Identification of Chromosomal Regions Involved in decapentaplegic Function in Drosophila

Corresponding author: William M. Gelbart, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, gelbart{at}morgan.harvard.edu (E-mail).

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Signaling molecules of the transforming growth factor ß (TGF-ß) family contribute to numerous developmental processes in a variety of organisms. However, our understanding of the mechanisms which regulate the activity of and mediate the response to TGF-ß family members remains incomplete. The product of the Drosophila decapentaplegic (dpp) locus is a well-characterized member of this family. We have taken a genetic approach to identify factors required for TGF-ß function in Drosophila by testing for genetic interactions between mutant alleles of dpp and a collection of chromosomal deficiencies. Our survey identified two deficiencies that act as maternal enhancers of recessive embryonic lethal alleles of dpp. The enhanced individuals die with weakly ventralized phenotypes. These phenotypes are consistent with a mechanism whereby the deficiencies deplete two maternally provided factors required for dpp's role in embryonic dorsal-ventral pattern formation. One of these deficiencies also appears to delete a factor required for dpp function in wing vein formation. These deficiencies remove material from the 54F-55A and 66B-66C polytene chromosomal regions, respectively. As neither of these regions has been previously implicated in dpp function, we propose that each of the deficiencies removes a novel factor or factors required for dpp function.

SIGNALING molecules of the transforming growth factor ß (TGF-ß) superfamily are present in numerous organisms where they elicit a variety of cellular responses in a number of different tissues. The best genetically characterized member of this superfamily is the product of the decapentaplegic (dpp) gene in Drosophila melanogaster (PADGETT et al. 1987 ). DPP participates in several events during Drosophila development including oogenesis (TWOMBLY et al. 1996 ), dorsal-ventral patterning (IRISH and GELBART 1987 ), patterning of the mesoderm (FRASCH 1995 ; STAEHLING-HAMPTON et al. 1994 ), morphogenesis of the larval midgut (IMMERGLUCK et al. 1990 ; PANGANIBAN et al. 1990 ), adult appendage development (SPENCER et al. 1982 ), morphogenetic furrow progression in the developing eye (HEBERLEIN et al. 1993 ), and wing vein formation (SEGAL and GELBART 1985 ; YU et al. 1996 ). We have taken a genetic approach to identify factors required for dpp function in Drosophila with the expectation that their identification will shed light on the molecular mechanisms underlying dpp's diverse roles and those of other TGF-ß superfamily members.

Recent work has significantly advanced our knowledge of the molecular mechanisms underlying the functions of various TGF-ß family members, but our understanding of these complex processes remains incomplete. TGF-ß family members are initially synthesized as pro-proteins which undergo cleavage to yield mature ligands which consist of both homo- and heterodimeric forms (reviewed in MASSAGUE 1990 ). Several proteins are known to function extracellularly to antagonize TGF-ß activities (reviewed by SASAI and DE ROBERTIS 1997 ; SIVE and BRADLEY 1996 ). One such molecule has been identified in Drosophila as the product of the short gastrulation (sog) gene (FRANCOIS et al. 1994 ). The inhibitory action of SOG is in turn antagonized by the product of the tolloid (tld) gene which has been shown to proteolytically cleave SOG in complexes containing DPP (MARQUES et al. 1997 ). TGF-ß family members initiate responses in target cells by binding to heteromeric complexes of transmembrane serine/threonine kinase receptors (reviewed by ATTISANO and WRANA 1996 ; MASSAGUE 1996 ). Two Drosophila genes, thick veins (tkv) and saxophone (sax) encode type I receptors for DPP (BRUMMEL et al. 1994 ; NELLEN et al. 1994 ; PENTON et al. 1994 ; XIE et al. 1994 ). A third Drosophila gene, punt, encodes a type II receptor for DPP (LETSOU et al. 1995 ; RUBERTE et al. 1995 ). A family of related molecules function downstream of the receptor complex as elements of the signal transduction pathway (DERYNCK and ZHANG 1996 ; MASSAGUE 1996 ; WRANA and ATTISANO 1996 ; NEWFELD et al. 1997 ). The founding member of this family is the product of the Drosophila gene Mothers against dpp (Mad) (SEKELSKY et al. 1995 ). Finally, CrebB-17A (ERESH et al. 1997 ) and the products of the schnurri (shn) (ARORA et al. 1995 ; GRIEDER et al. 1995 ; STAEHLING-HAMPTON et al. 1995 ) extradenticle (exd) (MANN and ABU-SHAAR 1996 ) and vrille (vri) (GEORGE and TERRACOL 1997 ) genes have been suggested as candidates for DPP-responsive transcription factors in Drosophila.

