Metamorphosis of the Central Nervous System of Drosophila James W. Truman Department of Zoology NJ-15, University of Washington, Seattle, Washington 98195

SUMMARY The study of the metamorphosis of the central nervous system of Dvosophifa focused on the ventral CNS. Many larval neurons are conserved through metamorphosis but they show pronounced remodeling of both central and peripheral processes. In general, transmitter expression appears to be conserved through metamorphosis but there are some examples of possible changes. Large numbers of new, adult-specific neurons are added to this basic complement of persisting larval cells. These

cells are produced during larval life by embryonic neuroblasts that had persisted into the larval stage. These new neurons arrest their development soon after their birth but then mature into functional neurons during metamorphosis, Programmed cell death is also important for sculpting the adult CNS. One round of cell death occurs shortly after pupariation and a second one after the emergence of the adult fly.

Insects that go through metamorphosis are faced with two “embryonic” periods, an initial one that produces the larval stage and a second one at metamorphosis that transforms the larva into the adult. In the case of the central nervous system (CNS), the metamorphic phase includes many of the same cellular processes that are used during embryogenesis such as neurogenesis, axon guidance, and synaptogenesis. However, these processes are not used to make a nervous system from scratch but rather they are involved in transforming a CNS adapted to an animal of one body form into one suitable for an animal of radically different form and behavior. Consequently, at metamorphosis one needs to consider the disassembly of the larval system as well as the construction of that of the adult. Studies on the ventral CNS of large insects such as the moth, Manduca sexla, show that one can readily identify specific neurons and lineages and follow them through metamorphosis. This ability to identify and track individual cells has been invaluable for defining the main processes involved

in CNS metamorphosis: the conservation and remodeling of larval neurons (Levine and Truman, 1985; Truman and Reiss, 1988), production and differentiation of adult-specific neurons (Booker and Truman, 1987), and programmed cell death (Weeks and Truman, 1985; Truman, 1983). Although the nervous system of a large insect like Munduca is useful for cellular and physiological approaches, studies of the genetic and molecular events that mediate these changes are difficult. By contrast, Drosophilu provides a wealth of molecular and genetic techniques that can be brought to bear on these problems but its small size and fused nervous system make the cellular studies difficult. Despite the problems of working with identified neurons in Drosophila there have been a number of encouraging advances in this direction. Also, information gathered on larger flies such as Calliphora is readily applicable to Drosophila. Even in the case of Manduca, it is possible to identify neurons and lineages that are homologues in the moth and the fruitfly (Thomas, Bastiani, Bate, and Goodman. 1984) so that the two systems can supplement each other. Although the development of certain specialized regions of the brain such as the optic lobes and the mushroom bodies have received attention (Technau and Heisenberg, 1982),

Received June 15, 1990; accepted June 20, 1990 Journal ofNeurobiology, Vol. 21, No. 7. pp. 1072-1084 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/07 1072-13$04.00

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Drosophila Metamorphosis this review will focus primarily on the ventral segmental nervous system. At this time these studies in Drosophilu are still primarily descriptive but it is hoped that they will pave the way for future molecular and genetic approaches. METAMORPHOSIS OF DROSOPHILA AND ITS CNS A summary of the developmental changes during the life history of Drosophila can be found in Ashburner ( 1989). Like the larvae of all other higher flies, those of Drosophilu are specialized for life in semiliquid environments. The head is greatly reduced and inverted into the thorax, the only sclerotized head structures being a pair of retractable mouth hooks. The body is limbless and has a reduced set of sensory structures. During the 100 h of larval life. the insect goes through 3 larval stages ( instars j and increases in size by approximately 300-fold. Through this time, the larval cells do not divide but growth occurs by cell enlargement and most cells become highly polyploid by the time growth is complete. During larval growth, however, preparations are also being made for metamorphosis. Diploid cells proliferate in various imaginal primordia and these cells will then be used to build most of the tissues of the adult. The most obvious of these primordia are the imaginal discs that give rise to adult epidermis and cuticle. The transition from the larva to the adult begins when the third instar larva crawls out of the food, attaches itself to the substrate, and transforms into an immobile puparium. The latter is a specialization of higher flies in which the larval cuticle hardens and tans to form a protective case for the animal within. The larval cells then begin to degenerate and the imaginal discs evert and secrete a thin pupal cuticle. This process culminates with the expulsion of the mouth hooks and the eversion of the head at 12- 1 3 h after pupariation (Bainbridge and Bownes, 198 1 ) . The subsequent transformation of this pupal stage into the adult can be followed by features of the forming adult visible through the puparial wall (Bainbridge and Bownes, 1981 j , The time from pupariation to the emergence of the adult is approximately 100 h at 25°C. Insect sensory neurons arise in the periphery and their cell bodies remain near their site of birth while their axons navigate into the CNS. In flies, the sensory neurons are born during two main penods-during embryogenesis ( Hartenstein, 1988)

