Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
*Author for correspondence (e-mail: kbhat{at}cellbio.emory.edu)
Accepted 14 January 2002
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SUMMARY |
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Key words: patched, Head, Drosophila
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INTRODUCTION |
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Of all the adult structures, morphogenesis of the Drosophila adult head capsule is a poorly understood developmental process. The eye-antennal discs primarily contribute to the formation of adult head, while the labial discs give rise to most of the proboscis. In the early larvae, the two contralateral eye-antennal discs that develop into the two halves of the adult head, are connected by a thin layer of squamous cells (Madhavan and Schneiderman, 1977). The proliferation of the eye-antennal disc begins only after 14 hours into the first instar larval stage (Madhavan and Schneiderman, 1977
). With the progression of metamorphosis, the disc cells spread out to fuse with the contralateral disc along the midline. This results in a contiguous epidermal layer and a lumen forming the head sac. At about 12 hours after puparium formation (APF), the head sac evaginates, exposing the primordial eye, the antenna and the head capsule cells to the surface, each of which continue to develop and differentiate into respective structures. An extensive clonal analysis by Haynie and Bryant (Haynie and Bryant, 1986
) has revealed a detailed fate map of the eye-antennal disc and the primordia of different head structures. The antennal half has imaginal cells for the three segments of the antenna, which are arranged in three concentric rings, for the maxillary palp and part of the head capsule. The cells surrounding the eye primordium together with the peripodial cells form the rest of the head capsule including the ocellar and the frontal regions. This patterning of the eye-antennal disc to give rise to adult structures occurs much later during larval-pupal transition stage.
Despite these studies, very little is known about the regulation of formation of the adult head structures. Only a few genes that regulate the events that lead to formation of the head capsule from the eye-antennal disc have been identified (Weaver and White, 1995; Royet and Finkelstein, 1996
; Amin et al., 1999
). For example, we are not yet certain even how many segments contribute to it. The dissection of the developmental events that lead to the formation of the head capsule has been complicated mainly because of the fact that the head capsule is a difficult and complex structure. Moreover, the genes that might be required for the development of the head are likely to be also required for embryonic development. Mutations in these genes will lead to embryonic lethality, and one never gets to analyze their role in post-embryonic stages of development in a genetically straightforward manner, thus, contributing to the difficulty in analyzing the adult head development.
Previous results indicate that several segment polarity genes such as hedgehog, wingless and orthodenticle are involved in the patterning of the ocellar and frontal regions of the head capsule (Royet and Finkelstein, 1996; Amin et al., 1999
). Over the past few years, we have been examining the role of one of the segment polarity genes, patched (ptc), during the development of the nervous system (Bhat, 1996
; Bhat and Schedl, 1997
; Bhat, 1999
). The ptc gene, which encodes a transmembrane protein, regulates a number of developmental events in both invertebrates and vertebrates. In vertebrates, loss of Ptc activity leads to nevoid basal cell carcinoma, medulloblastoma, spina bifida and several other developmental defects (Hahn et al., 1996
; Johnson et al., 1996
; Goodrich and Scott, 1998
; Bhat, 1999
). The Ptc protein has been shown to be a receptor for Hedgehog (Hh). Interaction of Hh with Ptc relieves the Ptc-mediated suppression of Smoothened (Smo), a G-protein coupled seven-pass transmembrane molecule, allowing Smo to activate downstream target genes. In the absence of Hh activity, Ptc functions as a repressor of Smo and thus, the repressor of downstream target genes.
