anteroposterior, and periodically spaced segmental gene expression boundaries. At certain points of intersection of these boundaries, the combination of genes expressed is such as to switch a cluster of cells into the imaginal disc pathway.
In molecular terms this corresponds to switching on expression of imaginal-disc-defining regulatory genes. In most of the discs, the gene Distal-less is switched on. This codes for a gene regulatory protein that is essential for the sustained growth required to create an elongated appendage such as a leg or an antenna with a proximodistal axis. In its absence, such appendages fail to form, and when it is artificially expressed at abnormal sites, misplaced appendages can be produced. Distal-less is expressed in a similar fashion in the developing limbs and other appendages of most species of invertebrates and vertebrates that have been examined (Figure 21-51). For the eye disc, another gene, eyeless (together with two closely related genes), performs the corresponding role; it too has homologues with homologous functions—the Pax-6 genes that drive eye development in other species, as discussed in Chapter 7.
Expression of Distal-less in developing legs and related appendages of various species. (A) A sea-urchin larva. (B) A moth larva. (A, from G. Panganiban et al., Proc. Natl. Acad. Sci. USA 94:5162–5166, 1997. © National Academy of Sciences; (more...)
From the outset, the cluster of cells forming the imaginal disc has the rudiments of an internal pattern, inherited from the earlier patterning process. For example, the cells in the posterior half of the wing-disc rudiment (and of most of the other imaginal-disc rudiments) express the segment-polarity gene engrailed, while those in the anterior half do not. The initial asymmetries lay the foundations for a subsequent more detailed patterning, just as in the egg and early embryo.
The sectors of the wing disc defined by these early differences of gene expression correspond to specific parts of the future wing. The posterior, engrailed-expressing region will form the posterior half of the wing, while the region that does not express engrailed will form the anterior half. Meanwhile, the dorsal part of the wing disc expresses a gene called apterous, while the ventral half does not. At metamorphosis, the disc folds along the line separating these domains to give a wing whose dorsal sheet of cells is derived from the apterous-expressing region and whose ventral sheet is derived from the region that does not express apterous. The wing margin, where these two epithelial sheets are joined, corresponds to the boundary of the apterous expression domain in the disc (Figure 21-52).
Gene expression domains in the wing imaginal disc, defining quadrants of the future wing. The wing blade itself derives from the oval-shaped domain toward the right, and it is divided into four quadrants by the expression of apterous and engrailed, as (more...)
The cells of the disc, having switched on expression of the genes that mark them as anterior or posterior, dorsal or ventral, retain this specification as the disc grows and develops. Because the cells are sensitive to these differences and selective in their choice of neighbors, sharply defined boundaries are formed between the four resultant sets of cells, with no mixing at the interfaces. The four corresponding quadrants of the disc are called compartments, because there is no exchange of cells between them (Figure 21-53).
Compartments in the adult wing. (A) The shapes of marked clones in the Drosophila wing reveal the existence of a compartment boundary. The border of each marked clone is straight where it abuts the boundary. Even when a marked clone has been genetically (more...)
Along each of the compartment boundaries—the anteroposterior boundary defined by engrailed and the dorsoventral boundary defined by apterous—cells in different states confront one another and interact to create narrow bands of specialized cells. These boundary cells produce new signals to organize the subsequent growth and more detailed patterning of the appendage.
Cells in the posterior wing compartment express the Hedgehog signaling protein, but cannot respond to it. Cells in the anterior compartment can respond to Hedgehog. Because Hedgehog acts only over a short distance, the signal reception pathway is activated only in the narrow band of cells just anterior to the compartment boundary, where anterior and posterior cells are juxtaposed. These boundary cells respond by switching on expression of another signaling molecule, Dpp—the same protein that we encountered previously, in the dorsoventral patterning of the early embryo (Figure 21-54). Dpp acts in its new context in much the same way as before: it is thought to diffuse (or somehow spread its effects) outward from the boundary cells, setting up a morphogen gradient to control the subsequent detailed pattern of growth and gene expression.