Mutations in many of the genes mentioned above interact genetically with dpp mutations. Mad and Med were first identified in a screen for mutations which act as dominant maternal enhancers of recessive embryonic lethal dpp mutations (RAFTERY et al. 1995 ). This screen took advantage of two properties of dpp function during early dorsal-ventral patterning. First, while dpp expression at this stage is strictly zygotic, many of the other factors in the dpp dorsal-ventral patterning pathway are expressed maternally. Second, specification of cell fates along the dorsal-ventral axis is exquisitely sensitive to levels of dpp activity (FERGUSON and ANDERSON 1992A ; WHARTON et al. 1993 ). The strategy, therefore, was to look for mutations which reduced dpp function by decreasing the level of a maternally provided product required for dpp's role in embryonic dorsal-ventral patterning. When such a mutation is placed in combination with an appropriate dpp mutation, there is insufficient dpp function to specify sufficient numbers of the most dorsal cell fates, and lethality results.

The screen which identified Mad and Med used ethyl methane sulfonate to generate lesions which were then screened for the ability to maternally enhance recessive embryonic lethal dpp mutations. While this screen identified multiple alleles of both of these loci, the limited number of mutagenized chromosomes tested suggested the possibility that additional loci remained which could be identified using this approach. We therefore employed a similar strategy to identify additional chromosomal regions containing genes required for dpp function. We surveyed a collection of deficiencies for the ability to maternally enhance dpp^hr4. We identified two regions not previously implicated in dpp function, 54F-55A and 66B-66C. We have found that these interactions are dependent on both the deficiency and the dpp mutation, and that the lethality correlates with a defect in dorsal-ventral patterning. We also show that deficiencies of the 54F-55A region can interact with dpp at at least one other stage of development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Stocks and culture conditions:
All crosses were carried out at 25° on cornmeal-agar medium. The dpp rescue construct, P{dpp-Sal20}332.19, is as described in HURSH et al. 1993 and the dpp transgene bearing balancer, CyO23, is as described in WHARTON et al. 1993 . Additional balancers used are as follows: CyO, Cy¹ dp^lv1 pr¹ cn², TM3 Sb¹ e¹ Ser¹, and SM6a, al² Cy¹ dp^lv1 cn^2P bw^k1 sp². All other strains are as described in FlyBase (FLYBASE 1996 ). Deficiency-bearing stocks were obtained from the Bloomington Indiana Stock Center with the exception of Df(1)wy26 which was obtained from D. PAULI (Geneva, Switzerland) and deficiencies in the 78 cytological region which were obtained from A. CARPENTER (Cambridge, UK). The Tp(3;3)P47, bx^34e- and Tp(3;3)bxd100-bearing stocks were obtained from E. LEWIS (Pasadena, CA). A collection of ethyl methane sulfonate-induced lethal mutations were obtained from S. BRAY (Cambridge, UK) and are as described in BRAY and KAFATOS 1991 .

Deficiency screen:
Females bearing deficiencies on the third and X chromosomes were crossed to net dpp^hr4/CyO. In order to distinguish all classes of progeny, females bearing second chromosome deficiencies were crossed to either net dpp^hr4/Pin or net dpp^hr4/Tft males. Several of the second chromosome deficiencies were also outcrossed to a common CyO balancer prior to testing for maternal enhancement. Multiple broods of each cross were scored such that a minimum of 100 progeny were counted for any given cross. For each cross, the recovery of mutant progeny relative to expectations was calculated as the ratio of the number of individuals recovered for each of two dpp mutant classes to the number of individuals recovered for a non-dpp mutant control class. For deficiencies of the X chromosome, the number of females carrying the deficiency and dpp^hr4 was compared to the number of females carrying the deficiency and CyO. The number of females carrying the X chromosome balancer and dpp^hr4 was compared to this same control class. Likewise, for deficiencies of the second chromosome, the number of progeny carrying the deficiency and dpp^hr4 was compared to the number of progeny carrying the deficiency and CyO, and the number of progeny carrying the second chromosome balancer and dpp^hr4 was compared to this same control class. For deficiencies of the third chromosome, the number of progeny carrying both the deficiency and dpp^hr4 was compared to the number of progeny carrying both the deficiency and CyO, and the number of progeny carrying the third chromosome balancer and dpp^hr4 was compared to this same control class.

Tests of overlapping deficiencies in the 78A-78C interval for maternal enhancement activity:
Females bearing the following deficiencies were crossed to dpp^hr4-bearing males: Df(3L)D-5rv6, Df(3L)ME14, Df(3L)Pc-9a, Df(3L)Pc-12h, Df(3L)Pc-14d, Df(3L)Pc-101, Df(3L)Pc-810, Df(3L)Pc-2q, Df(3L)ME1325. Enhancement activity was assessed as described above for other third chromosome deficiencies.

