Parallel Molecular Genetic Pathways Operate during CNS Metamorphosis inDrosophila

MCN

Molecular and Cellular Neuroscience 11, 134–148 (1998) Article No. CN980683

Parallel Molecular Genetic Pathways Operate during CNS Metamorphosis in Drosophila Linda L. Restifo1 and Wanda Hauglum ARL Division of Neurobiology, University of Arizona, Tucson, Arizona 85721-0077

Insect metamorphosis provides a valuable model for studying mechanisms of steroid hormone action on the nervous system during a dynamic phase of functional remodeling. The Drosophila Broad Complex (BRC) holds a pivotal position in the gene expression cascade triggered by the molting hormone 20-hydroxyecdysone (20E) at the onset of metamorphosis. We previously demonstrated that the BRC, which encodes a family of zinc-finger transcription factors, is essential for transducing 20E signals into the morphogenetic movements and cellular assembly that alter the CNS from juvenile to adult form and function. We set out to examine the relationship of BRC to two other genes, IMP-E1 and Deformed (Dfd), involved in the metamorphic transition of the CNS. Representatives of the whole family of BRC transcript isoforms accumulate in the CNS during the larval-to-pupal transition and respond directly to 20E in vitro. IMP-E1 is also directly regulated by 20E, but its induction is independent of BRC, revealing that 20E works through at least two pathways in the CNS. DFD expression is also independent of BRC function. Surprisingly, BRC and DFD proteins are expressed in distinct, nonoverlapping subsets of neuronal nuclei of the subesophageal ganglion even though both are required for its migration into the head capsule. This suggests that the segment identity and ecdysone cascades operate in parallel to control region-specific reorganization during metamorphosis. Key Words: ecdysone; steroid hormones; Broad Complex; Deformed; IMP-E1; neurogenetics; transcription factors; gene expression; in vitro culture

INTRODUCTION Steroid and thyroid hormones, acting through nuclear receptors, play essential regulatory roles in CNS devel1 To whom correspondence should be addressed at ARLDN, University of Arizona, 611 Gould-Simpson Building, Tucson, AZ 85721-0077. E-mail: [email protected].

134

opment and plasticity in many species (reviewed in Truman, 1988; Levine et al., 1991; Forrest, 1994; GarciaSegura et al., 1994). Because they have nuclear receptors that are ligand-dependent transcription factors (Meier, 1997) and because protein synthesis inhibitors can block their effects on the nervous system (e.g., Parsons et al., 1981; Luine and McEwen, 1983; Weeks et al., 1993), these hormones are believed to exert many of their effects by changing patterns of gene expression. Insect metamorphosis provides a dramatic and experimentally accessible system for studying hormoneregulated anatomical and physiological modification of the nervous system. Under the coordinating influence of the steroid molting hormone 20-hydroxyecdysone (20E), a preexisting, functional larval nervous system is reorganized into one that can serve the novel behavioral needs of the adult (Levine et al., 1995). During CNS metamorphosis, persistent neurons undergo extensive remodeling and neurochemical alteration, new neurons and glia are born and differentiate, and apoptosis is used sparingly to eliminate obsolete cells, while complex morphogenetic movements change the overall shape of the nervous system (reviewed in Pipa, 1973; Weeks and Levine, 1992; Tublitz, 1993; Truman, 1996). Previous work from this laboratory and others has begun to identify the molecular and genetic mediators of remodeling and hormonal responsiveness of the CNS during metamorphosis in Drosophila (Restifo and White, 1991; Robinow et al., 1993; Restifo and Merrill, 1994; Truman et al., 1994; Restifo et al., 1995). The data reported here address the relationships among three of these: Broad Complex (BRC) and IMP-E1, which are 20E-regulated genes, and Deformed (Dfd), a homeotic selector gene. In the CNS of Drosophila, receptors for 20E (EcR, the ecdysone receptor; Koelle et al., 1991) are widespread during the metamorphic transition, with different isoforms that may predict distinct developmental fates (Truman et al., 1994). In the best characterized example, 1044-7431/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

CNS Metamorphosis in Drosophila

doomed neurons that undergo apoptosis in response to the decline in 20E levels at the end of metamorphosis are marked for death by the expression of high levels of isoform EcR-A (Robinow et al., 1993). Although a number of primary response genes have been cloned and characterized (Thummel, 1996), to date only the BRC has been shown to be essential for CNS metamorphosis (Restifo and White, 1991). The BRC sits at the top of a genetic hierarchy critical for many aspects of Drosophila metamorphosis (reviewed in Bayer et al., 1996b). BRC transcript accumulation is inducible by 20E in whole larval organs, salivary glands, and imaginal discs in a manner characteristic of a primary response to the hormone (Chao and Guild, 1986; Karim and Thummel, 1992; Bayer et al., 1996a). The gene encodes a small family of zinc-fingercontaining transcription factors (DiBello et al., 1991; von Kalm et al., 1994; Bayer et al., 1996a) that control expression of many loci, including both primary and secondary response genes, in the ecdysone cascade (Lepesant et al., 1986; Guay and Guild, 1991; Karim et al., 1993; Dubrovsky et al., 1994, 1996; Hodgetts et al., 1995). Thus, the spatial and temporal profiles of BRC expression in the CNS are important for understanding how this gene regulates metamorphosis of this complex tissue. In the absence of BRC function, larvae develop relatively normally (the salivary glands providing a notable exception), but after wandering fail to initiate anatomical or behavioral features of the metamorphic transition (Stewart et al., 1972). Partial loss-of-function mutations define three lethal complementation groups (Belyaeva et al., 1980), causing death during prepupal or pupal life with a diverse array of developmental defects affecting both larval and imaginal tissues (Fristrom et al., 1981; Kiss et al., 1988; Restifo and White, 1991, 1992). Mutants of each complementation group (reduced bristles on the palpus, rbp; broad, br; and 2Bc) manifest distinct, partially overlapping subsets of phenotypes. In the CNS, three morphogenetic defects are common to mutants of all BRC complementation groups: failure of separation of the subesophageal ganglion (SEG) from the thoracic ganglion (TG), failure of fusion of the brain in the midline (the ‘‘split-brain’’ defect), and faulty rotation of optic lobe ganglia. In contrast, disorganization of visual system neuropil is a ‘‘restricted’’ phenotype, found predominantly and in severe form in 2Bc mutants, as well as in mild form in br mutants, but not at all in rbp mutants (Restifo and White, 1991). The three distinct BRC functions revealed by complementation studies are provided by the three most abundant BRC protein isoforms. All BRC proteins share

