DEVELOPMENTAL

BIOLOGY

148,174-194

(1991)

Mutations in a Steroid Hormone-Regulated Gene Disrupt the Metamorphosis of the Central Nervous System in Drosophila LINDA L. RESTIFO~~ANDKALPANAWHITE Department

of Biology, Brandeis

University,

Accepted August 6,

Waltham, Massachusetts

02254

1991

The actions of steroid hormones on vertebrate and invertebrate nervous systems include alterations in neuronal architecture, regulation of neuronal differentiation, and programmed cell death. In particular, central nervous system (CNS) metamorphosis in insects requires a precise pattern of exposure to the steroid molting hormone 20-hydroxyecdysone (ecdysterone). To test whether the effects of steroid hormones on the insect nervous system are due to changes in mutants of the ecdysterone-regulated locus, the Broad Complex patterns of gene expression, we examined Drosophila (BR-C). This report documents aspects of CNS reorganization which are dependent on BR-Cfunction. During wild-type metamorphosis, CNS components undergo dramatic morphogenetic movements relative to each other and to the body wall. These movements, in particular, the separation of the subesophageal ganglion from the thoracic ganglion, the positioning of the developing visual system, and the fusion of right and left brain hemispheres, are deranged in BR-C mutants. In addition, a subset of mutants shows disorganization of optic lobe neuropil, both within and among optic lobe ganglia. Optic lobe disorganization is found in mutants of the brand Z(1)ZBc complementation groups, but not in those of the rbp complementation group. This suggests that the three complementation groups of this complex locus represent distinct but overlapping functions necessary for normal CNS reorganization. This study demonstrates that ecdysteroneregulated gene expression is essential for CNS metamorphosis, illustrating the utility of Drosophila as a model system for investigating the genetic basis of steroid hormone action on the nervous system. o ISSI Academic ureas, ~nc.

tion of “intermolt” genes and initiates a cascade of gene activation, beginning with a small number of “early” During metamorphosis, holometabolous insects ungenes, whose protein products then activate a large numdergo dramatic alterations in external appearance and ber of “late” genes (Ashburner et aZ., 1974). Molecular behavioral repertoire. This developmental process ocgenetic analyses of several early genes provide support curs by a combination of programmed cell death, cell for this view of early gene function (Burtis et ah, 1990; proliferation and differentiation, and remodeling of old Segraves and Hogness, 1990; Urness and Thummel, structures for new functions, along with major morpho1990; DiBello et ah, 1991). genetic movements (reviewed in Riddiford, 1985). MetaCentral nervous system (CNS) reorganization during morphosis is triggered and orchestrated by the steroid insect metamorphosis represents a fascinating strategy molting hormone, 20-OH-ecdysone (ecdysterone) (refor dealing with the distinct but overlapping neural reviewed in Granger and Bollenbacher, 1981). A considerquirements of larvae and adults. Unlike the complete able body of work on insect salivary gland model sysreplacement of the epidermis, CNS metamorphosis is tems (reviewed in Richards, 1980;Ashburner, 1990) demcharacterized by reconstruction and reorganization, inonstrates that the actions of ecdysterone are mediated volving neurogenesis and neuronal differentiation, regulargely (although not necessarily exclusively) by alter- lated cell death, and changes in neuronal architecture ations in patterns of gene expression. Other avenues of and function (Edwards, 1969; Pipa, 1973). These compoinvestigation, in particular, using imaginal discs and nents of CNS metamorphosis have been shown, in a varitissue cultures cell lines, further indicate that these ef- ety of insect systems, to be under hormonal control fects occur via interaction of ecdysterone with a specific (reviewed in Weeks and Levine, 1990). Furthermore, receptor protein (reviewed in Yund and Osterbur, 1985). steroid hormones (especially reproductive steroids) regThe studies on Drosophila salivary gland polytene chro- ulate neuronal birth, death, and structural alteration in mosomes gave rise to a model in which ecdysterone, via vertebrate nervous systems as well (Pfaff and McEwen, a hormone-receptor complex, represses the transcrip1983; Arnold and Gorski, 1984; Meyer, 1985). Taken together, the documented effects of ecdysterone on the nervous system and on gene activity lead to ’ To whom correspondence should be addressed. the hypothesis that some aspects of CNS reorganization * Current address: ARL Division of Neurobiology, University of Ariduring metamorphosis are mediated by the hormonal zona, Tucson, AZ 85721. INTRODUCTION

0012-1606/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

174

RESTIFO AND WHITE

Drosophila

regulation of gene expression in the nervous system. A corollary of this hypothesis is that some mutations in ecdysterone-regulated genes should alter CNS metamorphosis. To test this corollary, we conducted a histological analysis of mutants of an ecdysterone-regulated gene, the Broad Complex (BR-Q. This gene was chosen for investigation in part because of our preliminary findings that BR-C transcripts are detectable throughout the larval and adult CNS (Restifo and White, 1988). Here we show that lethal BR-C mutants, despite apparently normal larval CNS structure, exhibit multiple defects of CNS maturation during metamorphosis. Located in the ecdysterone-inducible early puff at cytological position 2B3-5 (Belyaeva et al., 1987), the BR-C represents the only early gene candidate for which extensive genetic information is available (Fig. 1). Null mutations and deletions of the BR-C result in lethality at the end of the third and final larval instar, attesting to the absolute requirement of BR-c” function for normal metamorphosis (Stewart et al, 1972; Kiss et al, 19’76a; Belyaeva et ab, 1980; Kiss et al., 1988). A large number of mutants retaining partial BR-C function die at various points during prepupal and pupal life (Belyaeva et al., 1980; Zhimulev et al., 1982; Kiss et a,l., 1988). Several lines of evidence demonstrate that these developmental failures are not due to ecdysterone deficiency, but rather to defects in the response to the hormonal signal. The mutant phenotype cannot be rescued by wild-type ring gland transplantation into mutant larvae (Kiss et al, 19’78), by transplantation of imaginal discs into wild-type hosts (Stewart et al., 1972; Kiss et al., 1976a; Murphy et al., 1977; Fristrom et ah, 1981), or by in vitro culture with metamorphic concentrations of ecdysterone (Stewart et al., 1972; Kiss et al., 1976a; Murphy et al., 1977; Kiss and Molnar, 1980; Fristrom ef al., 1981). Furthermore, in genetic mosaics, wild-type epidermis undergoes normal pupariation, whereas mutant regions remain larval, indicating a cell-autonomous requirement for BR-c” function (Kiss et ah, 197613,1978). Complementation testing of a number of BR-C mutants demonstrates three lethal complementation groups: broad (br), reduced bristles on the palpus (rbp), and Z(1)2Bc (Belyaeva et al., 1980; Kiss et ah, 1988) (see Fig. 1C). Existing alleles include hypomorphic and amorphic mutations of the entire locus [e.g., 1(1)2Bab’ and np,rP, respectively], as well as hypomorphic and amorphic mutations of individual complementation groups (see Table 1 and references therein). Phenotypic analysis of imaginal disc development revealed two distinct functional domains within the BR-C, one required primarily for imaginal disc eversion and elongation (represented by Zrr and rbp mutants) and the other required for fusion of individual imaginal disc derivatives to

175

CNS Metarnorphasisis

form a contiguous adult cuticle [represented by 1(1)2Bc mutants] (Kiss et al., 1988). In addition, only members of the b complementation group interact with mutations at the Stubble-stubbloid locus (Beaton et al., 1988). The pattern of CNS defects seen in this study also confirms similarities and differences among mutants of the three complementation groups. Given the compelling genetic evidence that the BR-C encodes trans-acting factor(s) which alter the expression of many other ecdysterone-responsive loci (Belyaeva et al., 1981; Zhimulev et al., 1982; Dubrovsky and Zhimulev, 1988; Crowley et al., 1984; Lepesant et al., 1986; Vijay Raghavan et al., 1988; Galceran et al., 1990; Guay and Guild, 1991), the phenotype patterns seen in BR-C mutants may provide clues to the genetic regulatory hierarchy underlying normal CNS metamorphosis. MATERIALS