Recombinational mapping:
Recombinants for Df(2R)Pcl-11B were generated by first crossing to y¹ w^67c23. Individual recombinant chromosomes were recovered and balanced by crossing to y¹ w^67c23; Bc¹ Egfr^E1/CyO. Recombinants for Df(3L)66C-G28 and Df(3L)Pc-MK were generated by crossing to Canton S and individual recombinant chromosomes were recovered and balanced by crossing to y¹ w^67c23; D³ gl³/TM3. All recombinant lines were assayed for enhancement activity by crossing either to z¹ w^11E4; dpp^hr4 TE52/CyO males for the second chromosome deficiency, or to net dpp^hr4/CyO males for the third chromosome deficiencies. The presence of the deficiency was determined for the Df(2L)Pcl-11B-derived lines by crossing to cn¹ thr¹ bw¹ sp¹/CyO and assaying the viability of the recombinant chromosome over thr¹. The presence of the deficiency in each of the remaining recombinant lines was similarly determined by crossing the Df(3L)66C-G28-derived lines to Df(3L)66C-I65/TM3 and the Df(3L)Pc-MK-derived lines to P{ry^+t7.2 = PZ}l(3)04063⁰⁴⁰⁶³ ry⁵⁰⁶/TM3.

Rescue of enhancement by an additional copy of dpp⁺:
Females of the general genotype, y¹ w^67c23; Df/+ were crossed to y¹ w^67c23; dpp^hr4 TE52/+; P{dpp-Sal20}332.19/Brd¹ males.

Tests for maternal enhancement of other dpp alleles:
Unbalanced, deficiency-bearing individuals were crossed to either males or females of the following genotypes: dpp^hr56 cn¹ bw¹/CyO, Cy¹ dp^lv1 pr¹, net¹ dpp^hr4/CyO, or z¹ w^11E4; net¹ dpp^hr27 ed¹/CyO.

Tests for interactions with dpp during imaginal disc development:
Females of each of the four following genotypes: y¹ w^67c23; In(2L)dpp^s4, ast¹ dpp^s4 dpp^d-ho ed¹ dp^ov1 cl¹/SM6a, y¹ w^67c23; ast¹ dpp^s6 dpp^d-ho ed¹ dp^ov1 cl¹/SM6a, y¹ w^67c23; dpp^d5/SM6a, y¹ w^67c23; In(2L)dpp^d6, dpp^d6/SM6a, were crossed to males of each of the three following genotypes: z¹ w^11E4; dpp^hr4 TE52/+; Df(3L)66C-G28 Brd¹/+, net¹ dpp^hr4 Df(2R)Pcl-11B/CyO23, and net¹ dpp^hr4 Df(2R)Pcl-7B/CyO23. Additionally, females of the genotype net¹ dpp^hr4/CyO were crossed to males of each of two genotypes, ast¹ dpp^s6 Df(2R)Pcl-11B dp^ov1 b pr¹/SM6a and ast¹ dpp^s6 Df(2R)Pcl-7B/SM6a.

Cuticle and wing preparations:
Differentiated embryos were collected from crosses between Df/+ females and y¹ w^67c23; net¹ dpp^hr27 ed¹/CyO-wg P{lacZ} males. Cuticles were prepared as described in ASHBURNER 1989 . Wings were mounted in Euparal (ASCO Laboratories, Gordon, UK). All photomicrographs were made with bright-field optics on an Olympus BHS microscope (Olympus Corp., Lake Success, NY). Images were assembled into figures using Adobe Photoshop (Adobe Systems Inc., San Jose, CA) and Canvas (Deneba Software Inc., Miami, FL).

Tests of existing mutations in the 54F-55A and 66B-C intervals for maternal enhancement activity:
The following mutants reported to be in the region of the enhancing deficiency, Df(2R)Pcl-11B, were tested for maternal enhancement by crossing females bearing these mutations to dpp^hr4-bearing males: stau¹, P{PZ}l(2)06850, P{PZ}l(2)04548, P{PZ}l(2)03091. A collection of ethyl methane sulfonate-induced lethal mutations in the 54F-55A cytological region (BRAY and KAFATOS 1991 ) were also tested for maternal enhancement activity with respect to dpp^hr4. This collection included multiple alleles of five lethal complementation groups plus an additional seven lethal mutants which failed to complement both Df(2R)Pcl7B and Df(2R)Pcl11B (S. LAZAR and S. BRAY, personal communication).