In a modifier screen for genes that interact with ptc (see Materials and Methods), we isolated a mutation in the ptc gene, which, in combination with various loss-of-function alleles of ptc, showed severe head capsule defects. In this paper, we characterize the requirements of Ptc-mediated signaling during head morphogenesis. Our results reveal a non-canonical Ptc-mediated signaling pathway that regulates head morphogenesis. In this pathway, Ptc positively interacts with Smo and activates Baboon (Babo), the Activin type I receptor, to promote cell proliferation. Thus, when Ptc or Smo activities are eliminated, cell proliferation in the eye-antennal disc is affected and the adults show severe head capsule defects. Moreover, reducing the dose of smo in ptc background enhances the head defects. However, gain-of-function Hedgehog interferes with the activation of Babo by Ptc and Smo, causing head defects. That the role of Ptc-Smo signaling in head morphogenesis is to promote cell proliferation via activation of Babo is indicated by the fact that expression of an activated form of Babo in the ptc domain in a ptc mutant background completely rescues the head defects. These results provide insight into head morphogenesis and reveal an unexpected positive role for Ptc-Smo signaling in promoting cell proliferation.
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MATERIALS AND METHODS |
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Other fly stocks and genetics
Other stocks used were Df (2R) NP3/CyO (eliminates both babo and ptc), Df (2R) NP1 (eliminates babo but not ptc), babo32 (null allele), babo7737, punt10460, rpr, wrinkler, grim [Df (3l)H99], hs-hh and various ptc alleles (see Tables 1 and 2). The temperature-sensitive hh allele we used was hhts2 (Ma et al., 1996). Appropriate markers were used to identify the mutant versus the balancer-bearing individuals.
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Sequencing of the ptc gene in ptchdl allele
ptchdl homozygous embryos were identified by the presence of CNS defects (visualized by Eve staining) and lack of balancer-specific staining. Genomic DNA from 12 homozygous embryos were individually prepared and genomic DNA corresponding to ptc exons was amplified from these individual DNA preparations. A total of 14 nested pairs of oligos were used. Embryos that are homozygous for the balancer as well as wild-type Canton S flies were used as control. The amplified DNA fragments were then sequenced in both directions.
In vitro culture of eye-antennal discs
Late 3rd instar (getting immobile or nearly immobile with only its head showing slight movement) larval discs from mutant and wild type were dissected in insect cell culture medium CCM3 (HyClone). It is very important to dissect the discs out in the culture medium itself and transfer to the fresh medium with the mouth hook, brain and leg discs intact so that the structures are least disturbed. The discs are incubated with the CCM3 medium in a sterile 24-well tissue culture plate (the wells were thoroughly cleaned with alcohol). The culture plate was placed in a clean, alcohol cleaned humid box, and incubated at 25°C overnight. As control, we used ptchdl/CyO or ptcH84/Cyo and wild type (Canton-S). The discs were then fixed in 4% paraformaldehyde for 30 minutes and stained with FITC-conjugated Phalloidin (1:50 dilution). The mouth hook and the brain were cleaned off and the discs were mounted in Vector Shield mounting medium. Note that discs from wandering 3rd instar larvae did not show any significant signs of differentiation in vitro. It seems therefore that the stage of larval development is important for in vitro culturing of discs. Also, the leg discs seem to respond the most in vitro since we could make out the leg-primordium following overnight culture. Discs for the in vivo analysis were dissected out from late third instar larvae. In both cases the discs were subsequently fixed in freshly made 4% paraformaldehyde and then stained with FITC-conjugated Phalloidin (1:50 dilution) and examined by confocal microscopy.
BrdU incorporation experiment and staining
BrdU incorporation was as described elsewhere (Truman and Bate, 1988) and the staining was as described elsewhere (Prokop and Technau, 1994
) with slight modifications. Mid third instar larvae were fed with 1 mg/ml BrdU in their cornmeal media and also in yeast paste. They were grown in BrdU-containing food for 12 hours and then in normal medium for 6 hours before sacrificing. The dissected eye-antennal discs were fixed in Cornoys fixative (3:1 ethanol:acetic acid), after rehydration, treated with 2 N HCl for 30 minutes. The discs were incubated with Biotinylated anti-BrdU (Becton-Dickinson) at 1:200 dilution for 36 hours at 4° C and developed by HRP reaction.