Strains

AND

METHODS

and Stocks

A Canton-S strain of wild-type Llrosophjila melanogasfrom J. Hall (Brandeis University), was used for comparison with mutants. For the purpose of collecting wild-type or mutant individuals for analysis, larvae were reared at low density on cornmeal/yeast/ agar medium (1% agar, 7% inactivated yeast, 7% cornmeal, 0.2% Lexgard mold inhibitor) at 25”C, 60-70% relative humidity. White prepupae were selected from the walls of their culture vials, separated by sex, and placed on moistened ashless filter paper (Whatman No. 42) in glass petri dishes, and the time was noted. (“Regular” filter paper, e.g., Whatman No. 1, resulted in a high frequency of lethality, especially at the prepupal-pupal boundary.) Rearing was continued under the conditions noted above. Individuals were subsequently staged according to anatomical criteria, as described by Bainbridge and Bownes (1981). The prepupal stages are recognized as follows: Pl, white prepupa; P2, brown prepupa; P3, bubble develops in abdominal region; and P4, prepupa becomes buoyant, followed by rapid anterior movement of the bubble. True pupation takes place during the moving bubble stage (at about 12 hr after puparium formation when animals are raised at 25°C). The pupal stages ensue and are identified as follows: P5, the Malpighian tubules migrate and become visible as white structures in the dorsal anterior abdomen; P6, Malpighian tubules turn green; P7, “yellow body” (actually dark green) becomes visible between the Malpighian tubules; P8, eyes become yellow; P9, eyes become amber; PlO, eyes become red; Pll, head bristles, followed by thoracic bristles, darken; P12, wings become grey; P13, wings become black; P14, leg bristles and claws darken; P15, meconium visible in posterior abdomen, followed ter, obtained

1’76

DEVELOPMENTAL BIOLOGY TABLE

VOLUME 1481991 1

ALLELES AND CHROMOSOMESUSED IN THIS STUDY Mutant

Mutant

class”

broad, 1(1)2Ba

reduced bristles on palpus, 1(1)2Bb l(@Bab 1(1)2Bc nonpupariating,

1(1)2Bd

Current designation

Alternative designation(s)

br’ bvJ bP ti’ rbp’ rb;vl 1(1)2Bab’ 1(1)2Bc’ 1(1)2BBc2 nprl’ nprY

tYr 1t35 It103 or In(1)2B3-4;3Cl 1t99 lt358 1t4 1t10; 1(1)pp-1’ It76 l(l)d.norm.-12 or nprl Tp(1;3)2B5;61F3-4 Chromosomal

Designation

~967~ g YSZ280 Df(IJS.39 Df(l)mal-1.3

alleles

Chromosome Dp(l;Y)y%7g*** Dp(l;Y)#Sz280* Df(1)S.U we spl v f car* Df(l)mal-13, sc8 B****

duplications

Origin* and type”

Chromosome br’* or y br’ ybrJ* !4 @* y b? w* y rbp’* y rbp4* y l(l)PBab’* y 1(1)2Bc’* y 1(1)2BP y nprl’ w* or y nm-1 w sn’** y npr1’ w*

Span EMS EMS DEB EMS EMS EMS EMS EMS EMS DEB

(1)

(2) C46) (3)

(2) (2) (2) (2) (2) (4) (3)

and deficiencies

Cytology Dp(l;Y)lA;2B17-18 and 20A3 to base of X Dp(l;Y)lA;2Cl-2, with internal Df(1)2B3-4;2B7-8 Df(l)lEl-2;2B5-6 Df(l)lSA to base of X

Comments

Ref.

Covers BR-C Uncovers BR-C Uncovers BR-C Uncovers proximal portion of X duplicated in $Y67g

(2, 5, 6)

0 Based on Lindsley and Zimm (1985). * Spon, spontaneous; EMS, ethylmethane sulfonate; DEB, diepoxybutane. ’ v, viable; 1, lethal; n, null; h, hypomorphic. * Obtained from J. Fristrom, University of California, Berkeley. ** Obtained from Drosophila stock center, Umea, Sweden. *** Obtained from V. Raghaven, CalTech. **** Obtained from T. Tully, Brandeis University. Note. References (1) Morgan et al., 1925; (2) Belyaeva et al., 1980; (3) Kiss et al., 1988; (4) Kiss et al., 1976a; (5) Craymer Belyaeva et al., 1982; (7) Belyaeva et al., 1987; (8) Schalet and Lefevre, 1976.

by eclosion. See Bainbridge and Bownes (1981) for additional details of staging. Although strictly speaking “pharate adult” refers to all stages following the separation of pupal cuticle and adult epidermis (approximately P7-P8), for the sake of general discussion, the term is used to refer to individuals who have attained red eye pigmentation and darkened bristles (i.e., in the final day of metamorphosis, Pll-P15). Mutant strains were obtained from the sources indicated in Table 1. See Lindsley and Grell (1968) for descriptions of markers. Chromosomes bearing lethal BR-C alleles were maintained in females, in combination with the Binsn balancer chromosome (Lindsley and Zimm, 1990), and/or in males, in combination with a Y-borne duplication [Dp(l;Y)gY67g; see Fig. lB], in a stock with attached-X females. Viable mutants were usually maintained as homozygous stocks and as males

(v, h) (1, n) (1, h) (v, h) (1, h) (1, h) (1, h) (1, n) (1, h) (1, n) (1, n)

Ref.

(67) (2) (8)

and Roy, 1980; (6)

over duplication. To generate females bearing heteroallelic combinations of BR-C alleles, the following scheme was generally used: y allele a/Binsn

virgin females X

y allele b/$Y67g

males.

The majority of crosses were carried out at 25°C. However, because of the temperature dependence (both cold and heat sensitivity) of some BR-C mutant phenotypes (Kiss et al., 19SS), some crosses were done at 18°C or 29°C to maximize or minimize lethality. Selection and Staging of Mutants

Mutants were generally selected at the wandering third larval instar stage, by virtue of sex and yellow (y) mouthhooks and denticle belts, and placed on soft larval

RESTIFO

AND WHITE

Drosoph iln CNS Metu mwphosis

food (0.4% agar, 5% sucrose, 5% yeast extract, 2% inactivated yeast, with propionic acid:phosphoric acid::9:1, 0.5 ml per 100 ml total, added as mold inhibitor; Valles and White, 1986). After pupariation, the mutant individuals were placed on filter paper as above. The selected mutants were allowed to continue development at their original rearing temperature. In the majority of cases, the mutants developed more slowly, and with more variable rates, than did wild-type pupae. This necessitated scoring the developmental stages of individual mutants, according to anatomical criteria (Bainbridge and Bownes, 1981), for comparison with the corresponding wild-type stage. The use of time alone (e.g., number of hours after puparium formation) would have resulted in significant staging errors. In some cases, mutants showed regional mosaicism in development, defying attempts at scoring. For example, right and left sides of the head, or the head and thorax, achieved different developmental endpoints. In those cases, the external appearance was described as fully as possible, and the mutant was processed individually. In some instances, internal structures, e.g., development of indirect flight muscles (consistently discernible at P7), helped establish a minimum stage of the specimen. In one class of mutants, the internal phenotypes themselves confounded the staging. Specifically, in mutants bearing alleles of the l(@?Bc complementation group, the Malpighian tubules and “yellow body” never appear in the dorsal abdomen (Restifo and White, in preparation). This resulted in a long time window, between the beginning of stage P5 and the onset of P8 (a period of approximately 36 hr in wild type, and even longer in the mutants), during which there was no clue from external examination as to developmental stage. Histology

In most cases, mutant prepupae or pupae were dissected and fixed once they had attained their maximum developmental potential, Dissections were performed in Droso$iZa buffered saline (130 mM NaCl, 4.7 mM KCl, 1.8 mMCaCl,, 0.74 mMKH,PO,, 0.35 mMNa,HPO,, pH 7). Larvae were anesthetized with cold and/or ether. Larvae and prepupae were fixed whole after careful removal of the anterior- and posterior-most cuticle. For preparation of pupae, the pupal case (i.e., the sclerotized larval cuticle) was entirely removed, and the posterior end of the animal was opened. In the case of late pupae, the cellophane-like pupal cuticle was peeled off. For late pupae and adults, the legs and wings were removed and, frequently, the distal proboscis was also removed. In the early phase of this study, because of concerns about accessibility of the CNS to the fixative, the entire abdo-