The following mutants reported to be in the region of the enhancing deficiency, Df(3L)66C-G28, were tested for maternal enhancement activity with respect to dpp^hr4: P{hsneo}105, P{lacw}l(3)0139, T(2;3)WT(3;4)Antp, P{PZ}l(3)04111, P{lacW}l(3)j1c7, In(3LR)269, In(3LR)283, l(3)SG10^m27, l(3)SG11^5m33, l(3)SG12^j51, l(3)SG13^e20, P{PZ}l(3)01323, P{PZ}l(3)02067, P{PZ}l(3)03928, P{PZ}l(3)07217, P{PZ}l(3)08223, Tp(3;3)P47-bx^34e, Tp(3;3)bxd100, and Df(3L)66C-I65, T(2;3)TE35B-SR401, P{w⁺ = *}30, T(2;3)E(da).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Survey of deficiencies for enhancement activity:
We tested 129 deficiency-bearing stocks for the ability to maternally enhance the recessive embryonic lethal mutation dpp^hr4. Together the deficiencies contained in these stocks uncover approximately 55% of the euchromatic genome and provide an efficient means to systematically search for regions containing genes which are required for dpp function. The dpp allele we used reduces dpp activity yet still confers sufficient activity to support normal development in heterozygous individuals (WHARTON et al. 1993 ). The strategy we employed was designed to identify deficiencies that acted maternally to reduce dpp function in these heterozygous animals to a level which was no longer sufficient to support the development of viable individuals.

Maternally enhancing deficiencies were identified as those that generate a substantially reduced number of dpp mutant progeny relative to their non-dpp-mutant siblings in crosses of deficiency-bearing females to dpp mutant males. This was determined by comparing the number of individuals in each of the two dpp mutant classes to the number of individuals in a nonmutant class from such a cross.

Maternally acting deficiencies could be distinguished from those which act strictly zygotically or those which require both maternal and zygotic activities. Maternally acting deficiencies result in a diminution of both classes of dpp mutant progeny in crosses of deficiency-bearing females to dpp mutant males. In contrast, zygotically acting deficiencies or deficiencies that require both maternal and zygotic activities for enhancement result in a reduction only in the class of dpp mutant progeny that also carry the deficiency.

The deficiency-bearing stocks we used represent a wide variety of genetic backgrounds and this, we believe, is the basis for considerable variability in the relative survival of dpp mutant progeny that we observe in these crosses. For this reason we chose to pursue only those stocks which showed clear maternal enhancement; namely those deficiencies that could support the development of dpp mutant progeny at a frequency less than 25% of their nonmutant siblings.

Our search for deficiencies that maternally enhanced dpp^hr4 initially identified five candidate cytogenetic intervals. The results of these crosses are summarized in Figure 1and details are provided in the APPENDIX. Two of the enhancing deficiencies, Df(2R)Pcl-11B and Df(3L)66C-G28, appear to identify novel loci involved in dpp function. One deficiency, Df(3R)Scr, appears to interact due to the deletion of a previously identified gene involved in dorsal-ventral patterning. The maternal enhancement activity in two of the deficiency-bearing lines, Df(1)JA26 and Df(3L)Pc-MK, did not appear to result from disruption of a discrete element within the deficiency.

View larger version (27K): In this window In a new window Download PPT slide   Figure 1. Results of a deficiency survey for maternal enhancement of dpp. Each of the five major chromosome arms is represented with numbered cytological divisions. The approximate extent of each of the deficiencies tested is represented as a box below the appropriate chromosome arm. , deficiencies that showed no enhancement activity. , deficiencies that showed enhancement activity with respect to dpphr4. , deficiencies that initially showed enhancement activity but were later ruled out based on criteria described in the text.

Df(1)JA26 and Df(3L)Pc-MK:
The enhancement activity in both of these stocks segregated with the deficiency-bearing chromosome. However, in neither of the stocks does it does appear to result from disruption of a single locus within the deficiency. Four other deficiencies together span the entire region deleted by Df(1)JA26 and none of these show maternal enhancement activity with respect to dpp (see Figure 1 and APPENDIX). The source of the enhancement activity in this stock must therefore reside on the deficiency-bearing chromosome outside the deficiency, or require deletion of multiple loci within D1(1)JA26, which are not together deleted in one of the overlapping deficiencies.

Similarly none of a collection of deficiencies, which together span the entire region deleted by Df(3R)Pc-MK showed any maternal enhancement activity with respect to dpp (see MATERIALS AND METHODS and data not shown). Additionally, the enhancement activity in this stock appeared to be recombinationally separable from the deficiency (Table 1). This evidence suggests that all or part of the enhancement activity present on this chromosome lies outside the region affected by the deficiency.

Df(3R)Scr:
This deficiency acts not as a maternal enhancer, but as a zygotic enhancer of dpp (see APPENDIX), and appears to do so due to deletion of the previously identified gene zerknüllt (zen). Maintenance of zen expression during dorsal-ventral patterning has been shown to be regulated by dpp (RAY et al. 1991 ). Furthermore, hyperploidy for dpp⁺ has been shown to rescue the lethality of hypomorphic zen mutations (FERGUSON and ANDERSON 1992B ). Additionally, we have observed that zen⁷ can interact zygotically with dpp^hr4 in a manner similar to this deficiency (data not shown). We suggest that the deletion of zen in this stock is at least in part responsible for the activity we observe; however, Df(3R)Scr also removes the gene labial (lab) whose expression in the developing midgut is regulated by dpp (IMMERGLUCK et al. 1990 ; PANGANIBAN et al. 1990 ). Absence of lab or perhaps an additional gene within the deficiency could also be contributing to the interaction between Df(3R)Scr and dpp.

