p-element mutations affecting embryonic peripheral...

Coppight 0 1995 by the Genetics Society of America

P-Element Mutations Affecting Embryonic Peripheral Nervous System Development in Drosophila melanogaster

Artur Ma,* Adi Salzberg," Manzoor Bhat,* Diana D'Evelyn," Yuchun He,* Istvan Kisst and Hugo J. Bellen*

*Howard Hughes Medical Institute, Department of Molecular and Human Genetics, Division of Neuroscience, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, and +Institute of Genetics,

Biological Research Center of the Hungarian Academy of Sciences, H-6701, Szeged, Hungary Manuscript received October 18, 1994

Accepted for publication December 29, 1994

ABSTRACT The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study

the molecular mechanisms governing neural development. To identify genes controlling PNS develop ment, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the Pelement insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.

T HE embryonic peripheral nervous system (PNS) of Drosophila mlanogaster offers an excellent model

system to study nervous system development. It is composed of -650 neurons that innervate sensory organs (SO) whose anatomy has been well described (DAMBLY- CHAUDIERE and GHYSEN 1986; GHYSEN et al. 1986; BODMER and JAN 1987; HARTENSTEIN 1988). Three main types of embryonic PNS neurons can be distinguished: those innervating the external sensory (ES) organs, those innervating chordotonal (Ch) organs, and the multiple dendritic (MD) neurons. The ES organs consist of two cells exposed to the external environment: an external sensory cell and a socket cell. The sensory information is relayed to a subepidermally located neuron that is ensheathed by a glial cell. The ES organs probably transduce mechanosensory, chemosensory and possibly stretch information (DAMBLY-CHAUDIERE and GHYSEN 1986). The Ch organs are located in the body wall and are composed of a neuron and three support cells: the cap, ligament and sheath cells. The Ch organs are thought to function as stretch or propri- oreceptors (JAN and JAN 1993). The MD neurons do not have specific accessory cells and have been proposed to function as touch-, proprio- or chemoreceptors (KRAMER and KUWADA 1983; BODMER and JAN 1987). In addition to these body wall sense organs, the larval head contains

many other organs, some of which are involved in pho- toreception (JURGENS et al. 1986; BELLEN et al. 1992a).

The development of the larval SOs involves initial specification of sensory organ precursors (SOP) from among a group of ectodermal cells. Once determined, the SOPs delaminate into the subectodermal layer and undergo a series of two or three divisions (HARTENSTEIN 1988) such that all cells within a SO seem to be related by lineage (BODMER et al. 1989). The next step in SO morphogenesis involves differentiation of their cells. For example, following germ band retraction, peripheral neurons begin axonogenesis and pathfinding toward the central nervous system (CNS).

Genetic and phenotypic characterization of the pro- cesses involved in determination and differentiation of the SO components is well advanced [e.g., SALZBERG et al. 1994; reviewed by JAN and JAN (1993) and GHYSEN et al. (1993)l. The genes controlling SO development can be divided into seven classes depending on the specificity of their action. The ear4 pepattenzing genes (1) are involved in speclfylng body axes (reviewed by JAN and JAN 1993), hence providing positional cues for establishment of the expression pattern of the next group of genes, called the poneural genes (2). This group includes the genes of the achae&scu& complex (VILLAEES and CA- BREW 1987; MARTINEZ and MODELELL 1991), atonal OAR-

Corresponding authm Hugo Bellen, Howard Hughes Medical Insti- MAN et al. 1993) and daughterless (CAUDY et al. 1988). The

tute, Department of Molecular and Human Genetics, Baylor College proneural genes are expressed in a group Of

of Medicine, Houston, TX 77030. E-mail: [email protected] cells endowing them with the capability to become SOPS.

Genetics 139 1663-1678 (April, 1995)

1664 A. Kania et al.

The cells in these proneural clusters interact with each other through the gene products of the neurogenic gene (3), leading to specification of cells destined to become SOPs (reviewed by CAMPOS-~RTEGA 1993). The next group of genes, the neuronal precursor gena (4), is expressed solely by committed SOPs and is thought to en- dow them with early neuronal characteristics. This group includes asense (ALONSO and CABRERA 1988; GONZALEZ et al. 1989) and prospro (DOE et al. 1991; VAESSIN et al. 1991). The SO typesekctorgenes are involved in specifylng the type of SO (5) that will develop from the individual SOP. The members of this group include cut (BLOCH- LINGER et al. 1988) and pox-neuro (DAMBLY~HAUDIERE et al. 1992). The second group of SO cell fate selector genes (6) determines the cell identity within particular SO lineages. This group includes the gene numb, mutation of which results in a change in cell fates among the progeny of a SOP (UEMURA et al. 1989). Following determination of cell identities within an SO, the individual cells begin expression of genes involved in their further dzffmentia- tion (7). This class probably includes many genes that may be required for growth cone formation, axon guidance, fasciculation, target recognition, synapse formation, and other aspects of neuronal function. The above model for neural development, as well as the availability of a large number of markers of neuronal differentiation, makes the embryonic PNS an excellent model system to further unravel the molecular mechanisms of neuronal development. To isolate new genes that control neuronal determination and differentiation, we have previously screened the third chromosome of Drosoph- ila using a chemical mutagen (SALZBERG et al. 1994). However, the availability of large collections of Pelement enhancer detector insertions (e.&, TOROK et al. 1993) allows a more rapid though less systematic screening to identify potentially interesting novel genes. Given the advantages of Pelement enhancer detectors (e.&, BELLEN et al. 1989; WILSON et al. 1989), we screened the collection generated by TOROK et al. (1993) and herein present the identification and preliminary genetic and phenotypic characterization of 29 genes on the second chromosome of Drosophila that play a role in PNS development and have not been previously described.

MATERIALS AND METHODS

Stocks: All stocks were maintained on standard Drosophila medium at room temperature (ASHBURNER 1989). Genetic nomenclature is as outlined by LINDSIXY and ZIMM (1992). Mutant stocks were generated as described by TOROK et al. (1993) and are of the y w; P{lacZ,w+]/CyO genotype. The P{lacZ,w+] is an enhancer detector (BELLEN et al. 1989; O'KANE and GEHRINC 1989) described in BIER et al. (1989). The second chromosome deficiencies and relevant mutant strains were obtained from the Indiana Drosophila Stock Cen- ter and are listed in Table 1.

Immunocytochemistry: Three- to 16-hr-old embryos were collected and processed for whole mount antibody stains according to standard protocols (BIER et al. 1989; BELLEN et al.

1992b). The primary antibodies, MAb 22C10 (FUJITA et al. 1982; GOODMAN et al. 1984; ZIPURSKY et al. 1984) or anti$- galactosidase (Promega), were adsorbed at 4" overnight at 1 : l O O and 1:2000, respectively. The secondary antibody was a biotinylated horse anti-mouse IgG (Vector). The primary- secondary antibody complex was detected using the Vectas tain ABGHRF' kit (Vector) using diaminobenzidine (Sigma) and hydrogen peroxide (Sigma). Stained embryos were mounted in 70% glycerol and carefully examined with a Zeiss Axiophot microscope.

