Cell_Biology_Alberts

Cell_Biology_Alberts. In the posterior part of the presomitic mesoderm, the expression of certain genes oscillates in time. Snapshots of gene expression taken by fixing embryos for analysis at different times in the oscillation cycle reveal what is happening, and the oscillations can now also be observed in time-lapse movies of embryos containing fluorescent reporters of individual oscillating genes. One new somite pair is formed in each oscillation cycle, and, in mutants where the oscillations fail to occur, somite segmentation is disrupted: the cells may still break up, belatedly, into separate clusters, but they do so in a haphazard, irregular way. The gene-expression oscillator controlling regular segmentation is called the segmentation clock. The length of one complete oscillation cycle depends on the species: it is 30 minutes in a zebrafish, 90 minutes in a chick, 120 minutes in a mouse.


Cell_Biology_Alberts

Cell_Biology_Alberts. As cells emerge from the presomitic mesoderm to form somites—in other words, as they escape from the influence of the FGF and Wnt signals—their oscillation stops. Some become arrested in one state, some in another, according to the phase of the oscillation cycle at the time they leave the presomitic region. In this way, the temporal oscillation of gene expression in the presomitic mesoderm leaves its trace in a spatially periodic pattern of gene expression in the maturing mesoderm; this in turn dictates how the tissue will break up into physically separate blocks, through effects on the pattern of cell–cell adhesion (see Figure 21–38B).


Cell_Biology_Alberts

Cell_Biology_Alberts. How does the segmentation clock work? The first somite oscillator genes to be discovered were Hes genes, which are key components of the Notch signaling pathway. They are directly regulated by the activated form of Notch, and they code for inhibitory transcription regulators that inhibit the expression of other genes, including Delta. As well as regulating other genes, the products of Hes genes can directly regulate their own expression, creating a remarkably simple negative feedback loop. Autoregulation of certain specific Hes genes (depending on species) is thought to be the basic generator of the oscillations of the somite clock. Although the machinery has been modified in various ways in different species, the underlying principle seems to be conserved. When the key Hes gene is transcribed, the amount of Hes protein product builds up until it is sufficient to block Hes gene transcription; synthesis of the protein ceases; the protein then decays, permitting transcription to begin


Cell_Biology_Alberts

Cell_Biology_Alberts. the amount of Hes protein product builds up until it is sufficient to block Hes gene transcription; synthesis of the protein ceases; the protein then decays, permitting transcription to begin again; and so on, cyclically (Figure 21–39). The period of oscillation, which determines the size of each somite, depends on the delay in the feedback loop. This equals the sum of the gestation delays and accumulation delays (that is, the molecular lifetimes) of the Hes mRNA and protein molecules, according to the additive principle discussed earlier. Mathematical modeling (see Chapter 8) allows us to relate these basic molecular parameters to the cycle time of the segmentation clock: to a first approximation, the cycle period is simply equal to twice the total delay in the negative feedback loop, and thus twice the sum of the delays occurring at each step of the loop.


Cell_Biology_Alberts

Cell_Biology_Alberts. The feedback loop just described is intracellular, and each cell in the presomitic mesoderm can generate oscillations on its own. But these oscillations at the single-cell level are somewhat erratic and imprecise, reflecting the fundamentally noisy, stochastic nature of the control of gene expression, as discussed in Chapter 7. A mechanism is needed to keep all the cells in the presomitic mesoderm that will form a particular somite oscillating in synchrony. This is achieved through cell–cell communication via the Notch signaling pathway, to which the Hes genes are coupled. The gene regulatory circuitry is such that in this context Notch signaling does not drive neighboring cells to be different, as in lateral inhibition, but does just the opposite: it keeps them in unison. In mutants where Notch signaling Figure 21–39 Delayed negative feedback giving rise to oscillating gene expression.


Cell_Biology_Alberts

Cell_Biology_Alberts. (a) a single gene, coding for a transcription regulator that inhibits its own expression, can behave as an oscillator. For oscillation to occur, there must be a delay (or several delays) in the feedback circuit, and the lifetimes of the mrNa and protein (which contribute to the delay) must be short compared with the total delay. the total delay determines the period of oscillation. It is thought that a feedback circuit like this, based on a pair of redundantly acting genes called Her1 and Her7 in the zebrafish—or their counterpart, Hes7, in the mouse—is the pacemaker of the segmentation clock governing somite formation. (B) the predicted oscillation of Her1 and Her7 mrNa and protein, computed using rough estimates of the feedback circuit parameters appropriate to this gene in the zebrafish. concentrations are measured as numbers of molecules per cell. the predicted period is close to the observed period, which is 30 minutes per somite in the zebrafish (depending on temperature).


Cell_Biology_Alberts

Cell_Biology_Alberts. fails, including mutants defective in Delta or Notch itself, the cells drift out of synchrony and somite segmentation is again disrupted. This leads to gross deformity of the vertebral column—an extraordinary display of the consequences of the noisy temporal control of gene expression at the single-cell level, writ large in the structure of the vertebrate body as a whole. Intracellular Developmental programs can help Determine the time-course of a cell’s Development Although signaling between cells plays an essential part in driving the progress of development, this does not mean that cells always need signals from other cells to prod them into changing their character as development proceeds. Some of these changes are intrinsic to the cell (like the ticking of the segmentation clock) and depend on intracellular developmental programs that can operate even when the cell is removed from its normal environment.


Cell_Biology_Alberts

Cell_Biology_Alberts. The best-understood example is in the development of neural precursor cells, or neuroblasts, in the embryonic Drosophila central nervous system. These cells, as we saw, are initially singled out from the neurogenic ectoderm of the embryo by a typical lateral-inhibition mechanism that depends on Notch, and they then proceed through an entirely predictable series of asymmetric cell divisions to generate ganglion mother cells that divide to form neurons and glial cells (see Figure 21–36). The neuroblast changes its internal state as it goes through its set program of divisions, generating different cell types with a reproducible sequence and timing. These successive changes in neuroblast specification occur through the sequential expression of specific transcription regulators. For example, most embryonic neuroblasts sequentially express the transcription regulators Hunchback, Krüppel, Pdm, and Cas in a fixed order (Figure 21–40). When a neuroblast divides, the set of transcription


Cell_Biology_Alberts

Cell_Biology_Alberts. most embryonic neuroblasts sequentially express the transcription regulators Hunchback, Krüppel, Pdm, and Cas in a fixed order (Figure 21–40). When a neuroblast divides, the set of transcription regulators expressed at that time is inherited by the ganglion mother cell and its neural progeny; thus, the differentiated neural cells are endowed with different characters according to their time of birth.


Cell_Biology_Alberts

Cell_Biology_Alberts. Remarkably, when neuroblasts are taken from an embryo and maintained in culture, isolated from their normal surroundings, they step through much the same stereotyped developmental program as if they had been left in the embryo. Moreover, many of the neuroblast transitions occur even when cell division is blocked. The neuroblasts seem to have a built-in timer that determines when each of the transcription regulators is expressed, and this timer can continue to run in the absence of cell division. The molecular basis of the timing is largely unknown; in part, at least, it must depend on the time taken for gene switching, as described above; but it may well also depend on slow progressive changes in chromatin structure. These too can serve to measure the passage of time in the embryo. Figure 21–40 Temporal patterning of neuroblast fate in Drosophila.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 21–40 Temporal patterning of neuroblast fate in Drosophila. hunchback, Krüppel, pdm, and cas are transcription regulators that are expressed consecutively in the cell lineage of neuroblasts during development of the Drosophila nervous system. at successive time steps, correlated with cell division, the neuroblast switches its pattern of gene expression. each neuroblast division produces one daughter that remains a neuroblast and expresses the updated set of genes, and one ganglion mother cell that maintains the expression of this gene set and differentiates into specific cell types accordingly. (after B.J. pearson and c.Q. Doe, Nature 425:624–628, 2003.With permission from Macmillan publishers.) cells rarely count cell Divisions to time their Development


Cell_Biology_Alberts

Cell_Biology_Alberts. Many specialized cells in animals develop from proliferating progenitor cells that stop dividing and terminally differentiate after a limited number of cell divisions. In these cases, differentiation is coordinated with withdrawal from the cell cycle, but it is usually not known how the coordination is achieved. It has often been suggested that the cell-division cycle might serve as an intracellular timer to control the timing of cell differentiation. The cell cycle would be the ticking clock that sets the tempo of other developmental processes, with maturational changes in gene expression being dependent on cell-cycle progression. Most of the evidence, however, indicates that this tempting idea is wrong. Although there are examples where cells change their maturation state with each division and the change depends on cell division, this is not the general rule. As we just saw for neuroblasts in the Drosophila embryo, cells in developing animals often carry on with their normal


Cell_Biology_Alberts

Cell_Biology_Alberts. division and the change depends on cell division, this is not the general rule. As we just saw for neuroblasts in the Drosophila embryo, cells in developing animals often carry on with their normal timetable of maturation and differentiation even when cell division is artificially blocked; necessarily, some abnormalities occur, if only because a single undivided cell cannot differentiate in two ways at once. But it seems that most developing cells can change their state without a requirement for cell division. Developmental control genes can switch the cell-division-cycle machinery on or off, and it is the dynamics of these genes, rather than the cell cycle, that sets the tempo of development.


Cell_Biology_Alberts

Cell_Biology_Alberts. Genetic screens are useful for tracking down the genes involved in almost any biological process, and they have been used to search for mutations that alter developmental timing. Such screens were performed in the nematode Caenorhabditis elegans (Figure 21–41). This worm is small, relatively simple, and precisely structured. The anatomy of its development is highly predictable and has been described in extraordinary detail, so that one can map out the exact lineage of every cell in the body and see exactly how the developmental program is altered in a mutant. Genetic screens in C. elegans revealed mutations that disrupt developmental timing in a particularly striking way: in these so-called heterochronic mutants, certain cells in a larva at one stage of development behave as though they were in a larva at a different stage of development, or cells in the adult carry on dividing as though they belonged to a larva (Figure 21–42).