Morphogenetic signals created at compartment boundaries in the wing imaginal disc. (A) Creation of the Dpp signaling region at the anteroposterior compartment boundary through a Hedgehog-mediated interaction between the anterior and posterior cells. In (more...)
Analogous events occur at the dorsoventral compartment boundary (see Figure 21-54). Here, at the future wing margin, short-range communication mediated by the Notch pathway creates a band of boundary cells that produce another morphogen, the Wingless protein—the same signaling factor, belonging to the Wnt family, that acted earlier in the anteroposterior patterning of each embryonic segment. The Dpp and Wingless gradients, together with the other signals and asymmetries of gene expression that we have discussed, combine to drive expression of other genes at precisely defined locations within each compartment.
One of the most mysterious and ill-understood aspects of animal development is the control of growth: why does each part of the body grow to a precisely defined size? This problem is exemplified in remarkable way in the imaginal discs of Drosophila. By induced somatic recombination, one can, for example, create a clonal patch of cells that proliferate more rapidly than the rest of the cells in the developing organ. The clone may grow to occupy almost the whole of the compartment in which it lies, and yet it does not overstep the boundary of the compartment. Astonishingly, its rapid growth has almost no effect on the compartment's final size, its shape, or even the details of its internal pattern (see Figure 21-53). Somehow, the cells within the compartment interact with one another to determine when their growth should stop, and each compartment behaves as a regulatory unit in this respect.
A first question is whether the size of the compartment is regulated so as to contain a set number of cells. Mutations in components of the cell-cycle control machinery can be used to speed up or slow down the rate of cell division without altering the rate of cell or tissue growth. This results in abnormally large numbers of abnormally small cells, or the converse, but the size—that is, the area—of the compartment is practically unchanged. Thus, the regulatory mechanism seems to depend on signals that indicate the physical distance between one part of the compartment and another, and on cellular responses that somehow read these signals so as to halt growth only when the tissue has attained the correct area.
One clue to how the system works comes from the observation that flies with genetic defects in the signaling pathway mediated by insulin and insulin-like factors are small, with cells that are both small and reduced in numbers, while overactivity of this pathway can produce giant flies, with more and bigger cells. Localized misexpression of the same genes in a single compartment has similar effects on just that compartment. Since insulin is widely used in animals as a regulator of responses to nutrition, the mechanisms controlling the sizes of compartments and organs may have evolved from mechanisms that control cell growth and proliferation according to nutritional conditions.
The deeper problem remains, however: what mechanism ensures that each little piece of the pattern within a compartment grows to its appropriate size, despite local disturbances in growth rate or starting conditions? The morphogen gradients (of Dpp and Wingless, for example) create a pattern by imposing different characters on cells in different positions. Could it be that the cells in each region can somehow sense how close the spacing of the pattern is—how steep the gradient of change in cell character—and continue their growth until the tissue is spread out to the right degree? A striking demonstration of the phenomenon that needs to be understood is seen in the intercalary regeneration that occurs when separate parts of a Drosophila imaginal disc or of a growing cockroach leg are surgically grafted together. After the graft, the cells in the neighbourhood of the junction proliferate and fill in the parts of the pattern that should normally lie between them, continuing their growth until the normal spacing between landmarks is restored (Figure 21-55). The mechanisms that bring this about are a mystery, but it seems likely that they are similar to the mechanisms that regulate growth during normal development.
Intercalary regeneration. When mismatched portions of the growing cockroach leg are grafted together, new tissue (green) is intercalated (by cell proliferation) to fill in the gap in the pattern of leg structures, restoring a leg segment of normal size (more...)
