The Homeobox Gene cut Interacts Genetically With the Homeotic Genes proboscipedia and Antennapedia

Corresponding author: Karen Blochlinger, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop A2-025, P.O. Box 19024, Seattle, WA 98109-1024, kblochli{at}fred.fhcrc.org (E-mail).

Communicating editor: T. W. CLINE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cut locus (ct) codes for a homeodomain protein (Cut) and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify ct-interacting genes, we observed that flies containing a hypomorphic ct mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different ct mutant backgrounds. We show that this phenotype is accompanied by pronounced changes in the expression patterns of both ct and pb in labial discs. Furthermore, a significant proportion of ct mutant flies that are heterozygous for certain Antennapedia (Antp) alleles have thoracic defects that mimic loss-of-function Antp phenotypes, and ectopic expression of Cut in antennal discs results in ectopic Antp expression and a dominant Antp-like phenotype. Our results implicate ct in the regulation of expression and/or function of two homeotic genes and document a new role of ct in the control of segmental identity.

THE creation of stable cell fates relies on the initial specification of cellular identities and the subsequent persistence of committed cell states through cell divisions and other developmental processes. In the peripheral nervous system of Drosophila, the cut gene (ct) has been shown to be necessary for establishing the fates of external sensory organs (es organs, chemosensory and mechanosensory): in the absence of ct activity all es organs develop morphologically and antigenically as internal chordotonal organs (ch organs, proprioceptive), whereas the reciprocal effect is observed after ectopic expression of ct in ch organs (BODMER et al. 1987 ; BLOCHLINGER et al. 1991 ). Ectopic expression studies have also implicated cut in the maintenance of es organ fates (BLOCHLINGER et al. 1991 ). In addition, ct is essential for the development of tissues outside the nervous system, including the wing margin, the Malpighian tubules and the ovary; however, its role in these tissues is not as clearly understood (BLOCHLINGER et al. 1990 ; LIU et al. 1991 ; BLOCHLINGER et al. 1993 ; JACKSON and BLOCHLINGER 1997 ).

The ct locus codes for a nuclear protein (Cut) containing a homeodomain and three dispersed copies of a motif (cut repeat; BLOCHLINGER et al. 1988 ). The cut repeat was first identified in Cut and has since been found in several vertebrate proteins with structural and functional similarities to Cut (ANDRES et al. 1992 ; BLOCHLINGER et al. 1988 ; NEUFELD et al. 1992 ; VALARCHE et al. 1993 ; LUDLOW et al. 1996 ). cut repeats have DNA-binding activities in vitro (ANDRES et al. 1994 ; AUFIERO et al. 1994 ; HARADA et al. 1994 ; FAN and K. BLOCHLINGER, unpublished results) and several of the vertebrate Cut-like proteins have been shown to influence gene expression in cultured cells (LIEVENS et al. 1995 ). For these reasons, it is inferred that Cut is involved in the transcriptional regulation of target genes that, in the peripheral nervous system, direct cells to assume the properties of es organs. We performed a screen to identify potential target genes and/or genes that function together with ct. We report here that ct interacts genetically with two homeotic genes, proboscipedia (pb) and Antennapedia (Antp).

The homeotic genes are responsible for defining segmental identities in Drosophila (LAWRENCE and MORATA 1994 ). Loss of expression or ectopic expression of homeotic genes in cells causes them to assume fates characteristic of an inappropriate segment. Most of the homeotic genes are organized into two clusters, the Antennapedia complex (ANT-C; KAUFMAN et al. 1980 ) and the Bithorax complex (LEWIS 1978 ), and encode homeodomain proteins that regulate the expression of target genes through their interaction with DNA (WOHLBERGER 1996 ). The Antp gene is required for the development of thoracic segments. In mutants homozygous for loss-of-function Antp alleles, cuticle structures on the thorax are transformed into structures characteristic of more anterior segments (LEWIS et al. 1980 ; STRUHL 1981 ; WAKIMOTO and KAUFMAN 1981 ; SCHNEUWLY and GEHRING 1985 ; ABBOTT and KAUFMAN 1986 ). Dominant Antp alleles, in which Antp is ectopically expressed in more anterior segments, result in the replacement of the adult antennae by thoracic leg structures (e.g., SCHNEUWLY and GEHRING 1985 ). The pb gene is also located within the ANT-C and is required for the formation of mouthparts (BRIDGES and DOBZHANSKI 1933 ; KAUFMAN 1978 ; PULTZ et al. 1988 ). Labial palps are transformed to prothoracic legs in mutants lacking pb function or to antennae in mutants retaining partial pb function.

