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The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal–distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.

The transcription factor E74 is one of the early genes induced by ecdysteroids during metamorphosis of Drosophila melanogaster. Here, we report the cloning and hormonal regulation of E74 from the tobacco hornworm, Manduca sexta (MsE74). MsE74 is 98% identical to that of D. melanogaster within the DNA-binding ETS domain of the protein. The 5′-isoform-specific regions of MsE74A and MsE74B share significantly lower sequence similarity (30–40%). Developmental expression by Northern blot analysis reveals that, during the 5th larval instar, MsE74B expression correlates with pupal commitment on day 3 and is induced to maximal levels within 12h by low levels of 20-hydroxyecdysone (20E) and repressed by physiologically relevant levels of juvenile hormone I (JH I).Immunocytochemical analysis shows that MsE74B appears in the epidermis before the 20E-induced Broad transcription factor that is correlated with pupal commitment (Zhou and Riddiford, 2001). In contrast, MsE74A is expressed late in the larval and the pupal molts when the ecdysteroid titer has declined to low levels and in the adult molt just as the ecdysteroid titer begins to decline. This change in timing during the adult molt appears not to be due to the absence of JH as there was no change during the pupal molt of allatectomized animals. When either 4th or 5th instar larval epidermis was explanted and subjected to hormonal manipulations, MsE74A induction occurred only after exposure to 20E followed by its removal. Thus, MsE74B appears to have a similar role at the onset of metamorphosis in Manduca as it does in Drosophila, whereas MsE74A is regulated differently at pupation in Manduca than at pupariation in Drosophila.

Hormones and trophic factors provide cues that control neuronal death during development. These developmental cues in some way regulate activation of apoptosis, the mechanism by which most, if not all, developmentally programmed cell deaths occur. In Drosophila, apoptosis can be induced by the expression of the genes reaper, grim, or head involution defective. We demonstrate that prior to the death of a set of identifiable doomed neurons, these neurons accumulate transcripts of the reaper and grim genes, but do not accumulate transcripts of the head involution defective gene. Death of these doomed neurons can be suppressed by two manipulations: by increasing the levels of the steroid hormone 20-hydroxyecdysone or by decapitation. We have investigated the impact that these two manipulations have on reaper expression. Steroid treatment prevents the accumulation of reaper transcripts, whereas decapitation results in the accumulation of lower levels of reaper transcripts that are not sufficient to activate apoptosis. These data demonstrate that in vivo, reaper, and grim transcripts accumulate coordinately in a set of identified doomed neurons prior to the onset of apoptosis. These observations raise the possibility that products of the reaper and grim genes act in concert in postembryonic neurons to induce apoptosis. That reaper transcript accumulation is regulated by the steroid hormone titer and by the presence of the head is evidence that developmental factors control programmed cell death by regulating the expression of genes that induce apoptosis.

The development ofDrosophilamidline glia during larval and pupal stages was characterized by localizing β-gal expression in enhancer trap lines, as well as with BrdU incorporation and pulse–chase experiments. At hatching about 40 to 50 glial cells are present along the midline of the ventral nerve cord (2 to 3 dorsal and 1 to 2 ventral cells per neuromere). The cells proliferate during the third larval instar and spread dorsoventrally within the midline, increasing in number to about 230 or more (around 20 cells per neuromere). Cell divisions cease shortly after pupariation, and the cells persist for the first half of pupal life with no apparent changes in numbers or positions. Between 50 and 80% of metamorphosis, however, virtually all of the midline glia undergo programmed cell death. Tissue culture experiments indicate that the peak of ecdysteroids occurring at pupariation is required for the cessation of proliferation of midline glia and their subsequent degeneration. Midline glia in central nervous systems (CNS) cultured with low or no ecdysteroids survive and continue to divide, whereas they cease proliferating and later degenerate with high ecdysteroids levels. The midline glial may play a role during CNS metamorphosis similar to that of their progenitors in the embryo, in stabilizing outgrowing neurites that cross or run along the midline.

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.

When abdomens were isolated from larvae during a critical period of the molting cycle, only parts of the abdominal epidermis had seen sufficient ecdysone to render them capable of forming a new cuticle independent of the prothoracic glands. By isolating abdomens at various times, it was found that the epidermis of a segment gains independence according to a reproducible pattern. The pattern of independence seen for a larval molt is roughly the reverse of that seen for a pupal molt. The presence or absence of juvenile hormone commits the epidermis towards the synthesis of larval or pupal cuticle, respectively. Under the influence of juvenile hormone, the pattern by which the various areas of the epidermis become committed to make larval cuticle is very similar to the larval pattern of independence from the prothoracic glands. This commitment to larval differentiation occurs at approximately the time when the epidermis becomes independent. In the absence of JH (in preparation for the pupal molt), the pattern by which the epidermis is committed to synthesis of pupal cuticle is the reverse of that seen for the larval molt. Moreover, the epidermis becomes committed to pupal differentiation 2 days prior to the time that it actually becomes able to secrete a pupal cuticle in the absence of the prothoracic glands.

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.