Df(2R)Pcl-11B:
The stock bearing Df(2R)Pcl-11B shows substantial reductions in the relative numbers of dpp mutant progeny recovered in crosses of females bearing this deficiency to dpp^hr4 mutant males (see APPENDIX). We have carried out a number of experiments in an attempt to test this initial observation and to explore the dependence of this interaction on both the deficiency and the dpp mutation.

We attempted to recombinationally separate the deficiency from the enhancement activity present on the Df(2R)Pcl-11B-bearing chromosome. However, all 94 recombinant lines showed cosegregation of the enhancement activity and the deficiency (Table 1). This result is consistent with the deficiency being the source of the enhancement activity, although a tightly linked independent enhancer cannot be ruled out. Furthermore, Df(2R)Pcl-7B, which has limits similar to Df(2R)Pcl-11B (54E8-F1;55B9-C1 for Df(2R)Pcl-7B as compared to 54F6-55A1;55C1-3 for Df(2R)Pcl-11B), also shows maternal enhancement activity with respect to dpp^hr4. Compared to controls, 14% of the expected number of dpp^hr4 mutant progeny are recovered from crosses of Df(2R)Pcl-7B females to dpp^hr4 males, whereas 84% of the expected number of dpp^hr4 mutant progeny are recovered from crosses of Df(2R)Pcl-7B males to dpp^hr4 females.

We took two approaches to test the dependence of the interaction on the dpp mutation. First we looked for similar interactions between Df(2R)Pcl-11B and other recessive embryonic lethal dpp mutations that were in different genetic backgrounds. We found that this deficiency enhanced other recessive embryonic lethal dpp mutations in accordance with the known severity of those dpp mutations (Figure 2) (WHARTON et al. 1993 ). If the original interaction between the deficiency and the dpp^hr4 bearing chromosome were due to something other than the dpp mutation on that chromosome, it is unlikely that the Pcl-11B deficiency would show a similar interaction with other dpp mutations in other genetic backgrounds. It is even less likely that these interactions would correlate with the severity of the dpp allele. In all cases these interactions are dependent on the deficiency being present maternally. When deficiency-bearing males are crossed to dpp mutant females no enhancement is observed (Figure 2). We also observe all of these same phenomena when Df(2R)Pcl-7B-bearing individuals are crossed to these dpp mutations (data not shown).

View larger version (33K): In this window In a new window Download PPT slide   Figure 2. Deficiencies maternally enhance other recessive embryonic lethal dpp alleles in accordance with the known severity of those alleles.

Second, if the interaction between Df(2R)Pcl-11B and dpp^hr4 is dependent on the dpp mutation, then it should be possible to rescue the enhanced dpp mutant progeny with an additional dpp⁺ present on another chromosome. We first confirmed that the dpp⁺ transgene we were using for this purpose could function in this manner using the well-characterized maternal enhancer of dpp, Mad (Table 2). When we included this transgene in crosses of Df(2R)Pcl-11B-bearing females to dpp^hr4 mutant males, we recovered dpp mutant progeny at a frequency comparable to that of their nonmutant siblings only when the dpp mutant progeny also carried the dpp⁺ transgene. Mutant progeny which did not also carry the dpp⁺ transgene were recovered at a much lower frequency comparable to that seen previously for crosses between deficiency-bearing females and dpp mutant males (Table 2). The ability of an additional wild-type copy of dpp to rescue the enhanced dpp mutant progeny provides further support for the assertion that the interaction between Df(2R)Pcl-11B and dpp^hr4 is dependent on the dpp mutation and is not the result of another element or elements on that dpp^hr4-bearing chromosome.

If the reduction in the number of dpp mutant progeny that we observe in crosses between dpp mutant males and Df(2R)Pcl-11B-bearing females is the result of an increase in the severity of a defect caused by the dpp mutation, then it should be possible to identify lethal phenotypes among the enhanced progeny which are similar to those observed for more severe dpp alleles. For crosses involving dpp^hr4 males and Df(2R)Pcl-11B-bearing females, the existence of such a correlation remains unclear. We find that enhanced individuals from these crosses die during both embryonic and larval development, and inspection of the cuticles of enhanced embryos reveals no consistent gross abnormalities (data not shown). These observations are consistent with enhancement activity that interferes with dpp's role in dorsal-ventral patterning, but causes subtle defects that manifest themselves in lethality spread over an extended developmental period. Alternatively, these observations are consistent with enhancement activity that interferes with several of dpp's roles, and this interference at multiple stages is the basis for the broad phase of lethality.