Analysis of gain of function effects of hh
Second instar to early third instar hs-hh transgenic larvae were given series of heat shocks at 37°C for 20 minutes with an interval of 12 hours in between, and were continuously grown at 29°C during the intervals. The uneclosed pharate adults were dissected out of the pupal case to check for the head phenotype.
Rescue of the head capsule phenotype with activated babo
A constitutively activated form of Babo (Baboact) was ectopically expressed with two different gal4 drivers in a ptc mutant background. UAS-baboact carries a constitutively active form of Babo in which glutamine at position 302 is replaced by aspartic acid (Wieser et al., 1995). In the first set of experiments, UAS-baboact (on the III chromosome) was crossed to the ptc deficiency, Df (2R) NP3/Cyo. The individuals bearing Df (2R) NP3 and UAS-baboact were then crossed to ptcgal4. In the second set of crosses, ptcH84/+; UAS-baboact/+ flies were crossed to ptchdl/+; P {w+mW.hs=GawB} 69B-GAL4/+ (Gal4 in this line is expressed in embryonic epidermis and in all the imaginal discs, Bloomington Stock number 1772: w*; P{w+mW.hs=GawB} 69B/TM3,Sb1). The embryos were grown at 16.5°C until early second instar larval stage in order to avoid early lethality caused by ectopic expression of activated Babo (Gal4 activity is very low at 16.5°C), and then shifted to 25°C for the rest of their developmental period.
Immunostaining, whole-mount RNA in situ
For immunostaining the discs were dissected in cold PBS, fixed on ice for 45 minutes in freshly made 4% paraformaldehyde in PBS with lysine and sodium periodate. They were incubated with primary antibody in 0.05% PBSTx containing 10% natural goat serum. Anti-Ptc antibody was used at 1:5 dilution and anti-phosphohistone3 antibody (Upstate Biotechnology) at 1:1000 dilution. FITC-conjugated anti-mouse IgG, and Cy5-conjugated anti-rabbit IgG were used as secondary antibody for anti-Ptc and anti-phosphohistone 3, respectively. The cytoplasmic actin was marked using FITC-Phalloidin (Molecular Probes). Whole-mount RNA in situ was carried out using the standard procedures.
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RESULTS |
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The eye-antennal discs are affected in ptc mutant individuals
As shown schematically in Fig. 2A,B, the head capsule in Drosophila is generated by the eye-antennal disc (Haynie and Bryant, 1986; Weaver and White, 1995
; Royet and Finkelstein, 1996
; Karim and Rubin, 1998
; Amin et al., 1999
). While the eye and the antennal primordia develop into respective adult structures, the marginal regions of these primordia along with part of the peripodial layer give rise to the head capsule structures. As the ptchdl/ptc-null individuals suffer from head capsule defects, first we examined the expression of Ptc in the 3rd instar eye-antennal disc with anti-Ptc antibody. As shown in Fig. 2, Ptc is expressed in cells from the regions that contribute to the head capsule (Fig. 2C,D compare with 2A,B).
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baboon, which encodes an Activin type I receptor, genetically interacts with ptc during head capsule development
We next addressed how this Ptc-Smo pathway regulates cell proliferation. Previous results indicate that in Drosophila, the Activin pathway mediated by Babo, which is a type I Activin receptor, regulates cell proliferation in larval tissues such as the brain and wing discs (Brummel et al., 1999). We sought to determine if the Ptc-Smo pathway promotes cell proliferation via the Activin pathway. This was first addressed genetically by examining whether individuals that are mutant for babo show the head capsule phenotype. Indeed, as shown in Fig. 5C and Table 2, individuals that are transheterozygous for babo and a deficiency that eliminates babo [babo/Df (2R) NP1 this deficiency leaves ptc intact] showed the head capsule phenotype, indicating that babo is required for head morphogenesis (neither of the two babo alleles survive to pupal stages as homozygotes or transheterozygotes). This result is also consistent with the fact that babo is expressed in all the cells of the eye-antennal disc (Brummel et al., 1999
). In order to explore the possibility that the Ptc-Smo pathway interacts with the Activin pathway to promote cell proliferation, we next determined if the head capsule phenotype can be observed in individuals transheterozygous for ptc and babo (ptc, +/+, babo). As shown in Fig. 5D and Table 2, we indeed observed the head capsule phenotype in individuals that were transheterozygous for ptc and babo. Moreover, while the number of ptcgal4/ptcnull individuals showing the head capsule phenotype is 4%, the penetrance was enhanced to 67% in ptcgal4/ptcnull, + /babonull individuals [Df (2R) NP3 was used to eliminate ptc and babo; see Table 2] and fully penetrant in ptcnull/+, babo/babonull [babo7737 or babo32/Df (2R) NP3] individuals (Table 2).