In situ hybridization to polytene chromosomes: Males het- erozygous for the mutant chromosome were crossed to wild type (Canton S ) or y w females. Salivary glands of the progeny larvae were dissected in Ringer's insect saline and fixed with acetate. The pretreatment and hybridization to the chromosomes were essentially as outlined by LANCER-SAFER et al. (1982). The probe used was P{ZwB] (WILSON et al. 1989) DNA labeled with digoxigenin using the Genius kit (Boehringer Manheim) . Following hybridization, the probe was detected using an antidigoxigenin antibody conjugated to alkaline phosphatase (Boehringer Manheim) and nitrozole blue/5- bromo-4chlore3indolyl phosphate substrates (Boehringer Manheim). The chromosomes were then stained with Giemsa (Sigma), mounted in Permount (Fisher) and examined with a Zeiss Axiophot microscope.

Isolation of revertants: The isolation of revertant chromosomes was essentially as described by TOROK et aZ. (1993).

RESULTS

Structure of the embryonic PNS: MAb 22C10 (Fu- JITA et al. 1982; GOODMAN et al. 1984; ZIPURSKY et al. 1984) is an excellent marker for the developing embryonic PNS (Figure 1) and has been used successfully to screen for mutations that affect embryonic PNS development (SALZBERG et al. 1994). MAb 22C10 stains a total of 44 neurons in the PNS of each abdominal hemi- segment: 12 in the dorsal cluster, 12 in the lateral cluster, 9 in the dorsal portion of the ventral cluster and 11 in the ventral portion of the ventral cluster (CAMPOS ORTEGA and HARTENSTEIN 1985; DAMBLY-CHAUDIERE and GHSEN 1986; GHY~EN et al. 1986; BODMER and JAN 1987). In addition to labeling cell bodies, MAb 22C10 stains axonal projections of all neurons in the PNS including the intersegmental (ISN) and the segmental (SN) nerve fascicles. Furthermore, the highly specific immunoreactivity of MAb 22C10 reveals subtle morphological attributes of the labeled neurons such as axon thickness and shape of dendritic projections.

Genetic screen: We screened a collection of 2000 strains carrying second chromosome Pelement insertions that cause lethality or semilethality (TOROK et al. 1993). Embryos from each strain were stained immuno- cytochemically with MAb 22C10 and examined using light microscopy, and 92 strains exhibiting PNS defects were retained for further analysis. To establish whether the mutations are caused by Pelement insertions, we excised the P elements as described by TOROK et al. (1993). Briefly, females harboring mutant chromosomes were crossed to males bearing a source of A2- 3 transposase (COOLEY et al. 1988), and flies with the Pelement and the A2-3 transposase were crossed back

Mutations Affecting Embryonic PNS 1665

TABLE 1

Deficiencies and point mutations used in this study

Deficiency Cytology Reference

Df(2L)3OA; C Df(2L))ast2 Df(2L) b87e25 Df(2L)ed2 Df(2L)esclO Df(2L)H20 Df(WJ2 Df(2LY39 Df(2L)Mdh Df(2L)osp29 Df(2L)PMF

Df(2L)prdl. 7 Df(2L)pr76 Df(2L)rlO Df(2L)S2 Df2L)sclP8 Df(2L) TW161 Df(2L)TW50 Df(2L) TW84 Df(2L) VAI 7 Df(2R)vg135 Df(2R)44CE Df(2R)AAZl Df(2R) bwm2L Df(ZR)cn9 Df(2R) CXl Df(2R)en28 Df(2R)m30 Df(2R)en-A

Df(2R)me

Df(2L))pr-A14

Df(2R)a-B

Df(2R)JPl Df(2RYP8 Df(2R)Ml73 Df(2R)PC4 Df(2R)Pcll7B Df(2R)Pu-D17 Df(2R)trix

29F7-30A1; 3OC2-5 21D1-2; 22B2-3 34B12-Cl; 35BlO-Cl 24A34; 24D34 33A8B1; 33B2-3 36A8-9; 36E1-2 31B; 32A

30DF; 31F 31A-B; 32D-E

35B1-3; 35E6 21A1; 21B7-8 37D2-7; 39A47 33B2-3; 34A1-2 37D; 38E

21C6D1; 22A6-Bl

38A6-Bl; 40A4B1 36E4F1; 38A6-7 37F538A1; 38B2-Cl

48GD; 49D 44C; 44E14

59D6-El; 60GD 42E; 44C

35El-2; 36A6-7

24C2-8; 25C2-8

37B9-Cl; 39F5-38A1

56F; 57D-12

49C1-4; 50C23-Dl-2 48A1-2; 48BC1 48A34; 48C6-8

47E3; 48B2 47D3; 48A5-6

46C3-4; 46C9-11 51C3; 52F5-9 52F5-9; 52F10-53Al 56F 55A; 55F 54E8F1; 55B4C1 57B4; 58B 51A24; 51B6

LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) ASHBURNER et al. (1990) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) MLODZIK et al. (1990) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) SAXTON et al. (1991) SAXTON et al. (1991) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992) LINDSLEY and ZIMM (1992)

Bloomington stock Mutations center number Cytological location Reference

abbreviated (abb) 396 59EF LINDSLEY and ZIMM (1992) abero ( a h ) 206, 207 54E LINDSLEY and ZIMM (1992) big brain (bib) 30F LINDSLEY and ZIMM (1992) Bristle (Bl) 238, 404 38A LINDSLEY and ZIMM (1992) expandzd (ex) 295 21c LINDSLEY and ZIMM (1992) heavy vein (hv) 274 59D LINDSLEY and ZIMM (1992) knot (kn) 316 51EF LINDSLEY and ZIMM (1992)

longitudinals lacking (lola) 47A SEEGER et al. (1993) mastermind (mum) 50C LINDSLEY and ZIMM (1992) mini (mi) 350 59E LINDSLEY and ZIMM (1992) numb (numb') 30B LINDSLEY and ZIMM (1992) smooth (sm) 399, 400 56E LINDSLEY and ZIMM (1992) spitz (spi' ) 1859 38A LINDSLEY and ZIMM (1992)

l ( 2 M H ) 297, 418 58A-F LINDSLEY and ZIMM (1992)


FIGURE 1.-Lateral view of three abdominal segments of a wild-type embryo stained with MAb 22C10. Approximately 40 PNS neurons are visible in each segment dc, dorsal cluster; IC, lateral cluster; v'c, ventral' cluster; vc, ventral cluster; lch5, five lateral chordotonal neurons. Anterior is left and dorsal is up.

to the original stock. The progeny were then examined for the presence of whiteeyed non-Cy flies indicating reversion of lethality and precise or near precise excision of the Pelement. As shown in Table 2, the lethality in 63% of the Pelement insertions is revertible. This percentage does not accurately reflect the number of P-element insertions responsible for the observed PNS defects since some of the chromosomes carry other lethal mutations in addition to the Pelement insertions. Therefore, all mutant strains displaying a reproducible PNS phenotype were retained for further study, even if lethality was not revertible.