In our screen for dose-sensitive genes that interact with ct, we found that ANT-C deletions identified a candidate gene, which we then mapped to pb. We subsequently found that ct also interacts genetically with Antp. Corresponding changes in expression of these homeotic genes were observed as a consequence of altered ct expression. We therefore speculate that ct is involved, directly or indirectly, in the control of expression and/or function of at least two homeotic genes. This provides the first line of evidence for the involvement of ct in pathways regulating segment identity.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly stocks:
Table 1 lists the stocks used in this study. Stocks were maintained on standard cornmeal/yeast/molasses/agar food at 20°. ct^L188, ct⁶, ct^K, ct^C145, hsCut and UCut25-9 have been described (BLOCHLINGER et al. 1990 ; BLOCHLINGER et al. 1991 ; LUDLOW et al. 1996 ). Deficiency and ANT-C stocks were obtained from the Bloomington Stock Center (Bloomington, IN). Lines 4A.3dpp.GAL4 and 40C.6dpp.GAL4 were a gift from M. HOFFMANN (STAEHLING-HAMPTON et al. 1994 ). All crosses were maintained at 24°, unless otherwise indicated.

In our screen, ct^L188 virgins were crossed to males containing heterozygous deficiencies of the second or third chromosomes over marked balancers. ct^L188; Df/+ progeny males were examined for morphological defects and/or lethality compared to their siblings containing the balancer chromosome. To examine the pb and Antp phenotypes in ct^L188 or ct^L188/ct^C145 females, ct^L188; D/TM3 Sb virgins were crossed to males containing heterozygous pb or Antp alleles over marked balancers and ct^L188; Antp(pb)/D(TM3 Sb) progeny males were subsequently crossed to either ct^L188 or ct^C145/FM6 virgins.

Flies of genotype w P[mini-w⁺; hs-NM]8A,P[ry⁺; neo^R; FRT] 18A(18 NM) and w P[ry⁺; neo^R; FRT] 18A; MKRS P[mini-w⁺; hsp70-FLP] (18-1F) were gifts from G. RUBIN (XU and RUBIN 1993 ). y w ct^C145 was recombined onto the 18-NM chromosome using standard genetic methods to produce the strain y w ct^C145 NM/FM6 (JACKSON and BLOCHLINGER 1997 ).

Morphological analysis:
To characterize mutant cuticular phenotypes, adult flies were immersed in 5 M KOH at 95° for 5–10 min, mounted in Faure mounting medium (ASHBURNER 1989 ), and examined on a Zeiss Axiophot (Carl Zeiss, Inc., Thornwood, NY) using Nomarski optics. Pseudotracheal rows were counted and scored for the presence of antennal material (arista) and leg material (sex combs, bracted bristles, transverse rows of bristles, claws, pulvilli). Approximately 5% of labial palps did not evert completely and were not scored.

To examine thoracic outgrowths, 20 nm of gold/palladium alloy was deposited on headless flies and preparations were viewed and their images recorded using a JEOL 5800 scanning electron microscope (JEDL, Boston, MA).

Mosaic analysis:
Cell clones lacking ct function were generated using the FLP-FRT system (XU and RUBIN 1993 ). Recombination was induced in progeny from a cross between 18-1F males and y w ct^C145 NM/FM6 females by heat-shock for 1 hr at 37° during all three larval instars. The expression of the c-myc epitope tag was induced by an additional heat-shock at 37° for 1 hr before dissection of wandering third instar larvae.

Immunocytochemistry:
Embryos were fixed and processed for immunocytochemistry according to BLOCHLINGER et al. 1990 . Imaginal discs were dissected, fixed, and processed for immunocytochemistry according to JOHNSTON and SCHUBIGER 1996 .

The antibodies, their concentrations used and sources were: rat polyclonal (1:250) or mouse monoclonal (1:10) anti-Cut F2 (BLOCHLINGER et al. 1990 , Developmental Studies Hybridoma Bank), rabbit polyclonal anti-Pb E9 (1:75; CRIBBS et al. 1992B ), mouse monoclonal anti-myc Ab-1 (1:25; Oncogene Research Products, Boston, MA), monoclonal anti-Antp 4C3 (1:100; GLICKSMAN and BROWER 1988 ). Primary antibody binding was detected using FITC-conjugated anti-rat and Texas-Red-conjugated anti-mouse or anti-rabbit antibodies (Jackson Immuno-Research Labs., Inc., West Grove, PA).

Preparations were viewed on a MRC-600 confocal microscope (Bio-Rad, Richmond, CA) and processed using Adobe Photoshop.