135 a common amino-terminal region (‘‘BRcore’’; DiBello et al., 1991) that includes a domain (called ‘‘BTB’’ or ‘‘POZ’’) implicated in protein–protein interactions (Bardwell and Treisman, 1994; Zollman et al., 1994), and each isoform contains one of four carboxy-terminal pairs of zinc fingers (Z1–4; DiBello et al., 1991; Bayer et al., 1996a). The three abundant isoforms, BRC-Z1, -Z2, and -Z3, mediate rbp1, br1, and 2Bc1 functions, respectively (Crossgrove et al., 1996; Bayer et al., 1997; Sandstrom et al., 1997; Liu and Restifo, 1998). Studies to date point toward several predictions concerning BRC expression in the CNS. First, since all BRC subfunctions are required for CNS metamorphosis, all major BRC isoforms should be expressed in the CNS at some time during the metamorphic transition. Second, CNS expression of BRC should be hormonesensitive and independent of de novo protein synthesis, consistent with a primary response to 20E. Third, all regions of the CNS associated with a mutant phenotype should express BRC protein(s) and BRC proteins should be localized to the nucleus. The CNS phenotypes of BRC mutants are believed to arise from the misregulation of BRC target genes (Restifo and White, 1991; Liu and Restifo, 1998). Two genes were proposed as candidate BRC targets on the basis of a shared phenotype (Deformed) or overlapping expression patterns (IMP-E1), but correlative evidence revealed that neither gene fulfilled criteria expected of a direct BRC target (Restifo et al., 1995). One goal of the current study was to determine definitively whether Dfd and IMP-E1 are independent of BRC function in the CNS. 20E induces IMP-E1 in a subset of nonneuronal cells of the CNS (the perineurium and the interface glial scaffold) at the onset of metamorphosis (Natzle et al., 1988; Restifo et al., 1995). Unlike BRC, E74, and E75, which encode transcription factors, IMP-E1 represents a distinct class of primary response (‘‘early’’) genes. Its transcripts appear on membrane-bound polysomes, hence its product is secreted or membrane-associated. We suggested IMP-E1 as a candidate BRC target gene because both are expressed in interface glia and BRC expression precedes IMP-E1 induction. We found that IMP-E1 induction in the CNS in vivo is normal in BRC mutants of all three complementation groups (Restifo et al., 1995). However, functional redundancy among the isoforms (von Kalm et al., 1994) could mask a BRC requirement for normal regulation. Hence, while no individual BRC isoform is essential for the in vivo induction of IMP-E1 in the CNS at the larval–prepupal transition, we could not conclude that IMP-E1 is indepen-

136

Restifo and Hauglum

dent of BRC function without testing null mutants, and this required using in vitro organ culture. Dfd, a homeotic gene in the Antennapedia complex, is required for segment identity in the embryonic and adult head (McGinnis et al., 1990a, b) as well as for maturation of the SEG (Restifo and Merrill, 1994), the sole CNS region in which it is expressed (Mahaffey et al., 1989; Diederich et al., 1991). However, Dfd transcript accumulation is indifferent to 20E levels in vivo or in vitro (Restifo et al., 1995). This suggested that Dfd expression is BRC-independent, but that regulation of common target genes by the two transcription factors could explain their similar roles in SEG maturation. We therefore predicted that BRC and DFD proteins should colocalize in SEG nuclei.

RESULTS AND DISCUSSION A Family of BRC Transcripts Accumulates in the Late Larval CNS Using an antisense cRNA probe that detects all BRC transcripts, we found five bands in poly(A)1 RNA from wandering third instar larvae (Fig. 1, far left). The pattern is similar to those reported in earlier studies done with lower resolution and sensitivity (Karim and Thummel, 1992; Karim et al., 1993; Andres et al., 1993; Bayer et al., 1996a), except that it reveals the largest transcripts as two distinct bands. At this stage the CNS expresses the full complement of BRC transcripts (4.6, 6, 8.5, 10.5, and .12 kb; Fig. 1, middle), with the 4.6- and 8.5-kb forms being most abundant. The same pattern is seen in poly(A)1 CNS RNA (data not shown). In contrast, the same probe detects primarily the smallest BRC transcript in salivary glands (see also Chao and Guild, 1986), and this salivary gland transcript averages about 100 nt smaller (4.5 kb) than its CNS counterpart (based on five independent RNA preparations; data not shown). These data show that transcript heterogeneity and tissue-specific profiles may be obscured by wholebody RNA analysis. Probes specific for BRC zinc-finger variants detect all of them in the larval CNS (Fig. 2), consistent with the CNS requirement for all BRC genetic functions (Restifo and White, 1991) and with protein expression data (Emery et al., 1994). Note that the variants differ markedly in size representation and relative abundance. BRC-Z1 transcripts are abundant and found exclusively in the 4.6-kb form. Z2 and Z3 are each found in a trio of transcripts, of which the largest (10.5 kb) and smallest (6 kb) are rare. The intermediate Z3 form is slightly larger (8.5 kb) and far more abundant than that containing Z2

FIG. 1. BRC expression in the late larval CNS. Northern blot analysis of RNA from wandering third instar larvae. The panels show autoradiograms from three independent experiments, so signal strengths between panels are not directly comparable. However, all three gels were run approximately the same distance. Df(BRC), 1.5 µg poly(A) 1 RNA from wandering larvae lacking all BRC genomic DNA; WT (wild-type) larvae, 1.0 µg poly(A) 1 RNA from Canton-S wandering larvae; CNS, total RNA from 20 Canton-S CNSs; SG, total RNA from 20 pairs of salivary glands. In the middle panel, the CNS and SG RNA came from the same 20 larvae. In each case the blot was probed for BRcore plus Z1 (‘‘BRC’’) or Z3 plus 38-UTR (‘‘Z3’’) and subsequently reprobed for actin or rp49 transcripts. The specificity of the BRC probe is confirmed by the absence of signal in the Df(BRC) lane. The CNS expresses all BRC transcript sizes found in whole larvae. The major salivary gland transcript is slightly smaller than the smallest CNS transcript (4.5 kb vs 4.6 kb). Z3-4.5, prominent in RNA from whole larvae, is not detectable in the CNS.

(8.0 kb). The major Z4 transcript is rare and similar in size to this intermediate class. Smaller (6–6.5 kb) and larger (.10 kb) Z4 transcripts were occasionally detected. Thus, the CNS pattern of BRC transcripts (Fig. 1, middle) can be interpreted as follows: the abundant 4.6-kb transcript is exclusively Z1; the rare 6.0-kb form has Z2, Z3, and probably Z4 contributions; the abundant 8.5-kb band contains mostly Z3 transcripts, with small amounts of Z2 and Z4; and the rare 10.5-kb transcripts are a blend of Z2–4. The BRC zinc-finger representation in CNS RNA differs from that in whole-body larval RNA. In the CNS, members of the Z2 trio are equally abundant (Fig. 2), whereas in whole larvae and imaginal discs the smaller two forms predominate (Bayer et al., 1996a). For BRC-Z3

CNS Metamorphosis in Drosophila

137 likely results from the lower sensitivity of the previous experiments, which used only 10 µg of total RNA [cf., 1 µg of poly(A) 1 RNA in our experiments]. Interestingly, while Z3-4.5 and -3.6 transcripts were never detected in CNS RNA in vivo, they did appear in the CNS following in vitro culture with 20E and cycloheximide (see Fig. 3 below). Note also that the Z3-3.6 transcript (Fig. 1, right), although polyadenylated, does not contain BRcore sequences, since it is not detected with the common probe (Fig. 1, left). As discussed below, variation in BRC-Z3 transcript profiles may reflect tissue-specific differences in the dynamics of posttranscriptional processing.