The lethal phases of the corresponding strains were determined as described by TOROR et al. (1993). As shown in Table 2, the majority of the isolated mutations causes first instar or embryonic lethality.

Phenotypic grouping: Based on the MAb 23210 phenotype, the 92 strains were divided into five phenotypic classes essentially as described by SALZBERG et al. (1994) : gain of neurons, loss of neurons, pathfinding/fasciculation defects, organizational defects and morphological defects. As shown in Table 2, the frequency of these defects is distributed approximately equally among all the mutants except for the gain of neurons defects, which are less frequent.

To determine whether the isolated mutations were allelic to each other or to previously isolated genes, we

TABLE 2

S u m m a r y of the chromosome-2 P-element screen

Percentage of saved

Numbers strains

Lethal strains screened 2000 Number of saved strains 92 100 Number of revertible strains 52 62 a Mutations allelic to previously

42 46

76 83 4 4 1 1

10 11 1 1

isolated mutations Lethality'

Embryonic Larval lethal Pupal Adult ND

PNS phenotypes, observed w i t MAb 22C10"

Gain of neurons Loss of neurons Pathfinding Organization Morphology

17 (17)d 18

30 (16) 33 29 (9) 31

28 (8) 31 -. 23 (6) 25

"Percentage based on the number of known revertible strains per total number verified (n = 83). Because of wild- type looking adult escapees, we were not able to determine the revertability of seven strains. One homozygous viable strain exhibited bristle defects; hence we were able to assess its revertability. Two embryonically lethal strains were not examined for revertability.

Lethal phase based on data of TOROK et al. (1993). "Note that some strains exhibit a complex phenotype and

dNumbers in parentheses indicate alleles of previously are included in more than one category.

identified mutations.

performed a series of complementation tests. First, all mutations that cause a similar phenotype were crossed to each other and separate complementation matrices were established. Second, we determined that all mutations that cause the presence of extra neurons were allelic to mastermind (15 alleles) (SMOLLER et al. 1990) or big brain (2 alleles) (RAO et al. 1992), implying that no new neurogenic genes were isolated in this screen. Third, through complementation analysis of strains exhibiting pathfinding defects, we established that there was an insertional hotspot in longitudinals lacking (lola) (SEEGER et al. 1993; GINIGER et al. 1994): 19 of the 92 saved mutations failed to complement mutations in lola. Hence, 36 mutations identify only three previously characterized genes and were not studied further (Figure 2). Fourth, we determined the Pelement mapping position in the remaining mutant strains and carried out complementation tests with candidate mutations in the corresponding chromosomal region (see below). The results of these complementation tests demonstrated that we isolated mutations in two other previously characterized genes: spitz (2 alleles) (NOSSLEIN-VOLHARD et al. 1984) and numb (4 alleles) (UEMURA et al. 1989). The re-


w

FIGURE 2.-Cytological location of 96 insertions on the second chromosome (four strains carry two insertions mapping at different cytological positions). Black dots represent single independent insertions except for the indicated genes (numbers in parentheses indicate the number of insertions). Columns of dots indicate multiple insertions in the same gene.

maining 50 mutations were grouped as shown in Tables 3-5. These 50 mutations identify 29 different complementation groups involved in PNS development. Eight of these genes are identified by multiple alleles, whereas 21 are identified by one allele. Mutations were assigned to complementation groups solely on the basis of complementation data and not on the basis of similar P- element mapping positions or phenotypes.

Cytological and genetic mapping: The P elements present in 56 (92 less 36 insertions in mum, bib, lola) strains were mapped using standard cytological mapping techniques and P{lwB/ DNA (WILSON et al. 1989) as a probe. The mapping position of all P elements are diagrammatically shown in Figure 2 and listed in Tables 3-5. Furthermore, to independently confirm the mapping position and to assess whether the P-element insertion is indeed the cause of lethality, whenever possible, we crossed the mapped strains to strains harboring deficiencies uncovering the cytological location of the insertion and tested for complementation.

The results of the deficiency mapping data of the 50 mutant strains are summarized in the “Complements” and “Fails to complement” column of Tables 3-5. Based on these data as well as the reversion analysis, we conclude that 26 mutations are caused solely by the P- element insertion and are not associated with another lethal mutation on the same chromosome. Another nine mutant chromosomes may carry P elements that cause the mutant phenotype as they fail to complement deficiencies uncovering the regions to which the Pele- ments map. The inability to recover wild-type revertants is probably due to another lethal mutation on the same chromosome or a damaged P element. The remaining 15 mutations fall in different categories but several are allelic to other revertible P-element mutations (see below gutfeeling and bawen) . All genes identified by revertible P elements have been named on the basis of one of the phenotypic characteristics. We also named genes

identified by Pelement insertion mutations that are un- covered by deficiencies but were not revertible in the above genetic test. Hence, it should be emphasized that these mutations may not map to the same position as the Pelement. A number of genes identified by a single mutation were not named since we do not know where they map or whether the P element is responsible for the observed phenotype.

Phenotypic characterization: All 29 complementation groups identified in the screen were categorized on the basis of their MAb 22C10 phenotype into the following four phenotypic classes: loss of neurons, organization defects, fasciculation defects and morphological defects. As mentioned previously, all mutations that cause a gain of neurons were found to be allelic to mum and bib and were not included in this study. The number of mutations identified in each category are summarized in Table 2 and are discussed below.

Mutations that cause loss of neurons: Since the phenotypic description of mutants is solely based on h4Ab 22C10 staining, the term “loss of neurons” generally refers to lack of MAb 22C10 immunoreactivity and not necessarily a genuine decrease in cell number. We also included in this group mutations that cause an increase in cell size with a concomitant loss of neurons. As outlined in Table 3, there are eight complementation groups in the loss of neurons category: obelix, bawen, 67/12,82/55,88/7, gutfeeling, vegetableand 99/18. The mutations are ordered by their cytological mapping in- terval, starting with the telomere on the left arm of the chromosome.