177

men was removed from late pupae and adults and the dorsal midline of the thorax was split. It quickly became clear that this was not necessary and, for the majority of such samples, the thoracic cuticle was left intact and only the posterior one-quarter of the abdomen was removed. Dissected samples were fixed in “FAAG” (Campos et al., 1985), essentially FAA (85% ethanol, 4% formaldehyde, 5% acetic acid; Strausfeld, 1976) with the addition of 25% glutaraldehyde (electron microscopy grade) to a final concentration of approximately 1% . Some samples were fixed in Alcoholic Bouin (Bouin-Duboscq; Humason, 1979); these are indicated in the figure legends. Fixation was done at 4°C overnight. Samples were dehydrated by washing in a graded ethanol series (85, 95, lOO%), ending with four 30-min washes in absolute ethanol. Samples were cleared in xylene (three 30-min intervals) and transferred to Paraplast-Plus (Monoject) embedding media at 58°C. After several changes of Paraplast-Plus at lo-min intervals, infiltration was done in a vacuum oven at 58°C for 2-16 hr. Samples were oriented for sectioning in the horizontal, sagittal, or frontal plane. Six-micron serial sections were cut with disposable razor blades (Schick Platinum-Plus) on a Sorvall JB-4 microtome and mounted on gelatin-coated slides. The majority of slides were stained with Mayer hematoxylin, followed by the rapid trichrome method of Pollak (Humason, 1979). A small number of slides were stained with Mayer hematoxylin alone or with toluidine blue; these are indicated in the figure legends. Photomicrographs depicting sections cut in the sagittal plane are always oriented such that anterior is to the right. The terms anterior, posterior, dorsal and ventral are all used with respect to the axis of the whole animal. Sections were viewed by light microscopy using bright-field and phase-contrast optics (the latter was essential for optic lobe organization assessment). All photomicrographs are of bright-field images unless otherwise indicated in the figure legends. Photography was done with Kodak Panatomic X film, processed and printed according to the manufacturer’s instructions. Analysis

of Phenotypes

Serial sections of pupal, pharate adult, and adult samples were examined and scored with respect to the following phenotypes (see Table 2): position of the subesophageal ganglion relative to the head capsule and rest of the CNS; presence, size, and shape of the cervical connective; extent of midline fusion of the brain; position and organization of the optic lobes. The data are summarized in Table 2. Note that, for a given genotype,

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DEVELOPMENTAL BIOLOGY

VOLUME 148,199l

TABLE 2 MUTANT CNS PHENOTYPES SEG-TG

Genotype”

Number scored

Max stage reached

separation

SEG: posterior deviation

failure

CCt: short and wide

Dorsal brain: Midline “split”

Optic lobes abn position

abn organization

Percentage with some CNS phenotype

br W/npr17 bP bF/Df. Y* W/bP7 b?’ rb;D rbp’ rbp’/Df. Y* rbp*/Qr* rbp’ i-bp’ rb$ rbp’A(ll1Bub’ 1(1)2Bab l/l)PBab’ 1(1)3Bc l(l)LBc’ l(l)~BBc’/l(l)~B’Bnb’ l(l)gBB%

18 16 17 24 10

Late pupa Mid/late pupa Mid/late pupa Late pupa/adult Viable

22% 25% 47% 61% 0%

(18) (15) (17) (23) (10)

12% 7% 24% 52% 0%

24 26 23 37 22 15

Mid/late pupa Mid/late pupa Early pupa Mid/late pupa Mid/late pupa Late pupa/adult

56% 62% 29% 86% 90% 80%

(23) (26) (17) (37) (21) (15)

38% (24) 46% (26) N/A 83% (36) 89% (18) 47% (15)

18

Early

92% (13)

12 24 29

Early/mid pupa Late pupa/adult Mid pupa

25% (12) 33% (24) 46% (28)

pupa

(16) (15) (17) (21) (10)

45% 37% 64% 14% 14%

(11) (16) (14) (21) (7)

12% 67% 78% 73% 0%

(16) (12) (14) (15) (10)

35% 47% 64% 19% 0%

(17) (15) (14) (21) (10)

61 81 82 88 10

33% (12) 73% (11) N/A 50% (14) 33% (12) 78% (9)

86% (21) 73% (22) N/A 95% (19) 89% (18) 100% (10)

0% 0% 0% 0% 0% 0%

(23) (24) (23) (35) (22) (15)

88 81 22 92 100 100

N/A

N/A

N/A

0% (17)

70

12% (8) 21% (24) 32% (28)

86% (7) 61% (23) 61% (28)

100% (12) 17% (24) 96% (23)

100 79 93

N/A 27% (22) 41% (22)

Note. The numbers in parentheses represent the number of mutants of a given genotype which could be scored for a particular phenotype. CNS, central nervous system; Max, maximum; SEG, subesophageal ganglion; TG, thoracic ganglion; CCt; cervical connective; abn, abnormal; N/A, not applicable a Genotypes designated with one X chromosome were examined either as hemizggous males with a normal Y chromosome or as homozygous females. * Df. Y refers to Dp(l;Y)~~YSz280. ** Qf refers to Df(l)S%?

the number of samples scored (i.e., the number in parentheses) varies among the phenotypes. The inability to score all samples for all phenotypes resulted from several biological and/or technical limitations. First, the developmental stage of a specimen determines what structures can be scored. For example, mature optic lobe position is not attained until mid- to late-pupal stages (PlO). However, the majority of 1(1)2Bc’/Y individuals die at the prepupal-pupal boundary (P4/P5), rarely reaching early P8; hence, mutants of this genotype could not be scored for optic lobe position. Similarly, unless the phenotype was very severe (e.g., Figs. 3C and 3D), mutants dying as early pupae could not be scored for subesophageal ganglion position and cervical connective morphology because in wild-type early pupae these structures are intermediate between larval and adult in appearance. Second, the plane of section limited the scoring of some phenotypes. For instance, in the sagittal plane it was impossible to score the midline fusion of the brain reliably, although the impression of a split was frequently noted. In the frontal plane, subesophageal ganglion position was not reliably storable. Third, some

samples (about 10% overall) showed global loss of tissue integrity, suggesting that these mutants were already dead at the time of dissection. Within this group, scoring of optic lobe organization was not possible. Care was also taken to avoid false positives due to errors of histological technique. For example, if a wildtype specimen sectioned in the sagittal plane is mounted obliquely, the optic lobes on one side will appear too dorsal, and the optic lobes on the contralateral side will appear too ventral. Thus, for mutants sectioned in the sagittal plane, optic lobe position was scored as abnormal only if the defect was bilateral. However, since horizontal views clearly show that the optic lobe position defect can be unilateral, this conservative criterion for scoring sagittal sections may have led to an underestimate of penetrance of this phenotype. Finally, the mechanical influence of one phenotype might cause alterations in the appearance of another structure of interest. For example, in individuals with massive persistent larval salivary glands in the head (Restifo and White, in preparation), the positions of the optic lobes were sometimes distorted in bizarre ways. In

179

RESTIFO AND WHITE

these cases, structures influenced effects were not scored.