Temporal Features and Hormone Sensitivity of BRC Transcript Accumulation

FIG. 2. Zinc-finger distribution of BRC transcripts in the late larval CNS. Northern blot analysis of total RNA from CNS of Canton-S wandering third instar larvae. Each lane represents 20 CNS equivalents from blue-gutted (B) or clear-gutted (C) larvae. For Z1–3, the B and C samples each came from a single large RNA prep, and 20 CNS equivalents were loaded in each lane of one gel. The membrane was cut into triplicate strips and each was hybridized with a zinc-fingerspecific BRC riboprobe (for Z3, the 3’-UTR probe was used) and subsequently hybridized for actin transcripts. The autoradiographic exposures shown for Z1–3 are the same. The Z4 panel represents an independent experiment in which the bottom of the blot was hybridized for rp49 transcripts. Transcript size estimates are averages of several independent experiments. The table shows changes in transcript abundance between the blue- and the clear-gutted stages (number of independent experiments indicated in parentheses). The values are means of ratios of C to B, relative to actin or rp49, calculated after logarithmic transformation of the ratios, followed by transformation back to a linear scale (Sokal and Rohlf, 1981). Note that only Z1-4.6 and Z2-6.0 increase in abundance during the wandering interval.

transcripts in whole-body larval RNA, smaller forms are more abundant, and novel bands of about 4.5 and 3.6 kb are detected (Fig. 1, right). This difference in the BRC-Z3 transcript pattern between CNS and whole-larval RNA was detected with three different Z3 probes—Z3 region only, Z3 plus 38-untranslated region, and 38-untranslated region only (see Experimental Methods for details). This result contradicts a previously reported finding that all 4.5-kb BRC transcripts represent Z1 isoforms (Bayer et al., 1996a). This inconsistency most

Effect of the premetamorphic 20E peak. The paired lanes in Fig. 2 represent third larval instar CNS RNA at the beginning and end of the wandering interval. Larvae were reared on dye-supplemented food, and gut contents were used to assess developmental stage (see Experimental Methods). Blue-gutted (‘‘B’’) and cleargutted (‘‘C’’) stages correspond to times (i) prior to the premetamorphic rise in 20E levels and (ii) approaching the hormone peak, respectively (Huet et al., 1993; Andres and Thummel, 1994), and differ by 7–8 h (Restifo et al., 1995). BRC transcript levels in the CNS change relatively little during this interval (Fig. 2, bottom) despite the rapid rise in ecdysteroid levels (reviewed in Riddiford, 1993) and expression of EcR in many CNS regions (Truman et al., 1994). In fact, while Z1-4.6 and Z2-6.0 increase modestly, several transcripts decline in abundance. These findings contrast with the behavior of IMP-E1 transcripts which are induced fivefold in the CNS during this same interval (Restifo et al., 1995). These data suggest that BRC transcript levels in the CNS rise earlier during the third instar, and we have confirmed this for BRC-Z2 (Liu and Restifo, 1998). The sensitivity of BRC transcription in whole-body preparations to relatively low doses of 20E (Karim and Thummel, 1992) and the likelihood of low-amplitude elevations in 20E levels earlier in the third instar (see discussion in Thummel, 1996) are consistent with this interpretation. Nonetheless, these data do not rule out hormone sensitivity of BRC expression in the CNS of wandering larvae. Given that larger BRC transcripts likely represent partially processed intermediates and that 20E can regulate BRC posttranscriptionally as well as transcriptionally (Bayer et al., 1996a), in vivo transcript levels may not reveal the response to the hormone. Since the literature has focused on responses to the late larval

138 ecdysteroid peak, and since BRC mutants die because they cannot respond properly to the hormonal signal, it was important to pursue this question further. Induction by 20E in vitro. To determine whether the late larval CNS can respond to 20E by upregulating BRC expression, transcript accumulation was examined following primary organ culture using methods developed by Thummel and colleagues (Karim and Thummel, 1992; see Experimental Methods). Larvae were selected at the onset of wandering and whole-body organ preparations were cultured for 9 h, during which they were treated with 20E or 20E plus cycloheximide for varying times, or with unsupplemented medium. At the end of the culture interval, the CNS was dissected for RNA extraction and Northern blot analysis. The whole-organ culture method has the advantage of minimizing trauma to the CNS, which is extensively connected to other tissues. In addition, general effects of in vitro culture on gene expression are equalized by incubating all samples for the same duration. The dose of 20E used (5 3 1026 M) is sufficient to induce BRC in isolated salivary glands (Chao and Guild, 1986) and imaginal discs (Bayer et al., 1996a) and represents the high end of the dose–response curve for BRC induction in whole-larval organs (Karim and Thummel, 1992). Incubation in the presence of 20E alone resulted in the induction of all previously detected BRC-Z1, -Z2, and -Z3 transcripts in the CNS (Fig. 3). Peak accumulation occurred with 4–6 h of hormone exposure in vitro and declined at 8 h. When cycloheximide (7 3 1025 M) was included with 20E in the culture medium, BRC transcript levels rose more briskly and continued to increase after 6 h of hormone treatment. Thus, despite the modest changes in BRC transcript abundance during the premetamorphic rise of 20E in vivo, the CNS responds to 20E in vitro with enhanced BRC expression. Both the time course of this effect and the lack of protein synthesis requirement are typical of a primary response to the hormone. The enhanced accumulation of BRC transcripts in the presence of cycloheximide could result from stabilization of RNA under conditions of protein synthesis blockade. However, it may also indicate that BRC expression is normally downregulated by newly synthesized proteins after its initial induction by 20E. Such a negative feedback loop was inferred from salivary gland puffing studies (Ashburner et al., 1974) and would limit the response to any given rise in 20E level, allowing a gene to come to a new baseline from which to respond to the next hormone pulse. In the presence of 20E alone, the relative abundance of members of the Z2 and Z3 trios did not change, nor was

Restifo and Hauglum

FIG. 3. Induction of BRC transcripts in the larval CNS by 20E in vitro. Northern blot analysis of total RNA (10 µg per lane) from CNS dissected from larval tissues cultured in vitro for 9 h. Df(BRC) denotes a sample of 1 µg poly(A) 1 RNA from deficiency larvae (not cultured), included as a negative control for hybridization. Dextran sulfate (6.25%) was used during hybridization with the Z2-specific probe. The headings above the lanes reflect the number of hours the tissues were incubated in the presence of 20E (5 3 1026 M) or 20E plus cycloheximide (CH; 7 3 1025 M). Note that the 0-h lane represents CNS cultured for 9 h without 20E or CH. The blot was probed sequentially with zinc-finger-specific BRC riboprobes (for Z3, the 38-UTR probe was used). The lower part of the blot was probed for rp49 transcripts. All BRC transcripts are 20E-inducible, with peak accumulation at 4–6 h. The addition of CH causes a striking enhancement of hormonal induction, without the decline at 8 h. Moreover, when protein synthesis is inhibited, 20E causes a change in the relative abundance of Z3-containing transcripts, with a shift toward the smaller (6.0-kb) form and the appearance of a 4.5-kb form; on longer exposures, a 3.6-kb form was also seen.

there evidence of an isoform switch (to Z1 predominance) as occurs in imaginal discs (Bayer et al., 1996a). However, in the presence of 20E and cycloheximide, the relative abundance of Z3 transcripts shifted toward the smallest member of the original trio. Moreover, Z3-4.5 appeared with 4 h of hormone treatment and increased in abundance with longer treatments (Fig. 3). On longer autoradiographic exposures, Z3-3.6 was also detectable (data not shown). Note that the small Z3 transcripts appearing in the CNS when protein synthesis is blocked (Fig. 3) are similar in size to those seen in whole larvae

CNS Metamorphosis in Drosophila

FIG. 4. BRC transcripts in the CNS during the larval-to-pupal transition in vivo. Northern blot analysis of total RNA from larval, prepupal, and pupal CNS (20 per lane). The feeding 3L sample came from randomly selected foraging third instar larvae considerably smaller in size than wandering larvae. Prepupae were selected at pupariation (WPP denotes white prepupae), aged in humid chambers and restaged at specific times thereafter (2 h: P2, brown prepupae; 4 h: P3, bubble prepupae; 8 h: P4i, buoyant prepupae). Pupae were selected at head eversion and restaged 1–2 h (P5i) or 5 h (P5ii) later. Any animals not displaying the typical features expected at these times were discarded. Only the most abundant Z3 transcript is shown. The Z2 data are from a separate experiment and were intentionally overexposed. Z1-4.6 and Z3-8.5 increase modestly during prepupal stages and then decline to lower but readily detectable levels in young pupae. Df(BRC) provides a negative control. Note that npr13 mutant CNS (which contain no BRC protein; Emery et al., 1994; data not shown) does nonetheless accumulate BRC transcripts. However, the relative signal strengths of Z3-8.5 and Z1 are reversed, and the Z1 transcript is larger than in wildtype (Canton-S).