Cell_Biology_Alberts

Cell_Biology_Alberts. Genetic analyses showed that the products of the heterochronic genes act in series, forming regulatory cascades. Unexpectedly, two genes at the top of their respective cascades, called Lin4 and Let7, were found to code not for protein but instead for microRNAs (miRNAs)—short, untranslated, regulatory RNA molecules, 21 or 22 nucleotides long. These act by binding to complementary sequences in the noncoding regions of mRNA molecules transcribed from other heterochronic genes, thereby repressing their translation and promoting their degradation, as discussed in Chapter 7. Increasing levels of Lin4 miRNA govern the progression from first-stage larva cell behaviors to third-stage larva cell behaviors. 1.2 mm Figure 21–41 Caenorhabditis elegans. a side view of an adult hermaphrodite is shown. (From J.e. Sulston and h.r. horvitz, Dev. Biol. 56:110–156, 1977. With permission from academic press.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 21–42 Heterochronic mutations in the Lin14 gene of C. elegans. Only the effects on one of the many altered lineages are shown. a loss-of-function (recessive) mutation in Lin14 causes premature occurrence of the pattern of cell division and differentiation characteristic of a late larva, so that the animal reaches its final state prematurely and with an abnormally small number of cells. the gain-of-function (dominant) mutation has the opposite effect, causing cells to reiterate patterns of cell divisions characteristic of the first larval stage, continuing through as many as five or six molt cycles. the cross denotes a programmed cell death. Green lines represent cells that contain Lin14 protein (which binds to DNa), red lines those that do not. (adapted from V. ambros and h.r. horvitz, Science 226:409–416, 1984. With permission from the authors; and p. arasu, B. Wightman and G. ruvkun, Genes Dev. 5:1825–1833, 1991. With permission from the authors.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Increasing levels of Let7 miRNA govern the progression from late larva to adult. In fact, Lin4 and Let7 were the first miRNAs to be described in any animal: it was through developmental genetic studies in C. elegans that the importance of this whole class of molecules for gene regulation in animals was discovered. More generally, in many animals, miRNAs help regulate the transitions between different stages of development. For example, in flies, fish, and frogs, the maternal mRNAs that are loaded into the egg in the mother are removed during early development when the genome of the embryo begins to be transcribed; at this stage, the embryo begins to express specific miRNAs that target many maternal mRNAs for translational repression and degradation.


Cell_Biology_Alberts

Cell_Biology_Alberts. Thus, miRNAs can sharpen developmental transitions by blocking and removing mRNAs that define an earlier developmental stage. But how is the timing of miRNA expression itself controlled? In the case of the miRNAs that disable maternal mRNAs in frogs and fish, expression is activated at the end of the series of rapid, synchronous divisions that cleave the fertilized egg into many smaller cells. As the division rate of these blastomeres slows, widespread transcription of the embryo’s genome begins (Figure 21–43). This event, where the embryo’s own genome largely takes over control of development from maternal macromolecules, is called the maternal-zygotic transition (MZT), and it occurs with roughly similar timing in most animal species, with the exception of mammals.


Cell_Biology_Alberts

Cell_Biology_Alberts. One trigger for the MZT appears to be the nuclear-to-cytoplasmic ratio. During cleavage, the total amount of cytoplasm in the embryo remains constant, but the number of cell nuclei increases exponentially. As a critical threshold is reached in the ratio of cytoplasm to DNA, the cell cycles lengthen and transcription is initiated. Thus, haploid embryos undergo the MZT one cell cycle later than diploid embryos, which contain twice as much DNA per cell. According to one Figure 21–43 The maternal-zygotic transition in a zebrafish embryo.


Cell_Biology_Alberts

Cell_Biology_Alberts. Maternal mrNas are deposited by the mother into the egg and drive early development. these mrNas are degraded during different stages of embryogenesis, including blastula and gastrula stages, but a relatively abrupt change occurs at the maternal-zygotic transition (MZt). Before this, the embryonic (zygotic) genome is transcriptionally inactive; afterward, zygotic model, the nuclear-to-cytoplasmic ratio might be measured through the titration of a transcription repressor against the increasing amount of nuclear DNA. The total amount of repressor would stay constant during cleavage divisions, but the amount of repressor per genome would decrease, falling by a half with each round of DNA synthesis, until loss of repression allowed the zygotic genome to become transcriptionally active. The newly synthesized transcripts include the miRNAs that recognize many of the transcripts deposited in the egg by the mother, directing their translational repression and rapid degradation.


Cell_Biology_Alberts

Cell_Biology_Alberts. hormonal Signals coordinate the timing of Developmental transitions


Cell_Biology_Alberts

Cell_Biology_Alberts. We have so far emphasized timing mechanisms that operate locally and separately in the different parts of the embryo, or in specific subsystems of the molecular control machinery. Evolution has tuned each of these largely independent processes to run at an appropriate rate, matched to the needs of the organism as a whole. For some purposes, however, this is not enough: a global coordinating signal is required. This is especially true where changes have to occur throughout the body in response to a cue that depends on the environment. For example, when an insect or amphibian undergoes metamorphosis—the transition from larva to adult—almost every part of the body is transformed. The timing of metamorphosis depends on external factors such as the supply of food, which determines when the animal reaches an appropriate size. All the bodily changes have to be triggered together at the right time, even though they are occurring in widely separated sites. The coordination in such cases is


Cell_Biology_Alberts

Cell_Biology_Alberts. animal reaches an appropriate size. All the bodily changes have to be triggered together at the right time, even though they are occurring in widely separated sites. The coordination in such cases is provided by hormones—signal molecules that spread throughout the body.


Cell_Biology_Alberts

Cell_Biology_Alberts. The metamorphosis of amphibians provides a spectacular example. During this developmental transition, amphibians switch from an aquatic to a terrestrial life. Larva-specific organs such as gills and tail disappear, and adult-specific organs such as legs form. This dramatic transformation is triggered by thyroid hormone, produced in the thyroid gland. If the gland is removed or if thyroid hormone action is blocked, metamorphosis does not occur, although growth continues, producing a giant tadpole. Conversely, a dose of thyroid hormone given to a tadpole by an experimenter can trigger metamorphosis prematurely.


Cell_Biology_Alberts

Cell_Biology_Alberts. The thyroid hormone is distributed through the vascular system and induces changes throughout the animal by binding to intracellular nuclear hormone receptors, which regulate hundreds of genes. This does not mean, however, that target tissues all respond in the same way to the hormone: organs differ not only in their levels of thyroid hormone receptors and levels of extracellular proteins that locally regulate the amount of active hormone, but also in the sets of genes that respond. Thyroid hormone induces muscle in the limbs to grow and muscle in the tail to die. The timing of the responses also differs: for example, the legs form early in response to a very low concentration of circulating hormone, but it requires a high level of the hormone to induce resorption of the tail.


Cell_Biology_Alberts

Cell_Biology_Alberts. A surge of thyroid hormone triggers metamorphosis, but how is the timing of the surge controlled? One mechanism depends on coupling hormone synthesis to the size of the thyroid gland, which reflects the size of the tadpole. Only when the gland attains a certain size does it produce enough thyroid hormone to initiate metamorphosis. However, environmental cues other than nutrition also play a part: conditions such as temperature and light are sensed by the nervous system, which regulates the secretion of another tier of hormones (neurohormones) that stimulate the secretion of thyroid hormone. Thus, tadpole-intrinsic factors such as size combine with environmental factors to determine when metamorphosis begins. environmental cues Determine the time of Flowering


Cell_Biology_Alberts

Cell_Biology_Alberts. environmental cues Determine the time of Flowering Another striking example of environmentally controlled developmental timing is the flowering of plants. Flowering involves a transformation of the behavior of the cells at the growing apex of the plant shoot—the apical meristem. During ordinary vegetative growth, these cells behave as stem cells, generating a steady succession of new leaves and new segments of stalk. In flowering, the meristem cells switch to making the components of a flower, with its sepals and petals, its stamens carrying pollen, and its ovary containing the female gametes.


Cell_Biology_Alberts

Cell_Biology_Alberts. To time the switch correctly, the plant has to take account of both past and present conditions. One important cue, for many plants, is day length. To sense this, the plant uses its circadian clock—an endogenous 24-hour rhythm of gene expression—to generate a signal for flowering only when there is light for the appropriate part of the day. The clock itself is influenced by light, and the plant in effect uses the clock to compare past to present lighting conditions. Important parts of the genetic circuitry underlying these phenomena have been identified, including the phytochromes and cryptochromes that act as light receptors (discussed in Chapter 15). The flowering signal that is carried from the leaves to the stem cells via the vasculature depends on the product of Flowering locus T (Ft).


Cell_Biology_Alberts

Cell_Biology_Alberts. But this signal will trigger flowering only if the plant is in a receptive condition from prior long-term cold exposure. Many plants need winter before they will flower—a process called vernalization. Cold over a period of weeks or months progressively reduces the level of expression of a remarkable gene called Flowering locus C (Flc). Flc encodes a transcriptional repressor that suppresses expression of the Ft flowering promoter. How does vernalization shut down Flc so as to lift the block to flowering? The effect involves a noncoding RNA called Coolair that overlaps with the Flc gene and is produced when the temperature is low (Figure 21–44). Together with cold-induced chromatin modifiers, including Polycomb-group proteins, Coolair coordinates the switching of Flc chromatin to a silent state (discussed in Chapters 4 and 7). The degree of silencing depends on the length of cold exposure enabling the plants to distinguish the odd chilly night from the whole of winter.


Cell_Biology_Alberts

Cell_Biology_Alberts. The effect on the chromatin is long lasting, persisting through many rounds of cell division even as the weather grows warmer. Thus vernalization creates a persistent block in production of Flc, enabling the Ft signal to be generated when day length is sufficiently long. Figure 21–44 Temporal control of flowering in Arabidopsis. the Flc gene is active and blocks flowering when plants have been grown without exposure to winterlike temperatures. exposure to a prolonged period of cold leads to the production of the noncoding rNa Coolair, which overlaps with the Flc gene. coolair induces long-term chromatin changes that turn off Flc. these changes persist after the end of the cold period and allow the plant to flower when other environmental conditions are favorable for flowering.