We do observe phenotypes consistent with an increase in the severity of the dpp mutation among the dpp mutant progeny from crosses of Df(2R)Pcl-11B-bearing females to dpp^hr27 males. The dpp^hr27 allele, on its own, results in a relatively low level of dominant lethality (WHARTON et al. 1993 ) and correspondingly few heterozygous individuals with herniated head skeletons (Table 3). In crosses of Df(2R)Pcl-11B-bearing females to dpp^hr27 males we observe a substantial increase in the fraction of progeny with herniated head skeletons (Table 3; see also Figure 3 for an example of phenotype). The phenotypes of these individuals are very similar to the phenotypes observed in individuals heterozygous for null alleles of dpp (WHARTON et al. 1993 ) and are consistent with a mechanism whereby Df(2R)Pcl-11B causes synthetic lethality in combination with dpp^hr27 by further reducing dpp function during dorsal-ventral patterning.

View larger version (112K): In this window In a new window Download PPT slide   Figure 3. Cuticular defects observed among maternally enhanced progeny. (A) Phenotypically wild-type cuticle of a first instar larva. (B) An example of the herniated head phenotype observed in the progeny of crosses between Df(3L)66C-G28 females and dpphr27 males. This phenotype is also observed among the progeny of crosses between Df(2R)Pcl-11B females and dpphr27 males.

We tested the ability of Df(2R)Pcl-11B to interact with dpp during other stages of development (see MATERIALS AND METHODS). The mutation dpp^s6 disrupts dpp function during wing vein formation (SEGAL and GELBART 1985 ; YU et al. 1996 ). The transheterozygous combination of dpp^s6 and dpp^hr4 further reduces dpp function at this stage but results in relatively few wings with abnormal venation (Figure 4). The addition of either Df(2R)Pcl-11B (Figure 4) or Df(2R)Pcl-7B (data not shown) in this background results in an increased percentage of wings with abnormal venation (84% as compared to 29% for controls). The ability of these deficiencies to interfere with dpp function during wing vein formation is consistent with the gene or genes that they delete being involved with dpp function during multiple stages of development.

View larger version (115K): In this window In a new window Download PPT slide   Figure 4. Df(2R)Pcl-11B enhances dpp wing vein phenotypes. (A) dpps6/dpphr4 individuals exhibit a largely normal pattern of adult wing veins, with only 29% (n = 44) of wings exhibiting any interruption of the fourth longitudinal wing vein, whereas (B) 84% (n = 91) of dpps6 Df(2R)Pcl-11B/dpphr4 individuals exhibit interruptions of the fourth longitudinal wing vein (arrow).

Finally, we tested for interactions between Df(2R)Pcl-11B and alleles of several loci involved in dpp signaling. We found that Df(2R)Pcl-11B acted as a strong maternal enhancer of a gain-of-function allele of screw, scw^E1. Two percent of the expected number of scw^E1 progeny are recovered from crosses of Df(2R)Pcl-11B-bearing females to scw^E1-bearing males. This interaction is consistent with the fact that scw^E1 itself acts as a strong zygotic enhancer of recessive embryonic lethal alleles of dpp (RAFTERY et al. 1995 ). The fact that Df(2R)Pcl-11B does not enhance scw^E1R1, a loss-of-function allele that does not act as an enhancer of dpp, supports the assertion that this interaction is the result of the combined activities of Df(2R)Pcl-11B and scw^E1, each of which independently decreases the activity of a dpp-dependent pathway. We also tested for interactions between Df(2R)Pcl-11B and mutants in tolloid, Mad, Med, shrew, and 60A and found none.

Df(3L)66C-G28:
The stock bearing Df(3L)66C-G28 also shows substantial reductions in the relative numbers of dpp mutant progeny when females bearing this deficiency are crossed to dpp^hr4 mutant males (see APPENDIX). We carried out a number of experiments similar to those described for Df(2R)Pcl-11B in an attempt to support this initial observation and to demonstrate its dependence on both the deficiency and the dpp mutation.

We tested 77 recombinant lines for segregation of enhancement activity and the Df(3L)66C-G28 deficiency, and complete cosegregation was observed (Table 1). As with Df(2R)Pcl-11B, these data are consistent with the deficiency Df(3L)66C-G28 being the source of the enhancement activity in this stock.

We also established that the interaction between Df(3L)66C-G28 and dpp^hr4 was dependent on the dpp mutation. Crosses between Df(3L)66C-G28 and other recessive embryonic lethal dpp mutations showed that Df(3L)66C-G28 could interact with other dpp mutations in other genetic backgrounds. Furthermore, these interactions showed the same correlation with the severity of the dpp mutation, and the same maternal dependence that we observed for the interactions between Df(2R)Pcl-11B and these alleles (Figure 2). The interaction between Df(3L)66C-G28 and dpp^hr4 was also rescuable with an additional wild-type copy of dpp, as was the case for the Mad¹²-dpp^hr4 and Df(2R)Pcl-11B-dpp^hr4 interactions (Table 2). The observations that Df(3L)66C-G28 interacts with other recessive embryonic lethal dpp mutations in other genetic backgrounds and that its interaction with dpp^hr4 is rescuable with an additional wild-type copy of dpp⁺ are both consistent with the dpp dependence of this interaction.