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and during metamorphosis with the eversion and differentiation of the imaginal discs (Palka, Schubiger, and Murray. 1984). The CNS, by contrast, contains only motoneurons and interneurons. It is worthwhile considering a brief overview of the changes that occur in the CNS through metamorphosis before considering the various steps in detail. The larval CNS consists of two spherical brain lobes that are broadly attached via the circumesophageal connectives to the ventral nervous system (Fig. 1 ). The latter is made up of the fused segmental ganglia (termed npzmvneres) including those from the subesophageal. thoracic (Tl-T3), and abdominal (A 1-A9 ) segments. At hatching, the thoracic and abdominal regions contain about 300 neurons per neuromere (Poulson, 1950). These larval neurons differ from other larval cells in that they remain diploid rather than becoming polyploid during larval growth. The larval CNS also possesses a large number of neuronal stem cells, neuroblasts, that are concentrated primarily in the brain and thoracic regions of the CNS. The neuroblasts divide repetitively through most of larval life generating discrete clusters of immature, postmitotic neurons. The latter cells do not appear to contribute to the functioning of the larval CNS

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Figure 1 Ventral view of nervous systems from a wandering third stage larva (left) and from an adult (right) of Drosophilu melunoguster. Dashed lines outline some of the major areas of neuropil. ABD = abdominal neuromeres; BR = brain; SEG = subesophageal neuromeres; OL = optic lobes; OLA = optic lobe anlagen;TH = thoracic neuromeres; TI = prothoracic, T2 = mesothoracic. T3 = metathoracic neuromeres.

but are reserved for future use in the adult. Hence, these cells will be referred to as “adult-specific” neurons. Metamorphosis brings about a profound reorganization of the CNS (Fig. 1 ) . The first 12- 14 h after pupariation are devoted to the removal of larval elements. Some larval neurons die at this time. Degeneration is seen throughout the ventral CNS but is most extreme in the abdominal regions. For the neurons that survive, this period may also be a time to prune back larval dendntic and axonal arbors. A constriction appears between the subesophageal and thoracic portions of the CNS by 12 h after pupariation and the CNS begins to lose its larval shape and to acquire the shape characteristic of the adult. By a day after pupariation, adult differentiation in the CNS is well underway. Larval neurons, that will be used in the adult, show extensive outgrowth of neurites and axons. Similarly, the arrested adult-specific cells begin elaboration of processes. The result of this cell growth is a marked expansion of the brain and thoracic neuropils that occurs from 12 to 36 h after pupariation. Overall, the ventral CNS shows the enlargement of the thoracic neuromeres and a severe reduction in the abdominal CNS. This emphasis on the thorax is associated with the shift during metamorphosis of locomotor control from the larval abdomen to the thorax of the adult.

FATE OF LARVAL NEURONS Pattern of Neuronal Survival Through Metamorphosis Because there is relatively little death among larval neurons in the brain and thoracic regions of the CNS, it had been thought that many of these cells survive through metamorphosis. The small size of Urosophila neurons and the fused nature of their CNS, however, have made it difficult to follow individual cells through this transition. In recent years, immunocytochemical approaches have been used to identify small subsets of neurons and to follow them in successive animals through time. This approach has bcen most fruitful for sets of neurons that contain biogenic amines or neuropeptides. For example, Valles and White ( 1988) showed that the larval brain and ventral CNS contain a small number of neurons that show serotonin immunoreactivity. The same number of cells in the appropriate locations were also found in the