The limbs of vertebrates seem very different from those of insects. The insect wing, for example, consists mainly of two elaborately patterned sheets of epithelium, with very little tissue in between. In contrast, a limb of a vertebrate consists of an elaborately patterned system of muscles, bones and other connective tissues inside a thin and much more simply structured covering of epidermis. Moreover, the evolutionary evidence suggests that the last common ancestor of insects and vertebrates may have had neither legs, nor arms, nor wings, nor fins and that we have evolved these various appendages independently. And yet, when we examine the molecular mechanisms that control vertebrate limb development, we find a surprising number of similarities with the limbs of insects. We have already mentioned some of these resemblances, but there are many others: almost all the molecules we have already mentioned in the fly wing have their counterparts in the vertebrate limb, although these are expressed in different spatial relationships.
The parallels have been most thoroughly studied in the chick embryo. As we saw earlier, each leg or wing of a chick originates from a tongue-shaped limb bud, consisting of a mass of embryonic connective tissue cells, called mesenchyme cells, encased in a jacket of epithelium. In this structure, one finds expression of homologs of almost all the genes that we have mentioned in our account of Drosophila wing patterning, including Distal-less, wingless, Notch, engrailed, dpp, and hedgehog, mostly performing functions that seem more or less similar to their functions in the Drosophila wing disc (Figure 21-56).
Molecules that control patterning in a vertebrate limb bud. (A) A wing bud of a chick embryo at 4 days of incubation. The scanning electron micrograph shows a dorsal view, with somites (the segments of the trunk of the embryo) visible to the left. At (more...)
The Hox genes likewise make an appearance in the limbs of both insects and vertebrates. In the insect appendage, the anterior and posterior compartments are distinguished by expression of different genes of the Hox complex—a result of the serial expression pattern of these genes along the anteroposterior axis of the body as a whole. In the vertebrate limb, genes of two of the vertebrate Hox complexes (HoxA and HoxD) are expressed in a regular pattern, obedient to the usual rules of serial expression of genes in these complexes. They help, in conjunction with other factors such as the Tbx proteins mentioned earlier (see Figure 21-9), to regulate differences of cell behavior along the proximodistal limb axis.
According to one view, these molecular resemblances between developing limbs in different phyla reflect descent from a common ancestor that, while lacking limbs, had appendages of some sort built on similar principles—antennae, perhaps, or protruding mouthparts for snatching food. Modern limblike appendages, from the wings and legs of the fly to the arms and legs of a human, would then have evolved through activation of the genes for appendage formation at new sites in the body, as a result of changes in gene regulation.
We now pick up again the thread of development in the Drosophila imaginal disc and follow it through to the final step at which cells become terminally differentiated. Narrowing our focus further, we take as our example the differentiation of just one type of small structure that arises in the imaginal disc epithelium: the sensory bristle.
The bristles that cover the body surface of an insect are miniature sense organs. Some respond to chemical stimuli, others to mechanical stimuli, but they are all constructed in a similar way. The structure is seen at its simplest in the mechanosensory bristles. Each of these consists of four cells: a shaft cell, a socket cell, a neural sheath cell, and a neuron (Figure 21-57). Movement of the shaft of the bristle excites the neuron, which sends a signal to the central nervous system.
The basic structure of a mechanosensory bristle. The lineage of the four cells of the bristle—all descendants of a single sensory mother cell—is shown on the left.
The cells of the bristle of the adult fly derive from the imaginal disc epithelium, and all four of them are granddaughters or great-granddaughters (see Figure 21-57) of a single sensory mother cell that becomes distinct from the neighboring prospective epidermal cells during the last larval instar (Figure 21-58). (A fifth descendant migrates away from the rest to become a glial cell.) To account for the pattern of bristle differentiation, we have to explain first how the genesis of sensory mother cells is controlled and then how the five descendants of each such cell become different from one another.
Sensory mother cells in the wing imaginal disc. The sensory mother cells (blue here) are easily revealed in this special strain of Drosophila, which contains an artificial LacZ reporter gene that, by chance, has inserted itself in the genome next to a (more...)