(Fig. 1). These data suggest that 20E-plus-cycloheximide treatment allows the appearance of more mature Z3 transcripts whose accumulation in the CNS in vivo (Figs. 1 and 2) is undetectable because of their very short half-lives. The metamorphic transition in vivo. During the larval-to-pupal transition, BRC-dependent morphogenetic events in the CNS are underway (Restifo and White, 1991; L.L.R., unpublished observations). All the previously detected BRC transcripts continue to accumulate throughout prepupal development (WPP–P4i), with Z1-4.6 and Z3-8.5 levels increasing somewhat (Fig. 4). All transcripts decline after pupation (P5i and P5ii), a developmental transition triggered by a medium-sized

139 20E peak about 10 h after puparium formation (Riddiford, 1993). Z3-8.5 remains the predominant Z3 form, with no Z3 transcripts smaller than 6 kb detectable (data not shown). Taken together, the in vivo and in vitro data are consistent with a direct role for 20E in regulating BRC expression in the late larval–prepupal CNS. In reconciling the clear effect of 20E in vitro with the in vivo transcript profiles, one must consider that many of the BRC transcripts are incompletely processed (see above) and that posttranscriptional effects of 20E are possible (Bayer et al., 1996a). Transcript levels represent the net difference between synthesis and degradation. If 20E increases both transcription and turnover rates in vivo, then overall transcript abundance could remain stable as hormone levels rise, or one might see a delayed increase—as in the in vivo larval-to-pupal profile (Fig. 4). The large size of the BRC (100 kb; DiBello et al., 1991) would also delay increases in transcript levels. In addition, hormone exposure in vitro is more abrupt than that which occurs in vivo and, given the 20E dose, may represent a supraphysiological stimulus to the tissues. CNS maturation continues after adult emergence, with programmed cell death in the thoraco-abdominal ganglion in response to declining levels of 20E (Kimura and Truman, 1990; Robinow et al., 1993) and DNA synthesis in the mushroom body neuroblasts (Technau, 1984). As a first step toward determining whether BRC plays a role in posteclosion development, BRC transcripts were examined by in situ hybridization and Northern blotting using young adult flies. Adult heads contain BRC transcripts of all major zinc-finger isoforms (Fig. 5) and at least some of these come from the CNS (Fig. 6). Remarkably, all adult head transcripts are very large; in the case of Z1 and Z2 they are .10.5 kb. This suggests that transcript metabolism is different in the adult CNS, perhaps with processing much slower than in younger CNS tissue. In contrast, BRC transcripts in adult bodies have size distributions similar to those in larvae (Fig. 5). As in larvae (Restifo et al., 1995), all major cellular regions of the adult CNS express BRC transcripts (Fig. 6). However, in adults, optic lobe expression levels are noticably lower than those in the brain and SEG. There are also ‘‘hot-spots,’’ for example, a cluster of cells at the top of the cervical connective as it joins the SEG (Fig. 6C), and cells surrounding the mushroom body calyces (Fig. 6A). Immunostaining of adult brains with an anti-BRcore antibody reveals a similar pattern, with clusters of brightly labeled cells and with optic lobes having an overall much lower staining intensity (data not shown). Thus, either the huge transcripts detectable

140

FIG. 5. Persistence of BRC transcripts in adults. Northern blot analysis of poly(A)1 RNA from heads (5 µg; representing about 1000 heads) and bodies (10 µg; about 180 bodies) of 1- to 2-day-old Canton-S adults. Aliquots of head and body RNA were loaded in triplicate and run on the same gel. Poly(A) 1 RNA (3.5 µg) from Df(BRC) larvae was included as a negative control. The blot was cut into three panels that were hybridized in parallel using zinc-fingerspecific probes (for Z3, the 38-UTR probe was used). The lower part of the blot was probed for rp49 transcripts. Adult bodies express BRC transcripts, in sizes similar to those seen in larval stages. Adult heads also express BRC transcripts, but only of very large sizes. A Z3-4.5 band in body RNA is obscured by an unfortunate splotch of spurious labeling of the blot membrane, but has been seen in independent experiments with the Z3 1 38-UTR probe.

in adults by Northern analysis are translated or those BRC proteins have persisted from an earlier time in development. These data place BRC transcription factors in the adult brain where they could be instrumental in mediating neural plasticity. For example, BRC transcripts are abundant in the vicinity of the adult mushroom bodies, where DNA synthesis and experience-dependent changes in axon number have been reported (Technau, 1984). Mushroom body neurons undergo reorganization during metamorphosis (Technau and Heisenberg, 1982) and 20E may promote these events (Kraft et al., 1997). They are likely to be modified, at least functionally, during adult life because of their involvement in learning and memory (reviewed in Davis, 1993). It is tempting to speculate that BRC transcription factors play a modulatory role in these developmental and adult functions.

IMP-E1 Induction Is Independent of BRC Function IMP-E1 is directly inducible by 20E in imaginal discs (Natzle, 1993) and the CNS (Restifo et al., 1995) in vitro.

Restifo and Hauglum

We used mutants lacking all BRC function (npr13, a null allele) to confirm that IMP-E1 induction is independent of BRC transcription factors. Short-term in vitro organ culture was essential for this experiment because of several problems regarding staging and tissue integrity of BRC null mutant animals. First, BRC2 wandering larvae have highly variable rates of gut clearing (L.L.R., unpublished observations) and they do not pupariate (hence npr denotes nonpupariating). Thus, whereas we had previously used blue-gutted larvae, clear-gutted larvae, and white prepupae to examine induction of IMP-E1 in vivo (Restifo et al., 1995), this is not possible in null mutants. Second, the CNS and other internal tissues of BRC2 larvae degenerate during wandering (Restifo and White, 1991; L.L.R., unpublished observations). Finally, in vitro culture methods ensured that mutant and wild-type tissues experienced identical hormone levels. Mutant larvae were selected just as they commenced wandering, when they still have normal vigor, and their organs were cultured (as above) with or without 20E (5 3 1026 M) for 5 h (total culture time was 6 h; see Experimental Methods). Canton-S organs were tested in parallel as wild-type controls. Thereafter, CNS RNA was harvested for Northern analysis. Basal in vivo levels of IMP-E1 transcripts in BRC 2 [npr13 or Df(BRC)] CNS were comparable to those of wild-type CNS (Fig. 7). In two independent in vitro culture experiments, IMP-E1 transcript induction in BRC null mutant CNS was within the wild-type range (wildtype: 7.0- and 1.5-fold increase; BRC null: 3.3- and 5.6-fold increase). The magnitude of in vitro induction closely mirrors the in vivo rise in the CNS of wandering larvae (2- to 10-fold, with an average of 5-fold; Restifo et al., 1995). Thus, 20E-induced upregulation of IMP-E1 expression in the CNS occurs in the absence of all BRC transcription factors. This feature distinguishes IMP-E1 from a number of other primary response genes (e.g., E74 and E75) whose full induction by 20E requires BRC 1 function (Karim et al., 1993). These data indicate that there are at least two 20E-regulated genetic pathways operating in the CNS during the metamorphic transition, one BRC-dependent and the other BRC-independent.

