broad

DEVELOPMENTAL BIOLOGY

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, BMP signaling was analyzed in the FCs of 16 Drosophila species that span 45 million years of evolution. The spatial patterns of BMP signaling in the FCs were found to be dynamic and exhibit a range of interspecies variation. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. These results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning (Niepielko, 2011).

In FCs of D. melanogaster, dynamics of BMP signaling are regulated by the dynamics of tkv. In most Drosophila species, a correlation between tkv expression and BMP signaling dynamics was found. Remarkably, ectopic expression/depletion of tkv was sufficient to diversify BMP signaling in D. melanogaster. These perturbations successfully transformed the pattern of BMP signaling found in D. melanogaster into the diverse patterns of BMP signaling naturally found across Drosophila species, supporting the claim that tkv plays a major role in specifying the distribution of BMP signaling in the FCs. In D. melanogaster, a similar mechanism restricts the distribution of BMP signaling in wing and haltere imaginal discs (Niepielko, 2011).

In D. melanogaster, the dynamics of tkv are regulated jointly by BR and BMP signaling. Thus, the late patterns of BR, TKV, and P-MAD are observed in a similar group of cells. Surprisingly, in some species, the pattern of P-MAD correlated with the pattern of tkv; however, these patterns did not fully overlap the BR domain. Specifically, in addition to overlapping the BR domain, tkv was expressed in the adjacent cells that lacked BR expression. It is proposed that, in addition to being regulated by BR and BMP signaling, tkv is also regulated by transcription factors that are expressed in the adjacent domain such as Jra and Fos/Kayak (Niepielko, 2011 and references therein).

Of particular interest are species with three DAs' eggshell, due to the absence of tkv from the BR domain. It is suggested that this signaling pattern provides an example for decoupling of the regulation of tkv by BR from its regulation by BMP signaling. A model is proposed by which tkv is regulated by BMP signaling (anterior); however, it lost its regulation by BR. Of note, the mechanism governing tkv patterning is still unknown, and the established tkv enhancer trap in the Drosophila wing failed to recapitulate the patterns of tkv in the FCs. Thus, the proposed modifications in tkv regulation remain to be experimentally validated (Niepielko, 2011).

In D. melanogaster, early BMP signaling appears as an anterior stripe, reflecting the anterior secretion of the ligand DPP that signals through a uniformly expressed tkv receptor. Thus, in species from the virilis repleta groups, the uniform expression of tkv could account for the anterior stripe domain of late BMP signaling. Indeed, ectopic expression of tkv in all FCs prevented the dorsal anterior repression of BMP signaling in D. melanogaster, which was consistent with the pattern found in flies from the virilis repleta groups (Niepielko, 2011).

In the first three patterning classes, spatial modifications in the late patterns of tkv provided a reasonable explanation for the diversity in late patterns of BMP signaling. In the virilis repleta groups, late expression of tkv was uniform in all FCs; at the same time, BMP signaling was patterned. While the possibility cannot be excluded that a second copy of tkv is present in the non-sequenced species, it is proposed that in these species other BMP components have evolved to gain control of BMP signaling dynamics. In D. melanogaster, the disruption of saxophone (sax), a type I BMP receptor, deformed operculum size and DAs' morphologies. Thus, SAX is a potential regulator of the BMP signaling dynamics in the virilis repleta groups. Also, additional mechanisms were shown to regulate BMP signaling across animals including intracellular and extracellular inhibitors, co-receptors, levels of ligand expression, combinations of ligands, and interactions with other signaling pathways. These mechanisms should be studied systematically in order to determine which BMP component controls the DV phase of signaling in these species (Niepielko, 2011).

In D. melanogaster, the early phase of BMP signaling prevents the anterior domain of the follicle cells from acquiring DA cell fate. This mechanism is based on the inhibition of br expression by the anterior BMP signaling. Thus, it is not surprising that disruption of early BMP signaling is associated with eggshell deformations including modifications in the numbers and shapes of DAs'. Due to the high sensitivity of the eggshell's structures to changes in BMP signaling, it is speculated that small differences in early BMP signaling could guide the natural variations in numbers and shapes of DAs found across Drosophila species (Niepielko, 2011).