The phenotype of dpp mutant progeny that are maternally enhanced by Df(3L)66C-G28 is similar to that of progeny enhanced by Df(2R)Pcl-11B. When Df(3L)66C-G28-bearing females are crossed to dpp^hr4 males, no consistent gross cuticular defects are observed among the enhanced progeny, and lethality occurs during both embryonic and larval stages (data not shown). However, when Df(3L)66C-G28-bearing females are crossed to dpp^hr27 males, we observe a substantial increase in the number of progeny with herniated head skeletons as compared to controls (Table 3 and Figure 3). The increased number of individuals with herniated head skeletons among the progeny of this cross suggests that Df(3L)66C-G28 is acting in combination with the dpp mutation to decrease the level of dpp activity below the threshold required for viable dorsal-ventral pattern formation in the enhanced embryos.

We tested several mutant combinations for interactions between Df(3L)66C-G28 and dpp during other stages of development (see MATERIALS AND METHODS). We were unable to identify an increase in the severity of a dpp-like phenotype in the presence of the deficiency in any of the mutant combinations we tested. While it is possible that the dpp mutant combinations we tested were not sufficiently sensitive to detect such an interaction, it is also possible that this deficiency only affects dpp function during dorsal-ventral patterning.

As with Df(2R)Pcl-11B, we found that Df(3L)66C-G28 acted as a strong maternal enhancer of scw^E1. No scw^E1 progeny were recovered from crosses of Df(3L)66C-G28 females to scw^E1 males. We also failed to observe interactions between Df(3L)66C-G28 and mutants in tolloid, Mad, Med, srw, and 60A.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

Our survey of deficiencies identified three regions that contain putative dominant enhancers of recessive embryonic lethal alleles of dpp. One of these deficiencies deletes zen, a gene previously shown to be regulated by dpp in dorsal-ventral pattern formation (RAY et al. 1991 ). The other two deficiencies, Df(2R)Pcl-11B and Df(3L)66C-G28, delete regions not previously implicated in dorsal-ventral pattern formation or dpp function. Our screen did not identify loci previously implicated in dpp function. As has been observed for loci which act as dominant modifiers of ras (KARMIN et al. 1996 ), many of the loci which interact with dpp do so only as antimorphic gain-of-function alleles, e.g., tld (CHILDS and O'CONNOR 1994 ; FINELLI et al. 1994 ), sog (FERGUSON and ANDERSON 1992B ; FRANCOIS et al. 1994 ), sax (BRUMMEL et al. 1994 ; NELLEN et al. 1994 ; XIE et al. 1994 ), tkv (NELLEN et al. 1994 ; PENTON et al. 1994 ), put (LETSOU et al. 1995 ; RUBERTE et al. 1995 ), shn (ARORA et al. 1995 ; GRIEDER et al. 1995 ; STAEHLING-HAMPTON et al. 1995 ), and vri (GEORGE and TERRACOL 1997 ). As would be expected for loss-of-function alleles of these loci, no interactions were observed in cases where one of these loci were deleted by a tested deficiency. The two loci that do show strong interactions with dpp as nulls, Mad (RAFTERY et al. 1995 ; SEKELSKY et al. 1995 ) and Med (RAFTERY et al. 1995 ), were not deleted by any of the tested deficiencies.

We have shown that the interactions between both Df(2R)Pcl-11B and dpp, and Df(3L)66C-G28 and dpp require the dpp mutation and are not the result of interactions with other lesions on the dpp mutant chromosome. We have demonstrated that the enhancement activity present in the Df(2R)Pcl-11B and Df(3L)66C-G28 stocks lies within 1.1 and 1.3 map units of the respective deficiencies. We have also provided phenotypic evidence that, in both cases, these mutant combinations disrupt normal dorsal-ventral pattern formation. This evidence is consistent with the lethality in each case resulting from a decrease in the level of a maternally provided product required for dpp function at this stage.

Several possibilities exist for the molecular identities of factors which would be required for dpp function during dorsal-ventral patterning. All of the elements of the dpp signaling pathway identified to date are required for dpp function during multiple stages of development (BRUMMEL et al. 1994 ; NELLEN et al. 1994 ; PENTON et al. 1994 ; XIE et al. 1994 ; ARORA et al. 1995 ; GRIEDER et al. 1995 ; LETSOU et al. 1995 ; RUBERTE et al. 1995 ; SEKELSKY et al. 1995 ; STAEHLING-HAMPTON et al. 1995 ). We have demonstrated that two overlapping deficiencies [Df(2R)Pcl-11B and Df(2R)Pcl-7B] of the 54F-55A cytological region can interact with dpp at at least one other developmental stage. This observation is consistent with these deficiencies deleting a gene fundamentally involved in dpp function at multiple stages.