BRC Protein Expression in the CNS in Vivo: Relation to DFD Using an anti-BRcore antibody (Emery et al., 1994), we found BRC proteins localized to nuclei in all major regions of the late larval CNS (Fig. 8). Nonneuronal nuclei, such as those of peripheral nerve glia (Fig. 8E)

CNS Metamorphosis in Drosophila

141

FIG. 6. BRC transcripts in the adult CNS. In situ hybridization, using a tritiated riboprobe representing BRcore plus Z1 sequences, to paraffin-embedded sections of heads from 1- to 2-day-old Canton-S adults. (A–C) A trio of horizontal sections from the same head (12-week exposure). Approximate planes of section are indicated in the frontal-view drawing in (D). A tracing of the outline of the CNS is shown on the left; the inner dotted line represents the boundary between the neuronal cell body layer (generally around the periphery) and the neuropil. The corresponding dark-field photomicrographs are shown on the right. Note that silver grains are prominent in all cell body regions of the CNS, with somewhat lower density in optic lobe areas. Asterisks indicate the location of silver grains overlying nonneural tissue. (D) Tracing of a frontal section showing approximate locations of horizontal sections in (A–C) and of enlargements in (E–I). (E–I) High-magnification bright-field photomicrographs from the single frontal section drawn in (D). 6-week exposure, independent experiment from that in A–C. Bar (in I), 10 µm. Silver grains can be seen overlying neuronal cell bodies (E) flanking the dorsal protocerebrum (DP) near the mushroom body calyx (ca); (F) flanking the subesophageal ganglion (SEG); (G) between the lateral horn of the dorsal protocerebrum and the dorsal portion of the lobula (Lo); (H) between the ventrolateral protocerebrum (VLP) and the ventral portion of the lobula; and (I) in the optic lobes (OL) between the medulla (Me) and the lobula complex (Lo and Lop). e, esophageal canal.

142

FIG. 7. Expression of IMP-E1 in wild-type and BRC mutant CNS in vivo and in vitro. Northern blot analysis of total RNA; 10 CNS per lane. Two independent experiments are shown. In both cases, basal levels of IMP-E1 in the CNS of blue-gutted wandering third instar larvae (B) are similar in Canton-S and BRC null mutants [left, Df(BRC); right, npr13 ]. The other samples represent CNS RNA harvested after larval organs were incubated in vitro for 5 h with (1) or without (2) 20E (5 3 1026 M). For the in vitro culture samples, all of the mutants were npr13. The bottom of each blot was probed for rp49 transcripts. In vitro induction of IMP-E1 (relative to rp49) is indicated (‘‘Relative D IMP-E1’’) under each pair of lanes, and, for BRC-null CNS, is within the wild-type range.

and the perineurium (data not shown), also express BRC. The midline of the ventral ganglion contains many BRC-expressing cells (Fig. 8D), which may be neurons (Robinow and White, 1991) or glia (Ito et al., 1995). Expression in the optic lobes, which undergoes BRCdependent developmental events (Restifo and White, 1991), was most extensive, with the vast majority of cells staining for BRC (Fig. 8B). In contrast, the brain, SEG, and TG show clusters of stained cells, often surrounding a central zone devoid of staining (Fig. 8C). The pattern of ‘‘holes’’ is reminiscent of the positions of proliferating neuroblasts (Truman and Bate, 1988; Ito and Hotta, 1992). Overall, the geographic pattern of anti-BRcore staining in the CNS remains stable during the larval– pupal transition, although optic lobe staining intensity and uniformity decline (Figs. 8–10). The identical SEG phenotypes in BRC and Dfd mutants have raised the question of whether these two genes act in concert to control SEG maturation. Since Northern analysis had revealed that Dfd transcript accumulation is normal in BRC null mutant CNS (data not shown) and since normal function of Hox-type transcription factors requires specific patterns of expression along the anterior–posterior axis (Keynes and Krumlauf, 1994), we asked whether BRC regulates spatial features of DFD expression (Diederich et al., 1991). In BRC null mutant CNS (Fig. 9A) the DFD expression pattern appears wild type—a discrete trapezoidal array of neuronal nuclei in a portion of the SEG just posterior to the esophageal foramen. Thus, SEG–TG separation failure in BRC mutants cannot be due to ectopic expression of DFD.

Restifo and Hauglum

We tested the hypothesis that BRC and DFD are coexpressed in neuronal nuclei of the SEG, where we postulated that they share common target genes involved in separation of the SEG from the TG (Restifo and Merrill, 1994). This model was appealing because both BRC and DFD have interaction domains for other proteins (Bardwell and Triesman, 1994; Sharkey et al., 1997). However, when we examined wild-type larval CNS double-labeled for BRC and DFD, we found that the two transcription factors were localized to nonoverlapping subsets of SEG neurons (Fig. 9B). In fact, the DFD(1) and BRC(1) cells are generally located in adjacent domains, with relatively little intermingling. Similarly, no overlap was detected in young pupae, just prior to the onset of SEG migration and cervical connective formation (Fig. 10B), or in adults (data not shown). The zonal separation between DFD- and BRCexpressing neurons suggests that they have different developmental histories. DFD expression begins during embyrogenesis (Chadwick and McGinnis, 1987) and most of the DFD(1) larval and pupal nuclei are deep and close to the neuropil, consistent with an embryonic origin (Truman and Bate, 1988). In contrast, most of the BRC(1) neurons are more superficial within the neuronal somata region. Thus, BRC may be restricted to postembryonically derived neurons of the SEG.

Concluding Remarks We have deciphered several pathways that operate in the Drosophila CNS during the early stages of the metamorphic transition. BRC, which is critical for CNS reorganization, is inducible by 20E in that tissue even during protein synthesis inhibition, indicating a primary response to the hormone. However, BRC function is not needed for hormonal induction of IMP-E1, a member of the primary response gene set that encodes ‘‘worker proteins.’’ Thus, in the larval CNS the ecdysone cascade splits relatively early into two limbs, one BRCdependent and the other BRC-independent. Deformed, a homeotic gene required for SEG maturation, is not coexpressed with BRC in neuronal nuclei of that structure, nor does its highly regulated spatial pattern of expression require the presence of BRC proteins. Therefore, BRC and Dfd may direct SEG morphogenesis by independent molecular means. It remains possible that products of their target genes interact to induce separation of the SEG from the TG. For example, a target gene of one may encode a secreted molecule, whose receptor is encoded by a target gene of the other.

CNS Metamorphosis in Drosophila

143

FIG. 8. BRC distribution in the larval CNS. Projections of optical sections, obtained by confocal microscopy, of immunostained CNS from a wild-type (Canton-S) wandering third instar larva, labeled with anti-BRcore and FITC-conjugated goat anti-mouse. (A) Low-magnification (103) view of the whole CNS. Note the dense labeling of the optic lobes (OL) and the clustered cells in the brain (BR), subesophageal ganglion (seg), and thoracoabdominal ganglion (TG). Asterisk indicates the approximate position of the peripheral nerves shown in (E). (B–E) Higher magnification of the same sample. Subsets of optical sections are shown to highlight specific features. (B) Sections through the optic lobe and lateral brain (403; 5 sections; 2-µm Z steps). Note the almost uniform staining of densely packed neuronal nuclei in the optic lobe and the clusters of cells in the adjacent brain. (C) Sections through the brain (403; 12 sections; 1-µm Z steps), highlighting the stained neuronal nuclei surrounding unstained zones, some of which are marked by a plus sign. (D) Sections through the middle of the abdominal region of the TG (203; 9 sections; 1-µm Z steps). Note the intense staining of midline cells and the neuronal cell body layer surrounding the unlabeled neuropil (np). (E) Nuclear staining of peripheral nerve glia lateral to the TG (403; 28 sections; 2-µm Z steps).