In D. melanogaster, the late phase of BMP signaling is associated with the repression of br mRNA in the dorsolateral patches; however, its role in eggshell morphology was not explicitly explored. Depletion of BMP signaling from the BR domain did not affect early BR patterning and operculum size; however, this perturbation deformed DAs' morphology possibly due to late migration of BR cells. Interestingly, similar morphologies were found by disrupting Cad74A, a cadherin gene regulated by BMP signaling that was found to be important for proper eggshell morphology. BMP signaling regulates cadherins in the pupal retina of D. melanogaster and in the human renal epithelial cells. It is proposed that late BMP signaling is involved in the morphological processes of DAs' formation by affecting cell adhesion molecules. Other cadherin genes are expressed or repressed in the DAs' forming cells, and it will be interesting to study how their regulation by BMP signaling affects DAs' morphogenesis (Niepielko, 2011).

Effects of Mutation or Deletion

Deformed (Dfd) is a homeotic selector gene required for segment identity in the head. broadis a steroid hormone-regulated locus required for metamorphosis of the epidermis and multiple internal tissues. Dfd and BR-C mutants dying during metamorphosis share defects of CNS reorganization, ventral adult head development, and adult salivary gland morphogenesis. Specifically, the shared phenotypes are: (1) failure to separate the subesophageal ganglion (SEG) from the thoracic ganglion (TG), (2) structural and functional abnormalities of the proboscis and maxillary palps, innervated by the SEG, and (3) failure of the adult salivary glands to extend into the thorax. Experiments performed with a conditional allele demonstrate that Dfd+ function during either larval life or metamorphosis is sufficient to rescue the SEG-TG separation phenotype. BR-C/Dfd double mutants show synergistic enhancement of the ventral head defects. This genetic interaction suggests that the segment identity and steroid hormone-sensitive regulatory hierarchies intersect during postembryonic development (Restifo, 1994).

Broad transcription factors regulate muscle attachment in Drosophila. In Brc mutants of the rbp complementation group, dorsoventral indirect flight muscles (DVM) are largely absent and the dorsal longitudinal indirect flight muscles, tergotrochanteral muscles (TTM), and remaining DVM often select incorrect attachment sites. Heat induction of BRC protein containing the Z1 zinc-finger pair rescues the thoracic muscle defects of rbp completely. BRC-Z4 induction can also rescue the thoracic musculature, but BRC-Z2 and -Z3 can not. Thus, the effect is specific to BRC-Z1 and its closest relative, BRC-Z4. Formation of muscle primordia from imaginal myoblasts appears normal in rbp mutants. However, the myotendinous junctions linking the DVM to the dorsal epidermis are weak; the muscles detach during pupal life and subsequently degenerate. rbp mutations disrupt the cell-cell interactions between developing muscles and epidermal tendon cells as they recognize and attach to one another. Using a BRC-Z1-specific monoclonal antibody, it has been shown that both the developing muscles and epidermal tendon cells express BRC-Z1 at the time of pupation, before mutant muscles begin to attach. One striking aspect of the rbp phenotype is that dorsal attachments are defective while ventral attachments seem normal. This phenomenon has also been observed in the TTM of bendless and myospheroid mutant adults. It is also noted that all the susceptible attachment sites are derived from the wing imaginal disc, while the unaffected sites are derived from leg discs. Analysis of the Derailed receptor tyrosine kinase suggests that muscles use different molecules for making attachments at dorsal and ventral ends of their fibers. Derailed is localized to the ventral ends of a subset of embryonic muscles; derailed mutations cause ectopic ventral attachment of these muscles. The stripe dorso-longitudinal indirect flight muscle phenotype resembles the rbp DVM phenotype, in that normal initial development is followed by degeneration and disappearance. Although stripe does not appear to be under rbp control at head eversion, the two genes may regulate overlapping subsets of downstream targets (Sandstrom, 1997).

broad and E74 are induced directly by ecdysone and encode families of transcription factors that regulate ecdysone primary- and secondary-response genes. Genetic analyses have revealed that mutations in BRC and E74 are lethal during metamorphosis; these mutations also cause some similar lethal phenotypes and alterations in secondary-response gene transcription. To examine whether BRC and E74 function together during development, representative alleles from each BRC and E74 complementation group have been combined. Analysis of the morphological and molecular phenotypes of the double-mutant animals reveals that BRC and E74 alleles act together to produce both novel and synergistic effects. BRC and E74 share functions in puparium formation, pupation and early gene induction. In addition, the BR-C and E74 transcription factors may directly interact to regulate the expression of salivary gland glue and late genes (Fletcher, 1995).