The 81/42 insertion chromosome carries a mutation defining the obelix locus. Homozygous obelix embryos exhibit an increase in neuronal size, with a concomitant decrease in the number of neurons, as well as fasciculation defects. The overall division of the PNS into clus ters is relatively well preserved in obelix embryos. Given the fact that 81/42 is revertible with respect to lehality


TABLE 3

Mutations causing loss of neurons

Name Cytological Lethal Fails to Phenotypic ~~ ~~

(abbreviation) location Stock(s) Reversion" phase' complement Complements description/remarks LacZ expressionc

obelix (obx)

[99/181

barren (barr)

vegetable ( veg)

31EF 5OC6-12

36E/F

38A

38A

38A 38A

38A

38A 38A

39BC

43D 56F/57A

44F

48E6-12

48E 48E

53GE

53C-E

53C-E 51A

2 1A

81/42

99/18

8/2

140/14"

140/28" 3/10

139/3

48/5 49/ 12

67/12

82/55

881'7

95,' 1 2e

95/40' 118/3

34/2

72/28

170/10

12/6

Y

YJ

Y

Y

Y Y

Y

N N

N

N

N

N

N Y

N

Y

N

N

A

E

A

E

E E

E

E E

E

E

E/L

E

E E

E

E

E

E

Df(2L)J39 masta ind Few and large Df(2L)J2d neurons

Df(2R)PC4 Loss of chordotonals, Df(2R)MI 73 MD and ES smooth neurons

Df(2L)p-A14 Bristle Lch, MD ES missing

Pathfinding and organization defects

Df(2L)p-A14 Bristle, spitz MD and Lch missing Df(2L)TW84

Pathfinding and organization defects, neuronal loss

Df(2L) TW84 Severe loss Severe loss

Df(2L)VAI 7 Few and large Df(2L))TwIGl neurons

Df(ZR)cn9 As above

Df(2R)AA21 smooth

Df(2R)44CE Few and large neurons

Df(2R)en-B Loss of Neurons Df(2Rjen28 Faint staining

Loss of neurons, Lchs moved, fasciculation defects

Severe loss of neurons, fasciculation

Df(2R)trix Loss of neurons, Df(2RYPI fasciculation Df((ZRYP8 defects Df(2L)PMF d and v' neurons

missing

Most tissues, complex

Basic

H, gut, Ap

CNS, gut

Basic, H H, CNS

Subset of PNS and CNS, glia

H, PS, Ph Weak overall

Basic. M

Basic Weak overall

Basic

hG, strong mG As above As above

Subset CNS Brain Basic

Basic AS, SG Strong Ep

Basic

CNS, brain, M, Basic I

' Revertability: Y, revertible; N, nonrevertible. * Lethal phase according to TOROK et al. (1993): A, adult; E, embryonic; L, larval; P, pupal. Pattern determined by staining with anti-@galactosidase antibodies. A P , anal pads; AS, anterior spiracles; Ep, epidermis; H,

head; mG; midgut; M, muscles; Ph, pharynx; PS, posterior spiracles; SG, salivary glands. Basic pattern refers to staining in the hindgut, midgut, pharynx and ectodermal stripes. Complex pattern is characterized by LacZ expression in many tissues.

dViable over insertion but adults have rough eyes. "Insertions might be members of the same cluster and not genuine alleles [see TOROK et al. (1994)l. 'The revertability refers the lethality and not the neuronal loss phenotype. For more details see the RESULTS.

and fails to complement Df(2L)J39, it is likely that obelix chordotonal (Lch) neurons. Homozygous 8/2 adults maps at 31EF. are sterile, have rough eyes and have decreased bristle

The barren (bum) gene is defined by seven allelic inser- numbers. Genetic and phenotypic characterization re- tions at cytological position 38A. All bum alleles cause veals an allelic series ordered as shown in Table 3. The neuronal loss of varying extent. Embryos homozygous 49/12 mutation is the most severe allele causing a sub- for the mildest allele, 8 / 2 , display loss of some of the stantial neuronal loss such that only a few neurons are dorsal MD and ES neurons and most of the lateral left in each abdominal segment. Phenotypes caused by

Mutations Affecting Embryonic PNS

TABLE 4

Mutations causing organization defects

1669

~

Name Cytological Lethal Fails to Phenotypic (abbreviation) location Stock(s) Reversion“ phaseb complement Complements descriptionjremarks LacZ expression‘

I -

jloater ( jltr)

[84/ 121

hoi-polloi (hoip)

[4/241

gluon (glu)

rubberneck (rubr)

[54/221

bumper-to- bumper ( btb)

quo uadas (quo)

unchained (unch)

anarchist (ana)

chrowded (chr)

21E

21EF

30BC

30C

30D

36A-E

44E

46B3-13

47AB

47D3-6

49BC

54F

59E

95/30’

95/31‘ 69/21

84,’ 12

71/4

4/24

88/19

25/7

54/22

99/ 1

168/3 161/31

44/12

155/1

78/5

69/8

ND

ND Y

Y

N

N

N

Y

N

Y

Y Y

Y

N

N

Y

E Df(2L)ast2

ND E Df(2L))ast2

E Df(2L)jOA; C

E Df(2L)3OA; C

E Df(2L)J39d

E Df(2L)H20

E Df(2R)44CE

ND

E

E E

E Df(2R))m-B

E Df(2R)CXl

E Df(2R)Pcll7B

PA Df(2R))”mezL

Df(2L)S2 61 / I Df(2L)SZ Df(2L)S2 61 / I

numb, hoipolloi

Df(2L)Mdh lola, numb 84/15 4/24

Df(2L)Mdh numb hoa-polloi

Df(2L)rlO

lola

bib, mam anarchist Df(2R)me 61/1

lola

lola Df(2R)en-A

Df(2R)vg135

54/22 bib mam abero

lola abbreviated heavy vein

Lchs moved dorsally

Fewer Lch Pathfinding

Lch moved dorsally

Organization and fasciculation defects

Organization defects

Organization defects

Organization and fasciculation defects

Scattered neurons

Connectivity defects, Lch stalled

Variable Misplaced and

abnormal Lchs General Organization and

fasciculation defects

Lch moved dorsally

Scattered neurons

Lch organization, thick axons

ND

ND CNS midline Basic

Basic

Strong mG and hG Early PNS

Complex

M, Ph, hG

Basic, M, Ps

Basic, M Early PNS

Basic CNS. M

Strong, complex ND

Basic Weak

Basic

Basic, M

Basic Early and late M

‘“*See Table 3. Additional abbreviation: hG, hindgut; ND, not determined.

the different alleles vary depending on the position of the insertions with respect to the locus (M. BHAT and H. J. BELLEN, unpublished data).

Embryos homozygous for the 67/12 chromosome exhibit an increase in neuronal body size and a concomitant reduction in the total number of neurons. The MAb 22C10 positive cell bodies in the PNS as well as in the CNS appear enlarged and fused (Figure 3B). Attempts to excise the 67/12 P insertion or to map the mutation using deficiencies were unsuccess- ful. Given the increase in cell size, it is possible that the mutation on the 67/12 chromosome affects a gene playing a role in cell division (SALZBERG et al. 1994).

82/55 carries a mutation that causes an enlargement of neurons in the PNS and CNS. The size and morphology of the PNS neurons are not affected as severely as in 67/12 mutants and the overall organization of the PNS in 82/55 mutants is closer to wild type than in obelix embryos.

The 88/7 mutation defines yet another essential locus in which a mutation causes an increase in the neuronal cell body size. Homozygous 88/7 embryos exhibit large and fused neurons and abnormal neuronal out- growths. The total number of neurons in the PNS is decreased but not as severely as in 67/12 embryos. The overall neuronal morphology appears relatively normal.