144

EXPERIMENTAL METHODS Drosophila strains and stocks. Canton-S was the wild-type laboratory strain, as in previous studies (Restifo and White, 1991). Animals were cultured on standard medium (Elgin and Miller, 1978) at 25°C, 40–70% relative humidity. Egg-laying conditions were adjusted to ensure optimal larval density, which was critical to the reliable use of bromphenol blue-supplemented food (0.05%) for staging of wandering larvae (Maroni and Stamey, 1983) relative to the premetamorphic peak of 20E (Huet et al., 1993; Andres and Thummel, 1994). Under these conditions, blue-gutted larvae leaving the food will pupariate, on average, 9 h later, while cleargutted larvae pupariate on average 1.5 h later. Additional criteria were applied at the time of dissection to minimize variation within the two groups. ‘‘Blue’’ samples were required to have an uninterrupted column of blue gut contents, including the lumen of the proventriculus, as an indication of recent cessation of feeding. ‘‘Clear’’ samples were required to show salivary gland bloating due to glue secretion, a 20E-induced event (Boyd and Ashburner, 1977). Prepupae and pupae were staged according to Bainbridge and Bownes (1981) after timing from pupariation or pupation (head eversion). Mutant strains have been previously described (Restifo and White, 1991; Restifo et al., 1995). BRC null mutant larvae (y npr13 w sn3/Y) were selected on the basis of yellow body and mouthhooks and clear Malpighian tubules. Larvae carrying overlapping deficiencies [Df(1)S39 cho2 /y2YSz280] that remove all genomic BRC sequences (Belyaeva et al., 1987; Sampedro et al., 1989; DiBello et al., 1991) were selected based on chocolate Malpighian tubules. In vitro organ culture. Blue-gutted larvae were selected as they wandered out of the food, rinsed in oxygenated Robb’s phosphate-buffered saline (Robb, 1969), and inverted to expose internal tissues after removal of their posterior ends. Larval organs were cultured in oxygenated saline at room temperature (23 6 1°C) in an oxygenated chamber as described by Karim and Thummel (1992) for a total of 6 or 9 h. After the first hour of incubation, which was without hormone, the saline was replaced. At various times thereafter, the culture medium was changed to saline containing 20E (5 3 1026 M) or 20E plus cycloheximide (7 3 1025 M) and incubation continued for the remainder of the culture period. RNA preparation and analysis. Tissue dissection, RNA extraction, formaldehyde/agarose gel electrophoresis, transfer to nylon membranes, hybridization, and

Restifo and Hauglum

washes were done by standard methods as previously described (Restifo et al., 1995). Quantitation of autoradiograms was done using a video camera and NIH Image 1.44 software. Comparisons of transcript abundance were made relative to rp49, which provides an indicator of sample loading and blotting efficiency (Andres et al., 1993) or to actin. To generate figures, autoradiograms were scanned using DeskScan II (Hewlett Packard) software and images were assembled in CorelDraw. In situ hybridization of tritiated probes to 6-µm sections of paraffin-embedded heads was done according to the method of Ingham et al. (1985), with the modifications of Robinow and White (1988). Single-stranded cRNA probes were synthesized in vitro from bacteriophage promoters on linearized plasmid templates (Melton et al., 1984) using [ 3H]UTP or [ 32P]UTP and [ 32P]CTP. BRC cDNA clones (DiBello et al., 1991; Bayer et al., 1996a) were gifts from G. Guild (University of Pennsylvania) and C. Bayer (University of California, Berkeley). To detect all BRC transcripts, pqdm527 was linearized with HindIII, and T7 polymerase synthesized a 3.1-kb antisense cRNA representing BRcore as well as Z1 regions. To generate a Z1-specific riboprobe, the same plasmid was cut with BamHI. To synthesize a Z2-specific riboprobe, HindIII and T7 polymerase were used on pqdm796. Several different Z3specific riboprobes were used. One represented the Z3 and 38-UTR in pqdm797 (NcoI, T3 polymerase), one represented only the 38-UTR in pqdm797 (AviII, T3 polymerase), and one represented only the Z3 region in subclone CB259 (BamHI, T3 polymerase). Under the conditions used, zinc-finger-specific probes do not crosshybridize. For IMP-E1, the template was a genomic clone (E1-8.0RI, provided by J. Natzle, University of California, Davis; Natzle et al., 1988) linearized with ApaI, and SP6 polymerase was used for cRNA synthesis. For actin, XhoI-digested paadm809 (a genomic subclone of Hd.19; Vigoreaux and Tobin, 1987) and T7 polymerase were used. Riboprobe length was checked by gel electrophoresis. The average IMP-E1 probe length was 4 kb, with a range of 2–8 kb; all other riboprobes were full-length. rp49 transcripts (O’Connell and Rosbash, 1984) were detected using random hexamer-primed, [ 32P]dUTP-labeled pHR0.6, a genomic subclone provided by C. Thummel (University of Utah). In CNS samples, Z1-4.6 and Z3-8.5 transcripts were far more abundant than any of the others, which hovered near the limits of detection on a ‘‘standard’’ Northern blot with RNA samples representing 20 CNSs (about 6 µg total RNA). Blots that had been probed previously for abundant transcripts were often unable to reveal the rare forms even when hybridized with

CNS Metamorphosis in Drosophila

FIG. 9. Deformed expression in the larval CNS: independent of BRC. Projection of optical sections, obtained by confocal microscopy, of immunostained CNS from wandering third instar larvae. (A) y npr13 wa sn3/Y labeled with anti-DFD and Cy3-conjugated goat anti-rabbit (203). The distribution of labeled nuclei in the SEG is indistinguishable from that in wild-type CNS. (B) Canton-S double-labeled with anti-DFD (red) and anti-BRcore (green). High-magnification (603; 16 sections; 1-µm Z steps) view of the SEG, comparable to the boxed region in (A). DFD and BRC proteins are found in nonoverlapping subsets of nuclei. Two ‘‘yellow’’ nuclei at the left lateral margin of the DFD(1) zone are in the shadow of overlying BRC(1) optic lobe or brain neurons.

145

FIG. 10. Broad Complex and Deformed proteins in the wild-type early pupal CNS. Projection of optical sections, obtained by confocal microscopy, of a double-immunolabeled CNS from a Canton-S P5i pupa (1 h after head eversion). (A) Low-magnification (103) view of the anti-BRcore labeling. The overall distribution is similar to that seen in the larval CNS, although the relative intensity and uniformity of optic lobe staining are reduced. (B) High-magnification view of the SEG (603; 19 sections; 1-µm Z steps), which is boxed in (A). Anti-DFD is shown in red; anti-BRcore in green. The overall pattern of DFD expression is very similar to that seen in larval CNS samples (see Fig. 9). DFD(1) and BRC(1) neuronal nuclei are intermingled and no nuclei stain for both proteins. The two faint yellowish zones result from partial overlap of different nuclei in different sections.

146 very-high-specific-activity riboprobes (estimated to be 2.5 3 109 cpm/µg). Thus, because of the considerable size overlap among the transcripts, it was sometimes impossible to assess the same RNA preparation for all BRC transcript forms. Immunocytochemistry. Sample preparation and confocal microscopy were performed as described in Sandstrom et al. (1997). Mouse monoclonal anti-BRcore (mAb25E9; Emery et al., 1994), a gift from G. Guild and V. Bedian (University of Pennsylvania), was used at 1:100 dilution. Anti-DFD, a rabbit polyclonal made against a fusion protein representing the open reading frame of cDNA pc41, was a gift from T. Kaufman (Indiana University) and used at 1:150 dilution. Images of optical sections were collected using Confocal Assistant software (T. C. Brelje) and figures were assembled using Corel PhotoPaint and CorelDraw. For the analysis of double-labeled samples, individual optical sections were merged to check for signal overlap.