Cell_Biology_Alberts

Cell_Biology_Alberts. Mutations affecting the regulation of Flc expression alter the time of flowering and thus the ability of a plant to flourish in a given climate. The whole control system governing the switch to flowering is thus of vital importance for agriculture, especially in an era of rapid climate change. The example of vernalization suggests a general point about the role of chromatin modification in developmental timing. The plant uses changes in chromatin to record its experience of prolonged cold. It may be that in other organisms—animals as well as plants—slow, progressive changes in chromatin structure provide long-term timers for those mysterious developmental processes that unfold slowly, over a period of days, weeks, months, or years. Such chromatin timers may be among the most important clocks in the embryo, but as yet we understand very little about them.


Cell_Biology_Alberts

Cell_Biology_Alberts. Developmental timing is controlled at many levels. It takes time to switch a gene on or off, and this time delay depends on the lifetimes of the molecules involved, which can vary widely. Cascades of gene regulation involve cascades of delays. Feedback loops can give rise to temporal oscillations in gene expression, and these may serve to generate spatially periodic structures. During vertebrate segmentation, for example, expression of the Hes genes oscillates, and one new pair of somites is formed during each oscillation cycle. Hes genes encode transcription repressor proteins that can act back on expression of the Hes genes themselves. This negative feedback generates oscillations with a period that reflects the delay in the autoregulatory gene switching loop. The period of oscillation of this “segmentation clock” controls the sizes of the somites. Notch signaling between neighboring cells synchronizes their oscillations: when Notch signaling fails, the cells drift out of synchrony


Cell_Biology_Alberts

Cell_Biology_Alberts. of this “segmentation clock” controls the sizes of the somites. Notch signaling between neighboring cells synchronizes their oscillations: when Notch signaling fails, the cells drift out of synchrony because of genetic noise in their individual clocks, and the segmental organization of the vertebral column is disrupted.


Cell_Biology_Alberts

Cell_Biology_Alberts. Timing does not always depend on cell–cell interactions; many developing animal cells have intrinsic developmental programs that play out even in isolated cells in culture. Neuroblasts in Drosophila embryos, for example, go through set programs of asymmetric divisions, generating different neural cell types at each division with a predictable sequence and timing, through a cascade of gene switching events. Studies in both vertebrates and invertebrates show that such programs are rarely governed by the timing of cell division and can unfold even when cell division is blocked. MicroRNAs produced at critical moments sharpen developmental transitions by blocking the translation and promoting the degradation of specific sets of mRNAs. Global coordination of developmental timing is achieved by hormones: as a tadpole grows, for example, thyroid hormone levels surge and trigger its metamorphosis into a frog. Environmental control of developmental timing is especially striking in plants and


Cell_Biology_Alberts

Cell_Biology_Alberts. hormones: as a tadpole grows, for example, thyroid hormone levels surge and trigger its metamorphosis into a frog. Environmental control of developmental timing is especially striking in plants and reveals the presence of molecular timers that act over the long term. In vernalization, for example, prolonged cold induces changes in chromatin that chart the passage through winter so as to allow flowering only in the spring. Slow, progressive changes in chromatin structure are likely to be important timers in the long-term programming of development in animals too.


Cell_Biology_Alberts

Cell_Biology_Alberts. The specialization of cells into distinct types at specific times is important, but it is only one aspect of animal development. Equally important are the movements and deformations that cells go through to assemble into tissues and organs with specific shapes and sizes. Like developmental timing, this process of morphogenesis (“form generation”) is less well understood than the processes of differential gene expression and inductive signaling that lead to cell-type specialization. The cell movements can be readily described, but the underlying molecular mechanisms that coordinate the movements are much harder to decipher.


Cell_Biology_Alberts

Cell_Biology_Alberts. In Chapter 19, we saw how cells cohere to form epithelial sheets or surround themselves with extracellular matrix to create connective tissues. We also discussed how the basic features of tissues, such as the polarity of epithelia, arise from the properties of individual cells. In this section, we consider how the rearrangements of cells during animal development give shape to the embryo and to all the individual organs and appendages of the body.


Cell_Biology_Alberts

Cell_Biology_Alberts. A small number of cell processes are basic to morphogenesis. Individual cells can migrate through the embryo along defined tracks. They can crawl over one another in a coordinated way to elongate, constrict, or thicken a tissue. They can segregate from their neighbors and form physically separate groups. They can change their shape so as to deform an epithelial sheet into a tube or a vesicle. By stretching out while holding on to their companions, specialized sets of cells can form growing tubular networks such as the system of blood or lymph vessels. Mass migrations, as occur in gastrulation, can transform the entire topology of the embryo. Underlying all these processes are changes in cell shape and changes in cell contacts—either with other cells or with extracellular matrix. We begin by considering the migration of individual cells. cell Migration Is Guided by cues in the cell’s environment


Cell_Biology_Alberts

Cell_Biology_Alberts. cell Migration Is Guided by cues in the cell’s environment The birthplace of cells is often far from their ultimate location in the body. Our skeletal muscles, for example, derive from muscle cell precursors, or myoblasts, in somites, from which they migrate into the limbs and other regions. The routes that the migrant cells follow and the selection of sites that they colonize determine the eventual pattern of muscles in the body. The embryonic connective tissues form the framework through which the myoblasts travel, and these tissues provide the cues that guide myoblast distribution. No matter which somite they come from, the myoblasts that migrate into a forelimb bud will form the pattern of muscles appropriate to a forelimb, and those that migrate into a hindlimb bud will form the pattern appropriate to a hindlimb. It is the connective tissue that provides the patterning information.


Cell_Biology_Alberts

Cell_Biology_Alberts. As a migrant cell travels through the embryonic tissues, it repeatedly extends surface projections that probe its immediate surroundings, testing for cues to which it is particularly sensitive by virtue of its specific assortment of cell-surface receptor proteins. Inside the cell, these receptors are connected to the cortical actin and myosin cytoskeleton, which moves the cell along. Some extracellular matrix molecules, such as the protein fibronectin, provide adhesive sites that help the cell advance; others, such as chondroitin sulfate proteoglycan, inhibit locomotion and repel immigration. The nonmigrant cells along the migration pathway may likewise have inviting or repellent macromolecules on their surface; some may even extend filopodia to make their presence known.


Cell_Biology_Alberts

Cell_Biology_Alberts. Among the many guiding influences, a few stand out as especially important. In particular, many types of migrating cells are guided by chemotaxis that depends on a G-protein-coupled receptor (called CXCR4), which is activated by an extracellular ligand called CXCL12. Cells expressing this receptor can snuffle their way along tracks marked out by CXCL12 (Figure 21–45). Chemotaxis toward sources of CXCL12 plays a major part in guiding the migrations of lymphocytes Figure 21–45 CXCL12 guides migrating germ cells. Zebrafish germ cells migrate to domains that express cXcL12. as the sites of cXcL12 expression change, cells follow the cXcL12 track and are guided to the region where the gonad develops at a later developmental stage. (a) at the 4-somite stage, germ cells move from a position that is close to the midline to more lateral regions where cXcL12 is expressed. (B) as the cXcL12 expression retracts, germ cells are guided to more posterior positions.


Cell_Biology_Alberts

Cell_Biology_Alberts. source of CXCL12 and various other white blood cells; of neurons in the developing brain; of myoblasts entering limb buds; of primordial germ cells as they travel toward the developing gonads; and of cancer cells when they metastasize. Detailed studies of primordial-germ-cell migration have shown that CXCL12 signaling does not induce cell migration per se but rather serves to control its direction. In the absence of CXCL12 signaling, germ cells still display the membrane blebbing associated with cell migration, but the position of the cell front where blebs form is randomly chosen (Figure 21–46); if CXCL12 signaling is intact, blebbing is more frequent on the side of the cell that faces the source of CXCL12, resulting in directional migration. the Distribution of Migrant cells Depends on Survival Factors


Cell_Biology_Alberts

Cell_Biology_Alberts. the Distribution of Migrant cells Depends on Survival Factors The final distribution of migrant cells depends not only on the routes they take, but also on whether they survive the journey and thrive in the environment they find at the journey’s end. Specific sites provide survival factors needed for specific types of migrant cells to survive.


Cell_Biology_Alberts

Cell_Biology_Alberts. Among the most important sets of migrant cells in the vertebrate embryo are those of the neural crest. They arise from the border region between the part of the ectoderm that will form epidermis and the part that will form the central nervous system. As the neural ectoderm rolls up to form the neural tube, the neural crest cells break loose from the epithelial sheet along this border region and set out on their long migrations (see Figure 19–8 and Movie 21.5). They settle ultimately in many sites and give rise to a surprising diversity of cell types. Some lodge in the skin and specialize as pigment cells; still others form skeletal tissue in the face. Still others will differentiate into the neurons and glial cells of the peripheral nervous system—not only in the sensory ganglia that lie close to the spinal cord, but also, following a much longer migration, in the wall of the gut.


Cell_Biology_Alberts

Cell_Biology_Alberts. The neural crest cells that give rise to the pigment cells of the skin and those that develop into the nerve cells of the gut depend on a secreted peptide called endothelin-3, which is produced by tissues along the migration pathways and acts as a survival factor for the migrating crest cells. In mutants with a defect in the gene for endothelin-3 or its receptor, many of these migrating crest cells die. As a result, the mutant individuals have nonpigmented (albino) patches of skin and a deficit of nerve cells in the intestine, especially its lower end, the large bowel, which becomes abnormally distended for lack of proper neural control—a potentially lethal condition called megacolon. Figure 21–46 Directional migration by local blebbing. Germ cells migrate via protrusions that define the leading edge of the cell. the persistence and site of the protrusions are biased toward higher levels of cXcL12. thus, germ cells migrate up the cXcL12 gradient.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 21–47 Effect of mutations in the Kit gene. Both the baby and the mouse are heterozygous for a loss-of-function mutation that leaves them with only half the normal quantity of Kit gene product. In both cases, pigmentation is defective because pigment cells depend on the gene product as a receptor for a survival factor. (courtesy of r.a. Fleischman, from r.a. Fleischman et al., Proc. Natl Acad. Sci. USA 88:10885–10889, 1991.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Another important survival signal for many types of migratory cells, including primordial germ cells, blood cell precursors, and neural-crest-derived pigment cells, depends on a receptor tyrosine kinase called Kit. This is expressed on the surface of the migrant cells, and a protein ligand, called Steel factor, is produced by the cells of the tissue through which the cells migrate and/or in which they come to settle. Individuals with mutations in the genes for either of these proteins have deficits in pigmentation, blood cells, and germ cells (Figure 21–47). changing patterns of cell adhesion Molecules Force cells Into New arrangements


Cell_Biology_Alberts

Cell_Biology_Alberts. changing patterns of cell adhesion Molecules Force cells Into New arrangements Patterns of gene expression govern embryonic cell movements in many ways. They regulate cell motility, cell shape, and the production of proteins that guide migration. Importantly, they also determine the sets of adhesion molecules that the cells display on their surface. Through changes in its surface molecules, a cell can break old attachments and make new ones. Cells in one region may develop surface properties that make them cohere with one another and become segregated from a neighboring group of cells with different surface chemistry.