Ectopic expression of Cut:
Ectopic Cut expression was induced in an overnight collection of y w; C2/CyO embryos (Cut coding sequences inserted between hsp70 regulatory sequences; BLOCHLINGER et al. 1991 ) by a heat-shock treatment at 42° for 1 hr. Embryos were fixed and processed for Cut and Pb immunocytochemistry 30 min subsequent to the heat-shock.

Ectopic expression of Cut during postembryonic stages was induced by crossing either of two GAL4 lines, 4A.3dpp.GAL4 or 40C.6dpp.GAL4 to UCut25-9 (Cut coding sequences downstream of five GAL4 binding sites) flies.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic interaction of ct with pb:
To identify genes that interact with ct, we screened for lethality or morphological defects in males containing heterozygous deficiencies (Df) of the second and third chromosome and a hypomorphic ct allele, ct^L188, on the X chromosome. ct^L188 is a homozygous viable, temperature-sensitive allele discovered in a hybrid dysgenesis screen (LIU et al. 1991 ). At 18°, the hetero-allelic combination ct^L188 and ct^C145, a null allele, is essentially lethal, whereas at 24° ~50% of flies with this genotype eclose and appear morphologically normal (S. JACKSON and K. BLOCHLINGER, unpublished results). We have previously shown that the ratio of the two Cut-specific protein bands observed by Western analysis is altered in ct^L188 embryos (BLOCHLINGER et al. 1990 ); however, the molecular nature of the mutation is unknown. We hypothesized that the ct activity in ct^L188 flies is near a critical threshold required for viability and that heterozygous mutations in potential ct-interacting genes would lead to lethality or a visible phenotype.

Only 1 of 150 initial deficiencies spanning ~70% of the genome produced a fully penetrant phenotype in combination with ct^L188: in all flies with the genotype ct^L188/Y; Df(3R)Scr/+ the mouthparts were morphologically abnormal. There are two labial palps at the distal end of wild-type mouthparts, each with six pseudotracheal rows (e.g., Figure 1A) (BRYANT 1978 ). The number of pseudotracheal rows in ct^L188 flies is frequently slightly lower (Table 2). In ct^L188/Y; Df(3R)Scr/+ flies, the number of pseudotracheal rows is further reduced and structures characteristic of legs and/or antennae are present (Table 2), including aristal material specific to the antenna, and bracted bristles and sex comb teeth specific to legs. This phenotype resembles that of homozygous, or transheterozygous combinations of pb mutant flies, in which the labial palps are transformed to prothoracic legs or antennae (Figure 1, B–D; KAUFMAN 1978 ). Df(3R)Scr removes most of the ANT-C including pb. A similar phenotype is observed in ct^L188 males heterozygous for other deficiencies in which pb is deleted (Df(3R)MAP2 and Df(3R)MAP8) and in ct^L188 males heterozygous for any of four different pb loss-of-function alleles (pb¹, pb⁵, pb¹⁴ and pb²⁷) but not for a deficiency in the region that retains pb function (Df(3R)LIN). Therefore, we conclude that the ct-interacting locus is the pb gene.

View larger version (93K): In this window In a new window Download PPT slide   Figure 1. —Labial palp phenotype of ct and pb mutant flies. (A) pb1/+ labial palps showing normal morphology of labial palp (LP), maxillary palp (MP) and 12 pseudotracheal rows (PT, arrow); (B–D) two examples of pb1/pb5 labial palps. There are no pseudotracheae, and labial palps are transformed to leg and antenna. The maxillary palps are shorter and deformed. Bristles are misshapen because of the presence of Ki on the pb5 chromosome. Claws (arrowhead in B) and aristae (arrow in B) are apparent. The right palp has not everted (arrowhead in C) although leg structures are visible. D is a higher magnification view of distal leg structures in C to show leg-specific bracted bristles (arrow); (E) ctL188 labial palps showing incomplete pseudotracheal rows lacking either the dorsal (left arrow) or the ventral (right arrow) segment. The maxillary palps appear normal; (F–H) two examples of ctL188; pb5/+ labial palps showing loss of pseudotracheal rows (arrow in F) and leg-specific structures (arrow in G). H is a higher magnification view of leg structures in G showing bracted bristles as well as the yellow bristles of transverse rows and sex comb teeth (out of focus) characteristic of tibial and first tarsal leg segments. Bristles are misshapen because of the presence of Ki on the pb5 chromosome. Bar in A equals 100 microns for (A–C, E–F), 50 microns for (D, H).