by such mechanical

RESULTS

The decision to examine particular BR-Cmutants (Tables 1 and 2) was based on several considerations. CNS maturation was most reliably assessed in individuals which developed to stages beyond true pupation (see Kiss et al., 1988). Initial observations of a small number of mutant genotypes showed that BR-CCNS phenotypes are variable in penetrance and expressivity. To confirm the mapping of these phenotypes to the BR-C, the analysis was expanded to include a wide range of BR-C mutants, using alleles of independent origin. In addition to alleles deficient in both domains of BR-C function [qn-1 and Z(l,k’Bab], representatives of the three lethal complementation groups [br, rbp, 1(1)2Bc] were sampled to look for complementation group specificity of defects (see Fig. 1C). A pair of Y-borne duplications (Fig. 1B) was also used for mapping. Examination of serial sections of BR-C lethal mutants revealed derangements of some, but not all, features of CNS metamorphosis. These phenotypes include defects both in global aspects of CNS morphogenesis and in the internal organization of specific CNS regions. Before describing the CNS defects in the mutants, the wild-type metamorphosis of the brain, optic lobes, subesophageal ganglion, and thoracic ganglion are summarized (see also Hertweck, 1931; Power, 1952; Shatoury, 1956; Hinke, 1961; White and Kankel, 1978; Kankel et al., 1980; Campos-Ortega and Hartenstein, 1985). Wild-Type CNS Metamorph,osis Figures 2 and 4 depict the normal transition from the larval to the adult CNS, as seen in horizontal and sagittal planes of section, respectively. In conjunction with the differentiation of the retina, the optic lobes develop from imaginal primordia which are present in and grow during larval life (Fig. 2) (White and Kankel, 1978; Hofbauer and Campos-Ortega, 1990). During metamorphosis, the relative positions of the individual optic lobe neuropil regions change as a result of complex patterns of growth and movement within the head capsule (Shatoury, 1956; White and Kankel, 19’78; Campos-Ortega and Hartenstein, 1985; Hofbauer and Campos-Ortega, 1990). For example, in early pupal life the central axis of the medulla and lobula complex neuropil has a roughly anterior-posterior orientation when viewed in the horizontal plane (Fig. 2Ei). By the end of metamorphosis several days later, these neuropil regions have undergone an approximately 60” external rotation such that their axes now have a lateral-medial orientation (Fig.

B. y’YSz280 yzY67g 6

lJf(lJS39

C. Complementation

Map

br

rbp 102Bc

FIG. 1. The Blood Concp/r;c (B&C). (A) Diagrammatic representation of the tip of the X chromosome as seen in larval salivary gland polytene nuclei (based on Lefevre, 1976; Belyaeva cutul., 1987). Brackets indicate the cytogenetic location of the BR-C in region 2B3-5. (B) Diagrammatic representation of duplication- and deletion-bearing chromosomes used in this study. The straight black bars indicate portions of the X chromosome (compare to panel A) which are present. The jagged portions represent the heterochromatic Y chromosome. (C) Complementation map of the BR-C. This map indicates that (i) Dr and rbp mutations show partial complementation with each other, but fully complement I(lI2Bc mutations; and (ii) alleles of all three individual complementation groups fail to complement mutations of the overlying group, either fully (qkrl) or partially [I(I)2BBub]. This map is not intended to represent the physical relationships among the three complementation groups. It is a modification of that proposed by Kiss et ul. (1988), that being a revision of the original of Belyaeva rt trl. (1980).

2Fi). In addition, the lamina and medulla move relative to each other in several planes (compare Figs. 2Ei and 2Fi for horizontal views, and Figs. 6A and 6B for frontal views). The mature positioning of the optic lobe neuropil regions is complete by the end of the third day of metamorphosis (stage PlO). In larval life, the right and left sides of the brain are connected by a narrow band of fiber tracts just dorsal to the esophageal canal. With the ingrowth of sensory fibers during metamorphosis, right and left brain regions enlarge due to growth and differentiation of cortex and synaptic neuropil (Power, 1952). Furthermore, the number and size of decussating tracts increases and the neuronal cell body layer fuses in the midline (Hertweek, 1931) (Figs. 2 and 6). Similar changes have been

.- ..-

..->

180

RESTIFO AND WHITE

Drosophila

observed in explants of Drosophila CNS cultured in vitro with imaginal discs (Schneider, 1966) and in lepidopteran insects (Nordlander and Edwards, 1968). Thus, what had previously been two relatively independent brain spheres, ultimately become two massively interconnected hemispheres. The formation of the head and thorax, separated by a very narrow neck, necessitates repackaging of the CNS. During Droso&iZa (Hertweck, 1931) and other dipteran (Tung and Lee, 1968) metamorphosis, the subesophageal ganglion (SEG; designated with a star in all figures), which bridges the brain lobes with the subjacent ventral ganglion, moves anteriorly into the head capsule (Figs. 2 and 4). This process begins in early pupal life (P5) with the formation of a constriction between the SEG and the first thoracic segment of the ventral ganglion (Figs. 2B, BEii, and 4B). Concurrently, the SEG begins to move anteriorly and ultimately lies entirely within the head capsule, ventral to the supraesophageal ganglion (brain). During the separation, a cervical connective is elaborated and spans the neck, linking cephalic and thoracic regions of the CNS (Figs. 2C, 2F, and 4C). This process is completed during the second day of metamorphosis (P6-P7). As a result of cell division during larval life (Truman and Bate, 1988), the ventral nervous system of the mature larva already shows enlargement of the three thoracic segments relative to the eight abdominal ones (Fig. 2Diii). During metamorphosis, the thoracic segments continue to expand, while the abdominal segments merge into a single fused neuromere (Hertweck, 1931; Figs. 2 and 4). Larval

CNS of BR-C Null Mutants

Wandering third instar larvae lacking all BR-C function (e.g., nprP/Y) initially show normal CNS histology (Fig. 3A). Specifically, the inner and outer proliferative centers, the furrow at the entry site of the optic nerve, and the medulla and lobula neuropil are wild-type in

CNS Metarr~nrphosis

181

appearance. However, during the next l-2 days of prolonged wandering, the mutant larvae become sluggish and histological examination shows widespread loss of tissue integrity. The CNS assumes a polygonal profile, presumably because tissue softening allows adjacent structures to leave their imprint on the CNS (Fig. 3B). Although the optic nerve appears to widen in some dying npr13/Y individuals, it is not clear whether this represents continued photoreceptor ingrowth. Overall, there is little evidence of CNS development during the period of prolonged larval life. The histological appearance of these mutants is associated with gradual loss of staining with the neuropil-specific antibody, SYN-1 (White et aZ., 1983), consistent with progressive demise of a nonspecific nature (data not shown). Since larval imaginal discs from nprl individuals show some anatomical abnormalities at the mid-third instar stage (Fristrom et al, 1981), subtle abnormalities in the larval CNS of mutants were sought with reagents recognizing small subsets of neurons. The immunostaining patterns obtained with antisera against serotonin and the neuropeptide FMRFamide, and the histofluorescence pattern of catecholamine-containing neurons, were normal in CNS of npr13/Y third instar larvae (10 samples each; data not shown). Failure of Subesophageal Ganglion-Thoracic (SEG-TG) Separation

Ganglion

BR-Cmutants show abnormalities of SEG-TG separation, which result in either of two CNS configurations. In the most common version of this phenotype, the final SEG position is more posterior, relative to the head capsule and surrounding brain and optic lobes, than in wild-type individuals (Fig. 5). Faulty SEG-TG separation, with posterior SEG position, is seen in mutants bearing all examined alleles regardless of their stage of lethality (Table 2), but varies considerably in severity. In the most severe cases of this type, no separation from the TG occurs, and the SEG straddles the neck and ex-

FIG. 2. CNS metamorphosis in wild-type (Canton S) Drosophila. (A, D) White prepupa (Pl; hematoxylin); (B, E) early pupa (P5); (C, F) pharate adult (P15; hematoxylin). For each developmental stage, the cartoon drawing (A-C) depicts the lateral view of the specimen seen in the corresponding composite horizontal sections below (D-F, respectively). Within each composite panel (D-F), successive images go from dorsal to ventral, except that the abdominal portion of the thoracic ganglion (Ab) in the pharate adult generally tilts dorsally. Thus, the subesophageal ganglion (star) is always pictured between the brain and optic lobes (OL) anteriorly and dorsally, and the ventral/thoracic ganglion posteriorly and rentrally. The approximate planes of section are indicated by the horizontal lines in the cartoons. See Figs. 6A-6C for sagittal sections of comparable stages. Notable features of the developmental progression are listed here and described more fully in the text. The retina (R) develops from the larval eye-antenna disc (ea). The right and left sides of the brain fuse progressively (compare asterisks in E and F). The medulla (Me) and lobula complex (LoC) neuropil regions of the optic lobes undergo an external rotation (compare long arrows in Ei and Fi), sliding past the developing lamina (La). The subesophageal ganglion becomes separated from the thoracic ganglion (Tl, first thoracic neuromere), initially by a constriction (curved arrows in Eii), and later by the cervical connective (short straight arrows in Fii). The three thoracic neuromeres enlarge and the eight abdominal segments (Ab) condense into a single fused neuromere. Magnification is the same for D-F; scale bar = 50 pm.