Cell_Biology_Alberts

Cell_Biology_Alberts. Experiments done half a century ago on early amphibian embryos showed that the effects of selective cell–cell adhesion can be so powerful that they can bring about an approximate reconstruction of the normal structure of an early postgastrulation embryo after the cells have been artificially dissociated and mixed up. When these cells are reaggregated into a random mixture, the cells spontaneously sort themselves out according to their original germ-layer origins (Figure 21–48). As discussed in Chapter 19, cadherin proteins have a central role in the sorting process (see Figure 19–9). Cadherins belong to a large and varied family of Ca2+-dependent cell–cell adhesion proteins, and they and other cell–cell adhesion proteins are differentially expressed in the various tissues of the early embryo. Antibodies against these proteins interfere with the normal selective adhesion between cells of a similar type.


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Cell_Biology_Alberts. Changes in the patterns of expression of the various cadherins correlate closely with the changing patterns of association among cells during various developmental processes, including gastrulation, neural tube formation, and somite formation. These cell rearrangements are likely to be regulated and driven in part by


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Cell_Biology_Alberts. Figure 21–48 Sorting out by adhesion. cells from different parts of an early amphibian embryo will sort out according to their origins. In the classical experiment shown here, mesoderm cells (green), neural plate cells (blue), and epidermal cells (red) have been disaggregated and then reaggregated in a random mixture. they sort out into an arrangement reminiscent of a normal embryo, with a “neural tube” internally, epidermis externally, and mesoderm in between. (Modified from p.L. townes and J. holtfreter, J. Exp. Zool. 128:53– 120, 1955. With permission from Wiley-Liss.) the cadherin pattern. In particular, cadherins appear to have a major role in controlling the formation and dissolution of epithelial sheets and clusters of cells (see Movie 19.1). They not only glue one cell to another but also provide anchorage for intracellular actin filaments at the sites of cell–cell adhesion. In this way, the pattern of stresses and movements in the developing tissue is regulated according to


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Cell_Biology_Alberts. but also provide anchorage for intracellular actin filaments at the sites of cell–cell adhesion. In this way, the pattern of stresses and movements in the developing tissue is regulated according to the pattern of cell adhesions.


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Cell_Biology_Alberts. The different types of cadherins enable different types of cells to cohere selectively: cells expressing one type of cadherin will maximize their contact with cells expressing the same cadherin and thereby segregate from other cells, creating specific tissue boundaries. Cell mixing can be inhibited and boundaries created and maintained in another way as well: cells of different types can sometimes actively repel one another. The bidirectional activation of Eph receptors and ephrins discussed in Chapter 15 often mediates such repulsion, acting at interfaces between different groups of cells to keep the groups from mixing, and repelling invasion by inappropriate visitors. Ephrin–Eph signaling operates, for example, at the boundaries of the rhombomeres discussed earlier. Neighboring rhombomeres express complementary combinations of ephrins and Eph receptors, and this keeps the cells in adjacent rhombomeres strictly segregated, with a boundary between them that is sharply defined (Figure


Cell_Biology_Alberts

Cell_Biology_Alberts. express complementary combinations of ephrins and Eph receptors, and this keeps the cells in adjacent rhombomeres strictly segregated, with a boundary between them that is sharply defined (Figure 21–49).


Cell_Biology_Alberts

Cell_Biology_Alberts. Groups of Similar cells can perform Dramatic collective rearrangements Cadherin-mediated cell sorting and ephrin–Eph-mediated repulsion exemplify how differences in cell-surface properties can drive tissue arrangements, causing cells that express different sets of genes to separate from one another. However, groups of cells that are all similar can also undergo dramatic rearrangements. During frog gastrulation, for example, cells in one region of the surface epithelium invaginate and migrate as a sheet into the interior of the embryo and converge toward the embryonic midline. The movement is driven mainly by an active rearrangement of the migrating cells, called convergent extension. Here the cells crawl over one another in a coordinated way, displacing their neighbors as they migrate, causing the cell sheet to narrow along one axis (converge) and elongate along another (extend). Strikingly, small, square fragments of tissue from the


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Cell_Biology_Alberts. Figure 21–49 Sorting out by repulsion. ephrin–eph signaling in hindbrain segmentation in a chick embryo. each pair of rhombomeres (segments in the hindbrain) is associated with a branchial arch (a modified gill rudiment) to which it sends innervation. rhombomeres are distinguished from one another by expression of different Hox genes (see Figure 21–32). Mutual repulsion (red bars) between cells that express ephrinB2 in rhombomere 4 and epha4 in rhombomere 5 creates a sharp boundary. lamellipodia attempt to crawl on surfaces of neighboring cells, pulling them inward in direction of arrows appropriate region of the embryo, isolated in culture, will spontaneously narrow and elongate, just as they would in the embryo (Figure 21–50). The alignment of the cell movements depends on the same signaling pathway that is involved in generating planar cell polarity within developing epithelia, as we discuss next.


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Cell_Biology_Alberts. Cells within an epithelium always have an apical–basal polarity (discussed in Chapter 19), but the cells of many epithelia show an additional polarity at right angles to this axis: the cells are all arranged as if they had an arrow written on them, pointing in a specific direction in the plane of the epithelium. This type of polarity is called planar cell polarity. In the wing of a fly, for example, each epithelial cell has a tiny asymmetrical projection, called a wing hair, on its surface, and the hairs all point toward the tip of the wing. Similarly, in the inner ear of a vertebrate, each mechanosensory hair cell has a precisely oriented asymmetric bundle of actin-filled, rodlike protrusions called stereocilia sticking up from its apical plasma membrane as a detector of sound and of forces such as gravity. Tilting the bundle in one direction causes ion channels in the membrane to open, electrically activating the cell; tilting in the opposite direction has the opposite effect. For


Cell_Biology_Alberts

Cell_Biology_Alberts. such as gravity. Tilting the bundle in one direction causes ion channels in the membrane to open, electrically activating the cell; tilting in the opposite direction has the opposite effect. For the ear to function properly, the hair cells must be oriented correctly. Planar cell polarity is also important in the respiratory tract, where every ciliated cell must orient the beating of its cilia so as to sweep mucus upward, away from the lungs.


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Cell_Biology_Alberts. Screens for mutants with misoriented wing hairs in Drosophila have identified a set of genes that is critical for planar cell polarity. Some of these genes code for Figure 21–50 Convergent extension and its cellular basis. (a) Schematic diagram of cell behaviors that underlie convergent extension. the cells form lamellipodia, with which they attempt to crawl over one another. alignment of the lamellipodial movements along a common axis leads to convergent extension. the process depends on the Wnt–Frizzled/ planar-cell-polarity signaling pathway and is cooperative, presumably because cells that are already aligned exert forces that tend to align their neighbors in the same way.


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Cell_Biology_Alberts. (B–G) the pattern of convergent extension of dorsal mesoderm during zebrafish gastrulation at 8.8 (B, e), 9.3 (c, F), and 11.3 (D, G) hours after fertilization. cells that will give rise to the notochord are labeled in green, and cells that will give rise to somites and muscle are labeled in blue. the notochord and somite domains are spatially separate from the start of the recording (B, e), but their boundaries are at first barely visible and only a little later become obvious. convergence narrows the notochord domain to a width of about two cells at the last time point (D, G). (a, after J. Shih and r. Keller, Development 116:901–914, 1992; B–G, after N.S. Glickman et al., Development 130:873–887, 2003. With permission from the company of Biologists.)


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Cell_Biology_Alberts. Figure 21–51 Planar cell polarity. (a) Wing hairs on the wing of a fly. each cell in the wing epithelium forms a small, spiky protrusion or “hair” at its apex, and all the hairs point the same way, toward the tip of the wing. this reflects a planar polarity in the structure of each cell. (B) Sensory hair cells in the inner ear of a mouse similarly have a well-defined planar polarity, manifest in the oriented pattern of stereocilia (actin-filled protrusions) on their surface. the detection of sound depends on the correct, coordinated orientation of the hair cells. (c) a mutation in the gene Flamingo in the fly, coding for a nonclassical cadherin, disrupts the pattern of planar cell polarity in the wing. (D) a mutation in a homologous Flamingo gene in the mouse randomizes the orientation of the planar cell polarity vector of the hair cells in the ear. the mutant mice are deaf. (a and c, from J. chae et al., Development 126:5421–5429, 1999. With permission from the company of Biologists;


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Cell_Biology_Alberts. the planar cell polarity vector of the hair cells in the ear. the mutant mice are deaf. (a and c, from J. chae et al., Development 126:5421–5429, 1999. With permission from the company of Biologists; B and D, from J.a. curtin et al., Curr. Biol. 13:1129–1133, 2003. With permission from elsevier.) components of the Wnt signaling pathway, others code for specialized members of the cadherin superfamily, while the functions of others are uncertain. These components of planar-cell-polarity signaling are assembled at cell–cell junctions in the epithelium in such a way as to exert a polarizing influence that can propagate from cell to cell. Essentially the same system of proteins controls planar cell polarity in vertebrates; mice deficient in homologs of the Drosophila planar polarity genes have a variety of defects, including incorrectly oriented hair cells in the inner ear, making them deaf (Figure 21–51).