Two lines of genetic evidence indicate that the mutation in the ct locus (rather than elsewhere on the chromosome) is causally related to the dominant mouthpart phenotype in heterozygous pb flies (summarized in Table 2). First, the phenotype is observed in ct^L188/ct^L188; pb/+ and ct^L188/ct^C145; pb/+ flies but not ct^L188/+; pb/+ or in ct^C145/+; pb/+ flies, indicating that recessive alleles at the ct locus are required. Also, there is no phenotype in heterozygous pb flies combined with two alleles of ct that specifically affect the Cut expression pattern in the wing imaginal disc (ct⁶ and ct^K), but not during embryogenesis or in other imaginal discs. Secondly, we found that the labial phenotype invariably is associated with the presence of the ct^L188 allele in six individual recombinants between the chromosomes ct^L188 and y w ct⁶ sn³ in combination with pb/+. Therefore, the locus responsible for this interaction maps to a region between w (3C2) and sn (7D2). These results strongly suggest that the labial phenotype in ct^L188/Y; Df(3R)Scr/+ flies documents a specific interaction between pb and ct.

Complete loss of pb function transforms labium to leg, while partial loss of function results in a transformation of labium to antenna (KAUFMAN 1978 ; PULTZ et al. 1988 ). Interestingly, we observed the same spectrum of morphological abnormalities in all genetic combinations of ct and heterozygous pb mutants (referred to subsequently as ct; pb/+) that affect labial development. For example, the labial phenotype is similar using either null or hypomorphic pb alleles (Table 2, see Table 1 for definition of pb alleles). However, among ct; pb/+ flies of the same genotype, the expressivity of the phenotype varies considerably: although the number of pseudotracheal rows is reduced in 100% of the flies, the extent of labial transformation to antenna or leg or both is variable (Table 2). Homozygous pb null flies (pb⁵/pb⁵) did not eclose under our culture conditions, so pb¹/pb⁵ transheterozygotes were used to compare the phenotype to ct; pb/+ flies. pb¹ is a temperature-sensitive hypomorphic allele, which results in a labial palp to antenna transformation at 18° and a labial palp to leg transformation at 29°. In pb¹/pb⁵ flies at 24°, the distal labia are transformed to a mixture of antenna and distal prothoracic legs. Of these, 46% include claws and some display fully defined tarsal segments (Figure 1C and Figure D). In contrast, fully defined leg segments or claws are never seen in the labia of any ct; pb/+ combinations, although bracted bristles and transverse rows indicative of the basitarsus are frequently observed. Finally, the maxillary palps of pb¹/pb⁵ transheterozygotes are reduced in size and deformed into an ovoid shape, suggestive of a maxillary palp to antenna transformation (KAUFMAN 1978 ), but the maxillary palps of ct; pb/+ flies are only occasionally slightly misshapen. Therefore, the transformation phenotype in ct; pb/+ flies is less severe than in pb homozygous flies.

We also tested the interaction of ct^L188 with pb⁵ at 18°, but found no difference in either the penetrance or the severity of the labial phenotype compared to that obtained at 24° (data not shown).

ct and pb expression is altered in ct; pb/+ discs:
The adult mouthparts are produced from the labial imaginal discs (reviewed in BRYANT 1978 ). pb protein (Pb) is expressed in nuclei of labial disc cells in third instar larvae (RANDAZZO et al. 1991 ; Figure 2A), and we find that Cut is expressed in a pattern that substantially overlaps with that of Pb and is also nuclear (Figure 2B and Figure C). In wild-type and ct^L188 discs, Cut and Pb are expressed throughout the entire disc, however in ct^L188; pb⁵/+ discs both the level and the pattern of expression of both proteins is altered (Figure 2, D–I). The level of expression of both proteins is significantly decreased overall and entirely lost in some of the cells. Where present, Cut expression appeared more punctate in comparison to wild-type discs (Figure 2F and Figure I). Moreover, the mutant discs were morphologically abnormal (Figure 2H). Staining of ct^L188; pb⁵/+ labial discs with acridine orange show no consistent increase in apoptotic cell death relative to that in control discs at this stage (data not shown). Finally, Pb expression is undetectable in pb¹/pb⁵ mutant labial discs and the pattern of Cut expression is altered (Figure 2, J–L) to resemble that of a leg imaginal disc, in which Cut is expressed in two rows of cells in the position of the future claw organ in the most distal segment (BLOCHLINGER et al. 1993 ).

View larger version (70K): In this window In a new window Download PPT slide   Figure 2. —Cut and Pb expression in mutant labial discs. Confocal images of labial discs labelled with both anti-Cut and anti-Pb antibodies. Middle panels show merged images of both channels, where Pb is red, Cut is green and the overlap is yellow. A–I are the same magnification, and J–L are the same magnification. Bars in A and J equal 50 microns. (A–C) Wild-type labial disc showing expression of Pb and Cut in the nuclei of all cells; (D–I) two ctL188; pb5/+ labial discs showing, reduced levels and abnormal pattern of Cut and Pb expression. There is not complete overlap of Cut and Pb expression; (J–L) pb1/pb5 labial disc showing absence of Pb expression and reduced levels and abnormal pattern of Cut expression, specifically in the distal region of the disc, which corresponds to the transformed region of the labial palps. Labial discs are expanded in size compared to wild type or ctL188; pb5/+.