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FIG. 3. Horizontal sections through the CNS of early-dying RR-C mutants. (A, B) Level of esophageal canal (e); (C, D) ventral to esophageal canal. (A) nprl’/Y, wandering third instar larva (hematoxylin). Note the normal appearance of eye-antenna discs (EA), optic nerves (on), brain and optic lobe anlagen, including the inner and outer proliferation centers (ipc and opt, respectively), and proventriculus (pv). Short arrows point to the furrows in the opt adjacent to the site of entry of the optic nerves. (B) nprl’/Y, after 1 day of prolonged wandering (hematoxylin). The eye-antenna discs are dilated; the CNS, especially the optic lobes (OL), has a distorted contour; and the proventriculus is shrunken and degenerating. (C) Z(I)Z&zb’/Y, early pupa (abnormal P4/5; hematoxylin). The SEG (star) is inside the newly formed head capsule, but there is no constriction between the SEG and the TG, and the first thoracic neuromere (Tl) is in the neck. (D) rw/Y, early pupa (P5/6). A constriction (curved arrows) has formed between the SEG and TG, but the SEG has not moved anteriorly into the head capsule. The developing antenna1 lobes (An) are posteriorly displaced into the neck. Note the normal organization of the OL neuropil regions. R, retina; La, lamina; Me, medulla; Lo, lobula; Lop, lobula plate. Magnification is the same throughout; scale bar = 50 pm.

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FIG. 4. Lateral views of wild-type CNS metamorphosis. Sagittal sections through the CNS of Canton-S (A) wandering third instar larva (hematoxylin), (B) early pupa (P5), and (C) pharate adult (P15; hematoxylin). In A, the ventral ganglion is seen in midsagittal section in the plane of the esophagus (e), and the brain and optic lobe (OL) are seen in parasagittal section. In comparing the three stages, note the progressive anterior movement of the subesophageal ganglion (star) as it separates from the ventral/thoracic ganglion via a constriction (curved arrows in B) and, later, via the cervical connective (straight arrows in C). In B, the small clump of darkly staining cells in the ventral head capsule is a fragment of degenerating larval salivary

tends into the anterior thorax (Fig. 5C). More commonly the anterior movement of the SEG appears incomplete, but some evidence of a constriction or cervical connective can be found (Figs. 3D, 5A, and 5B). The second type of abnormality is seen only in mutant individuals dying as early pupae. In this variant, the SEG moves into the head capsule, but no constriction forms and the first thoracic neuromere moves anteriorly as well, and comes to reside in the developing neck (Fig. 3C). If cuticle development proceeds further, the anterior-most thoracic ganglion gets trapped in the head and pinched by the neck (data not shown). Because of the physical association between the SEG and the cervical connective, and because the movement of the former is normally associated with the formation of the latter, one might expect that abnormalities of the SEG position and the cervical connective structure would be closely associated in BR-C mutants. In the extreme case this is true: if no cervical connective forms, then the SEG position is profoundly abnormal. However, in cases with intermediate severity, the relationship between the SEG and the cervical connective is not simple. For example, in the samples depicted in Figs. 5A and 5B, the cervical connective is present but short. However, in Fig. 5B, the SEG does not underlie the supraesophageal ganglion at all, whereas in Fig. 5A, the SEG has almost attained its usual anterior position. The formation of a cervical connective of a given length need not be associated with a proportionate degree of anterior movement of the SEG. The abnormalities of SEG-TG separation were seen in lethal mutants representing all three complementation groups. Overall, the penetrance of the SEG phenotype was higher in mutants of the rbp complementation group (70%) than in those of the br (41%) or Z(l)ZBc (38%) complementation groups. However, penetrance and expressivity (severity) were not associated. In other words, severe and mild abnormalities were found in similar proportions (approximately 25 and ‘75%, respectively) among the affected individuals of the three complementation groups. A very high incidence of SEG-TG separation failure (92%) was also seen in l(l)ZBab’ mutants, which are partially deficient in all BR-Cfunctions (Belyaeva et ah, 1980; Kiss et ab, 1988). Failure

of Midline

Brain Fusion

BR-C mutants show defects in the fusion between right and left brain hemispheres (Fig. 6). After the midglands (sg). Tl, first thoracic neuromere; pv, proventriculus; An, antenna] lobe; ab, abdominal portion of thoracic ganglion; DLM, dorsal longitudinal muscles. Magnification is the same throughout; scale bar = 50 fim.

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pupal stage, wild-type individuals frequently have some apparent right-left discontinuity in dorsal-most horizontal sections due to variability in the dorsal contour of the brain (Fig. 6B). However, in wild-type individuals (Canton-S or mutant allele/duplication) this discontinuity never extends more than 18 pm below the dorsal rim of the brain. Thus, samples were scored as abnormal with regard to this phenotype only if they showed rightleft discontinuity of 36 pm or greater. (See Fig. 6C for a horizontal section through a wild-type brain 36 pm below the dorsal rim.) This conservative criterion may have resulted in an underestimate of penetrance of the fusion-failure phenotype. Detailed qualitative aspects of right-left connectivity were not investigated. The brain fusion defects were quite variable in severity, with the most severely affected individuals showing no right-left connection dorsal to the fan-shaped body of the central complex. Such severely affected individuals also showed failure of midline fusion of anterior and posterior cortices at all levels dorsal to the esophageal canal (Fig. 6E). Generally, the defect was limited to more dorsal regions (e.g., Fig. 6D). The lack of right-left fusion was not accompanied by any gross lack of central brain structures, i.e., central complex and mushroom bodies, although different staining methods will be needed for higher resolution assessment of these neuropi1 regions. The brain fusion defects were not necessarily associated with any of the other CNS phenotypes. Again, lethal mutants in each of the three complementation groups were affected (see Table 2), with the rank order of penetrance as follows: 1(1)2Bc (64%) > rbp (51%) > br (35%). Rarely (one of seven samples), a viable bm individual showed mild midline fusion failure. Abnormal

FIG. 5. SEG-TG separation failure in BR-C mutants. Sagittal sections through the CNS of BR-C pharate adults, showing the range of phenotypic severity. (A) Mild: rbp’/rbpl (P14/15); short cervical connective (arrows) with mild posterior deviation of SEG (star). (B) Moderate: rbp’/rb$ (P14/15); short cervical connective (arrows) with significant posterior deviation of SEG. (C) Severe: rbp’/Y (P12); no cervical connective (arrows) between thoracic ganglion and SEG, which straddles the neck. Note the abnormal positions of the antenna1 lobes (An) relative to those in wild-type shown in Fig. 4C. Tl, first thoracic neuromere; ab, abdominal portion of thoracic ganglion; asterisk, persistent larval salivary gland; pv, proventriculus. Magnification is the same throughout; scale bar = 50 pm.