Two genes, called achaete and scute, are crucial in initiating the formation of bristles in the imaginal disc epithelium. These genes have similar and overlapping functions and code for closely related gene regulatory proteins of the basic helix-loop-helix class (see Chapter 7). As a result of disc-patterning mechanisms of the type we have already discussed, achaete and scute are expressed in the imaginal disc in the regions within which bristles will form. Mutations that eliminate the expression of these genes at some of their usual sites block development of bristles at just those sites, and mutations that cause expression in additional, abnormal sites cause bristles to develop there. But expression of achaete and scute is transient, and only a minority of the cells initially expressing the genes go on to become sensory mother cells; the others become ordinary epidermis. The state that is specified by expression of achaete and scute is called proneural, and achaete and scute are called proneural genes. The proneural cells are primed to take the neurosensory pathway of differentiation, but, as we shall see, which of them will actually do so depends on competitive interactions among them.
Cells expressing the proneural genes occur in groups in the imaginal disc epithelium—a small, isolated cluster of fewer than 30 cells for a big isolated bristle, a broad, continuous patch of hundreds or thousands of cells for a field of small bristles. In the former case just one member of the cluster becomes a sensory mother cell; in the latter case many cells scattered throughout the proneural region do so. In either case, each sensory mother cell becomes surrounded by cells that switch off expression of the proneural genes and become condemned to differentiate as epidermis instead. Experiments with genetic mosaics show that this is because a cell that becomes committed to the sensory-mother-cell pathway of differentiation sends a signal to its neighbors not to do the same thing: it exerts a lateral inhibition. If a cell that would normally become a sensory mother is genetically disabled from doing so, a neighboring proneural cell, freed from lateral inhibition, will become a sensory mother cell instead.
The lateral inhibition is mediated by the Notch signaling pathway. The cells in the cluster initially all express both the transmembrane receptor Notch and its transmembrane ligand Delta. Wherever Delta activates Notch, an inhibitory signal is sent into the Notch-expressing cell; consequently, all the cells in the cluster initially inhibit one another. However, receipt of the signal in a given cell is thought to diminish not only that cell's tendency to specialize as a sensory mother cell, but also its ability to fight back by delivering the inhibitory Delta signal in return. This creates a competitive situation, from which a single cell in each small region—the future sensory mother cell—emerges as winner, sending a strong inhibitory signal to its immediate neighbors but receiving no such signal in return (Figure 21-59). The consequences of a failure of this regulatory mechanism are shown in Figure 21-60.
Lateral inhibition. (A) The basic mechanism of Notch-mediated competitive lateral inhibition, illustrated for just two interacting cells. In this diagram, the absence of color on proteins or effector lines indicates inactivity. (B) The outcome of the (more...)
The result of switching off lateral inhibition during the singling-out of sensory mother cells. The photograph shows part of the thorax of a fly containing a mutant patch in which the neurogenic gene Delta has been partially inactivated. The reduction (more...)
The same lateral inhibition mechanism dependent on Notch operates repeatedly in the formation of bristles—not only to force the neighbors of sensory mother cells to follow a different pathway and become epidermal, and but also later to make the daughters, the granddaughters, and finally the great-granddaughters of the sensory mother cell express different genes so as to form the different components of the bristle. At each stage, lateral inhibition mediates a competitive interaction that forces adjacent cells to behave in contrasting ways. Using a temperature-sensitive Notch mutation, it is possible to switch off Notch signaling after the sensory mother cell has been singled out, but before it has divided. The progeny then differentiate alike, giving a cluster of neurons in place of the four different cell types of a bristle.
Like many other competitions, those mediated by lateral inhibition are often rigged: one cell starts with an advantage that guarantees it will be the winner. In the development of the different cell types of the sensory bristle, a strong initial bias is provided by an asymmetry in each of the cell divisions of the sensory mother cell and its progeny. A protein called Numb (together with certain other proteins) becomes localized at one end of the dividing cell, so that one daughter inherits the Numb protein and the other does not (Figure 21-61). Numb interacts with Notch, blocking its activity. Thus the Numb-containing cell is deaf to inhibitory signals from its neighbors while its sister remains sensitive. Since both cells initially express the Notch ligand Delta, the cell that has inherited Numb proceeds to become neural, while driving its sister toward a nonneural fate.