We did not observe any evidence for an interaction between Df(3R)66C-G28 and dpp at later stages of development. It is possible that the particular mutant combinations we tested were not sufficiently sensitive to detect such an interaction and that Df(3R)66C-G28 does, in fact, interact with dpp at multiple stages. It is also possible that Df(3R)66C-G28 and dpp only interact during dorsal-ventral pattern formation and that the relevant factor or factors deleted in Df(3R)66C-G28 are only required for dpp function at that stage. If this were the case, such a factor would be a candidate for one which mediates the specificity of the response to, or the activity of, dpp at this stage.

We chose to use deficiencies to identify regions that contain maternal enhancers because this approach had several advantages over other types of screens. Deficiencies provided a means of rapidly surveying a large portion of the genome. The relatively small number of stocks involved allowed us to be quantitative in our analysis of their enhancement activities. Our assay was therefore more sensitive than those in previous screens where such analysis is impractical, and all but the strongest enhancers would be discarded. The enhancing deficiencies we isolated yield a significant number of escapers, and this may be one reason they were not identified in previous screens. Our deficiency screen also immediately suggested a cytological location for the source of the enhancement activity in the interacting stocks, and therefore greatly reduced the amount of recombinational mapping required to localize the activity. With this approach, we could also target our search to new regions and thus avoid continued reisolation of previously identified enhancer loci. Finally, the use of deficiencies in this assay suggests that null mutations in identified loci are capable of interacting with dpp. A deficiency screen therefore potentially allowed us to avoid recovering spuriously interacting gain-of-function mutations in genes that are not normally involved in dpp function.

While our analysis of the interactions between dpp and the deficiencies was complicated by the fact that the deficiencies were generated on a number of different backgrounds, the primary disadvantage of our approach is the fact that the deficiencies disrupt multiple complementation groups. To further pursue the function and molecular identity of these maternal enhancers, we must first identify genetic lesions which selectively disrupt them. To this end, we have tested all available mutants in the 54F-55A and 66B-66C cytological intervals and found none that act as maternal enhancers of dpp (see MATERIALS AND METHODS).

A number of plausible explanations exist for why the source of the enhancement activity is not represented among the previously generated mutants in these regions. First, a complete collection of noncomplementing mutations is unavailable for either deficiency, and so it is possible that the sources of enhancement activity were simply not represented in the collection tested. This is a possibility for both Df(2R)Pcl-11B for which an extensive collection of ethyl methane sulfonate-generated noncomplementing mutations exist (BRAY and KAFATOS 1991 ) and Df(3L)66C-G28 for which far fewer genetic reagents are available. It is also possible that the source of the enhancement activity in one or both cases could be a non-vital gene that would not have been isolated in many of the strategies used to generate the mutations we tested. Yet another possibility is that, in one or both cases, the source of the enhancement activity is multigenic, requiring the simultaneous loss of multiple loci within the deficiency. If this is the case, it may not be possible to recreate the enhancement activity with a mutation in only a single locus. A precedent for identifying a deficiency containing multiple genes all involved in a particular process exists for the apoptotic genes reaper, head involution defective, and grim which are all deleted by Df(3L)WR10 (reviewed in MCCALL and STELLER 1997 ). These genes were identified in a deficiency screen for apoptotic mutants not unlike the one described here used to identify enhancers of dpp (WHITE et al. 1994 ).

In order to understand the basis of the interactions we describe, the source of the enhancement activity within Df(2R)Pcl-11B and Df(3L)66C-G28 will require further characterization both genetically and molecularly. Future work will be directed at providing this characterization. In this paper, we have presented evidence that strongly supports the contention that factors required for dpp function are encoded within the 54F-55A and 66B-66C cytological intervals. Until the sources of the enhancement activities are specifically identified, we propose to refer to the enhancer uncovered by Df(2R)Pcl-11B as E(dpp)55A and the enhancer ucovered by Df(3L)66C-G28 as E(dpp)66C. It is our hope that continued characterization of these loci will provide the foundation for the discovery of additional components of pathways regulating dpp or mediating dpp signaling and that their identification will further our understanding of the molecular mechanisms underlying dpp function specifically and TGF-ß function in general.

	ACKNOWLEDGMENTS

We thank ARIEL FREY for her assistance in screening the second chromosome deficiencies and ADELAIDE CARPENTER, DANIEL PAULI, ED LEWIS, and the Bloomington Stock Center for providing stocks. We thank STUART NEWFELD for helpful comments on the manuscript. This work was supported by a National Institutes of Health grant to W.M.G. R.E.N. was supported by a National Research Service Award genetics training grant.

Manuscript received October 1, 1997; Accepted for publication January 28, 1998.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX LITERATURE CITED

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