Optic Lobe Position

When viewed in the horizontal plane, optic lobe position of BR-Cmutant late pupae and pharate adults (Fig. 7B) is midway between that of wild-type early pupae (Fig. 2E) and that of wild-type pharate adults (Figs. 2F and 7A). This “intermediate” position might result if optic lobe rotation began normally but was curtailed, or if the process proceeded more slowly than usual. However, an additional abnormality is apparent in the sagittal plane: the positions of the mutant lamina and medulla neuropil are excessively dorsal with respect to the head capsule (Figs. 7C-7H). Thus, the position defect cannot be interpreted simply as incomplete or delayed optic lobe rotation. Rather, the unfolding of the curvilinear larval proliferation centers (Hertweck, 1931; Campos-Ortega and Hartenstein, 1985) is probably qualitatively abnormal. The abnormalities of optic lobe neuropil position were seen in lethal mutants representing all three comple-

RESTIFO AND WHITE

Drosophila CNS Metamorphosis

FIG. 6. Dorsal brain development in wildtype (Canton-S) and BR-C mutants. (A, B) Frontal sections through the heads of wild-type specimens. Note that in the early pupa (P6; panel A), the right and left sides of the brain are still separated (arrowhead) dorsal to the developing central complex (asterisk), whereas, in the pharate adult (P14115; panel B), right and left sides are fully joined across the midline (arrowhead). (C) Horizontal section through the head of a wild-type pharate adult (Pll; hematoxglin) at the level of the mushroom body calyces, also showing right-left contiguity (between arrows). (D, E) Horizontal sections through the heads of II112BcL/Ypharate adults (Pll and P14, respectively), showing the persistence of the midline split (arrow). C and D are each 36 pm ventral to the dorsal rim of the brain (corresponding approximately to the line marked “C, D” in panel B). E is 12 pm dorsal to the roof of the esophageal canal (corresponding approximately to the line marked “E” in panel B) and represents an extreme example of fusion failure in the anterior midline. La, lamina; Me, medulla neuropil; LoC. lobula complex neuropil; star, subesophageal ganglion. Magnification is the same throughout; scale bar = 50 Km.

mentation groups, with higher penetrance in rbp mutants (84%) than in br (55%) or l(l)ZBc (34%) individuals. These abnormalities are not necessarily associated with any other CNS phenotype. Since margins of optic lobe cortical regions are not easily identified in routine histological preparations, their positions could not be assessed in BR-C mutants. Abnormal

Optic Lobe Organization

The normal dipteran optic lobe consists of a group of interconnected but distinct synaptic neuropil regions

(Fig. 8A) (Meinertzhagen, 1973; Strausfeld, 1976; Fischbath and Dittrich, 1989) and their associated co&ices. Visual information is transmitted in a retinotopic manner via sequential synaptic connections from the retina to the lamina, medulla, and lobula complex. The internal organization within each neuropil region is quite precise, and even low-magnification histological examination reveals characteristic rows and columns. Some BR-C mutants display a striking anomaly of optic lobe organization in which the boundaries of neuropil regions are disrupted and the neuropil structure is jum-

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FIG. 7. Optic lobe position in wildtype (Canton-S) and BR-C mutants. (A, B) Horizontal sections at the level of the esophageal canal (E) through the heads of Canton-S (Pll; panel A) and r&*/Y (P12; panel B). As indicated by the long arrows, the overall angle of the medulla and lobula complex, relative to the head capsule, is different in the two specimens. Scale bar = 50 pm. (C-E) Series of parasagittal sections through the optic lobes, from lateral to medial, in the wild-type (PlO, hematoxylin). As the sections become more medial, the lamina and medulla attain more ventral positions. (F-H) Corresponding series of parasagittal sections through the optic lobes of rbp“/Y (Pll). Here, in contrast to the wild-type case, the lamina and medulla are situated very dorsally within the head capsule. C through H are all at the same magnification. R, retina; E, esophageal canal; La, lamina; Me, medulla; Lo, lobula; LOP, lobula plate; Vlp, ventrolateral protoeerebrum; A, antenna1 lobe. Scale bar = 50 Wm.

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Lh-osophilu CNS Metarwrphosis

‘k

‘!I?&S..‘..,-. _ -

-

FIG. 8. Optic lobe organization in wildtype and BR-C mutants. Phase-contrast photomicrographs of horizontal sections through the optic lobes. (A) Canton-S midpupa (P8; hematoxylin): note the distinct boundaries between individual optic lobe neuropil regions. (B) t#/,$YSz28!80 pupa (PlO): a thin ectopie fiber band traverses the lobula neuropil (arrow). (C) /{I,U?sP/Y pharate adult (Pll): the lamina looks relatively normal, but the medulla and lobula complex are deranged. Anteriorly, the medulla neuropil appears to be extending a projection into lobula terrain (straight arrow). Posteriorly, the medulla and lobula plate appear fused (curved arrow), and medially, the lobula plate embraces the lobula. (D) lIIILBc’/Ypupa (P8): the lamina is “kinked” (arrows), and the lobula and lobula plate neuropil show areas of fusion. R, retina; La, lamina; Me, medulla; Lo, lobula; LOP, lobula plate. Magnification is the same throughout; scale bar = 50 Wm.

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bled and deranged (Figs. 8C and 8D). Fibers from one neuropil region appear to “invade” neighboring areas. In particular, the posterolateral medulla is frequently fused to the adjacent lobula plate (Fig. 8C). In some cases, it was difficult to assign names to abnormal regions or to say where one neuropil region ends and another begins. The boundary abnormality and intraganglion disorganization are clearly distinguishable from nonspecific tissue degeneration associated with lethality. For instance, pupal lethal mutants of an unlinked gene not required for optic lobe development do not have these histological features (Restifo and Merrill, unpublished observations). On the other hand, some lethal mutant specimens (B&C, as well as others) do show generalized loss of internal structural detail in the optic lobes, which may reflect nonspecific postmortem changes (these were not scored as abnormal). In addition to gross neuropil fusion (Fig. 8D), more subtle abnormalities were also seen in some BR-C mutants. In particular, thin bands of “fibers” cut across neuropil at random orientations with respect to the normal organization of the structure (Fig. 8B). Such subtle defects involved small patches of tissue, lo-50 pm in diameter. The three features of optic lobe disorganization, (i) fusion of adjacent neuropil regions, (ii) disarray within individual ganglia, and (iii) thin fiber bundles traversing neuropil regions, are seen in BR-C mutants regardless of their stage of pupal lethality. Lesions were sometimes unilateral, and, when bilateral, they were often asymmetric. Among the optic lobe ganglia of BR-C mutants, the lobula complex is most frequently disrupted, the medulla less often, and the lamina least often. Optic lobe disorganization was not necessarily accompanied by abnormal optic lobe position (e.g., Fig. 8C), but both phenotypes were seen concurrently in some samples. Lethal alleles of the br complementation group cause optic lobe disorganization with variable, moderate penetrance (19-64%). In br mutants, the defects were subtle and often limited to small patches of tissue, whereas in 1(1)2Bc mutants the abnormalities were typically conspicuous, involving large fractions of optic lobe neuropil. 1(1)2Bab’ mutants did not show optic lobe disorganization. This allele is hypomorphic for br, rbp, and l(l)%‘Bc functions, although the lack of l(lJ2Bc activity is less prominent in imaginal disc development (Belyaeva et al, 1980; Kiss et al., 1988). Consistent with this view, l(1)2Bab1/l(l)2Bc’ transheterozygotes manifest partial complementation failure for optic lobe disorganization (17% penetrance). In contrast to the optic lobe position phenotype, disorganization of optic lobe neuropil is notable for its ab sence in rbp mutant individuals (O/142 samples or O/284

optic lobes) and its very high penetrance among 1(1)2Bc mutants bearing strictly lethal alleles (95%). Note that the rbp alleles used here are hypomorphic (Kiss et al., 1988). However, rbp’/Df(l) female pupae, which have less wild-type BR-C activity and, therefore, are more severely affected than rbp’ homozygous females or hemizygous males, also showed normal optic lobe organization. Thus, rbp+ function may be irrelevant for normal organization of optic lobe neuropil. The optic lobes (as well as the compound eyes) of BR-C mutants often appeared somewhat smaller than those of wild-type individuals, but no morphometric analysis was undertaken. The retinae of mutant individuals were sometimes collapsed at the time of dissection, perhaps due to the imminent or foregoing demise of the animal, thus making assessment of retinal integrity difficult. Collapsed retinae were also seen in the absence of optic lobe disorganization and/or optic lobe position defects. Less frequently, the retinae of mutants appeared vacuolated (Fig. 8B). CNS Abnormalities Fusim Defects