Three lethal insertions, 95/12, 95/40, and 118/3,


TABLE 5

Mutations affecting neuronal morphology

Name Cytological Lethal Fails to Phenotypic (abbreviation) location Stock(s) Reversion" phaseb complement Complements description/remarks LacZ expression'

43/6 N E Df(2L)edZ Df(2L)sclW

29EF 37E

bunched (bun) 33F

fondue (fond) 26A

38BC

35D

10/21 N E 35/14 N E

90/36 Y P

25/14 N E

50/2 N E

81/4 Y L

on-thwack (rack) 48A 150/ 1 Y

charlatan (chn) 51EF 42/18 Y

mindmelt (mm) 54A 71/3 Y

A

E

P/A

fata morgana ( fam) 5781-5 59EF 57B

57B

57B

26/8 Y A

75/5 Y E

77/ I Y E

124/5 Y E

Df(2L)osp29

Dff2WP1

Df(2R)bw""'

Df(2R)Pu- D l 7

Df(2R)Pu- D l 7

Df(2R)Pu- D l 7d

Df(2IJVAl7 Df(2L)TW5U Df(2L)pr-AI 4

Df(2L)prdl. 7 Df(2L)esclU

mini, smooth mindmelt lola Df(2L)pr76

Df(2L)rlO Df(212)b87e25

Df(2R)m28 Df(2R)m-B Df(2R)enXI Df(2R)er-A

knot

fondue

Elongated neurons, thick axons, fasciculation defects

Defects as above As above (more

severe)

Faint Lchs, disorganized

Fused, elongated lch

As above

Misplaced Lchs/disconnected

clusters

Loss of Lch, stretched Lch

Faint staining

Fused neurons

Lch stain faintly,

Same elongated

Same

Same bd neurons stain faintly

Basic CNS subset VMes, Tr, PNS

Basic, early PNS Basic, early brain

Basic, M, Tr

Basic, CNS and PNS

Brain, complex subset

Basic

Basic, CNS

Basic, strong Ec

Early M CNS subset Basic

Complex, CNS primordium

Basic, early PNS M Basic

Basic

a-pSee Table 3. Additional abbreviations: Ec, ectoderm; ND, not determined; Tr, trachea; VMes; visceral mesoderm.

cause a similar apparent loss of neuron phenotype when stained with MAb 22C10 and were found to be allelic to each other. This complementation group was named gutfeeling because all three insertions confer strong P-galactosidase expression in the gut and affect sensory organ development. As shown in Figure 3C, a decreased number of neurons is evident in each PNS cluster of gutfeezing mutant embryos when compared to wild type. In addition, the level of 22C10 immunoreactivity is significantly lower than normal in the remaining PNS (and CNS) neurons. The overall organization of the PNS appears normal in gutfeeling embryos, although aberrations in the position of specific neurons and their axonal connections are often observed.

A group of four allelic mutations that cause a loss of neurons identifies a new gene involved in PNS development that we named vegetable (veg). The 34/2 insertion chromosome carries an allele of ueg that causes a highly variable loss of neurons in the PNS. All types of neurons

are affected by veg mutations, but ES neurons seem to be the most affected. 34/2 embryos also exhibit intersegmental nerve pathfinding defects as well as a disruption of the overall CNS organization. The phenotype ob served in 72/28 embryos (Figure 3D) is more consistent than the one observed in 34/2 embryos: Lch neurons are usually missing, only 4-5 neurons are left in the dorsal cluster and the v' chordotonal is the only neuron remaining in the lateral region. Severe loss of neurons is also observed in the ventral PNS. In addition to the neuronal loss phenotype, 72/28 embryos frequently exhibit intercluster axom that fail to connect or are mis- routed. The 72/28 insertion is revertible to wild type. The 170/10 strain represents an even stronger allele of veg: loss of neurons as well as the CNS phenotype are more severe than that observed in 34/2 embryos. Some severe organization defects and disconnected neuronal clusters are also apparent in 170/10 embryos. The overall morphology of 170/10 embryos is abnormal, sug-

1671

wild typ

Mutations Affecting Embryonic PNS

67n 2 gutfeehg

FIGURE 3.-Mutations causing a loss of neurons. Three abdominal segments of embryos stained with MAb 22C10. (A) Wild type (Can- ton S, stage 16); (B) 67/12 embryo. Note the decreased number and very large size of the neurons. (C) gutfeeling (allele 118/3). Note the decreased number of neurons in the dorsal, lateral, and ventral’ cluster. (D) vegetable (allele 72/ 28). Note the severe lack of neurons and lack of organization; the bipolar and v’ chordotonal neurons (arrows) are still present. (E) Embryo from the 99/18 stock. Note the complete absence of the Lch neurons and the severe reduction in the number of neurons in the dorsal cluster. However, the v’ chordotonal neurons (arrows) are usually present.

gesting that the 170/10 chromosome either carries the most severe allele of vegetable or other mutations that cause embryonic defects. The fourth allele of veg is carried by the 12/6 chromosome. Its allelism is based on complementation data since 12/6 embryos do not dis play reproducible phenotypes similar to the ones found in the other vegetable strains. Homozygous 12/6 embryos exhibit neuronal loss only occasionally.

The 99/18 stock harbors a single revertible P insertion that maps at 36E/F. Embryos from this stock exhibit a severe loss of different neuronal types (Figure 3E). Other tissues such as CNS and muscles are not obviously affected (data not shown). Phenotypic analysis of the revertants reveals that the P element does not cause the observed phenotype since it persists in strains carrying the viable revertant chromosome. Preliminary genetic analysis suggests that the mutation causing the phenotype maps to the Cy0 balancer chromosome and that the P element is inserted in the L30 ribosomal gene (S. KOOYER and H. J. BELLEN, unpublished data). Interestingly, although none of the 99/18 mutants show a minute phenotype, M(2)36Fmaps to the same region.

Mutations that cause organization defects: As re-

vealed by MAb 22C10 (Figure l), the organization of the embryonic PNS is highly stereotyped and the position of specific neurons along the dorsoventral and anterioposterior axes is constant in most abdominal segments. The common phenotype of the mutants included in this group is a dislocation of neuronal clusters or lack of organization within specific clusters. A number of strains in this group also exhibit loss of neurons; however, because these mutations cause obvious organization defects, we assigned them to this phenotypic category. We also included mutations that cause a scattered neuron phenotype characterized by complete PNS disorganization as well as abnormal development of other tissues. It is possible that the PNS phenotype of such mutants is secondary to other defects. However, the observation that these mutants exhibit a high degree of random neurogenesis (i.e., scattered neurons) was the basis for their inclusion in this group. This category contains 12 complementation groups: $outer, 84/12 (see below glial cells missing), hoipolloi, 4/24, gluon, rub beneck, 54/22, bumper-to-bumper, quo vadis, unchained, anarchist and chrowded. The mutations in Table 4 are ordered by their cytological position.