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Cell_Biology_Alberts. Animals require specialized types of epithelial surfaces for many functions, including excretion, absorption of nutrients, and gas exchange. Where large surfaces are required, they are often organized as branching tubular structures. The lung is an example. It originates from epithelial buds that grow out from the floor of the foregut and invade neighboring mesenchyme to form the bronchial tree, a system of tubes that branch repeatedly as they extend. Endothelial cells that form the lining of blood vessels invade the same mesenchyme, thereby creating a system of closely apposed airways and blood vessels, as required for gas exchange in the lung (Figure 21–52). This whole process of branching morphogenesis depends on signals that pass in both directions between the growing epithelial buds and the mesenchyme. Genetic studies in mice indicate that FGF proteins and their receptor tyrosine kinases play a central part in these signaling processes. FGF signaling has various roles in


Cell_Biology_Alberts

Cell_Biology_Alberts. buds and the mesenchyme. Genetic studies in mice indicate that FGF proteins and their receptor tyrosine kinases play a central part in these signaling processes. FGF signaling has various roles in development, but it is especially important in the many interactions that occur between a developing epithelium and mesenchyme. Figure 21–52 The airways of the lung,


Cell_Biology_Alberts

Cell_Biology_Alberts. In the case of lung development, FGF10 is expressed in clusters of mesen-shown in a cast of the adult human bronchial tree. resins of different colors chyme cells that lie near the tips of the growing epithelial tubes, and its receptor is expressed in the invading epithelial cells. In FGF10-deficient mutant mice, a pri of the tree of airways. (From r. Warwick mary bud of lung epithelium is formed but fails to grow out into the mesenchyme and p.L. Williams, Gray’s anatomy, 35th ed. to create a branching bronchial tree. Conversely, a microscopic bead soaked in edinburgh: Longman, 1973.) two new centers of FGF10 made by cluster of mesenchyme cells cells at tip of FGF10 and placed near embryonic lung epithelium in culture will induce a bud to form and grow out from the epithelium toward the bead. Evidently, the epithelium invades the mesenchyme only by invitation, in response to FGF10.


Cell_Biology_Alberts

Cell_Biology_Alberts. But what makes the growing epithelial tubes of the lung branch repeatedly as they invade the mesenchyme? This depends on a Sonic hedgehog signal that is sent in the opposite direction, from the epithelial cells at the tips of the buds back to the mesenchyme, as shown in Figure 21–53. In mice lacking Sonic hedgehog, the lung epithelium grows and differentiates, but it forms a sac instead of a branching tree of tubules.


Cell_Biology_Alberts

Cell_Biology_Alberts. FGF signaling acts in a remarkably similar way in the formation of the air-exchange system of insects, which consists of a pattern of fine, air-filled channels called tracheae and tracheoles. These originate from the epidermis covering the surface of the body and extend inward to invade the underlying tissues, branching and narrowing as they go (Figure 21–54). The FGF acts on cells at the tips of the advancing tracheae, causing them to extend filopodia and migrate toward the source of the FGF signal. Because the tip cells remain connected to the remainder of the tracheal epithelium, the pulling force that they generate elongates the tracheal tube.


Cell_Biology_Alberts

Cell_Biology_Alberts. Initially, the pattern of FGF production in fly embryos is defined by the D-V and A-P patterning systems discussed earlier. In later stages of development, however, FGF expression is induced by transcription regulators called hypoxia-inducible factors (HIFs) that are activated by hypoxia (low oxygen levels). In this way, hypoxia stimulates the formation of finer and finer and more extensively branched trachea, until the oxygen supply is sufficient to stop the process. Hypoxia and HIFs have similar roles in vertebrates, especially in the development of blood vessels, as we shall see in the next chapter.


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Cell_Biology_Alberts. Figure 21–54 Branching morphogenesis of airways in a fly. (a) Drosophila embryonic tracheal system. (B) FGF (produced in Drosophila by the Branchless gene) signals from surrounding cells to the tracheal epithelium and activates its FGF receptors, leading to filopodia formation and tube elongation. [a, from G. Manning and M.a. Krasnow, in the Development of Drosophila (a. Martinez-arias and M. Bate, eds), Vol. 1, pp. 609–685. New York: cold Spring harbor Laboratory press, 1993.] Figure 21–53 Branching morphogenesis of the lung. how FGF10 and Sonic hedgehog are thought to induce the growth and branching of the buds of the bronchial tree. Many other signal molecules, such as BMp4, are also expressed in this system, and the suggested branching mechanism is only one of several possibilities.


Cell_Biology_Alberts

Cell_Biology_Alberts. as indicated, FGF10 protein is expressed in clusters of mesenchyme cells near the tips of the growing epithelial tubes, and its receptor is expressed in the epithelial cells themselves. the Sonic hedgehog signal is sent in the opposite direction, from the epithelial cells at the tips of the buds back to the mesenchyme. the patterns of gene expression and their timing suggest that the Sonic hedgehog signal may serve to shut off FGF10 expression in the mesenchyme cells closest to the growing tip of a bud, splitting the FGF10-secreting cluster into two separate clusters, which in turn cause the bud to branch into two. Figure 21–55 The forms of cell behavior an epithelium can Bend During Development to Form a tube or involved in tube formation. FoldingVesicle generates the neural tube, budding underlies the formation of lungs and


Cell_Biology_Alberts

Cell_Biology_Alberts. The creation of systems of tubes such as blood vessels and airways is a complex trachea, cord hollowing occurs during process, and it can involve various additional forms of cell behavior, as sketched the formation of mammalian salivary in Figure 21–55. glands, cell hollowing is involved in the As explained in Chapter 19, the process that converts an epithelial sheet into a formation of tracheal terminal cell tubes, and cell assembly generates the heart tube tube depends on contraction of specific bundles of actin filaments. With the help that forms at the earliest stage of heart of myosin motor proteins, actin filament bundles can shorten, causing the epithe development.


Cell_Biology_Alberts

Cell_Biology_Alberts. lial cells to narrow at their apex. These actin bundles are connected from cell to cell by adherens junctions, and if their contraction is coordinated along a specific axis, the result will be that the sheet bends and rolls up into a tube (Figure 21–56). The vertebrate neural tube, which we discuss in the last section of this chapter, originates in this way. sheet of epithelial cells INVAGINATION OF EPITHELIAL SHEET CAUSED BY AN ORGANIZED TIGHTENING ALONGadhesion belt with ADHESION BELTS IN SELECTED REGIONS OFassociated actin flaments CELL SHEET


Cell_Biology_Alberts

Cell_Biology_Alberts. sheet of epithelial cells INVAGINATION OF EPITHELIAL SHEET CAUSED BY AN ORGANIZED TIGHTENING ALONGadhesion belt with ADHESION BELTS IN SELECTED REGIONS OFassociated actin flaments CELL SHEET Figure 21–56 Bending of an epithelial sheet to form a tube. contraction of apical bundles of actin filaments linked from cell to cell via adherens junctions causes the epithelial cells to narrow at their apex. Depending on whether the contraction is oriented along one axis of the sheet or is equal in all directions, the epithelium will either roll up into a tube or invaginate to form a vesicle. (a) Diagram showing how an apical contraction along one axis of an epithelial sheet can cause the sheet to form a tube. (B) Scanning electron micrograph of a cross section through the trunk of a two-day chick embryo, showing the formation of the neural tube by the process diagrammed in (a). (B, courtesy of Jean-paul revel.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Animal development involves dramatic cell movements, including the guided migration of individual cells, the adhesion and repulsion of groups of cells, and the complex extension, branching, or rolling up of epithelial tissues. Migrant cells, such as those of the neural crest, break loose from their original neighbors and travel through the embryo to colonize new sites. Many migrant cells, including primordial germ cells, are guided by chemotaxis dependent on the receptor CXCR4 and its ligand CXCL12. In general, cells that have similar adhesion molecules on their surfaces cohere and tend to segregate from other cell groups with different surface properties. Selective cell–cell adhesion is often mediated by cadherins; repulsion is often driven by ephrin–Eph signaling. Within an epithelial sheet, cells can rearrange themselves to drive epithelial convergence and extension, as in gastrulation. Many movements are coordinated through a Wnt-dependent planar-polarity signaling pathway that is


Cell_Biology_Alberts

Cell_Biology_Alberts. cells can rearrange themselves to drive epithelial convergence and extension, as in gastrulation. Many movements are coordinated through a Wnt-dependent planar-polarity signaling pathway that is also responsible for orienting cells correctly in various types of epithelium. Elaborate branched tubular structures, such as the airways of the lung, are generated through bidirectional signaling between an epithelial bud and the mesenchyme that it invades, in a process called branching morphogenesis. Epithelial tubes and vesicles can originate in various ways, most simply by the rolling up and pinching off of a segment of epithelium, as in the formation of the neural tube.


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Cell_Biology_Alberts. One of the most fundamental aspects of animal development is one we know surprisingly little about—how the size of an animal or an organ is determined. Why, for example, do we grow to be so much larger than a mouse? Even within a species, size can vary greatly; a Great Dane, for instance, can weigh over 40 times more than a Chihuahua (Figure 21–57). Three variables define the size of an organ or organism: the number of cells, the size of the cells, and the quantity of extracellular material per cell. Size differences can arise from changes in any of these factors (Figure 21–58). If we compare a mouse with a human, for example, we find that the difference lies chiefly in the number of cells, there being roughly 3000 times more cells in a human, corresponding to a body that is roughly 3000 times more massive. Wild and cultivated species of food plants, on the other hand, often differ in body size chiefly because of differences of cell size.


Cell_Biology_Alberts

Cell_Biology_Alberts. The challenge, therefore, is to understand how cell numbers, cell size, and extracellular matrix production are regulated. First of all, we need to identify the signals that drive or inhibit growth. Then we need to discover how the signals themselves are regulated. In many cases, the size of an organ or of the body as a whole seems to be controlled homeostatically, so that the correct size is reached and maintained even in the face of drastic disturbances. This suggests that the developing structure somehow senses its own size and uses this information to regulate the signals for its own growth or shrinkage. In most cases, the nature of this feedback control remains a profound mystery.


Cell_Biology_Alberts

Cell_Biology_Alberts. In other cases, the duration of growth and the final size seem to be dictated by intracellular programs that take no cognizance of the size the structure has attained. These intracellular programs, too, present many mysteries, as we saw in our discussion of developmental timing. Very often, it seems, the sizes and proportions of body parts must depend on combinations of size-measuring feedback controls and intracellular programs, as well as on environmental influences such as nutrition.