Associated with Imaginal

Disc

Two other types of CNS abnormalities were seen in a small fraction of BR-C mutants deficient in Z(l)2Bc function [i.e., 1(l)2Bc1/ Y, l(l,MBab’/ Y, l(l@Bab’/ l(l)2Bc’, 1(1)2Bc’/l(l)2Bdq but never in br or rbp mutants (data not shown). In one variation, the CNS was partially or totally in the thorax. What distinguished these from the more typical posterior deviations associated with SEG-TG separation failure was the coincident location of both developing retinae-and entire head capsule-in the thorax. In other words, this abnormality appeared to result from failure of head eversion. A similar phenotype has been described in a pupal lethal mutant, introvert (Fischbach et al, 1989). In the second variation, parts of the CNS projected outside of the body wall. These individuals had overt holes in the cuticle, most likely a result of failure of fusion of imaginal disc derivatives, previously described in Z(1)2Bc mutants (Kiss et al., 1988). Perhaps head eversion failure was also secondary to such fusion defects. In any event, because of their bizarre geometry, these individuals could not be scored for the CNS phenotypes described above and were not included in this analysis. Genetic Mapping

of CNS Phenotypes

to the BR-C

Two lines of evidence support the conclusion that the phenotypes described here map to the BR-C. First, these phenotypes are uncovered by a small interstitial deletion (2B3-4 through 2B6) within a Y-borne duplication (1A to 2Cl-2), which retains no BR-C function

RESTIFO AND WHITE

Lkosophila

[Dp(l;y)y2YSx280; Belyaeva et al, 1982, 1987; Kiss et ah, 19881 (Fig. 1B). Phenotypes which could be scored in early-dying pupae were also uncovered by a larger deletion, Df(l)S39, which removes lEl-2 to 2B5-6 (Belyaeva et ah, 1980) (Fig. 1). A Y-borne duplication (1Al to 2B1’7-18), which includes all BR-C function [Dp(l;Y)gY67g; Belyaeva et ah, 1980; Kiss et al., 19881(Fig. lB), fully complements all BR-C CNS phenotypes. Specifically, rbp’/$Y67g and Z(l)ZBc’/y”Y67g (10 P14/15 individuals of each genotype) were indistinguishable from wildtype (Canton-S, 20 individuals). Since the gY67’g duplication also includes a portion of the proximal-most X chromosome (Craymer and Roy, 1980), several BR-C alleles [nprl’, rbp’, and Z(1)2Bc’] were placed in combination with a deletion of the proximal X chromosome [Df(l)maZ-13, which lacks 19A through the base of the X (Schalet and Lefevre, 1976)]. These flies were fully viable and show no abnormalities of wings, legs, or palps characteristic of BR-C mutants (data not shown). Thus, by deletion/duplication analysis, the observed CNS phenotypes map to a small region known to encompass the BR-C and two more proximal lethal complementation groups (Belyaeva et aZ., 1982). Second, independently isolated alleles (Table 1) show qualitatively similar defects of SEG-TG separation, midline fusion, and optic lobe position and organization (Table 2). Pairwise combinations of alleles in lethal transheterozygotes fail to complement for any of the phenotypes. Most of the mutants examined bore alleles of the “t series,” isolated in chemical mutageneses using the same parental chromosome (Belyaeva et al., 1980). However, similar results were obtained in transheterozygotes bearing alleles from different parental stocks (br’/npr17 and b?‘/b?; see Tables 1 and 2). Although viable w7 mutants have virtually no CNS defects, in combination with an amorphic allele of the br complementation group, b?, all of the CNS phenotypes are revealed. Thus, br;2’ is a weak hypomorph for CNS metamorphosis as well as for lethality. The finding of complementation failure with independently derived alleles confirms that the phenotypes are not due to non-BR-C mutations on chromosomes in the parental stock from which the “t series” alleles were derived. DISCUSSION

Central Nervous System

Metamorphosis

and the Broad

Complex

As in other holometablous insects, the Drosophila CNS undergoes dramatic changes in structure and function during metamorphosis. As a first step toward understanding the hormonal mediation of this process, we

CNS Metamorphosis

189

have identified several aspects of CNS reorganization dependent on ecdysterone-regulated gene activity. Pupae and pharate adults bearing lethal BR-C alleles have defects in the three-dimensional movement (optic lobes), midline fusion (brain), and separation (SEG-TG) of major CNS regions with respect to each other. In addition, disorganization within and among optic lobe ganglia is seen in BR-C mutants bearing br and Z(l)2Bc alleles, but not rbpalleles. As with BR-C-associated cuticular phenotypes (Fristrom et al., 1981), the CNS abnormalities appear to reflect failures of morphogenesis, rather than of cellular differentiation. Both cytoskeletal and extracellular matrix abnormalities have been reported in BR-Cmutant imaginal discs (Fristrom et al, 1981), and such defects in the CNS may well play a role in the phenotypes seen here. These defects in CNS morphogenesis appear after a normal period of larval CNS development, suggesting an abnormal response to ecdysterone by otherwise normal neural tissue. However, the BR-C has transcriptional activity (Chao and Guild, 1986; Galceran et ab, 1990) and nonvital functions (Crowley et al., 1984; Guay and Guild, 1991) prior to the end of larval life. Furthermore, mild abnormalities of BR-C imaginal discs are detectable at mid-third instar (Fristrom et al., 1981). Therefore, subtle defects may be present in BR-C larval CNS, and these could contribute to the abnormalities of CNS reorganization. During wild-type CNS metamorphosis, the majority of larval neurons persist (Power, 1952; White and Kankel, 1978; White et ab, 1986; Valles and White, 1988; Budnik and White, 1988;Tix et al., 1989), with some reorganization (Technau and Heisenberg, 1982); new neurons are born and differentiate (White and Kankel, 1978; Truman and Bate, 1988; Hofbauer and Campos-Ortega, 1990); and some cells die (Stocker et al., 1976; Fischbach and Technau, 1984; Hofbauer and Campos-Ortega, 1990; Kimura and Truman, 1990). The relationship between these cellular events and regional morphogenetic movements is not yet clear, but the former are ecdysterone dependent in other insects (e.g., Weeks and Truman, 1985; Booker and Truman, 1987; Levine, 1989). Ventral/thoracic ganglion development, which includes the condensation of the abdominal ganglia, is notably unaffected in BR-C mutants. In contrast, Cecropia prepupae treated with juvenile hormone show failure of abdominal ganglion fusion (Riddiford, 1972), and Ga.ZZeria nerve cord shortening during metamorphosis is ecdysteroid dependent (Pipa, 1969; Robertson and Pipa, 1973). TG development in Drosophila may also be hormone-dependent, but mediated by a different limb of the ecdysterone-initiated gene expression cascade.