nervous systems from both pupal (24 h postpupariation) and adult stages, indicating that the larval serotonergic neurons persist through metamorphosis. Indeed, the serotonergic system of the adult consists almost exclusively of these persistent larval cells with only a small number of adult-specific cells added to the optic lobe region of the brain. A similar situation was found for the catecholamine-containing cells (Budnik and White, 1988). Essentially all of these cells in the larval brain and ventral CNS appear to persist through metamorphosis and these make up the bulk of the adult catecholamine system with the addition of only a few new cell groups in the central brain and optic lobes. There is also a marked conservation of larval peptidergic cells. The most complete studies have been canicd out on neurons that express FMRFamide (Phe-Met-Arg-Phe-NH2) immunoreactivity. The larval nervous system contains a number of neurons that react to antisera raised against this peptide (White, Hurteau, and Punsal, 1986). The gene that encodes a family of FMKFamide-related peptides was isolated from Drosophilu ( Nambu, Murphy-Erdosh, Andrews, Feistner, and Scheller, 1988: Schneider and Taghert, 1988), and in situ hybridization studies show that the immunoreactive cells contain transcripts from this gene (Taghert, Schneider, and O’Brien. 1990). As with the biogenic amines, most of the cells in the adult that contain FMRFamide-related peptides correspond to neurons that persisted from the larval stage (White et al., 1986; Taghert et al., 1990). Many larval motoneurons also persist through metamorphosis into the adult stage. The most detailed studies have been carried out on the mesothoracic motoneuron, MN5, that innervates the dorsolongitudinal flight muscles, DLMS and 6 (Ikeda and Koenig, 1988j. The unique cell body position and axon trajectory of MN5 allowed M. Bate (unpublished) to identify the corresponding cell in the larva and to examine its morphology through metamorphosis by intracellular injection of lucifer yellow. During this transition, the neuron shows both a central reorganization and a change in its peripheral targets. Other motoneurons also persist through metamorphosis but have more complex problems. For example, the leg motoneurons of the adult also appear to be persistent larval cells but they lack peripheral targets in thc legless larva. These cells only develop peripheral processes at metamorphosis when they extend their axons into the leg imaginal discs and inner-

Drosophila hfetumorphosis

vate thc developing leg musculature (M. Bate, unpublished). Persisting larval neurons are estimated to make up no more than 7% of the cells on the thoracic CNS of the adult (Truman and Bate, 1988), yet they make a disproportionate contribution to certain classes of neurons. They are the major or sole source of the scrotonergic, catecholaminergic. and most of the FMRFamide-containing cclls as well as the adult motoneurons that have been examined so far. Thus the persisting larval cells seem to provide the core of the motor and neuromodulatory systems of the adult. What, then, are the functions of the adult-specific cells that are added at metamorphosis? Munducu shows a pattern of conservation of neuronal classes that is very similar to that seen in Drusophilu (Levine and Truman, 1985; Kent and Levine, 1988; J. Witten, unpublished). In this moth, the adult-specific cells are thought to be primarily interneurons devoted to processing the vast amount of information provided by the new adult sensory system (Truman and Booker, 1986). A similar situation most likely occurs in Drosophila. The Time Course of Changes in Conserved Cells The transformation of a cell from its larval to its adult form involves first the loss of larval-specific components followed by the outgrowth of' the adult elements. These changes are evident in Figure 2 which follows the changes in a set of ventral neurosecretory cells during metamorphosis. These neurons react with a number of peptide antisera including those raised against FMRFamide (White et al., 1986; Taghert et al., 1990), Substance P (White and Valles, 1985). gastrin/cholecystokinin (CCK) (Nassel, Ohlsson, and Cantcra, 1988), and the small cardioactive peptide B ( SCPB)(J. W. Truman, unpublished). These cells undergo pronounced changes in their peripheral neurohemal release sites during metamorphosis [Fig. 2 ( A ) ] (Nassel ct al., 1988; J. W. Truman. unpublished). In the larval stage. each of the thoracic cells project to a segmental neurohemal organ that extends dorsally from the midline of its neuromere (Nassel et al., 1988). In Drosophila the larval neurohemal structure stays intact up through the time of puparium formation. By 12 h after pupariation these structures show advanced degcneration but by 24 h new axonal growth cones are evident around the base of the degenerating larval structures. Over thc next