The jloater gene is defined by three insertions causing similar organizational defects. Embryos homozygous for the 95/30 insertion chromosome display defective organization of the dorsal cluster, Lch neuron migration defects as well as occasional fasciculation defects. The 69/21 allele ofjloater is characterized by absence of two of the five Lch neurons. The loss of neurons sometimes extends to the ventral region affecting one of the chordotonal neurons. Embryos homozygous for 69/21 also exhibit pathfinding problems as well as a collapsed CNS. Based on deficiency and cytological mapping of insertions in $outer, its mapping position appears to be very close to Star (LINDSLEY and ZIMM 1992). The phenotype of jloater embryos and the ,&galactosidase expression pattern of the 69/21 insertion strain are very similar to those described for Star. However, complementation data show that jloater complements a Star allele. Given the complex complementation data observed with Star and asteroid alleles (LINDSLEY and ZIMM, 1992), we cannot exclude that jloater is allelic to Star.

Another mutation causing neuronal disorganization is identified by the 84/12 insertion, which cause the Lch neurons to be located more dorsally than in wild type. SALZBERG et al. (1994) have proposed that the Lch neurons arise in the dorsal cluster, rotate 145" and descend to a more lateral position during PNS differentiation. The phenotype observed in 84/12 embryos suggests that this insertion defines a gene involved in the migratory but not the rotatory aspect of Lch development. 84/12 embryos also exhibit a lack of longitudinal tracts in the CNS. Complementation analysis indicates that the 84/ 12 insertion is allelic to glial cells missing (T. HOSOYA and Y. HOTTA, personal communication).

The hoipolloi locus is defined by a mutation carried by the nonrevertible insertion chromosome 71/4. Em- bryos homozygous for this mutation exhibit subtle organization defects characterized by slightly misplaced cells within specific clusters. This defect is frequently apparent in dorsal clusters as seen in Figure 4B. In addition to organization defects, hoi$olloi embryos frequently display mild fasciculation defects.

Although phenotypically similar to hoipolloi, the mutation carried by the 4/24 insertion chromosome is ge- netically distinct. When homozygous it also causes slight organizational defects in the dorsal cluster, abnormally positioned Lch neurons and occasional fasciculation defects.

The gluon gene is defined by a mutation on the 88/19 chromosome. The embryonic phenotype (Figure 4D) is characterized by the ventral and lateral PNS exhibiting subtle organization defects resulting in irregularly shaped clusters. Although we were unable to revert this mutation, the lethality of 88/19 over Df(2L)H20 suggests that it may be the cause of the phenotype.

The rubberneck gene is identified by the revertible insertion 25/7 that affects the location and organization of most clusters in addition to a loss of neurons in the

dorsal cluster (Figure 4F). Furthermore, homozygous 25/7 embryos also exhibit severe pathfinding defects as well as gaps in the CNS. In some 25/7 embryos, we observe that axons of Lch neurons do not follow a straight path to the CNS.

The mutation carried by the 54/22 chromosome appears to cause a severe disruption in development, resulting in embryos dotted with darkly staining MAb 22C10 positive cells in the periphery. Most neurons display abnormal morphology, although some of them send out axon-like projections. The overall morphology of 54/22 embryos is generally very poor, indicating that the 54/22 mutation affects other tissues than the nervous system. The phenotype is already apparent in relatively young embryos [stage 12; for staging see CAMPOS- ORTEGA and HARTENSTEIN (1985) ], suggesting that the defects originate early in development.

The bumper-tebumper (btb) gene is identified by the revertible 99/1 insertion. The phenotype observed among 99/1 embryos is distinguished by pathfinding and connectivity defects, mainly from the dorsal to the lateral cluster. Furthermore, the five Lch neurons fail to completely descend from the dorsal cluster and are stalled along the dorsoventral path of their migration, close to their final destination. Occasionally, this phenotype is more extreme and the five Lch neurons remain associated with the dorsal cluster as shown in Figure 4G. Complementation tests show that the 99/1 is one of the rare Pelement insertions that maps to 47A but complements a mutation in lola, an insertional hotspot.

The three insertions in the quo vadis (quo) locus cause similar defects in the migration of the five Lch neurons. These defects resemble those observed in bumper-to- bumper mutations. The stalled lateral chordotonal neurons often seem to die, resulting in small, MAb 22ClO positive, spherical cell bodies (Figure 4H). The 168/3 quo allele causes a low expressivity of the Lch migration defects. Most 168/3 embryos do not exhibit any defects, suggesting that it is the weakest of the three alleles of quo. In contrast to 168/3, the 161/31 allele causes fasciculation and connection defects, as well as a loss of different neuronal types in addition to the Lch neuron defects. The 44/12 allele primarily causes a loss of Lch neurons, loss of dorsal cluster neurons and Lch migration defects.

The 155/1 insertion that defines the unchained gene causes the Lch neurons to stall along the path of their migration. Frequently, the stalled neurons fail to extend their axons toward the CNS. Furthermore, the affected clusters sometimes consist of less than five neurons. Although the 155/1 chromosome cannot be reverted to wild type, the P insertion is probably the cause of the phenotype since it fails to complement a deficiency spanning its cytological location.

The anarchist allele 78/5 belongs to a group of mutations that cause a scattered neuron phenotype (Figure 45) much like the mutations found on chromosomes


wild type

wild type

rubbc" - -" bumper-to-bumper

anarchist

gluon

FIGURE 4.-Mutations causing positional and organizational defects. Abdominal segments of embryos stained with MAb 22C10. (A) Wild type (Canton S; focus on four dorsal clusters, stage 16); (B) hoi-polloi (allele 71/4). Note the abnormal organization of neurons in the dorsal cluster and the anomalous axonal pathways (arrows) of the descending nerve. (C) Wild type (Canton S; stage 16), focus on ventral clusters; (D) Similar view of a gluon (allele 88/19) embryo. The neurons of each cluster appear consistently bunched together. (E) Wild type (Canton S, stage 16); (F) r u b h e c k (allele 25/7). The Lch neurons (arrows) are located more dorsally than in wild-type embryos. Furthermore, neuronal loss and defective axonal pathways are clearly visible. (G) bumper-t&umper (allele 99/1). Arrows point to improperly positioned Lch neurons. Note that the Lch neurons that fail to descend do not rotate properly and point posteriorly instead of dorsally (see second segment). (H) quo vadis (allele 168/3). Arrows point to improperly positioned and poorly differentiated chordotonal neurons. (I) Wild type (Canton S; stage 14); (J) anarchist (allele 78/5; approximately stage 14). Note the completely disorganized PNS and the lack of differentiation.

61/1 and 54/22. However, in contrast to 54/22, the that causes crowding and abnormal appearance of the overall morphology of early embryos (up to stage 13) Lch neurons with a variable severity. Ventral chordoto- is relatively normal. As the other mutants of this group, nals as well as other neurons (ES and MD) appear to 78/5 embryos are spotted with cells labeling strongly be affected as well. Since we observe a great variability with MAb 22C10, some of which are able to connect to in the penetrance of the 69/8 mutation, including the others with axon-like projections. emergence of adult escapees, we propose that 69/8 is

The chrowded locus is defined by the 69/8 insertion a weak allele of chrowded.