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Kiss et al, 1988), BR-C mutants may also have disordered connections between the SEG and the periphery. MidZine fusion failure. The development of connections between right and left sides of the CNS are an important feature of sensory-motor integration, as demonstrated by a variety of behavioral studies following midline lesions (reviewed in Howse, 1975). Proper midline fusion during Drosophila metamorphosis requires BRA’+ functions encoded by all three complementation groups. Right-left fusion may require normal breakdown of the blood-brain barrier (Edwards, 1969; Pipa and Woolever, 1964; Lane and Swales, 1978), a known target of ecdysterone (Natzle et ab, 1988). It is not obvious from the present study which structures are affected by the persistent midline split, but by analogy with the Musca brain (Strausfeld, 1976), one might expect anomalies of the following fiber tracts, which normally cross the midline dorsal to the central complex (listed in order from ventral to dorsal): ocellar interneuron decussation, intertubercle tract, superior protocerebral commissures 1 and 2, commissure of the lateral horns, and anterior dorsal commissures. In “splitbrain” BR-C mutants these tracts may remain ipsilatera1 or cross at a more ventral level. Optic lobe position. The optic lobes of BR-C mutants demonstrate excessively dorsal positions of lamina and medulla neuropil and, in the horizontal plane, apparent incomplete rotation of medulla and lobula neuropil. A number of visual system mutants, including one with primary retinal defects (Glued: Meyerowitz and Kankel, 1978; Harte and Kankel, 1982) and one with primary CNS defects [1(3)optic ganglion reduced: Lipshitz and Kankel, 19851,show faulty optic lobe position. On the other hand, optic lobe malposition is not necessarily a hallmark of visual system mutants. Normal optic lobe position is seen in small optic lobes (Fishbach and Technau, 1984), Vacuolar medulla (Coombe and Heisenberg, 1986), and lob&a plateless (Heisenberg and Wolf, 1984). CNS Phenotypes in BR-C Mutants Potential causes of the BR-Coptic lobe position defect include abnormalities of cell migration, neuronal birth SEG-TG separation failure. Our results identify and differentiation, the movements of other CNS reBR-CY functions, encoded by all three complementation gions, head capsule morphogenesis, and/or glial cell begroups, as essential for proper SEG-TG separation. havior. Hence, defective SEG-TG separation could conAlong with cervical connective formation, SEG-TG septribute to optic lobe position defects, although it cannot aration is a general feature of metamorphosis in holometabolous insects (e.g., Hertweck, 1931; Pipa, 1963; provide a complete explanation, since the two phenoHeywood, 1965; Tung and Lee, 1968), but little is known types can be seen independently. Optic lobe organixatimz, The optic lobe derangements about the force-generating mechanisms that “pull” the of br and Z(l)2Bc mutants-misrouted fibers, neuropil SEG and TG apart. Since cervical connective formation fusion, and internal neuropil disarray-represent abnorand SEG position need not go hand-in-hand in BR-C malities of both interneuropil and intraneuropil developmutants (Fig. 5), SEG-TG separation may have several components which can be differentially disrupted. ment (see Meinertzhagen, 1973). The profound disorGiven their cuticular and bristle abnormalities of the ganization seen in early-dying mutants indicates that maxillary palps and proboscis (Belyaeva et al, 1980; the disrupted events occur relatively early in optic lobe The assignment of the BR-C to the ecdysterone-inducible early puff at 2B3-5 is based on considerable genetic evidence. An approximately lOO-kb genomic interval encompasses both the puff and the BR-C (Chao and Guild, 1986; Belyaeva et ab, 1987), contains the sites of chromosomal breakpoints and insertions of foreign DNA in BR-C mutants (Belyaeva et ah, 1987; Sampedro et ah, 1989;DiBello et al., 1991), and gives rise to multiple ecdysterone-regulated transcripts (Chao and Guild, 1986; Galceran et ah, 1990; DiBello et al, 1991). On the basis of this evidence, it seems reasonable to consider the BR-C the major ecdysterone-regulated occupant of the 2B3-5 early puff, with the caveat that other (presumably nonvital) ecdysterone-regulated loci may reside there as well. Thus, the BR-C mutant phenotypes presented here support the hypothesis that hormonal regulation of CNS metamorphosis is mediated, at least in part, by alterations in gene expression. Cytogenetic (Belyaeva et al, 1981; Zhimulev et al, 1982; Crowley et ah, 1984), molecular genetic (Crowley et al, 1984; Vijay Raghavan et al., 1988; Galceran et al, 1990; Guay and Guild, 1991), and biochemical studies (Dubrovsky and Zhimulev, 1988) of BR-C mutants indicate that BR-C product(s) play a role in transcriptional regulation. DNA sequence analysis of BR-C cDNA clones predicts a family of related proteins, each containing a pair of Zn2+-fingers (DiBello et al., 1991). Thus, by analogy with known Zn2+-finger-containing transcription factors (reviewed in Evans and Hollenberg, 1988), BR-C products are proposed to act as DNA-binding proteins to trans-activate many other loci (DiBello et al., 1991). Given a working model of BR-C encoded transregulatory products, the spectrum of CNS defects seen in BR-C mutants may result from misregulation of many ecdysterone-regulated genes, including nervous system-specific late genes.

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development. The BR-C phenotypes are reminiscent of ectopic fiber pathways and neuropil fusion described in mutants of irregular chiasm C which may be due to displacement of optic lobe pioneer neurons (Boschert et ah, 1990). Normal retinal projections are one prerequisite for normal optic lobe structure (e.g., Power, 1943; Meyerowitz and Kankel, 1978; Fischbach, 1983; Selleck and Steller, 1991). However, BR-Coptic lobe disorganization is unlikely due solely to disordered afferent input, since the gradient of severity (lobula > medulla > lamina) is the inverse of what is seen in mutants primarily affecting the eye. On the other hand, eyeless sine oculis mutants examined by silver and Golgi staining show fusion of medulla and lobula plate (Fischbach, 1983) which may be similar to that observed in BR-C mutants by hematoxylin/trichrome staining. It is possible that BR-C function is required in cells of both the optic lobe and the retina to achieve a completely normal optic lobe. Two genetic issues arise from the optic lobe organization phenotypes of BR-C mutants. First, recall that 1(1)2Bc mutants show high penetrance and severity, whereas br mutants show low penetrance and severity of optic lobe disorganization. Do the differences between br and 1(1)2Bc represent opposite ends of the expressivity spectrum of the same phenotype, or, rather, do they represent qualitatively distinct phenotypes? A preliminary analysis of br/l(l)2Bc, br/rbp, and rbp/ Z(l)2Bc transheterozygotes (all viable) reveals normal optic lobe organization in all three. This interallelic complementation suggests that the defects in br and Z(Z)2Bc mutants may result from deficiencies of distinct genetic functions. Second, the striking absence of optic lobe disorganization in rbp mutants raises interesting possibilities concerning BR-C-dependent late gene expression. The data are consistent with two distinct late gene classes necessary for optic lobe development. One of these classes should be required for optic lobe organization and regulated by br’ and 1(1)2Bc+ functions. The other class should be required for optic lobe rotation and regulated by rbp+, as well as by br’ and 1(1)2Bc+ functions. Analysis of BR-C-dependent late genes in the optic lobe development pathway(s) should allow this hypothesis to be tested. Finally, optic lobe disorganization in br but not in rbp mutants emphasizes that these two complementation groups do not share all genetic functions (Belyaeva et al., 1980; Kiss et ab, 1988; Guay and Guild, 1991). CNS Defects in Relation

to Other Phenotypes

The BR-C-associated CNS phenotypes presented here are part of a syndrome of defects, which includes persis-

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tence of larval salivary glands (Figs. 5A and 5B) and gut, reduction or lack of dorsal-ventral indirect flight muscles, abnormalities of proventriculus and adult salivary glands (Restifo and White, in preparation), as well as the previously described abnormalities of imaginal disc development (Kiss et ab, 1988; Beaton et al., 1988). Although the BR-C mutant pupae and pharate adults analyzed in this study had fairly normal cuticular features, qualitative aberrations in epidermal morphogenesis, e.g., head eversion, may contribute to the abnormalities of CNS morphogenesis. Like the CNS abnormalities, the other internal tissue phenotypes were also variable in penetrance. No CNS phenotype was dependent on the presence of a defect in a nonneural structure. For example, SEG-TG separation failure was seen in the absence of other mutant phenotypes (e.g., persistent larval salivary glands) which might conceivably have blocked the path of SEG movement. Conversely, normal SEG position and cervical connective formation were also seen in samples with prominent tissue masses in the neck region. Furthermore, a similar SEG phenotype has been mapped to the Deformed locus, recessive mutants of which lack other BR-C-associated internal tissue defects (Restifo and Merrill, unpublished observations). Therefore, mechanical hindrance cannot explain the SEG phenotype. In the face of the striking pleiotropy and variability in penetrance and expressivity seen in this study, we cannot claim that BR-C function is required in a tissue-autonomous manner for CNS metamorphosis. However, mosaic analyses have shown that BR-(7 activity is required in a cell-autonomous fashion for cuticle pupariation (Kiss et ab, 1976b) and salivary gland gene expression (Vijay Raghavan et al., 1988). Furthermore, BR-C transcripts are detectable throughout the CNS in late third instar larvae and adults (Restifo and White, 1988). Normal CNS metamorphosis may require BR-C expression both in the nervous system, including optic lobes, midbrain, SEG, and TG, and in nonneural tissues. The authors thank T. Tully for discussions during the course of this work; P. DiBello, P. Guay, and G. Guild for sharing unpublished data; G. Guild, R. Levine, 1’. Merrill, and S. Selleck for comments on the manuscript; J. Fristrom and A. Beaton for supplying fly stocks; and D. Correll for technical assistance. This work was funded by a Muscular Dystrophy Association postdoctoral fellowship and an NINCDS Clinical Investigator Development Award (NS01259) to L.L.R.

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