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24-36 h, the axons spread over the dorsal surface of the developing CNS and by 72 h after pupariation the cells have established the adult neurohema1 rclease site, which ramifies throughout the dorsal sheath. The time course of changes in the central neurites of these cells matches those seen in the periphery [Fig. 2( B)] . In the larval stage, SCPn immunoreactivity is evident in the major processes as well as in fine branches associated with lateral ncuropile. Fine processes disappear by 12 h after pupariation and by 24 h even some of the major processes are reduced in extent. Short processes then reappear by 36 h, and these continue to expand in extent such that by 72 h, regions of punctate staining are evident through selected regions of the dorsal neuropile. By this time the adult pattern of staining has been achieved. Some caution must be used in interpreting the changes in the central arbors because the loss of a stained process does not necessarily mean that the process itself is gone. However, the apparcnt loss of central neurites is coincident with the dramatic degeneration of the neurohemal processes in the periphery. Also, dye fills of identified motoneurons in both Drosophila (M. Bate, unpublished) and in Manduca (Truman and Reiss, 1988) show that some loss of larval branches typically preceeds thc onset of adult outgrowth. Consequently, it is likely that the changes in staining pattern depicted in Fig. 2 ( B ) represents a real loss and regrowth of fine neurites. Changes in Transmitter Expression During Metamorphosis As larval cclls go through metamorphosis, they may change their morphology and patterns of connectivity. but is transmitter expression also plastic? Jn Manduca some abdominal neurosecretory cells appear to switch the type of neuropeptides that they make during metamorphosis (Tublitz and Sylwester, 1990). Also, the cell bodies of many larval motoneurons express a peptide immunoreactivity that is then lost during the transition to the pupa (J. Witten, unpublished). Thus far, the data for Drosophila show considerable stability in the pattern of immunostaining through metamorphosis (Valles and White, 1988; Budnik and White, 1988; Taghert et al., 1990). There is, however, at least one case in which a shift may occur. As seen in Figure 2 ( B ) , the T2 region of the larval CNS has only onc pair of SCPB-positive cells. whereas that region of the adult CNS has two

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Truman

pairs. This new set ofcells isevident usingantibodies against substance P ( Nassel, Lundquist, Hoog. and Grimelius, 199O), FMRFamide (White et al.,

1986; Taghert et al., 1990), and gastrin/CCK (Nassel et al., 1988). This second set of cells begins to express immunoreactivity by 24 h after pupar-

A

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B

LARVA

P+12

P + 24

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Figure 2 Changes through metamorphosis in the immunostaining in a set of thoracic neurosecretory cells that react against an antibody raised against the molluscan neuropeptide, SCPB. ( A ) Dorsal view of the ventral CNS showing the loss of the larval neurohemal release sites and the growth of the adult sites. Dashed profiles are the location of the cell bodies of the neurons; the subesophageal cells are included in the first two pannels. ( H ) Ventral view showing the changes in the central arbors of these cells during metamorphosis. Note in the P + 36 individual that only one cell was present in the metathorax. Also, a second set of mesothoracic cells first appear as weakly immunopositive at P 24 (open outlines). P N refers to the number of hours after pupanation.

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Drosophila Metamorphosis

iation. l’heir staining at this time is weak and their cell body size is smaller than that of their T2 partner. Over the next 24 h both soma size and levels of immunoreactivity increase so that these cells become indistinguishable from the other mesothoracic set, a condition that is maintained into the adult. The question remains, howcvcr, as to the origin of these cells. Are they adult-specific cells or are they larval cells that shift putativc modulatory substances during metamorphosis? When postembryonically derived neurons are labelled by feeding larvae on diets containing the substituted nucleoside, 5-bromodeoxyuridine ( BUdR), and then the labelled neurons are examined after metamorphosis, the nuclei of these cells do not contain label (J. W. Truman, unpublished). This failure to label is consistent with the hypothesis that these cells are of embryonic origin but this conclusion must be considered tentative until the larval counterparts of these cells have been identified.