Finally, consistent with the finding that reducing the dose of smo did not suppress but, instead, enhanced the head capsule phenotype in ptc mutants, heterozygosing smo in a babo homozygous and ptc heterozygous condition [smo1, Df (2R) NP3/+, babo7737 ] did not suppress the head capsule defects either (Table 2). These results provide further evidence that Ptc, together with Smo, positively interacts with Babo to promote cell proliferation during head morphogenesis.
During Activin signaling, the Type II receptor binds to its ligand, which in turn promotes physical interaction between Type II and Type I receptors and the phosphorylation of Type I receptor (Wrana et al., 1994). We examined if ptc interacts in trans with punt, the Type II receptor (Childs et al., 1993
). However, unlike the interaction between ptc and babo, no such transheterozygous interaction was observed between ptc and punt (Table 2).
An activated form of Babo rescues the head capsule defects in ptc mutants
Previous results have indicated that substitution of glutamine at position 302 of the Babo protein with aspartic acid results in a constitutively active form of Babo, as assayed by its ability to interact with Smad2 protein (Wieser, et al., 1995; Brummel et al., 1999
). This suggests that for Babo to become active, specific conformational changes in the protein might be required. It is possible that Ptc-Smo signaling interfaces with Babo by promoting the activation of Babo or it might function upstream of the activation of Babo. First, we also examined if the transcription of babo is affected in ptc mutants. Whole-mount RNA in situ of the eye-antennal disc with a babo probe did not reveal any reduction in the levels of babo RNA compared with wild type (data not shown). This indicates that the Ptc-Smo pathway does not regulate babo at the transcription level. It is generally the case that if 50% reduction of two separate genes shows a phenotype not revealed in the individual heterozygous mutant, the transheterozygous interaction is very specific and indicative of a very close interaction (Artavanis-Tsakonas et al., 1995
; Winberg et al., 1998
). Given that ptc and babo show transheterozygous interaction (see above and Table 2), it is possible that Ptc and Smo interaction with Babo is at the activation of Babo level, although we cannot rule out the possibility that Ptc-Smo pathway regulates expression of one of the Activin-like ligands.
Finally, we sought to determine if the Ptc-Smo pathway during head morphogenesis operates upstream of activation of Babo by determining if an activated form of babo can rescue the head capsule defects in ptc mutants. We introduced a babo transgene that encodes the activated form of Babo into ptc background (UAS-baboact). The constitutively active form of the Babo has glutamine at position 302 replaced by aspartic acid (Wieser et al., 1995). Induction of babo in ptc mutant background in the exact Ptc expression domain using the UAS-baboact transgene and ptc-GAL4 driver (ptcgal4; we used this allele not only to generate the mutant combination but also to express baboact in the Ptc-domain), completely rescued the head phenotype in ptc mutant individuals (Fig. 5E,F; Table 2). Moreover, expression of UAS-baboact in ptchdl/ptcnull background using a disc-specific GAL4 driver (69B-GAL4) also rescued the head capsule defects (Table 2; Fig. 5G).