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Cell_Biology_Alberts. The variation in control strategies is nicely illustrated by some classic transplantation experiments. If several fetal thymus glands are transplanted into a developing mouse, each grows to its characteristic adult size. In contrast, if multiple fetal spleens are transplanted, each ends up smaller than normal, but collectively they grow to the size of one adult spleen. Thus, thymus growth is regulated by local mechanisms intrinsic to the individual organ, whereas spleen growth is controlled by a feedback mechanism that senses the quantity of spleen tissue in the body as a whole. In neither case is the mechanism known. Figure 21–57 Members of the same species can have dramatically different sizes. the chihuahua weighs 2–5 kilograms, whereas a Great Dane weighs 45–90 kilograms. (courtesy of Deanne Fitzmaurice.) Figure 21–58 Determinants of organ size. the proliferation, Death, and Size of cells Determine Organism Size


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Cell_Biology_Alberts. The nematode worm C. elegans illustrates the different ways in which size differences can arise. This creature follows an astonishingly precise and predictable developmental program. Each individual of a given sex is generated by almost exactly the same sequences of cell divisions and cell deaths, and consequently has precisely the same number of somatic cells—959 in the adult hermaphrodite (the sex of the majority of these animals)—although the number of germ cells is more variable from worm to worm. The stereotyped development makes it possible to trace somatic cell lineages in exhaustive detail. More than 1000 cell divisions generate 1090 somatic cells during hermaphrodite development, but 131 of these cells undergo apoptotic cell death. Thus, precise regulation of both cell division and cell death determines the final numbers of somatic cells in the worm. In fact, genetic screens in C. elegans identified the first genes responsible for apoptosis and its regulation—thereby


Cell_Biology_Alberts

Cell_Biology_Alberts. and cell death determines the final numbers of somatic cells in the worm. In fact, genetic screens in C. elegans identified the first genes responsible for apoptosis and its regulation—thereby revolutionizing our molecular understanding of this form of programmed cell death (discussed in Chapter 18).


Cell_Biology_Alberts

Cell_Biology_Alberts. The final number of somatic cells in the adult worm is already present at sexual maturity (around three days after fertilization), after which no more somatic cells are generated. Yet the worm continues to grow, doubling in size between sexual maturity and death 2–3 weeks later. This doubling results from somatic cell growth: although the cells no longer divide, they continue to go through rounds of DNA synthesis; this endoreplication of the genome makes the cells polyploid. As in all organisms, the size of a cell is proportional to its ploidy—that is, the number of genome copies that it contains: a doubling of ploidy roughly doubles cell volume. By artificial manipulation of somatic cell ploidy, and thereby somatic cell size, the size of the worm as a whole can be increased or decreased. Thus the worm’s final size is set by a combination of programmed cell divisions and cell deaths, along with regulation of the sizes of individual cells through changes in ploidy.


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Cell_Biology_Alberts. In plants, as in animals, cell size increases as ploidy increases (Figure 21–59). This effect has been exploited in the agricultural breeding of plants for large size: most of the major fruits and vegetables that we consume are polyploid.


Cell_Biology_Alberts

Cell_Biology_Alberts. The size of an animal or organ depends on both cell number and cell size—that is, on total cell mass. Remarkably, many animals and organs can somehow assess their total cell mass and regulate it, providing evidence for feedback controls of the sort highlighted earlier in our introductory account of general principles of growth control. In contrast with C. elegans, if cell size is artificially increased or decreased in these cases, cell numbers adjust to maintain a normal total cell mass. This has been beautifully illustrated by experiments done long ago in salamanders, where cell size can be manipulated by altering the animal’s ploidy. As shown in Figure 21–59E, salamanders of different ploidies end up being the same size with very different numbers of cells. The individual cells in a pentaploid salamander,


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Cell_Biology_Alberts. Figure 21–59 Effects of ploidy on cell size and organ size. In all organisms, from bacteria to humans, cell size is proportional to ploidy—the number of copies of the genome per cell. this is illustrated for (a–D) Arabidopsis flowers and (e) for salamanders. In each case, the upper panels show cells in a specific tissue [a petal for Arabidopsis, a pronephric (kidney) tubule for the salamander]; the lower panels show the gross anatomy—flowers for Arabidopsis, the whole body for the salamander. In the case of Arabidopsis flowers, increase in cell size increases organ size. By contrast, the salamander and its individual organs attain their normal standard size regardless of ploidy, because large cell size is compensated for by fewer cells. this indicates that the size of an organism or organ in this species is not controlled simply by counting cell divisions or cell numbers; size must somehow be regulated at the level of total cell mass. [a–D, from c. Breuer et al., Plant Cell


Cell_Biology_Alberts

Cell_Biology_Alberts. or organ in this species is not controlled simply by counting cell divisions or cell numbers; size must somehow be regulated at the level of total cell mass. [a–D, from c. Breuer et al., Plant Cell 19:3655–3668, 2007. With permission from the american Society of plant Biologists; e, adapted from G. Fankhauser, in analysis of Development (B.h. Willier, p.a. Weiss and V. hamburger, eds), pp. 126–150. philadelphia: Saunders, 1955.] for example, are about five times the size of those in a haploid salamander, but there are only one-fifth as many cells. This scaling operates not only in the body as a whole, but in its individual organs.


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Cell_Biology_Alberts. The imaginal discs of Drosophila provide another striking example of homeostatic size control. These are epithelial pouches that grow by cell proliferation during the larval period and, during the pupal stage, form the organs and extremities of the adult fly (Figure 21–60). Experiments have been chiefly done on the wing imaginal disc. Mutations in components of the cell-cycle control machinery can be used to speed up or slow down the rate of cell division in the disc. Remarkably, such mutations can result in an excessive number of abnormally small cells Figure 21–60 The imaginal discs in the Drosophila larva (below) and the structures in the adult (above) that 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.]


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Cell_Biology_Alberts. Figure 21–61 Pituitary dwarf and pituitary giant. the “giant” on the right is robert Ladlow (1914–1940), the tallest recorded man at 8 feet 11 inches (2.72 m), together with his father, who was almost 6 feet tall (1.82 m). the dwarf on the left is General tom thumb, which was the stage name of charles Sherwood Stratton (1838–1883). On his 18th birthday, he was measured at 2 feet 8.5 inches (82.6 cm) tall, and at his death, he was 3 feet 4 inches (102 cm).(Images from http://en.wikipedia.org/wiki/ File:robert_Wadlow.jpg. © Bettmann/cOrBIS.) or a reduced number of abnormally large cells, respectively, leaving the size (area) and patterning of the adult wing practically unchanged. Thus, the size of the disc is not regulated so as to contain a set number of cells. Instead, there must be a regulatory mechanism that halts growth when the disc’s total cell mass reaches the appropriate value, so that the size and pattern of the adult wing that develops from the disc are normal. Remarkably,


Cell_Biology_Alberts

Cell_Biology_Alberts. mechanism that halts growth when the disc’s total cell mass reaches the appropriate value, so that the size and pattern of the adult wing that develops from the disc are normal. Remarkably, developing discs—or even disc fragments, taken out of their normal context and transplanted into the abdomen of an adult female—will grow until they reach their normal size. Clearly, the mechanisms that regulate disc size are intrinsic to the disc.


Cell_Biology_Alberts

Cell_Biology_Alberts. We still have very little idea how organisms or organs assess their total cell mass or monitor their own growth. Nevertheless, we are beginning to understand 1 meter some of the signal molecules that drive or halt growth in response to the mysterious cues that convey information about the size attained.


Cell_Biology_Alberts

Cell_Biology_Alberts. We have already seen how some signals act systemically as hormones to regulate the development of the animal as a whole. Some of these serve to regulate growth. In mammals, for instance, growth hormone (GH) is secreted by the pituitary gland into the bloodstream and stimulates growth throughout the body: excessive production of growth hormone leads to gigantism, and too little leads to dwarfism (Figure 21–61). Pituitary dwarfs have bodies and organs that are proportionately small, unlike achondroplastic dwarfs, for example, whose limbs are disproportionately short, usually because of a mutation in a gene encoding an FGF receptor that disrupts normal cartilage development (Figure 21–62).


Cell_Biology_Alberts

Cell_Biology_Alberts. Growth hormone stimulates growth largely by inducing the liver and other organs to produce insulin-like growth factor 1 (IGF1), which acts mainly as a local signal within many tissues to increase cell survival, cell growth, cell proliferation, or some combination of these, depending on the cell type. Large breeds of dogs such as Great Danes owe their great size to high levels of IGF1, while miniature breeds such as Chihuahuas have low levels (see Figure 21–57).


Cell_Biology_Alberts

Cell_Biology_Alberts. Not all growth-regulating extracellular signals stimulate growth; some inhibit it, by promoting cell death or inhibiting cell growth, cell division, or both. Myostatin is a TGFβ family member that specifically inhibits the growth and proliferation of myoblasts—the precursor cells that fuse to form the huge, multinucleated cells of skeletal muscle. When the Myostatin gene is deleted in mice, muscles grow to be several times larger than normal. Remarkably, two breeds of cattle that were bred for large muscles have both turned out to have mutations in the Myostatin gene; whippet dogs mutant for Myostatin develop similarly (Figure 21–63).


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Cell_Biology_Alberts. Figure 21–62 Achondroplasia. this type of dwarfism occurs in one of 10,000–100,000 births; in more than 99% of cases it results from a mutation at an identical site in the genome, corresponding to amino acid 380 in the FGF receptor FGFr3 (a glycine in the transmembrane domain). the mutation is dominant, and almost all cases are due to new, independently occurring mutations, implying an extraordinarily high mutation rate at this particular site in the genome. the defect in FGF signaling causes dwarfism by interfering with the growth of cartilage in developing long bones. (From Velasquez’s painting of Sebastian de Morra. © Museo del prado, Madrid.) Figure 21–63 Myostatin limits muscle growth. a wild-type whippet dog and a bully whippet that lacks myostatin. (a, from http://www.merlinanimalrescue.co.uk/dogs/?m=201211; B, from http://animalslook.com/schwarzenegger-dog/.)


