lethal(1)optomotor-blind [l(1)omb] is defined by lack of com- plementation among over a dozen recessive lethal mutations that map to the omb gene locus.
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1199-1203, February 1992 Genetics
The lethal(l)optomotor-blind gene of Drosophila melanogaster is a major organizer of optic lobe development: Isolation and characterization of the gene (neurogenetics/complex gene/lethality/brain structure/behavior)
GERT 0. PFLUGFELDER*, HELMUT ROTH, BURKHARD POECK, STEFAN KERSCHER, HERBERT SCHWARZt, BEATE JONSCHKER, AND MARTIN HEISENBERG Institut fur Genetik und Mikrobiologie, Universitat Wurzburg, D 8700 Wurzburg, Federal Republic of Germany
Communicated by M. M. Green, October 18, 1991 (receivedfor review July 31, 1991)
mutation In(J)ombH3I and enhance the phenotype of the viable Qd and bi mutants. l(1)omb is therefore the central function in the omb locus. Here we characterize the main transcript from the omb locust and describe the defects in the adult brain caused by lethal mutations in the omb gene. The relationship between the l(J)omb complementation group and viable omb mutations is discussed.
The X-chromosomal complementation unit ABSTRACT lethal(1)optomotor-blind [l(1)omb] is defined by lack of complementation among over a dozen recessive lethal mutations that map to the omb gene locus. Mutations in l(l)omb also fail to complement viable mutations of three seemingly unrelated functions in this region: bifid (bh), manifesting defective wings, Quadroon (Qd), a semi-dominant mutation expressing abnormal tergite pigmentation, and In(1)omb3', giving rise to a normal external morphology but with discrete defects in the optic lobes and behavior. The locus encodes a 70-kilobase primary transcript that is spliced into a 6-kilobase mature RNA. cDNAs for this transcript were isolated and sequenced and the derived amino acid sequence was analyzed. Certain features of this sequence suggest that the I(1)omb gene product is a nuclear regulatory protein. The lethal phase of various apparent null mutants was determined and found to occur mainly in the pupal stage. A large proportion of all hemizygous mutant males develop to pharate adults that eclose only rarely but can be rescued from the pupal case. These animals show a severe maldevelopment of the optic lobes. In addition they have only rudimentary wings as well as a Quadroon-like abdominal pigmentation. Thus, in the lethal mutants those parts of the body are affected for which independent viable mutations have been previously described in the omb locus, such as optomotorblind, bifid, and Quadroon.
MATERIALS AND METHODS Drosophila Stocks. Flies generally were raised at 230C on standard Drosophila medium (cornmeal, agar, molasses, yeast, and Nipagin). In experiments where we wanted to identify male larvae bearing X chromosomes marked with the white mutation, larvae were reared on formula 4-24 instant Drosophila medium (Carolina Biological Supply) supplemented with 0.03% riboflavin (10). DfUl)ovo4' has been described in ref. 8 and was obtained from the author. 1(J)oMb2565 was obtained by ethyl methanesulfonate mutagenesis and will be described elsewhere. The sources of all other chromosomes referred to in this study are listed in ref. 9. Isolation of the rb13S Insertion Chromosome. The Df(l)rb13S chromosome (8), in addition to a deletion, also contains an insertion at -11 kilobases (kb) of the omb map. The insertion has been separated from the deficiency by recombination in Df(l)rbb3S/l(J)bil>4 females. The only viable male progeny from these females bear an X chromosome that has recombined between the 1(1)biD' point mutation and the deficiency. Recombination can occur distally and proximally to the rb135 insertion. Both types of recombinants have been obtained and were identified by Southern blot analysis. cDNA Library Screens. Embryonal cDNA libraries (4-8 hr and 12-24 hr) in the pNB40 vector were obtained from N. Brown and were screened as in ref. 11.
The first isolated omb mutation was the recessive and viable
In(J)ombH3' allele (1, 2). Adult mutant flies show specific defects in optomotor behavior (3-5). This behavioral defect correlates with, and very likely is the consequence of, the apparent absence of a subset of lobula-plate giant fibers in mutant brains (5). Subsequent mutational screens for the neuroanatomical defect among the progeny of ethyl methanesulfonate-mutagenized flies by a mass histological procedure (6) yielded additional but only lethal alleles. A number of lethal mutations with one chromosome break at the omb locus have been obtained also by other groups (7, 8). The cloning of the locus and the mapping of omb breakpoints indicated a minimum size of 80 kilobases (kb) for the omb gene (9). By complementation analysis of the various omb alleles, one lethal complementation group, l(J)omb, has been defined in the omb locus. Aside from affecting brain morphology and function, mutations in the unit can cause a number of different phenotypes. They range from altered wing venation [bifid (bi)] and increased pigmentation on the abdominal tergites [Quadroon (Qd)] to the pleiotropic defects seen in the pharate adults of hemizygous lethals. Alleles of the l(1)omb complementation group fail to complement the viable omb
RNA Isolation and Northern Blot Analysis. These procedures were carried out as described (9). DNA Sequencing and Analysis of Sequence Data. Genomic and cDNA fragments were subcloned in pKS+ (Stratagene). Unidirectional deletions were created by the method of Henikoff (12). Templates were sequenced by the dideoxy method (13) using T7 DNA polymerase (14) and 7-deazadGTP from a Pharmacia kit. cDNAs were sequenced in both strands and these sequences were further verified by sequencing across genomic DNA fragments encompassing exons. Sequence data were analyzed with the DNASIS (PharAbbreviation: ORF, open reading frame. *To whom reprint requests should be addressed. tPresent address: Department of Immunology, Scripps Clinic, 10066 North Torrey Pines Road, La Jolla, CA 92307. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M81796).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact.