Loss of ct activity and ectopic Cut expression do not affect Pb expression:
To determine whether complete loss of ct function in labial disc cells changes Pb expression, we used FLP-mediated mitotic recombination to generate ct null mutant patches in labial discs, marked by the presence of a myc epitope (Figure 3). Surprisingly, the level of Pb expression in ct mutant cells appears to be unaltered in all (n = 150) of the clones examined (Figure 3). To address the possibility that the effect of ct on pb is restricted to a defined period in development, we induced clones during all three larval instars and obtained similar results. The mutant clones contained between 4 to 30 cells each depending on their time of induction and were similar in size to their corresponding wild-type twin clones.

View larger version (89K): In this window In a new window Download PPT slide   Figure 3. —Pb expression in ct null clones in labial disc. (A) labial disc showing Pb expression (red) and a ctC145 homozygous clone, which has two copies of the myc epitope tag and is most brightly labeled with myc antibodies (green); (B) higher magnification view of the ct null clone in A showing unaltered Pb expression in ct null cells.

In a reciprocal experiment, we expressed Cut ectopically in imaginal discs using the GAL4 system (BRAND and PERRIMON 1993 ). A stripe of ectopic Cut expression along the anterior-posterior compartment boundary of the labial disc is activated in flies containing both a transgene expressing GAL4 from decapentaplegic disc regulatory sequences (dppGAL4; STAEHLING-HAMPTON et al. 1994 ) and a transgene containing the Cut coding sequences downstream of GAL4 binding sites (UCut25-9; LUDLOW et al. 1996 ). However, we observed no changes in Pb expression, nor morphological alterations of the labial palps in these animals (data not shown).

We also tested whether the expression of Pb is affected by changes in Cut expression during embryogenesis. In embryos, both Cut and Pb are expressed in the labial lobes (PULTZ et al. 1988 ; BLOCHLINGER et al. 1990 ). No alteration in the pattern or level of embryonic Pb expression was detected in ct null mutants, or after ectopic Cut expression from a heat-inducible transgene (data not shown).

Ectopic Cut expression results in a dominant Antp phenotype:
As described above, we did not observe any effects of ectopic Cut expression under dpp regulatory control on the pattern of Pb expression and the development of the labial discs. However, UCut/+; dppGAL4/+ flies are severely malformed because of the loss of structures that normally arise in the dpp expression domain in the eyes, legs and wings (data not shown). In addition, we noticed that in 43% of the flies that were transheterozygous for dppGAL4 and UCut, the antenna is transformed to leg. Specifically, structures such as claws and bracted bristles are present either at the distal end of the arista or entirely replace the arista (Figure 4A and Figure B). This phenotype resembles that produced by dominant Antp alleles as a result of ectopic Antp expression in antennal discs (JORGENSON and GARBER 1987 ; SCHNEUWLY et al. 1987 ). Immunocytochemistry of all eye-antennal discs with this genotype showed a stripe of ectopic Cut expression along the anterior-posterior compartment boundary of the antennal disc (Figure 4D). Unexpectedly, we found that Antp protein (Antp) is expressed ectopically and uniformly across the entire disc (Figure 4E and Figure F), although expression of Antp in the mesothoracic discs is normal. This ectopic expression pattern is similar to that observed in dominant Antp mutants, where Antp is expressed throughout the eye-antennal disc, resulting in transformation of antenna to mesothoracic leg (JORGENSEN and GARBER 1987; SCHNEUWLY et al. 1987 ). We infer from this that the antennal phenotype observed in UCut/+; dppGal4/+ flies represents a transformation of the antenna to mesothoracic leg.

View larger version (114K): In this window In a new window Download PPT slide   Figure 4. —Ectopic Cut expression in eye-antennal discs. (A) UCut 25-9/+; dppGAL4/+ fly showing arista partially transformed to distal leg (arrow). Contralateral antenna lacks an arista and is not transformed to leg (arrowhead). (B) Higher magnification view of transformed arista in A showing a leg-specific claw (arrowhead) next to the arista (arrow). (C) Wild-type eye-antennal disc showing normal pattern of Cut expression. (D) UCut25-9/+; dppGAL4/+ eye-antennal disc showing a stripe of ectopic Cut expression along the anterior-posterior compartment boundary of the disc (arrow). Magnification is same as in C; (E) UCut25-9/+; dppGal4/+ eye-antennal disc showing ectopic Antp expression throughout the eye-antennal disc with strong expression in the end knob, which gives rise to the arista. Magnification is same as in C; (F) higher magnification view of a UCut25-9/+; dppGAL4/+ eye-antennal disc showing strong ectopic Antp expression in the end knob. Bar is 50 microns.