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Cell_Biology_Alberts. Like TGFβ itself, myostatin acts through the Smad intracellular signaling pathway (see Figure 15–57) to inhibit muscle growth specifically. Another intracellular signaling pathway, called the Hippo pathway, inhibits organ and organism growth more generally. It was discovered in Drosophila, but it operates in vertebrates as well. It inhibits growth both by promoting cell death (by blocking an apoptosis inhibitor) and by inhibiting cell-cycle progression (by inhibiting the expression of the cell-cycle gene Cyclin E). Some components of the pathway in Drosophila are shown in Figure 21–64. The organs of animals that are abnormally resistant to Hippo repression can grow to a monstrous size (Figure 21–65).


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Cell_Biology_Alberts. It is important to note that in all species nutritional conditions also play a fundamental part in regulating the pace and extent of growth, and in animals they do so through hormonal signal networks that are highly conserved between vertebrates and invertebrates. Although we do not have space for details here, genetic experiments, especially in Drosophila, have begun to unravel the logic of these controls, and to indicate how they may operate alongside other machinery, such as the Hippo pathway, to determine final size. The sizes of animals and their organs vary widely and largely depend on total cell mass. This in turn depends on the size and number of cells, which are increased through cell growth and cell division, respectively. Cell numbers are reduced by programmed cell death. Each of these processes depends on both intracellular and


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Cell_Biology_Alberts. Figure 21–65 Overcoming Hippo repression increases organ size. (a) Livers from control and Yap-overexpressing mice. In these mice, hippo signaling is insufficient to block Yap. (B) adult heads from control and Yap-overexpressing flies. In the mutant flies, hippo signaling is unable to block Yap. (From J. Dong et al., Cell 130:1120–1133, 2007.With permission from elsevier.) Figure 21–64 Hippo pathway. hippo, a protein kinase, limits growth by phosphorylation and activation of the kinase Warts, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (called Yap in vertebrates). When unphosphorylated, Yorkie/Yap drives tissue growth: it activates the transcription of the growth-promoting gene Myc, the cell-cycle progression gene Cyclin E, the antiapoptotic gene Diap, and the microrNa Bantam. hippo-induced phosphorylation of Yorkie/Yap blocks this effect.


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Cell_Biology_Alberts. extracellular signals. The mystery is how these processes are regulated and coordinated to produce and maintain the characteristic final size of the adult organ or animal. Some signals such as survival factors, growth factors, and mitogens stimulate growth by promoting cell survival, cell growth, and cell division, respectively, while other signal molecules do the opposite. Although most of these signals operate locally to help sculpt the size and shape of the animal, its organs, and appendages, others act as hormones to regulate the growth of the animal as a whole. Nutrients can regulate growth through hormonal signals in the entire body. Many animals and organs can, by unknown mechanisms, assess their total cell mass and regulate it. If, for example, cell size is artificially increased or decreased in these cases, cell numbers adjust to maintain a normal total cell mass. Conversely, if cell numbers are artificially increased or decreased, cell size adjusts to compensate.


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Cell_Biology_Alberts. The development of the nervous system poses problems that have little parallel in other tissues. A typical nerve cell, or neuron, has a structure unlike that of any other class of cells, with a long axon and branching dendrites, both of which make many synaptic connections to other cells (Figure 21–66). The central challenge of neural development is to explain how the axons and dendrites grow out, find their right partners, and synapse with them selectively to create a neural network—an electrical signaling system—that functions correctly to guide behavior (Figure 21–67). The problem is formidable: the human brain contains more than 1011 neurons, each of which, on average, has to make connections with a thousand others, according to a regular and predictable wiring plan. The precision required is not so great as in a man-made computer, because the brain performs its computations in a different way and is more tolerant of vagaries in individual components. But the human brain


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Cell_Biology_Alberts. required is not so great as in a man-made computer, because the brain performs its computations in a different way and is more tolerant of vagaries in individual components. But the human brain nevertheless outstrips all other biological structures in its organized complexity.


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Cell_Biology_Alberts. The components of a typical nervous system—the various classes of neurons, glial cells, sensory cells, and muscles—originate in a number of widely separate locations in the embryo. Thus, in the first phase of neural development, the different parts of the nervous system develop according to their own local programs: neurons are born and assigned specific characters according to the place and time of their birth, under the control of inductive signals and transcription regulators, by mechanisms of the types we have already discussed. In the next phase, newborn neurons extend axons and dendrites along specific routes toward their target cells, guided by extracellular signals that attract or repel them. In the third phase, neurons form synapses with other neurons or muscle cells, setting up a provisional but orderly network of connections. In the final phase, which continues into adult life, the synaptic connections are adjusted and refined through mechanisms that usually depend on


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Cell_Biology_Alberts. up a provisional but orderly network of connections. In the final phase, which continues into adult life, the synaptic connections are adjusted and refined through mechanisms that usually depend on synaptic signaling between the cells involved terminal branches of axon make 25 µm synapses on target cells


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Cell_Biology_Alberts. Figure 21–66 A typical neuron of a vertebrate. the arrows indicate the direction in which signals are conveyed. the neuron shown is a basket cell, a type of neuron in the cerebellum. (adapted from S. ramón y cajal, histologie du Système Nerveux de l’homme et des Vertébrés, 1909–1911. paris: Maloine; reprinted, Madrid: c.S.I.c., 1972.) Figure 21–67 The complex organization of nerve cell connections. this drawing depicts a section through a small part of a mammalian brain—the olfactory bulb of a dog—stained by the Golgi technique. the black objects are neurons; the thin lines are axons and dendrites, through which the various sets of neurons are interconnected according to precise rules. (From c. Golgi, Riv. sper. freniat. Reggio-Emilia 1:405–425, 1875.) (Figure 21–68). At all stages, neurons are in intimate contact with various types of non-neuronal supporting cells—the glial cells. Neurons are assigned Different characters according to the time and place of their Birth


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Cell_Biology_Alberts. Neurons are assigned Different characters according to the time and place of their Birth We start our account here with the first phase of neural development: the generation of neural progenitors and their differentiation into hundreds of different neuronal subtypes, along with a much smaller number of glial types. Although the nervous system is exceptional in the extent of cell diversity, the process depends on the same principles that generate different cell types in other organs. We have already discussed some of the underlying machinery in the developing Drosophila nervous system. We turn now to vertebrates. The vertebrate spinal cord, the brain, and the retina of the eye together constitute the central nervous system (CNS). They all originate as parts of the neural tube, whose formation was described earlier (see Figure 21–56). The brain and eyes develop from the anterior neural tube and the spinal cord from the posterior.


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Cell_Biology_Alberts. The developmental anatomy is seen at its simplest in the spinal cord. As it develops, the epithelium forming the walls of the posterior neural tube becomes enormously thickened as the cells proliferate and differentiate, creating a highly organized structure of neurons and glial cells, surrounding a small central channel. Bands of neurons with different future functions—and expressing different genes—are laid out along the dorsoventral axis of the tube. Motor neurons (those that control the muscles) are located ventrally, whereas neurons that process sensory information are found dorsally. This pattern is established by opposing gradients of morphogens. These are secreted by specialized groups of cells that run the length of the ventral and dorsal midlines of the neural tube (Figure 21–69). The two morphogen gradients—consisting of Sonic hedgehog protein from the ventral source and BMP and Wnt from the dorsal source—help induce different groups of proliferating neural progenitor cells


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Cell_Biology_Alberts. The two morphogen gradients—consisting of Sonic hedgehog protein from the ventral source and BMP and Wnt from the dorsal source—help induce different groups of proliferating neural progenitor cells and differentiating neurons to express different combinations of transcription regulators. These regulators in turn drive the production of different combinations of neurotransmitters, receptors, cell–cell adhesion proteins, and other molecules, creating terminally differentiated neurons that will form synaptic connections selectively with the right partners and exchange appropriate signals with them.


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Cell_Biology_Alberts. genesis of neurons outgrowth of axons and dendrites synapse formation Figure 21–68 The four phases of neural development. refnement of synaptic connections 1200 Chapter 21: Development of Multicellular Organisms lumen of groups ofneural tube differentiatingfoor plate and notochord (ventricle) neurons Extracellular morphogen gradients, however, are not the only way to generate cell diversity. As we saw earlier in our discussion of Drosophila neuroblasts (see Figure 21–36), different cell types can also be generated by temporal patterning, in which an intracellular program changes the character of a progenitor cell over time, giving rise to different cell types as development progresses. This mechanism also seems to operate in vertebrate neurogenesis. The most striking illustration comes from study of another part of the CNS—the mammalian cerebral cortex.


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Cell_Biology_Alberts. Although the cerebral cortex is the most complex structure in the human body, it has a simple beginning—from the anterior neural tube. As in the spinal cord, the cells that form the walls of the tube proliferate, and the neuroepithelium thickens and expands as they divide. On a predictable schedule, the divisions of the neuroepithelial cells begin to produce a succession of cells committed to terminal differentiation as neurons. These future neurons are born close to the lumen (the central cavity) of the tube. From here, they migrate outward, losing attachment to the lumenal surface and crawling outward along neighboring cells that continue to span the full thickness of the neuroepithelium. These latter neuroepithelial cells do double duty, functioning as progenitors of neurons and glia, and as supporters of the epithelial architecture. They become stretched out as radial glial cells, forming a scaffold that continues to span the neuroepithelium even as this grows to an enormous


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Cell_Biology_Alberts. and as supporters of the epithelial architecture. They become stretched out as radial glial cells, forming a scaffold that continues to span the neuroepithelium even as this grows to an enormous thickness (Figure 21–70). At the same time, the radial glial cells continue to divide as neural precursors, giving rise to both neurons and glial cells—new radial glial cells as well as glial cells of other types. The newborn neurons, migrating along the radial glial cells, find their appropriate resting places in the developing cortex, where they mature, and from these sites they send out their axons and dendrites. The first-born neurons settle closest to their birthplace near the lumen, while neurons born later crawl past them to settle farther out (Figure 21–71). The successive generations of neurons thus build up as a series of cortical layers, ordered by birthdate and endowed with different intrinsic characters.