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DISCUSSION |
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Ptc-signaling promotes cell proliferation in the eye-antennal disc
Previous results indicate that Ptc is a repressor of cell proliferation. Our results, however, show that during head development in Drosophila, Ptc functions to promote cell proliferation. The loss of the head capsule in ptc mutants is not due to cell death, as we did not observe any inappropriate and massive cell death in the eye-antennal disc by the TUNEL assays. However, we observed a lack of BrdU incorporation as well as fewer phospho-histone-positive cells in the eye-antennal disc. Lack of differentiation of cells of the eye-antennal discs can also give rise to similar head capsule defects. For example, pharate adults mutant for the headcase gene show severe head capsule defects with resemblance to ptc mutants (Weaver and White, 1995). However, in headcase mutants, the morphology, the size and the shape of the eye-antennal discs are normal and the head capsule defects appear to be due to a failure in the differentiation of cells of the eye-antennal disc (Weaver and White, 1995
). In ptc mutants, our results indicate that the morphology, organization, and size of the eye-antennal disc are severely affected by late 3rd instar larvae and the primary cause for the head capsule defects is loss of cell proliferation. This conclusion is further supported by the fact that an activated form of Babo completely rescues the head capsule defects in ptc mutants. babo is a known player in promoting cell proliferation and it has been previously shown to be required only for cell proliferation but not for cell differentiation in the imaginal discs (Brummel et al., 1999
). Moreover, our in vitro culture of eye-antennal discs indicate that the differentiation per se is not affected in ptc mutants (see Fig. 3D). Therefore, we conclude that Ptc promotes cell proliferation in the eye-antennal disc during head development.
Ptc, together with Smo, promotes cell proliferation in the eye-antennal disc
Previous studies indicate that Ptc is likely to complex with Smo and repress Smo from activating downstream target genes. Binding of Hh to Ptc frees Smo from Ptc repression, which then goes on to activate downstream target genes. Thus, Ptc has been always viewed as a suppressor of gene activity via suppressing Smo. For example, during the development of the embryonic nerve cord, loss of ptc activity leads to missing RP2 neurons. This is due to the ectopic activation of Gsb in the neuroectoderm from which the RP2 precursor neuroblast (NB4-2, a row 4 NB) delaminates; ectopic Gsb prevents Wingless signaling from specifying NB4-2 identity and therefore the loss of RP2 neurons (Bhat, 1996; Bhat and Schedl, 1997
; Bhat et al., 2000
). Consistent with the possibility that Smo is downstream of Ptc, ectopic expression of Gsb in row 4 in ptc mutants and the consequent loss of RP2 neurons is rescued in ptc, smo double mutants (K. M. B., unpublished). If this signaling also occurs during the head development, loss of Ptc will lead to inappropriate activation of Smo, leading to the head capsule defects; loss of Smo activity in a ptc mutant background, therefore, should suppress the head capsule defects. However, reducing the dose of Smo in a ptc mutant background (smo/+, ptcgal4/ptcnull), instead of suppressing the head defects (or at the least reducing the severity), enhanced the head capsule defects. Moreover, our results show that loss of Smo activity leads to the same head capsule defects as in ptc mutants.
Previous results have indicated that Ptc might negatively regulate levels of Smo via vesicular trafficking of Smo from the cell surface (Denef et al., 2000; Ingham et al., 2000
; Martin et al., 2001
; Strutt et al., 2001
). Thus, in ptc mutants it has been inferred that the level of Smo on the membrane is high, leading to the inappropriate activation of downstream target genes. That a similar mechanism might operate during head capsule development is unlikely for the following reasons. First, we found that reducing the dose of smo in ptc mutant background enhances the phenotype. Second, in one of the ptc alleles, ptcS2, the mutation is an amino acid change from a charged to a neutral in the sterol-sensing domain (Martin et al., 2001
). We found that ptcS2 fully complements ptchdl and the transheterozygotes have no head capsule defects (Table 1). Moreover, in ptchdl/ptcnull mutant eye-antennal disc, the level of Smo is not upregulated (N. Mortimer and K. M. B., unpublished). Based on these results, we conclude that a positive signaling by Ptc and Smo regulates cell proliferation during head development.