Genetic interaction between ct and Antp:
Since ectopic Cut expression results in a dominant Antp phenotype, we tested for genetic interactions between ct and Antp by examining the external morphology of flies that were heterozygous for dominant or recessive Antp mutations in a ct mutant background.

Antp¹¹ is an ethyl methanesulfonate-induced, recessive-lethal, loss-of-function allele. The molecular nature of the mutation is unknown. In 22% of ct^L188 males in heterozygous combination with Antp¹¹, we observed ectopic structures projecting from the position of the anterior spiracle on the dorsal prothorax (Figure 5B and C; Table 3). These dorsal prothoracic outgrowths are sometimes bilateral (0.4%) but mostly unilateral (21%) and are typically associated with malformations or absence of the two macrochaetes on the humeral callus. A similar phenotype was described in flies with heterozygous combinations of Antp alleles, as an inferred consequence of loss of Antp activity during the development of the adult thorax (LEWIS et al. 1980 ; ABBOTT and KAUFMAN 1986 ).

View larger version (73K): In this window In a new window Download PPT slide   Figure 5. —Thoracic outgrowths in ctL188; Antp11/+ flies. Scanning electron micrographs of a dorsal thorax from a wildtype (A) and ctL188; Antp11/+ (B) fly. The anterior notopleural and presutural bristles are indicated by asterisks. (C) Higher magnification view of a thoracic outgrowth in a ctL188; Antp11/+ fly.

We tested whether the expression of the dorsal prothoracic phenotype is cold sensitive. An increase in the incidence of both unilateral and bilateral outgrowth in ct^L188; Antp¹¹/+ males is observed at 18° (to 37% and 3%, respectively), providing further evidence that the activity of ct^L188 is cold sensitive. Thoracic outgrowths also occur in ct^C145/+; Antp¹¹/+ females, at a frequency intermediate to that of ct^L188; Antp¹¹/+ males at 24° and at 18°. The highest incidence is observed in flies of the genotype ct^L188/ct^C145; Antp¹¹/+ at 24°, of which 63% have either unilateral or bilateral outgrowths.

Thoracic outgrowths are also observed when other heterozygous Antp alleles, both dominant and recessive, are combined with ct mutations (Table 4), however, at a lower frequency compared to Antp¹¹.

Antp and pb expression in ct mutant dorsal prothoracic discs:
Dominant defects in the dorsal prothoracic region, particularly the absence or malformation of the two large bristles on the humeral callus, have been previously observed in flies containing a mutant chromosome, Df(3R)SCB^XL2, in which most of the ANT-C is deleted. This phenotype was shown to be because of the ectopic expression of pb in prothoracic discs with the concomitant reduction in Antp expression (CRIBBS et al. 1992A ). Cut is expressed in all nuclei of prothoracic discs (data not shown). Since our results indicate that ct and pb interact during the formation of the labial palps, we tested whether reducing ct activity in dorsal prothoracic discs results in ectopic pb expression. In contrast to heterozygous Df(3R)SCB^XL2 dorsal prothoracic discs, where pb is ectopically expressed and Antp expression was reduced (Figure 6), we found that pb is not expressed in ct^L188/ct^C145 dorsal prothoracic discs (Figure 6).

View larger version (37K): In this window In a new window Download PPT slide   Figure 6. —pb and Antp expression in mutant dorsal prothoracic discs. Confocal image of dorsal prothoracic discs labeled with anti-Pb antibodies (green) and anti-Antp antibodies (red). Overlap between the expression of Pb and Antp is yellow. Anterior is up. Magnification is x400; (A) dorsal prothoracic disc from Df(3R)SCBXL2 larva showing ectopic expression of Pb and reduction in Antp expression; (B) dorsal prothoracic disc from ctL188/ctC145 larva showing normal Antp expression and absence of Pb expression.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The first ct mutant isolated was viable with scalloped wing margins (MORGAN et al. 1925 ). Many other viable and lethal ct alleles have been described since (LINDSLEY and ZIMM 1992 ) and the ct locus has been shown to be large and complex (JOHNSON and JUDD 1979 ; JACK 1985 ). Although ct appears to be critical for many developmental processes (WIESCHAUS et al. 1984 ; BODMER et al. 1987 ; BLOCHLINGER et al. 1990 ; BLOCHLINGER et al. 1991 ; JACKSON and BLOCHLINGER 1997 ), we know very little about its interactions with other genes. We have recently documented that ct interacts genetically with Notch and protein kinase A during oogenesis (JACKSON and BLOCHLINGER 1997 ). A genetic interaction between ct and Notch has also been described in cells along the developing wing margin (JACK and DELOTTO 1992 ; DE CELIS et al. 1996 ; DOHERTY et al. 1996 ; NEUMANN and COHEN 1996 ; MICCHELLI et al. 1997 ). In this report, we document a genetic interaction between ct and two homeotic genes in the ANT-C, pb and Antp.