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Cell_Biology_Alberts. Strikingly, single cortical progenitor cells isolated in culture generate distinct types of cortical neurons and glial cells, with the timing and characteristics appropriate to specific cortical layers. These observations suggest that the neural progenitors in the developing mammalian cortex, much like the Drosophila neuroblasts, step through an intracellular developmental program that generates the ordered succession of different nerve cell types. Figure 21–69 A schematic cross section of the spinal cord of a chick embryo, showing how cells at different levels along the dorsoventral axis acquire different characters. (a) Signals that direct the dorsoventral pattern. Sonic hedgehog protein from the notochord and the floor plate (the ventral midline of the neural tube) and BMp and Wnt proteins from the roof plate (the dorsal midline) act as morphogens to control gene expression.


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Cell_Biology_Alberts. (B) the resulting patterns of cell fates in the developing spinal cord. Different groups of proliferating neural progenitor cells (in the ventricular zone, close to the lumen of the neural tube) and of differentiating neurons (in the mantle zone, further out) express different combinations of transcription regulators. Neurons expressing different transcription regulators will form connections with different partners and may make different combinations of neurotransmitters and receptors. colors represent different cell types and combinations of regulatory proteins. outer surface of cell body of radial glial cell inner surface of developing neural tube 10 µm the Growth cone pilots axons along Specific routes toward their targets


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Cell_Biology_Alberts. According to the character assigned to it during its early development, a neuron will proceed to make connections with specific partners. This phase of neural development involves a type of morphogenesis unique to the nervous system, in which axons and dendrites extend along specific routes toward their target cells. A typical neuron sends out one long axon and many dendrites, which are usually shorter. The axon projects to distant target cells to which the neuron will eventually send signals. The dendrites will receive incoming signals from axon terminals of other neurons. Axons and dendrites extend by growth at their tip, where one sees an irregular, spiky enlargement called a growth cone (Figure 21–72 and Movie 21.6). The growth cone is both the engine that produces the crawling movement and the steering apparatus that directs the tip along the proper path. Cytoskeletal machinery in the growth cone creates active protrusions, in the form of filopodia and lamellipodia (see Chapter


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Cell_Biology_Alberts. and the steering apparatus that directs the tip along the proper path. Cytoskeletal machinery in the growth cone creates active protrusions, in the form of filopodia and lamellipodia (see Chapter 16 for details): when such a protrusion layers of cortical neurons


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Cell_Biology_Alberts. Figure 21–70 Migration of immature neurons. Before sending out axons and dendrites, newborn neurons often migrate from their birthplace and settle in another location. the diagrams are based on reconstructions from sections of the cerebral cortex (part of the neural tube) of a monkey and rely on a staining technique that picks out at random a small subset of the whole dense mass of neuroepithelial cells. the neurons go through their final cell division close to the inner, lumenal face of the neural tube (in the ventricular proliferative zone) and then migrate outward by crawling along radial glial cells that form a scaffold. each of these latter cells extends from the inner to the outer surface of the tube, a distance that may be as long as 2 cm in the cerebral cortex of the developing brain of a primate. the radial glial cells can be considered as persisting cells of the original columnar epithelium of the neural tube that become extraordinarily stretched as the wall of the tube


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Cell_Biology_Alberts. brain of a primate. the radial glial cells can be considered as persisting cells of the original columnar epithelium of the neural tube that become extraordinarily stretched as the wall of the tube thickens. they also serve as neural stem cells: depending on stage and region, the newborn neurons can be generated from radial glial cells that undergo mitosis while their nuclei are close to the inner surface of the tube, or they can be generated from a nearby class of specialized progenitors in the ventricular proliferative zone. (after p. rakic, J. Comp. Neurol. 145:61–84, 1972. With permission from John Wiley & Sons, Inc.)


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Cell_Biology_Alberts. Figure 21–71 Programmed production of different types of neurons at different times from dividing progenitors in the cerebral cortex of the brain of a mammal. close to one face of the cortical neuroepithelium, progenitor cells divide, in stem-cell fashion, to produce successive generations of neurons (colored here blue, green, red, orange, and black). the neurons migrate out toward the opposite face of the epithelium by crawling along the surfaces of radial glial cells, as shown in Figure 21–70. the first-born neurons settle closest to their birthplace, while neurons born later crawl past them to settle farther out. Successive generations of neurons thus occupy different layers in the cortex and have different intrinsic characters according to their birth dates.


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Cell_Biology_Alberts. contacts an unfavorable surface, it withdraws; when it contacts a more favorable surface, it persists longer, steering the growth cone in that direction. In this way, the growth cone is guided by subtle variations in the properties of the surfaces over which it moves. At the same time, it is sensitive to specific signaling molecules, which—as we discuss next—can either encourage or hinder its advance. a Variety of extracellular cues Guide axons to their targets


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Cell_Biology_Alberts. Growth cones generally travel toward their targets along predictable routes, according to programs stored in the memory of the particular neuron to which they belong (Movie 21.7). In the simplest case, a growth cone can take a route that has been pioneered by other neurites, which they follow by contact guidance. As a result, nerve fibers in a mature animal are usually found grouped together in tight parallel bundles (called fascicles or fiber tracts). Such crawling of growth cones along axons is partly mediated by homophilic cell–cell adhesion molecules— membrane glycoproteins that help a cell displaying them to stick to any other cell that displays the same molecules. As discussed in Chapter 19, many homophilic adhesion molecules fall into one of two main classes: they are members of either the immunoglobulin superfamily, such as N-CAM, or the Ca2+-dependent cadherin family, such as N-cadherin. Members of both families are generally present on the surfaces of growth cones, of axons,


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Cell_Biology_Alberts. the immunoglobulin superfamily, such as N-CAM, or the Ca2+-dependent cadherin family, such as N-cadherin. Members of both families are generally present on the surfaces of growth cones, of axons, and of various other cell types that growth cones crawl over, including glial cells in the central nervous system and muscle cells in the periphery of the body. Growth cones also migrate over components of the extracellular matrix. When tested with neurons growing in a culture dish, some of the matrix molecules, such as laminin, favor axon outgrowth, while others, such as chondroitin sulfate proteoglycans, discourage it. But exactly how the matrix functions to guide axons in intact animals remains to be discovered.


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Cell_Biology_Alberts. Growth cones are generally guided by a succession of different cues at different stages of their journey, as summarized in Figure 21–73. Many of these cues involve specific signaling molecules. Some of these are encountered in the extracellular matrix, while others are attached to the plasma membrane of cells that the growth cones touch. Another important part is played by chemotactic factors; these are proteins secreted from cells that act as beacons at strategic points along the path—some attracting, others repelling. The trajectory of commissural axons— axons that cross from one side of the body to the other—provides a well-studied example.


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Cell_Biology_Alberts. Commissural axons are a general feature of bilaterally symmetrical animals, such as us, because they are required to coordinate behavior of the two sides of the body. In the developing spinal cord of a vertebrate, for example, a large number of neurons send their axonal growth cones ventrally toward the floor plate (the same structure that we encountered earlier as a source of the morphogen Sonic hedgehog—see Figure 21–69). The growth cones cross the floor plate and then turn abruptly through a right angle to follow a longitudinal path up toward the brain, parallel to the floor plate but never again crossing it (Figure 21–74). The first stage of the journey depends on a concentration gradient of the signal protein Netrin, secreted by the cells of the floor plate: the commissural growth cones sniff their way toward its source.


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Cell_Biology_Alberts. Figure 21–72 Internal architecture of a neuronal growth cone, as seen in culture on a flat substratum. the growth cone forms as an expansion of the tip of the growing axon. (a) Image by interference-contrast microscopy. (B) Immunostaining to show microtubules (green). (c) Immunostaining to show actin filaments (red). (D) Diagram of the cytoskeletal machinery. Filopodia form and push forward by assembly of actin filaments at the leading edge of the growth cone. Microtubules stabilize the directional decisions made by the actin-rich protrusions. Filopodia adhering to the flat substratum contract and pull the growth cone forward. (Images by chi-hung Lin, paul Forscher Laboratory, Yale University, New haven, ct.)


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Cell_Biology_Alberts. If commissural growth cones are attracted to the floor plate, why do they cross it and emerge on the other side, instead of staying in the attractive territory? And having crossed it, why do they never cross back again? The answers lie in a change in the responsiveness of the growth cones during their journey. As the growth cones cross the midline, they lose sensitivity to Netrin and become sensitive instead to a signal protein called Slit (see Figure 21–74). Slit is also produced by the floor plate, but it has the opposite effect to that of Netrin: it repels the growth Figure 21–74 The guidance of commissural axons. (a) the pathway taken by commissural axons in the embryonic spinal cord of a vertebrate.


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Cell_Biology_Alberts. Figure 21–74 The guidance of commissural axons. (a) the pathway taken by commissural axons in the embryonic spinal cord of a vertebrate. (B) attraction to the midline. the growth cone is first attracted to the floor plate by Netrin, which is secreted by the floor-plate cells and acts on the receptor Dcc in the axonal membrane. (c) repulsion from the midline after crossing it. as the growth cone crosses the floor plate, Slit comes into play: it binds to its receptors robo1 and robo2 and acts as a repellent to keep the growth cone from re-entering the floor plate. In addition, it blocks responsiveness to the attractant Netrin. Before crossing the midline, the commissural neurons express robo3.1, an alternative splice form of robo3 that is related to robo proteins but blocks Slit signaling. as neurites cross the midline, robo3.1 is lost and growth cones become responsive to Slit and are repelled from the midline.


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Cell_Biology_Alberts. cones, preventing them from re-entering the midline territory. The responses of the growth cone depend on the receptors that it expresses: as commissural neurons approach the floor plate, the Slit receptors are kept inactive by an inhibitory protein (Robo3.1) in the same membrane, allowing the commissural axons to grow to the midline without being repelled. Robo3.1 is lost as the growth cones cross the midline; now the growth cones become sensitive to repulsion by Slit and are thereby prevented from crossing back to the other side. At the same time, signals from the Slit receptors interfere with those from the Netrin receptors, making the growth cones deaf to the signal that attracted them to the floor plate initially. A similar mechanism, using similar proteins, seems to govern midline crossing of commissural axons in other animals, including flies and worms.