Numb biases lateral inhibition during bristle development. At each division of the progeny of the sensory mother cell, Numb protein is asymmetrically localized, producing daughter cells that differ. Note that some of the divisions are oriented with the (more...)
For the Numb mechanism to operate, there must be machinery in the dividing cell to segregate the determinant to one side of the cell before division. In addition, as the cell enters mitosis the mitotic spindle must be aligned with this asymmetry so that the determinant is allocated to just one daughter cell, and not shared out to both daughters at the time of cell division. In the above case, the sensory mother cell, at its first division, regularly divides to give an anterior cell that inherits Numb and a posterior cell that does not. This type of polarity in the plane of the epithelium is called planar polarity (in contradistinction to apico-basal polarity, where the cellular asymmetry is perpendicular to the plane of the epithelium). It is manifested in the uniformly backward-pointing orientation of the bristles, giving the fly its wind-swept appearance (Figure 21-62).
Planar cell polarity manifest in bristle polarity on a fly's back: the bristles all point backwards. (Scanning electron micrograph courtesy of S. Oldham and E. Hafen, from E. Spana and N. Perrimon, Trends Genet. 15:301–302, 1999. © Elsevier.) (more...)
The planar polarity in the initial division of the sensory mother cell is controlled by a signaling pathway similar to the one that we encountered controlling asymmetric divisions in the nematode (see Figure 21-20), depending on the receptor Frizzled. Frizzled proteins have been discussed in Chapter 15 as receptors for Wnt proteins, but in the control of planar polarity—in flies and probably in vertebrates too—this pathway functions in a special way: the intracellular relay mechanism exerts its main effects on the actin cytoskeleton, rather than on gene expression. The intracellular protein Dishevelled, downstream from Frizzled, is common to the gene-regulatory and the actin-regulatory branches of the signaling pathway. Separate domains of the Dishevelled molecule can be shown to be responsible for the two functions (Figure 21-63). Frizzled and Dishevelled both take their names from the unkempt look of flies where bristle polarity is disordered.
The control of planar cell polarity. (A) The two branches of the Wnt/Frizzled signaling pathway. The main branch, discussed in Chapter 15, controls gene expression via β-catenin; the planar-polarity branch controls the actin cytoskeleton via Rho (more...)
The mechanisms we have described for controlling the genesis of neurons of sensory bristles operate also, with minor variations, in the genesis of virtually all other neurons—not only in insects, but also in other phyla. Thus in the embryonic central nervous system, both in flies and in vertebrates, neurons are generated in regions of expression of proneural genes akin to achaete and scute. The nascent neurons or neuronal precursors express Delta and inhibit their immediate neighbors, which express Notch, from becoming committed to neural differentiation at the same time. When Notch signaling is blocked, inhibition fails, and in the proneural regions neurons are generated in huge excess at the expense of non-neuronal cells (Figure 21-64).
Effects of blocking Notch signaling in a Xenopus embryo. In the experiment shown, mRNA coding for a truncated form of the Notch ligand Delta is injected, together with LacZ mRNA as a marker, into one cell of an embryo at the two-cell stage. The truncated (more...)
In many of these processes of neurogenesis, as in the development of the sensory bristle, asymmetric cell divisions play an important part, although the details, and the relationship to Notch signaling, are variable. Thus, in the embryonic central nervous system of Drosophila, the nerve-cell precursors, or neuroblasts, are singled out from the neurogenic ectoderm by a typical lateral-inhibition mechanism that depends on Notch, but then undergo asymmetric divisions in which the cells are polarized apico-basally. The localization of a set of neuronal cell-fate determinants at one end of the neuroblast is ultimately directed, in this case, by Bazooka, an apically localized protein that has a fundamental role in defining the apico-basal polarity of epithelia in general and is also involved in neurogenesis in the central nervous system of vertebrates. Bazooka is homologous to Par-3, one of the Par proteins that govern the asymmetric cell divisions in the early nematode embryo.