cut interacts with pb:
The phenotype of homozygous mutant pb flies is dose sensitive: complete absence of pb activity leads to transformation of mouthparts to prothoracic leg identity, whereas partial loss of pb function causes transformation to antenna or a mixture of leg and antennal identities (BRIDGES and DOBZHANSKI 1933 ; KAUFMAN 1978 ; PULTZ et al. 1988 ). In addition, recent ectopic expression studies have shown that segmental identity can be controlled by critical threshold levels of pb expression (CRIBBS et al. 1995 ). We found evidence for the transformation of labial structures to both antennal and leg structures in ct;pb/+ mutant flies, but the severity of the phenotype is always less than that observed in pb null mutant flies. Also, we found that the penetrance and the severity of the phenotype is similar in ct^L188/ct^C145; pb/+ and ct^L188; pb/+ flies. Nor were any differences observed in ct^L188; pb/+ flies at different temperatures, although ct^L188 is clearly temperature sensitive in its interaction with Antp. It is likely, therefore, that the levels of cut activity associated with the specific combinations of ct mutant alleles used or experimental conditions do not cause pb levels to vary beyond a threshold.

The patterns of expression of both Pb and Cut are substantially altered in ct^L188; pb/+ labial discs, suggesting a cross-regulatory relationship between ct and pb. It was therefore surprising that no changes in Pb expression were seen in ct^C145 mutant clones in mosaic flies. It is possible that the effect of ct on pb expression is restricted to a specific developmental time, earlier than the induction of recombination and the creation of mosaicism. However, we also did not detect any changes in Pb expression in ct^C145 mutant embryos, although these results could be explained by perdurance of Pb initiated by regulators other than ct. The absence of detectable alterations in the embryonic Pb expression in ct mutants or after ectopic Cut expression may indicate that the ct/pb interaction is limited to imaginal discs. Although it cannot be rigorously excluded at this point that the interaction between ct and pb is specific to ct^L188, the absence of allele-specificity in the interaction between ct and Antp (see below) does not support this hypothesis. Alternately, the effect of ct on pb expression could be nonautonomous, involving cell interactions or long-range signaling. In contrast to the autonomous role of ct in specifying cell fate in the peripheral nervous system (BODMER et al. 1987 ), ct has recently been shown to have nonautonomous activity during oogenesis, where reduction of ct activity in somatic follicle cells results in morphological defects in the germline-derived cells (JACKSON and BLOCHLINGER 1997 ) and during patterning of the wing margin (SANTAMARIA and GARCIA-BELLIDO 1975 ; MICCHELLI et al. 1997 ). Nonautonomous behavior has also been seen in mitotic Antp clones (STRUHL 1981 ) and the homeodomain of Antp is internalized by cells in culture to affect transcription of endogenous genes (JOLIOT et al. 1991 ; ROUX et al. 1995 ). Moreover, homeotic function or expression has been recently shown to be modulated by wingless (JOHNSTON and SCHUBIGER 1996 ) and by Ras1 (BOUBE et al. 1997 ), both of which are known components of signaling pathways.

ct interacts with Antp:
Evidence for nonautonomous functions of ct can also be found in its interaction with Antp, since ectopic Cut expression in a stripe along the anterior-posterior compartment boundary of both portions of the eye-antennal disc results in uniform activation of Antp throughout the disc.

The incidence of outgrowths in the dorsal prothoracic region of flies that are mutant for ct and heterozygous for Antp is highest in the presence of the recessive allele Antp¹¹, which is an EMS-induced null allele with normal cytology (ABBOTT and KAUFMAN 1986 ). However, thoracic outgrowths were consistently observed in combination with dominant alleles, such as Antp^Wu and Antp^R, both of which are associated with chromosomal inversions (LINDSLEY and ZIMM 1992 ). Many of the dominant Antp alleles are also recessive lethal (DENELL 1973 ; DUNCAN and KAUFMAN 1975 ; WAKIMOTO and KAUFMAN 1981 ), so the outgrowths seen in combination with these alleles can still be reconciled with a loss of Antp function. In fact, similar outgrowths were first observed (also with incomplete penetrance) in heterozygous revertants of the dominant allele Antp^Ns in combination with Polycomb (DENELL 1973 ). Since then, they have also been shown to occur with complete penetrance in flies containing combinations of hypomorphic, null or moderate dominant Antp alleles, along with a failure of anterior spiracle eversion at the onset of pupariation (LEWIS et al. 1980 ; ABBOTT and KAUFMAN 1986 ). We have previously documented the expression of ct in embryonic spiracles (BLOCHLINGER et al. 1990 ) and embryonic lethal ct mutations result in defective spiracle formation (WIESCHAUS et al. 1984 ; BODMER et al. 1987 ).