Mutations that cause fasciculation and pathfinding defects: We have retained one Pinsertion strain exhibiting prevalent pathfinding and fasciculation defects in the PNS. The mutation on the 25/11 chromosome causes mild pathfinding defects that are characterized by ISNs and SNs crossing segmental boundaries and fasciculating with neighboring nerves. Furthermore, 25/11 embryos sometimes display aberrant CNS longitudinal tracts and head involution defects. The embryonic phenotype is not fully penetrant and adult homozygous escapees with behavioral defects are routinely observed. Interestingly, in 25/11 embryos, P-galactosidase is expressed in CNS glia, ISN/SN exit glia as well as in peripheral glia. We have been able to obtain excisions of this Pelement that are lethal over a deficiency, suggesting that the 25/11 Pelement defines an essential locus involved in PNS development (A. KANIA, unpublished observations).

Mutations that cause abnormal neuronal morphology: This group is comprised of those mutations that mainly alter the shape or appearance of the cell body of neurons or expression of the 22C10 antigen in the PNS. Some of the strains included in this group also exhibit other defects such as loss of neurons or pathfinding defects, but we include them in this category due to their prominent morphological defects. This group is comprised of eight complementation groups: cyrano, bunched, fondue, 8 1 /4, on-the-rack, charlatan, rnind- melt, and fatu rnmganu. The mutations are ordered in Table 5 by their cytological mapping position.

The cyrano complementation group is defined by three unmapped and nonrevertible mutations. The mutation(s) on the 43/6, 10/21 and 35/14 P-insertion chromosomes fail to complement each other and also cause a very similar phenotype: elongated neuronal cell bodies, dark staining with MAb 22C10 (Figure 5B), and mild germ band retraction defects. Furthermore, cy embryos have thicker than wild type axon bundles and frequently exhibit minor pathfinding defects. Of the three alleles, 35/14 appears to be the most severe.

Embryos from the 90/36 stock display subtle morphological defects characterized by closely associated Lch neurons and a decreased level of MAb 22C10 staining. Based on the observed phenotype, we decided to name the gene bunched.

The mutations carried on the nonrevertible insertion chromosomes 25/14 and 50/2 define the locus fondue. The two mutations cause very similar phenotypes characterized by crowding and elongation of the Lch neurons (Figure 5E). These mutations also alter the overall organization of the PNS resulting in a less stereotyped pattern than in wild type.

The 81/4 insertion chromosome carries a mutation affecting Lch neuron organization and morphology. The Lch neurons often appear very close to each other and more elongated than in wild-type embryos. These neurons also frequently stain faintly. Unfortunately, as

opposed to other mutations described here, the penetrance of this phenotype is significantly lower than 100%; nevertheless, due to its similarity to other mutations affecting Lch neuron development, we included it in this study.

The on-the-rack locus is identified by the 150/1 insertion at 48A. Embryos of this mutant strain usually have less than five (two to four) Lch neurons. The mutation also affects the shape of the lateral cluster causing neurons to be associated more closely and to appear elongated. This mutation is complemented by deficiencies uncovering its cytological location. However, the phenotype can be reverted with precise or near precise excisions. It is possible that the P element is the cause of the phenotype and that the precise extent of the deficiency has not been mapped correctly. Alterna- tively, a mutation mapping elsewhere could be the cause of this phenotype.

The 42/18 insertion defines the charlatan locus. In charlatan embryos, the PNS neurons appear slightly enlarged but not as much as those observed in embryos from the large neuron group mutants (i.e., obelix, 67/ 12, 88/7). Some neurons also appear to stain faintly with MAb 22C10 and the Lch neurons are bunched.

The rnindrnelt (mrn) locus is characterized by a revertible Pelement on the 71/3 chromosome. Embryos de- rived from this strain frequently display Lch neurons that are associated more closely than in wild type, exhibit a mild loss of neurons, show abnormal neuronal morphology and fasciculation defects (Figure 5C). Al- though rnrn is similar in many aspects of its phenotype tofondue, complementation tests suggest they are genet- ically distinct.

The four insertions that identify the fata rnorgana (furn) locus cause embryonic phenotypes affecting primarily the development of the lateral and vr chordotonal neurons. The MAb 22C10 staining of these neurons is frequently absent or very faint. When stained, their cell bodies and dendrites appear elongated. In contrast to the Lch neurons, the ventral chordotonal neurons appear normal. The thoracic chordotonal neurons located in the dorsal cluster are affected in a manner similar to the Lch neurons. furn embryos also display subtle pathfinding defects. The 26/8 and 75/5 insertions cause similar phenotypes, whereas the 77/1 insertion seems to be a more severe allele causing more consistent morphological defects in the dorsal cluster. In 77/1 embryos, dorsal neurons are clustered tightly and do not form typical dendritic projections. The 124/ 5 allele affects lateral and vr chordotonal neurons to the same extent as the above alleles, but in addition, it causes the dorsal bipolar neuron to stain faintly with MAb 22C10. The overall organization and fasciculation patterns in 124/5 embryos suggest that it is possibly the most severe allele of farn.

Analysis of lad expression: Previous P-element enhancer detector screens focused mostly on searching

wild type

Mutations Mecting Embryonic PNS

-rano

1675

I L a

for tissue specific enhancer expression patterns (e.g., BELLEN et al. 1989; BIER et aL 1989). In the present screen we searched for specific PNS defects in a collection of second chromosome enhancer detector strains and were interested to establish whether the observed phenotypes could be correlated with the P-galactosidase expression patterns. Analysis of the ladexpression pattern demonstrates that the majority of the mutations are not associated with P elements that confer expression of lac2 in the nervous system. Sixty-four percent of saved strains (32/50) express P-galactosidase weakly in the pharynx, portions of the midgut and hindgut as well as in a striped pattern in the epidermis. Because these expression domains usually occur together, we believe this expression pattern is caused by sequences intrinsic to the P elements rather than those of genes next to the insertion. As outlined in Tables 3-5, we name this expression pattern the “basic pattern.” Only six of the remaining 18 strains express P-galactosidase in the PNS.

DISCUSSION

Screens based on enhancer detectors in Drosophila typically focus on the P-galactosidase expression pattern

FIGURE 5.-Mutations affecting neuronal morphology. Three abdominal segments of embryos stained with MAb 22C10. (A) Wild type (Canton S); (B) grano (allele 35/14). Neurons in cyano embryos stain dark. Note the thicker axons and the fasciculation defects. (C) mindmelt (allele 71/3). The Lch neurons (arrows) are somewhat larger in size and appear fused together when compared with wild type. The chordotonal neurons in the ventral cluster appear normal (arrow-

wild type heads). (D) Wild type (Canton s), focus on lateral chordotonal organs; (E) frmdue (allele 25/14). The Lch neurons are more crowded than in wild-type embryos.

fondue

conferred by the enhancer detector. Once strains with interesting expression patterns are identified, a detailed search for possible phenotypes associated with the loss of the gene of interest is often initiated. Many interesting and important genes have been isolated and characterized using this strategy (for review see WILSON et al. 1990). An alternative strategy is to use P-element enhancer detectors as mutagens and to screen for a mutant phenotype prior to analyzing their expression patterns. Enhancer detectors provide many significant advantages over traditional mutagenesis strategies as they allow rapid cloning of flanking DNA sequences, provide a means of assessing the expression pattern of the neighboring gene, permit reversion of the induced mutation, and allow isolation of additional alleles (BELLEN et al. 1989; WILSON et al. 1989). Unfortunately, this approach to screening for mutants is extremely time consuming because <lo% of all P elements cause visible or lethal phenotypes (COOLEY et al. 1988; BELLEN et al. 1989). For example, identlfylng 2000 homozygous lethal mutations on one of the major autosomes re- quires generation of -40,000 P-element insertion stocks on the second and third chromosomes. Subse- quently, the insertions can be assigned to an autosome.