The process of lateral inhibition and cell diversification, initiated by expression of proneural genes and mediated by Notch, has turned out to be crucial for the fine-grained patterning of an enormous variety of different tissues. In the fly, it controls the production not only of neurons but also of many other differentiated cell types—for example, in muscle, in the lining of the gut, in the excretory system, in the tracheae, and in the eye and other sense organs. In vertebrates, homologs of the proneural genes and of Notch and its ligands are expressed in the corresponding tissues and have similar functions: mutations in the Notch pathway upset the balance not only of neurons and non-neuronal cells in the central nervous system, but also of the different specialized cell types in the lining of the gut, of endocrine and exocrine cells in the pancreas, and of sensory and supporting cells in sense organs such as the ear, to give only a few examples.
In all these tissues, a balanced mixture of different cell types is required. Notch signaling provides the means to generate the mixture, by enabling individual cells expressing one set of genes to direct their immediate neighbors to express another set.
The final choice of a particular mode of differentiation is marked by expression of specific cell-differentiation genes in the aftermath of the interactions mediated by Notch. Each cell type must express a whole collection of genes to perform its differentiated function, but the expression of these genes is coordinated by a much smaller set of high-level regulators. These regulators are sometimes called “master regulatory proteins” (though even they can exert their specific effect only in combination with the right partners, in a cell that is adequately primed). An example is the MyoD family of gene regulatory proteins (MyoD, myogenin, Myf5, MRF4 and their homologs in invertebrates). These proteins drive cells to differentiate as muscle, expressing muscle-specific actins and myosins and all the other cytoskeletal, metabolic and membrane proteins that a muscle cell needs (see Figure 7-72).
The gene regulatory proteins that define particular cell types often belong (as do MyoD and its relatives) to the basic helix-loop-helix family, encoded by genes homologous to, and in some cases apparently identical to, the proneural genes that initiate the final phase of development. Their expression is often governed by the Notch pathway via complicated feedback loops.
Terminal cell differentiation has brought us to the end of our sketch of how genes control the making of a fly. Our account necessarily has been simplified. Many more genes than we have mentioned are involved in each of the developmental processes that we have described. Feedback loops, alternative mechanisms operating in parallel, genetic redundancy, and other phenomena complicate the full picture. Despite all this, the overriding message of developmental genetics is one of an unexpected simplicity. A limited number of genes and mechanisms, used repeatedly in different circumstances and combinations, are responsible for controlling the main features of the development of all multicellular animals.
We next turn to an essential aspect of animal development that we have so far neglected: controlled cell movements.
The external parts of an adult fly develop from epithelial structures called imaginal discs. Each imaginal disc is divided at the outset into a small number of domains expressing different gene regulatory proteins as a result of early embryonic patterning processes. These domains are called compartments, because their cells do not mix. At the compartment boundaries, cells expressing different genes confront one another and interact, inducing localized production of morphogens that govern the further growth and internal patterning of each compartment. Thus, in the wing disc, dorsal and ventral cells interact by the Notch signaling mechanism to create a source of Wingless (Wnt) protein along the dorsoventral compartment boundary, while anterior and posterior cells interact through short-range Hedgehog signaling to create a source of Dpp protein (a TGFβ family member) along the anteroposterior compartment boundary. All these signaling molecules have homologs that play similar parts in limb patterning in vertebrates.
Within each compartment, the morphogen gradients control the sites of expression of further sets of genes, defining patches of cells that interact with one another yet again to create the finest details of the ultimate pattern of cell differentiation. Thus, proneural gene expression defines the sites where sensory bristles will form, and Notch-mediated interactions among the cells of the proneural cluster, together with asymmetric cell divisions, force the individual cells of the bristle to follow different paths of terminal differentiation.
Each compartment of an imaginal disc, and each substructure within it, grows to a precisely predictable size, even in the face of seemingly drastic disturbances, such as mutations that alter the cell division rate. Although the morphogen gradients in the disc are clearly involved, the critical regulatory mechanisms that control organ size are not understood.
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