PRODUCTION OF ADULT SPECIFIC NEURONS Patterns of Neurogenesis The neurons for the larval CNS are born during a brief 4.5-h period during embryogenesis. The segmental neuroblasts segregate from the neuroectoderm starting at about 15% of embryonic development. Depending on their time of emergence, each then undergoes five to eight divisions before neurogenesis stops at about 40% (after stage 14; Hartenstein, Rudloff, and Campos-Ortega, 1987). At each division, the neuroblast divides unequally to produce a small ganglion mother cell (GMC), which in turn divides symmetrically to yield two daughter neurons. The final result is the 300 neurons per neuromere found at the time of hatching (Poulson, 1950). A second period of neurogenesis occurs during larval life and is more extensive than that seen in the embryo. The ’H-thymidine study by White and Kankel ( 19781 focused on neurogenesis in the brain but also noted extensive proliferation in the ventral CNS, especially in the thoracic region. More detailed descriptions of the pattern and time course of neurogenesis were later made possible by toluidine-blue staining, which selectively highlighted the neuroblasts, and by immunocytochemical detection of BUdR incorporation after

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chronic or acute treatments (Fig. 3) (Truman & Bate, 1988). The most detailed account of postembryonic neurogenesis is available for the thoracic neuroblasts (Fig. 3, 4 ) (Truman and Bate, 1988). By 12 h after hatching, the first neuroblasts become evident as small, toluidine-blue-positive cells. They enter their first S phase by 24 h, followed by enlargcment and the onset of divisions by 36 h. Each thoracic neuromere contains 47 neuroblasts (23 paired and 1 unpaired neuroblast) situated in stereotyped locations in the segment. As larval growth proceeds, the rate of neuroblast divisions increases such that by late in the third larval stage, a GMC is being produced every 50-55 min. In the embryo. the neuroblasts became smaller with successive divisions (Hartenstein et al., 1987), but in the larva they become larger as their cell cycle shortens (J. W. Truman, unpublished). By the time thc neuroblasts die at 12-18 h after pupariation each has produced an average of over 100 neurons. The pattern of postembryonic neurogenesis in other parts of the CNS varies according to region. Most abdominal segments contain only a few neuroblasts (three pairs) and the proliferative period is quite short as compared to those in the thorax. By contrast in the brain hemispheres, five neuroblasts are already active in thc newly hatched larva. These are then joined by other neuroblasts during the ensuing 12-36 h of larval life, and the brain ncuroblasts continue divisions well into the pupal stage (White and Kankel, 1978; Truman and Bate, 1988). Thus the nervous system adopts the same strategy that is used by the other organ systems in the fly. The larval period is a proliferative period during which immature cells are stockpiled for later use during metamorphosis. These cells, however, are found in discrete groups with each group being the lineage from a single neuroblast. The following two sections will address two questions: What are the origins of the larval neuroblasts and what are the fates of their progeny during metamorphosis? The Origin of the Larval Neuroblasts

In the segmental ganglia of insects with incomplete metamorphosis, such as grasshoppers, there is only a single period of neurogenesis that ends prior to hatching (Shepherd and Bate, 1990). How then does this pattern of neurogenesis relate to the twophase pattern evident in higher insects such as moths and flies? One possibility is that insects with

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complete metamorphosis acquired a new sct of neuroblasts that are dedicated to postembryonic neurogenesis. Alternatively, the embryonic neuroblasts might produce an initial lineage, become dormant, and then reactivate in the larva to produce the adult-specific cells. Direct obscrvations of

Drosophila embryos have not resolved this question because at the time the embryonic neuroblasts stop dividing, they can no longer be distinguished from their surrounding progeny (Hartenstein et al., 1987). Neuroblasts then cannot be unambiguously recognized until about 24 h later when they

Figure 3 Immunoperoxidase-stained whole mount of a Drosuphilu larval CNS showing the incorporation pattern of a 6-h pulse of HUdR given at 9 1 11 after hatching. Note the prominent labelling of discrete clusters in the thoracic region as well as a dark band of incorporation in the optic lobe region of the brain. (From Truman and Bate, 1988).