Recently, TOROK et al. (1993) undertook such a screen on the second chromosome, and we have screened this collection for mutations that affect the development of the PNS (this collection will soon be available from the Indiana Drosophila Stock Center; Berkeley Drosophila genome project; KATHY MATTHEWS, personal communication). Here, we report the mutants that were isolated and discuss some of the general properties of the mutant collection.

The majority of mutations are caused by P elements One of the major questions concerning any collection of P-element strains is in what fraction of the strains are the P elements responsible for the mutant phenotype? Although we cannot answer this question with great precision, these and other data (H. J. BELLEN, unpublished data) indicate that in 66% of the isolated insertions the lethality can be reverted through precise or near precise excision. In addition, 10% of the P elements cannot be reverted but cause lethality over a deficiency that uncovers their cytological location. Hence, in -76% of the mutant strains, the Pelements are probably the cause of the mutant phenotype. In addition, it should be mentioned that the chromosomes in which the mutations were generated were isogenized prior to the mutagenesis experiment (TOROK et al. 1993). We therefore conclude that most lethal mutations are on the same chromosome as the P elements but that they were most likely created because of expo- sure to A2-3 transposase. The A2-3 transposase is proposed to act as a mutagen because of high affinity for DNA (KAUFMAN et al. 1989) and because it causes double strand breaks and recombination in males (MCCARRON et al. 1994). As we found several mutations that fail to complement each other but have insertions in different cytological locations (e.g., alleles of cyrano, fondue, vegetable), it is possible that a P element inserted but excised immediately and imprecisely, leaving a lethal signature. More importantly, the absence of a heavy genetic load (no marker mutations) on these chromosomes has proved extremely useful as very subtle defects could be detected relatively easily in the vast majority of the strains given that few embryos exhibited embryonic defects. Screening in this background has proved to be significantly easier than in similar experiments in which chemical mutagens were used (SALZBERG et al. 1994). Hence, we believe that this collection of lethal and semi- lethal mutations provides an excellent opportunity to screen and identify mutations for -40% (TOROK et al. 1993; Berkeley Drosophila genome project, personal communication) of the essential genes on the second chromosome.

Mutagenic enhancer detectors are poor reporters of gene expression: As mentioned above, many enhancer detector screens (e.g., BELLEN et al. 1989; BIER et al. 1989) have focused on expression pattern rather than on phenotypes. By preselecting enhancer detectors that cause lethality and PNS defects, and subsequently stain-

ing for ,&galactosidase expression, we find that only six strains out of 50 (12%) express lacZ in the PNS. The majority of the strains shows no P-galactosidase expression pattern or a pattern that we termed basic pattern because it is virtually identical in most strains. Similar “repeat or basic patterns” were found in other enhancer detector strains and have been reported previously (BELLEN et al. 1989; GHYSEN and O’KANE 1989). We conclude that screening for defects in the PNS does not enrich a collection for enhancer detectors that report expression in the PNS since it was previously shown that 20% of all enhancer detector strains express lacZ in the PNS (BELLEN et al. 1989; BIER et al. 1989). This apparent discrepancy can be explained by the finding that the vast majority of insertions that have been characterized on the basis of their expression pattern has been shown to be inserted in the 5‘ regulatory sequences of the gene (e.g., WILSON et al. 1989; BELLEN et al. 1992a; KANIA et al. 1993; ELDON et al. 1994). We propose that insertion strains that are selected on the basis of a phenotype are probably located within exons or introns and are not properly positioned to be influ- enced by enhancer and promoter elements. Further characterization and mapping of these P elements will be required to test this hypothesis.

Insights into PNS development: Following the initial characterization we classified the complementation groups into the same five phenotypic groups as described previously (SALZBERG et al. 1994). Several mutants on the second chromosomes exhibit very similar phenotypes to those described on the third. For example, the phenotype of obelix appears very similar to the phenotype caused by mutations in the pauarotti gene; thus obelix may be required for cell division in the PNS and possibly elsewhere. Another example is the PNS phenotype exhibited by cyrano embryos that is very similar to that of homozygous hearty embryos. The morphology of Lch neurons in mindmelt embryos resembles morphology of neurons in the sticky chordotonals mutants (SALZBERG et al. 1994). The fact that many of the mutants isolated in this screen are phenotypically similar to the third chromosome mutants suggests that these mutations may lie in the same genetic pathways. The fact that some of the mutants from this screen have a “homologous” mutation isolated in the third chromosome screen also suggests that the genetic pathways governing neuronal development are relatively few and should soon be saturated with mutations. However, it should be noted that the similarities in phenotypes could be due to similar screening methods used in both screens.

Given the neuronal development model outlined in the introduction, we can derive a few insights into the nature of the mutations isolated in this screen. We do not believe that we have recovered any new neurogenic genes since none of the isolated mutants exhibit significant increases in neuronal cell numbers characteris-


tic of loss of function mutations in neurogenic genes. Although gain of function mutations in neurogenics do result in neuronal loss (e.g., Notch) (FOSTER 1975), we do not anticipate that our neuronal loss mutations affect new neurogenic genes since P-element insertions generally result in loss of function mutations.

Mutations affecting overall neuronal morphology are probably not disrupting the initial steps of neurogenesis such as specification of SOP numbers. Since in a few of these mutants the overall neuronal cell numbers as well as their position are not affected significantly, we propose that these mutations are probably disrupting genes involved in final steps of differentiation. An excellent example of such mutation is cyruno since it does not cause any obvious changes in neuronal numbers or position. cyruno embryos exhibit an increase in MAb 22C10 immunoreactivity and axonal pathfinding defects. Both of these phenotypes could be easily explained by defects in the final neuronal differentiation steps. However, to gain a more precise insight into the roles of most genes described in this study will require extensive genetic analyses, further phenotypic characterization, and molecular characterization of the genes of interest.

We thank COREY S. GOODMAN for generous supply of MAb 22C10. We also thank KATHY MATTHEWS and the Indiana Stock Center for promptly sending us so many fly strains,JUDI COI.EMAN for secretarial assistance, and JUAN BOTAS, KAREN SCHULZE and HUDA ZOGHBI for comments on the manuscript. AS. is a postdoctoral researcher of the Howard Hughes Medical Institute. H.J.B. is an assistant investigator of the H.H.M.I.

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Communicating editor: R. E. DENELI.

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