1199
1200
Genetics:
Pflugfelder et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
strongly curved lumps of tissue adjacent to the remaining optic lobe neuropil. Neither the outer nor the inner optic chiasm is properly formed. Of the three neuropil regions-medulla, lobula, and lobula plate-only the distal medulla can be tentatively identified, due to its regular columnar organization and the projections of the lamina monopolar fibers. In many instances this structure is found to be the most proximal layer of the remaining optic lobe neuropil. The rudiment of the optic lobes is dorsocaudally connected to the central brain via a stalk that is distinctly thinner than in wild type (not in the plane of section of Fig. 2). Nothing or very little is left of the anterior optic tract, which already in the viable allele In(1)ombH3' is reduced by about 200 fibers (17). Giant fibers of the lobula plate are not detected. Twenty percent of mutant animals show a stronger phenotype in which hardly any optic lobe neuropil remains. Other regions of the body are also affected in l(1)omb mutants. Most conspicuous is a reduction in wing size and an increased abdominal pigmentation. A more detailed description of external phenotypes will be presented elsewhere. The l(1)omb Transcription Unit. In the omb locus only two transcripts, T7 and T3, were found by Northern blot analysis to span the genetically defined lethal domain of l(1)omb (9). T7 is a smeared high molecular weight signal with an apparent length of >12 kb, which in the gel system used is close to the limiting mobility. It is detected with all probe fragments between -5 and -80 kb (Fig. 1). The ends of the T7 domain coincide with the first and the last exons of T3 as they have been found by Northern blot and cDNA analysis. The developmental expression profiles of T3 and T7 are indistinguishable (9). We therefore conclude that T7 is the precursor RNA for the mature T3 transcript, which is about 6 kb long. The genomic sequence in this region reveals a sufficient number of adenine runs so that T7 or fragments derived thereof would be oligo(dT)-selected even if they were not 3'-polyadenylylated. T3 is of relatively low abundance (see Fig. 4). cDNAs were isolated from two embryonic libraries (11). Two overlapping cDNA clones, pceII-52 and pcel-11, containing a large open reading frame (ORF) were analyzed
macia) and HUSAR (Deutsches Krebsforschungszentrum, Heidelberg, release 2, 1990) program packages. Histology. Paraffin-embedded heads were sectioned and stained by the Holme-Blest method (15). In Situ Hybridization of Central Nervous System Whole Mounts with Digoxigenin-Labeled DNA Probes. Our protocol followed that of Tautz and Pfeifle (16), with minor modifications.
RESULTS The l(1)omb Complementation Group. The l(1)omb complementation unit is defined by three lethal, apparent point mutations [l(1)omb182, I(1)omb9", and I(1)bk')] and three lethal mutations with chromosome rearrangement breakpoints [T(1;3)biD1, Tp(1)biDl, and T(1;2)biD2)], which fail to complement each other and the brain defect of the original In(1)ombR3l viable allele. Furthermore, there are a number of deficiencies that uncover still other lethal complementation groups. All l(1)omb alleles fail to complement the viable mutations bi and Qd. We therefore consider omb, bi, and Qd to be alleles of l(1)omb in spite of the mutual complementation of these three viable mutations. Df(1)ovoDIrG7 defines the maximal proximal extent and T(J;3)biD1 the minimal distal extent of this lethal complementation group (Fig. 1). Imaginal Brain Phenotype of 1(1)omb Mutants. l(1)omb mutants are pupal lethals. Sixty percent of hemizygous males die around head eversion, the remaining animals complete pupal development but fail to eclose. Since the viable In(1)ombH31 allele has a very specific defect in the adult optic lobes, heads of l(J)omb flies rescued from the pupal case were sectioned. In about 80%o of mutant animals the optic lobes, as judged by the histology of paraffin sections and reduced silver staining, are severely deformed (Fig. 2). Their shape varies among animals of the same genotype and between the right and left side of the same fly. The neuropil is greatly reduced in volume. The cartridges of the lamina do not form the normal compact sheath beneath the eye. They are only loosely associated with each other and often form several
Z-".Z.z /.-,- z Ilz zz
Z-ZL
,
ESS
E
ES
SEE SE EE
E 60
-7 0,
E.1 *.
-5C
SE E S
S -4C
III IV 1
E S EES .30
S -
E k
FE
SE
x
,
r
x
..
F
._
! _ --
-, i-'