ACKNOWLEDGMENTS We thank C. Bayer, J. Fristrom, G. Guild, J. Natzle, and C. Thummel for generously providing DNA clones; G. Guild and T. Kaufman for antibodies; F. Karim and C. Thummel for advice on organ culture; G. Guild and C. Bayer for discussing unpublished data; P. Jansma for help with confocal microscopy; W. Pott for help with computer graphics; P. Estes, K. Moore, and H.-B. Shen for assistance with experiments; and H. Foster and C. McGonigle for stock-keeping and media preparation. This work was supported by awards from NIH (NS30850) and the John Merck Fund (Scholarship Program in Developmental Disabilities in Children) to L.L.R.

REFERENCES Andres, A. J., Fletcher, J. C., Karim, F. D., and Thummel, C. S. (1993). Molecular analysis of the initiation of insect metamorphosis: A comparative study of Drosophila ecdysteroid-regulated transcription. Dev. Biol. 160: 388–404. Andres, A. J., and Thummel, C. S. (1994). Methods for quantitative analysis of transcription in larvae and prepupae. In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology (L. S. B. Goldstein and E. A. Fyrberg, Eds.), Vol. 44, pp. 565–573. Academic Press, San Diego. Ashburner, M., Chihara, C., Meltzer, P., and Richards, G. (1974). Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38: 655–662. Bainbridge, S. P., and Bownes, M. (1981). Staging the metamorphosis of Drosophila melanogaster. J. Embryol. Exp. Morphol. 66: 57–80. Bardwell, V. J., and Treisman, R. (1994). The POZ domain: A conserved protein–protein interaction motif. Genes Dev. 8: 1664–1677. Bayer, C. A., Holley, B., and Fristrom, J. W. (1996a). A switch in Broad-Complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177: 1–14.

Restifo and Hauglum

Bayer, C. A., von Kalm, L., and Fristrom, J. W. (1996b). Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis. In Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells (L. I. Gilbert, J. R. Tata, and B. G. Atkinson, Eds.), pp. 322–361. Academic Press, San Diego. Bayer, C. A., von Kalm, L., and Fristrom, J. W. (1997). Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Dev. Biol. 187: 267–282. Belyaeva, E. S., Aizenzon, M. G., Semeshin, V. F., Kiss, I., Koczka, K., Baritcheva, E. M., Gorelova, T. D., and Zhimulev, I. F. (1980). Cytogenetic analysis of the 2B3-4—2B11 region of the X-chromosome of Drosophila melanogaster. I. Cytology of the region and mutant complementation groups. Chromosoma 81: 281–306. Belyaeva, E. S., Protopopov, M. O., Baricheva, E. M., Semeshin, V. F., Izquierdo, M. L., and Zhimulev, I. F. (1987). Cytogenetic analysis of region 2B3-4—2B11 of the X-chromosome of Drosophila melanogaster. VI. Molecular and cytological mapping of the ecs locus and the 2B puff. Chromosoma 95: 295–310. Boyd, M., and Ashburner, M. (1977). The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro. J. Insect Physiol. 23: 517–523. Chadwick, R., and McGinnis, W. (1987). Temporal and spatial distribution of transcripts from the Deformed gene of Drosophila. EMBO J. 6: 779–789. Chao, A. T., and Guild, G. M. (1986). Molecular analysis of the ecdysterone-inducible 2B5 ‘‘early’’ puff in Drosophila melanogaster. EMBO J. 5: 143–150. Crossgrove, K., Bayer, C. A., Fristrom, J. W., and Guild, G. M. (1996). The Drosophila Broad-Complex early gene directly regulates late gene transcription during the ecdysone-induced puffing cascade. Dev. Biol. 180: 745–758. Davis, R. L. (1993). Mushroom bodies and Drosophila learning. Neuron 11: 1–14. DiBello, P. R., Withers, D. A., Bayer, C. A., Fristrom, J. W., and Guild, G. M. (1991). The Drosophila Broad-Complex encodes a family of related, zinc finger-containing proteins. Genetics 129: 385–397. Diederich, R. J., Pattatucci, A. M., and Kaufman, T. C. (1991). Developmental and evolutionary implications of labial, Deformed and engrailed expression in the Drosophila head. Development 113: 273–281. Dubrovsky, E. B., Dretzen, G., and Bellard, M. (1994). The Drosophila Broad-Complex regulates developmental changes in transcription and chromatin structure of the 67B heat-shock gene cluster. J. Mol. Biol. 241: 353–362. Dubrovsky, E. B., Dretzen, G., and Berger, E. M. (1996). The BroadComplex gene is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene. Mol. Cell. Biol. 16: 6542–6552. Elgin, S. R., and Miller, D. W. (1978). Mass rearing of flies and mass production and harvesting of eggs. In The Genetics and Biology of Drosophila (M. Ashburner and T. R. F. Wright, Eds.), Vol. 2a, pp. 112–121. Academic Press, New York. Emery, I. F., Bedian, V., and Guild, G. M. (1994). Differential expression of Broad-Complex transcription factors may forecast distinct developmental tissue fates during Drosophila metamorphosis. Development 120: 3275–3287. Forrest, D. (1994). The erbA/thyroid hormone receptor genes in development of the central nervous system. Semin. Cancer Biol. 5: 167–176. Fristrom, D. K., Fekete, E., and Fristrom, J. W. (1981). Imaginal disc development in a non-pupariating lethal mutant in Drosophila melanogaster. Roux’s Arch. Dev. Biol. 190: 11–21.

CNS Metamorphosis in Drosophila

Garcı´a-Segura, L. M., Chowen, J. A., Pa´rducz, A., and Naftolin, F. (1994). Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms. Prog. Neurobiol. 44: 279–307. Guay, P. S., and Guild, G. M. (1991). The ecdysone-induced puffing cascade in Drosophila salivary glands: A Broad-Complex early gene regulates intermolt and late gene transcription. Genetics 129: 169– 175. Hodgetts, R. B., Clark, W. C., O’Keefe, S. L., Schouls, M., Crossgrove, K., Guild, G. M., and von Kalm, L. (1995). Hormonal induction of Dopa decarboxylase in the epidermis of Drosophila is mediated by the Broad-Complex. Development 121: 3913–3922. Huet, F., Ruiz, C., and Richards, G. (1993). Puffs and PCR: The in vivo dynamics of early gene expression during ecdysone responses in Drosophila. Development 118: 613–627. Ingham, P. W., Howard, K. R., and Ish-Horowicz, D. (1985). Transcription pattern of the Drosophila segmentation gene hairy. Nature 312: 439–445. Ito, K., and Hotta, Y. (1992). Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster. Dev. Biol. 149: 134–148. Ito, K., Urban, J., and Technau, G. M. (1995). Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval nerve cord. Roux’s Arch. Dev. Biol. 204: 284–307. Karim, F. D., and Thummel, C. S. (1992). Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO J. 11: 4083–4093. Karim, F. D., Guild, G. M., and Thummel, C. S. (1993). The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis. Development 118: 977–988. Keynes, R., and Krumlauf, R. (1994). Hox genes and regionalization of the nervous system. Annu. Rev. Neurosci. 17: 109–132. Kimura, K., and Truman, J. W. (1990). Postmetamorphic cell death in the nervous and muscular systems of Drosophila melanogaster. J. Neurosci. 10: 403–411. Kiss, I., Beaton, A. H., Tardiff, J., Fristrom, D., and Fristrom, J. W. (1988). Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster. Genetics 118: 247–259. Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P., and Hogness, D. S. (1991). The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67: 59–77. Kraft, R., Levine, R. B., and Restifo, L. L. (1997). Ecdysone enhances the growth of Drosophila pupal mushroom body neurons in cell culture. Soc. Neurosci. Abstr. 23: 60. Lepesant, J. A., Maschat, F., Kejzlarova´-Lepesant, J., Benes, H., and Yanicostas, C. (1986). Developmental and ecdysteroid regulation of gene expression in the larval fat body of Drosophila melanogaster. Arch. Insect Biochem. Physiol. Supp. 1:133–141. Levine, R. B., Fahrbach, S. E., and Weeks, J. C. (1991). Steroid hormones and the reorganization of the nervous system during insect metamorphosis. Semin. Neurosci. 3: 437–447. Levine, R. B., Morton, D. B., and Restifo, L. L. (1995). Remodeling of the insect nervous system. Curr. Opin. Neurobiol. 5: 28–35. Liu, E., and Restifo, L. L. (1998). Identification of a Broad Complexregulated enhancer in the developing visual system of Drosophila. J. Neurobiol. 34: 253–270. Luine, V. N., and McEwen, B. S. (1983). Sex differences in cholinergic enzymes of diagonal band nuclei in the rat preoptic area. Neuroendocrinology 36: 475–482. Mahaffey, J. W., Diederich, R. J., and Kaufman, T. C. (1989). Novel