Gain-of-function Hh interferes with the positive signaling by Ptc-Smo in head morphogenesis
In the conventional Ptc-signaling, interaction of Hh with Ptc relieves the repression on Smo, thus allowing Smo to function. When Hh is ectopically expressed, it interacts with Ptc to relieve the repression on Smo. This in turn is thought to cause phenotypes in hh gain-of-function situations. Thus, in the CNS, for example, loss of Ptc activity from the RP2 neuronal precursor cell leads to missing RP2 neurons (see above); ectopic expression of Hh in adjacent rows of cells leads to loss of RP2 neuron via inappropriate activation of Gsb in the neuroectoderm from which NB4-2 is delaminated (K. M. B., unpublished). The results described in this paper, that during head development gain-of-function Hh mimics a loss of function ptc phenotype, are not inconsistent with the finding that Ptc, together with Smo, promotes cell proliferation. That is, ectopic expression of Hh will bind to Ptc and this will interfere with the positive signaling by Ptc and Smo. One possibility is that Ptc and Smo are physically associated with one another, and binding of Hh to Ptc will break this physical association, rending Ptc or Smo unable to positively regulate cell proliferation in the eye-antennal disc.
Interaction between Babo signaling and Ptc-Smo signaling
Our results indicate that Ptc-Smo signaling leads to the activation of Babo. During Activin signaling, Activin binds to Activin type II receptor, which promotes physical interaction between type II and type I receptors and the phosphorylation of type I receptor. Both type I and type II receptors are transmembrane serine/threonine kinases. Phosphorylation of the type I receptor results in the activation of its kinase activity and the phosphorylation of downstream transcription activators such as the Smad proteins, resulting in their nuclear localization (Wrana et al., 1994; Heldin et al., 1997
). In Drosophila, analysis of null mutants for the type I receptor babo, as well as analysis of babo germline clones, indicates that babo is not required during embryogenesis but is essential during pupal development and adult viability (Brummel et al., 1999
). The major defect in babo mutants is a reduction of cell proliferation in the imaginal discs and brain tissue. It has also been shown that in tissue culture experiments, a constitutively active form of Babo can signal to vertebrate TGF-ß/Activin, but not to BMP-responsive promoters (Brummel et al., 1999
). The activated Babo then interacts with Drosophila Smad2 to effect the nuclear localization of this transcription factor.
Our results, that expression of an activated form of Babo in the ptc-expression domain in the eye-antennal disc of ptc mutants completely rescues the head capsule defects, indicates that Ptc-Smo signaling ultimately leads to activation of Babo and promotes cell proliferation in the eye-antennal disc. As babo and ptc show transheterozygous interaction, it is tempting to speculate that the interaction between Ptc and Babo might be direct. A transheterozygous interaction is generally observed in several cases where the two proteins associate with one another, in cases such as the receptor-ligand pairs Notch and Delta (Artavanis-Tsakonas et al., 1995; Winberg et al., 1998
; Kidd et al., 1999
; Bashaw et al., 2000
). However, it is also possible that Ptc-Smo signaling and Babo signaling represent parallel pathways that converge at the point of cell cycle control. In this scenario, partial reduction in each could have a synergistic negative affect on cell proliferation, while overexpression of one (i.e. activated Babo) could compensate for loss of the other. Yet another possibility would be that the Pt-Smo pathway activates one of the Activin-like ligands. While our results indicate that there is no transheterozygous genetic interaction between ptc and punt (the inferred type II receptor for Activin), we cannot rule out the possibility that the Ptc-Smo pathway does not interact with Punt. This is due to the fact that a lack of transheterozygous interaction does not mean that the two players do not interact, as it actually depends on what is limiting. Nonetheless, our finding that Ptc, together with Smo stimulates cell proliferation and the interfacing of Ptc-signaling with Babo-signaling in this process provides new insight into the process of head development.
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ACKNOWLEDGMENTS |
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