Drosophila Metamorphosis

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Figure 4 Camera lucida drawings of the ventral view of the larval nervous system showing the progressive addition of cells that have incorporated BUdR during the course of larval fife. Thc vertical dashed line represents the ventral midline; TI to A8 indicate the segmental nerves. At 13 h. trachcal cells only are stained. .4few neuroblasts in the thoracic region incorporate label by 24 h. The neuroblasts subsequently enlarge and generate a steadily increasing cluster of progeny during the remainder of larval life. (Froin Truman and Bate. 1988).

become evident in the late first stage larva (Truman and Bate, 1988). Support for the hypothesis that the embryonic and larval neuroblasts are the same cells comes from comparative studies on the tsetse fly, Gfossina pullidipes ( J . W. Truman, unpublished). Tsetse larvae are produced one at a time and each is brooded in the maternal uterus until it reaches its full growth. After its birth, the larva immediately burrows into the soil and initiates melamorphosis. Compared to free-living larvae of other flies, such as Drosophilu, the newly hatched tsetse larva has severely retarded CNS development. Indeed, as seen in Figure 5, at hatching the neuroblasts are still actively dividing and are in their embryonic configuration. Moreover, the neuroblasts remain large and identifiable through the subsequent period of CNS condensation when they are rearranged into an array that is similar to that seen in larval Drosophila. Thus in Glossina, it is clear that the same neuroblasts are responsible for both the embryonic and postembryonic phases of neurogenesis. Experimental support for this hypothesis comes from recent experiments by Prokop and Technau ( 1989). They transplanted single cells from early

gastrula rtage embryos into the presumptive thoracic regions of similarly staged hosts. The donor cells were from a transformant strain that expressed beta-galactosidase and they were also injected with horseradish peroxidase (HRP). The HRP provided a cytoplasmic stain. but with repeated divisions it was diluted out. By contrast, the beta-galactosidase expression was only nuclear but marked all descendents from the transplanted cell. Two classes of marked cells were found when the hosts were subsequently examined as last stage larvae. Onc class consisted of a small number of doubly labelled cells. which could be identified as clusters of larval neurons. The other class of clones included a cluster of doubly labelled neurons associated with a beta-galactosidase-marked cluster of postembryonic neurons with its associated neuroblast. Importantly, they did not find clones consisting of only postembryonic cells. These results strongly argue that the postembryonic lineages are indeed extensions of the embryonic set. Fate of the Postembryonic Cells

At this time the fates of the adult-specific cells are best understood in Munducu. In this moth, as in

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Figure 5 Camera lucida drawings illustrating the progression of neurogenesis in embryos and larvae of the tsetse fly, Glossiria pallidipes. (Left) The gross morphology of the CNS during the various stages, highlighting neuromere T2 in black. (Right) Ventral view of segment T2

Drosophila Metamorphosis

Drusuphila, each thoracic ganglion possesses 24 distinct lineages that come from the 23 paired and 1 unpaired neuroblasts (Booker and Truman, 1987). The cells produced within a given lineage tend to share particular phenotypes. For example, the transmitter gamma-amino butyric acid (GABA) is produced by cells in only six lineages but by essentially all of the cells in each of these six lineages (Witten and Truman, 1988; unpublished). Comparison of the distributions of GABA-positive cells in the moth with those in the CNS of higher flies suggests that homologous lineages in the fly may generate similar classes of neurons (Witten and Truman, 1989). Indeed, it seems likely that information on lineage fates in the moth will be directly applicable to similar problems in Drosophila. Neurons that are born during the postembryonic phase of neurogenesis differ in one important aspect from those that are born in the embryo. The former cells do not immediately mature into functional neurons but, rather, become arrested in an immature, postmitotic condition. The characteristics of the cells in this arrested condition are as yet poorly defined. These cells have scant cytoplasm and poorly developed processes that extend into the neuropile. Also, they show no hint of transmitter production. This arrest is then broken at metamorphosis when the cells resume development and mature into functional adult neurons. The earliest changes shown by these cells as they break their arrest involve alterations in the level of expression of certain nuclear regulatory genes (M. Glicksman, J. W. Truman and M. Bate, unpublished). For example, some lineages of adult-specific neurons express the product from the homeotic gene Ultrabithorax ( Ubx) (White and Wilcox, 1985). In these lineages the level of expression, as indicated by antibody staining, is quite low while the cells are arrested. By about 6 h after pupariation, however, the Ubx levels in most of the lineages becomes elevated and remains high for the remainder of metamorphosis. Presumably, the enhanced expression of regulatory genes, such