It has been speculated that the outgrowths may be the product of a homeotic change (DENELL 1973 ). Although bristles are generally present on all the outgrowths we have characterized, they are generally malformed and we have not been able to identify any segment or appendage-specific features.

Curiously, the incidence of dorsal thoracic outgrowths observed with ct mutations in combination with null alleles other than Antp¹¹ is generally low or nonexistent, even in the presence of a deficiency that removes most of the ANT-C (Df(3R)Scr). Similarly, somatic clones of two different null alleles (Antp⁴ and Antp²⁵) and a hypomorphic allele (Antp²⁰) were associated with alterations of the cuticular morphology and loss of bristles in the region of the anterior mesonotum and humeral callus, but no outgrowths were observed (ABBOTT and KAUFMAN 1986 ). A solution to this conundrum may lie in the molecular nature of the Antp mutations, which currently is not known for most alleles.

Thoracic defects, including malformation of the spiracle and loss of bristles, have also been observed in flies containing the Df(3R)SCB^XL2 chromosome (CRIBBS et al. 1992A ). The phenotype in these flies was demonstrated to be because of ectopic pb expression in prothoracic discs which resulted in decreased Antp expression (CRIBBS et al. 1992A ). The defects in Df(3R)SCB^XL2 flies are unaffected by altering ct activity, and thoracic outgrowths are infrequent. Moreover, we showed directly that pb is not ectopically expressed in dorsal prothoracic discs of ct^L188/ct^C145 flies. We therefore conclude that ct is not exerting its effect on thoracic development by interfering with the pattern of pb expression.

A role for ct in the control of segmental identity?
Given the importance of homeotic genes in controlling segmental identity, it is intuitively pleasing to speculate that the role of ct in the processes regulated by pb and Antp is mechanistically similar to its role in the peripheral nervous system, where it acts as a switch between alternative cell fate choices. However, we have no experimental evidence to support this theory. The abnormalities observed as a consequence of the mutant interactions closely resemble pb and Antp loss-of-function phenotypes, from which we infer that ct acts as a positive regulator of the activity of these two homeotic genes. As the level of pb expression in labial discs is clearly reduced when ct activity is lowered, we further speculate that ct is directly or indirectly involved in maintaining the expression of pb, and perhaps also Antp. It is also possible that Cut has a role as a co-factor and that it interacts cooperatively with Antp and Pb to refine their target specifities, as has been shown for other homeodomain proteins, for example the product of the extradenticle locus (WILSON and DESPLAN 1995 ).

We did not observe any genetic interactions between ct and other homeotic loci in our screen, however, such interactions may have been missed because of incomplete penetrance or subtlety of the phenotype. In fact, the interaction between ct and Antp was only apparent following a careful study using a panel of Antp alleles, which was prompted by the unexpected Antp-like phenotype observed after ectopic expression of Cut in imaginal discs. Therefore, we cannot exclude that ct also interacts with other homeotic genes, in addition to pb and Antp.

	FOOTNOTES

¹ The order of these authors is alphabetical.

	ACKNOWLEDGMENTS

We thank GEROLD SCHUBIGER for pointing out the bracted bristles, PAUL TALBERT for identifying the dorsal prothoracic outgrowths, DAVID L. CRIBBS and THOMAS C. KAUFMAN for providing the Pb antibodies, DANNY BROWER for the Antp antibodies, STEPHEN M. JACKSON for performing the hsCut experiment, LIZ CALDWELL for the scanning electron micrograph images, the Image Analysis Lab (Fred Hutchinson Cancer Research Center) and the W.M. Keck Center for Advanced Studies in Neural Signaling (University of Washington) for the use of confocal microscopes, and DAVID L. CRIBBS and BARBARA WAKIMOTO for their critical comments on this manuscript. This work was supported by grants from the American Cancer Society (#DB-108), the Muscular Dystrophy Association and the Human Frontiers Science Program to K.B. and from the National Institutes of Health (F32 GM 17373) to L.A.J.

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