147 patterns of homeotic protein accumulation in the head of the Drosophila embryo. Development 105: 167–174. Maroni, G., and Stamey, S. C. (1983). Use of blue food to select synchronous, late third instar larvae. Drosophila Info. Serv. 59: 142–143. McGinnis, W., Jack, T., Chadwick, R., Regulski, M., Bergson, C., McGinnis, N., and Kuziora, M. A. (1990a). Establishment and maintenance of position-specific expression of the Drosophila homeotic selector gene Deformed. In Genetic Regulatory Hierarchies in Development (T. R. F. Wright, Ed.), Vol. 27, pp. 363–402. Academic Press, San Diego. McGinnis, N., Kuziora, M., and McGinnis, W. (1990b). Human Hox-4.2 and Drosophila Deformed encode similar regulatory specificities in Drosophila embryos and larvae. Cell 63: 969–976. Meier, C. A. (1997). Regulation of gene expression by nuclear hormone receptors. J. Recept. Signal Transduct. Res. 17: 319–335. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12: 7035–7056. Natzle, J. E. (1993). Temporal regulation of Drosophila imaginal disc morphogenesis: A hierarchy of primary and secondary 20-hydroxyecdysone-responsive loci. Dev. Biol. 155: 516–532. Natzle, J. E., Fristrom, D. K., and Fristrom, J. W. (1988). Genes expressed during imaginal disc morphogenesis: IMP-E1, a gene associated with epithelial cell rearrangement. Dev. Biol. 129: 428– 438. O’Connell, P., and Rosbash, M. (1984). Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res. 12: 5495–5513. Parsons, B., Rainbow, T. C., Pfaff, D. W., and McEwen, B. S. (1981). Oestradiol, sexual receptivity and cytosol progestin receptors in rat hypothalamus. Nature 292: 58–59. Pipa, R. L. (1973). Proliferation, movement, and regression of neurons during the postembryonic development of insects. In Developmental Neurobiology (D. Young, Ed.), pp. 105–129. Cambridge Univ. Press, Cambridge. Restifo, L. L., and White, K. (1991). Mutations in a steroid hormoneregulated gene disrupt the metamorphosis of the central nervous system in Drosophila. Dev. Biol. 148: 174–194. Restifo, L. L., and White, K. (1992). Mutations in a steroid hormoneregulated gene disrupt the metamorphosis of internal tissues in Drosophila: Salivary glands, muscle, and gut. Roux’s Arch. Dev. Biol. 201: 221–234. Restifo, L. L., and Merrill, V. K. L. (1994). Two Drosophila regulatory genes, Deformed and the Broad-Complex, share common functions in development of adult CNS, head, and salivary glands. Dev. Biol. 162: 465–485. Restifo, L. L., Estes, P. S., and DelloRusso, C. (1995). Genetics of ecdysteroid-regulated central nervous system metamorphosis in Drosophila. Eur. J. Entomol. 92: 169–187. Riddiford, L. M. (1993). Hormones and Drosophila development. In The Development of Drosophila melanogaster (M. Bate and A. Martinez Arias, Eds.), pp. 899–939. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Robb, J. A. (1969). Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media. J. Cell Biol. 41: 876–885. Robinow, S., and White, K. (1988). The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev. Biol. 126: 294–303. Robinow, S., and White, K. (1991). Characterization and spatial

148 distribution of the ELAV protein during Drosophila melanogaster development. J. Neurobiol. 22: 443–461. Robinow, S., Talbot, W. S., Hogness, D. S., and Truman, J. W. (1993). Programmed cell death in the Drosophila CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform. Development 119: 1251–1259. Sampedro, J., Galceran, J., and Izquierdo, M. (1989). Mutation mapping of the 2B5 ecdysone locus in Drosophila melanogaster reveals a long-distance controlling element. Mol. Cell. Biol. 9: 3588–3591. Sandstrom, D. J., Bayer, C. A., Fristrom, J. W., and Restifo, L. L. (1997). Broad-Complex transcription factors regulate thoracic muscle attachment in Drosophila. Dev. Biol. 181: 168–185. Sharkey, M., Graba, Y., and Scott, M. P. (1997). Hox genes in evolution: Protein surfaces and paralog groups. Trends Genet. 13: 145–151. Sokal, R. R., and Rohlf, J. F. (1981). Biometry. Freeman, New York. Stewart, M., Murphy, C., and Fristrom, J. W. (1972). The recovery and preliminary characterization of X chromosome mutants affecting imaginal discs of Drosophila melanogaster. Dev. Biol. 27: 71–83. Technau, G. M. (1984). Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience. J. Neurogenet. 1: 113–126. Technau, G., and Heisenberg, M. (1982). Neural reorganization during metamorphosis of the corpora pedunculata in Drosophila melanogaster. Nature 295: 405–407. Thummel, C. S. (1996). Flies on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genet. 12: 306–310. Truman, J. W. (1988). Hormonal approaches for studying nervous system development in insects. Adv. Insect Physiol. 21: 1–34. Truman, J. W. (1996). Steroid receptors and nervous system metamorphosis in insects. Dev. Neurosci. 18: 87–101.

Restifo and Hauglum

Truman, J. W., and Bate, M. (1988). Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev. Biol. 125: 145–157. Truman, J. W., Talbot, W. S., Fahrbach, S. E., and Hogness, D. S. (1994). Ecdysone receptor expression in the CNS correlates with stagespecific responses to ecdysteroids during Drosophila and Manduca development. Development 120: 219–234. Tublitz, N. J. (1993). Steroid-induced transmitter plasticity in insect peptidergic neurons. Comp. Biochem. Physiol. 105C: 147–154. Vigoreaux, J. O., and Tobin, S. L. (1987). Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster. Genes Dev. 1: 1161–1171. von Kalm, L., Crossgrove, K., Von Seggern, D., Guild, G. M., and Beckendorf, S. K. (1994). The Broad-Complex directly controls a tissue-specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis. EMBO J. 13: 3505–3516. Weeks, J. C., and Levine, R. B. (1992). Endocrine influence on the postembryonic fate of identified neurons during insect metamorphosis. In Determinants of Neuronal Identity (S. M. Shankland and E. R. Macagno, Eds.), pp. 293–322. Academic Press, New York. Weeks, J. C., Davidson, S. K., and Debu, B. H. (1993). Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm, Manduca sexta. J. Neurobiol. 24: 125–140. Zollman, S., Godt, D., Prive´, G. G., Couderc, J.-L., and Laski, F. A. (1994). The BTB domain, found primarily in zinc-finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91: 10717–10721. Received September 19, 1997 Revised March 5, 1998 Accepted March 18, 1997