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as Ubx, is an important prerequisite for the subsequent maturation of these adult-specific cells. The time course of maturation of the adult-specific cells has not yet been followed in detail in Drosophila. The marked expansion of the neuropile that occurs between 18 to 36 h after pupariation suggests that this is a major period of neurite growth for these cells. In Manduca adult-specific neurons begin to express their transmitters at about 30% of the way between pupal ecdysis and adult emergence. The corresponding time in Drosophila (considering head eversion as being equivalent to pupal ecdysis) is about 40 h after pupariation. Further work is obviously in order to establish the exact time course of transmitter acquisition in the fly. PROGRAMMED NEURONAL DEATH

Programmed cell death is used to remove unneeded larval neurons from the CNS. There are two bouts of cell death, one that occurs during the first 12-18 h after pupariation and a second wave soon after the emergence of the adult. This last bout of neuronal death is the only one that has received attention in Drnsophila (Kimura and Truman, 1990). At the end of metamorphosis, the fly shows a set of highly specialized behaviors that are used dunng its emergence from the puparium and the expansion of its new wings. These behaviors are not used again during the life of the fly, and the muscles and motoneurons dedicated to their performance rapidly degenerate (Fig. 6). Ecdysial muscles, such as the medial DIO muscles, begin degeneration at 6 h postecdysis and are gone by 12 h. The abdominal motoneurons show a pcak of degeneration at 6 h after ecdysis. Although the muscles and neurons show a coordinated degeneration, these deaths appear to be independently regulated (Kimura and Truman. 1990). A signal from the head of the animal that occurs prior to ecdysis appears to trigger the death of the muscles. The death of the

showing the distribution of neuroblasts; cells in adjacent segments are shown in dotted outline. ( A ) CNS from a shortened germ-band stage prior to dorsal closure showing the full array of embryonic neuroblasts. ( B ) Nervous system in the course ofgangliogenesis from an embryo shortly before hatching. (C) Nervous system characteristic of an early first stage larva in which the neuromeres are fused but ganglion condensation has not yet occurred. ( D ) Nervous system characteristic of second instar larvae in which the CNS is fully condensed. The neuroblast array can be followed through this entire transition.

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Figure 6 Normal time course of muscle breakdown (top) and neuronal death (bottom) in Drosophilu after adult eclosion. (Top) Each point represent the mean (+S.D.) score of the degeneration of the medial DIO muscle in the fourth abdominal segment. Numbers beside the points are counts of the flies examined. (Bottom) Iiach point represents the number of large (diameter of cell body > 5 pm) dying neurons in the fused ventral ganglion. The solid line is drawn through the median values. Inset is a camera lucida drawing of a 6-h nervous system showing the large number of pyknotic cclls in the abdominal region of the CNS. (From Kimura and Truman, 1990)

neurons, by contrast, is not triggered until after the emergence of the fly and can be delayed for hours by preventing the fly from performing the last behaviors associated with wing expansion. The mechanisms that mediate the death response are not known.

CONCLUSIONS When insects evolved complete metamorphosis, they had to modify the processes through which they constructed their adult nervous system. These developmental modifications presumably re-

quired new genetic control systems about which we know essentially nothing. One major modification has been to interrupt neurogenesis so that part of it occurs embryonically, whereas the remainder occurs during larval life. At this time it is not known what causes the neuroblasts to stop dividing in the embryo or to reactivate in the late first larval instar. Similarly, the developmental control over some of the progeny from the neuroblasts has had to be modified. It is not clear why the progeny of these cells arrest after their birth during the postembryonic period, whereas in the embryo they directly differentiate into functional neurons. Processes of neuronal remodeling and programmed

Drosophila Metumotphosis

neuronal death also play prominent roles at metamorphosis and should provide fruitful systems for genetic and molecular studies. The process of metamorphosis is under endocrine regulation, through the action of the steroid hormones, the ecdysteroids (Richards, 1980). Although it has not been emphasized in this review, a number of changes that occur in the CNS at metamorphosis are undoubtedly driven by the action of these steroids (Truman, 1988). Indeed, some of the early genes that are thought to mediate the action of ecdysteroids in Drosophila have recently been shown to be required for normal metamorphosis of the CNS (Restifo and White, 1989). Thus the fruitfly appears to provide the best opportunity to track the molecular changes that underlie various aspects of metamorphosis from the initial action of the controlling hormones through to the final differentiated response. Unpublished work reported in this review was supported by a grant from the National Institutes of Health (NS-13079).

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