During Drosophila third instar larval development, one or more pulses of the steroid hormone ecdysone activate three temporally distinct sets of genes in the salivary glands, represented by puffs in the polytene chromosomes. The intermolt genes are induced first, in mid-third instar larvae; these genes encode a protein glue used by the animal to adhere itself to a solid substrate for metamorphosis. The intermolt genes are repressed at puparium formation as a high titer ecdysone pulse directly induces a small set of early regulatory genes. The early genes both repress their own expression and activate more than 100 late secondary-response genes. broad is an early ecdysone-inducible gene that encodes a family of DNA binding proteins defined by at least three lethal complementation groups: br, rbp, and l(1)2Bc. BRC is critical for the appropriate regulation of all three classes of ecdysone-inducible genes. Both rbp and l(1)2Bc are required for glue gene induction in mid-third instar larvae. In addition, the l(1)2Bc function is required for glue gene repression in prepupae; in l(1)2Bc mutants the glue genes are re-induced by the late prepupal ecdysone pulse, recapitulating a mid-third instar regulatory response at an inappropriate stage in development. l(1)2Bc function is also required for the complete ecdysone induction of some early mRNAs (E74A, E75A, and BRC) and efficient repression of most early mRNAs in prepupae. Like the intermolt secondary-response genes, the late secondary-response genes are absolutely dependent on rbp for their induction. An effect of l(1)2Bc mutations on late gene activity can also be detected, but is most likely a secondary consequence of the submaximal ecdysone-induction of a subset of early regulatory products. These results indicate that BRC plays a key role in dictating the stage-specificity of the ecdysone response. In addition, the Ecdysone-receptor protein complex alone is not sufficient for appropriate induction of the early primary-response genes, but requires the prior expression of BR-C proteins. These studies define the BR-C as a key regulator of gene activity at the onset of metamorphosis in Drosophila (Karim, 1993).

The Broad-Complex early gene, which acts as a global regulator of tissue-specific responses to ecdysone, encodes a family of zinc-finger DNA binding proteins known as Z1, Z2, Z3, and Z4. Genetically the BR-C encodes three complementing functions: br, rbp, and 2Bc, and a class of npr1 alleles that fail to complement any of the other genetic functions. Using heat-inducible transgenes, the lethality associated with each of the complementing genetic functions was rescued and transcriptional activity of tissue-specific BR-C(+)-dependent target genes was restored. br+ function, including rescue of mutant lethality, is only provided by the Z2 isoform. Rescued br mutants have normal head and abdominal structures, but their legs are malformed, their wings are broad, and they die soon after eclosion. Z1 transgenes provide full rbp+ function, while Z4 provides partial function. In both rbp1 and rbp5 mutants the reduced bristle number phenotype is fully rescued. Likewise, while Z3 provides full 2Bc+ function, Z2 also provides partial function. Half of the treated 2Bc mutants advance to late pupal stages when rescued with Z3. In contrast to Z3, however, the 2Bc animals that eclose following Z2 expression have malformed legs and wings and die very soon after eclosing. npr1 mutant lethality is not rescued by any BR-C transgene. Dopa decarboxylase is rescued by Z2; Salivary gland secretion protein 4 is rescued by Z1, and Fat body protein 2 is rescued by Z3 and to an lesser extent by the other transgenes. These results indicate possible functional redundancy or regulatory dependence (via autoregulation) associated with the rbp+ and 2Bc+ functions. The establishment of these relationships between BR-C genetic functions and protein isoforms is an important step toward understanding the roles of BR-C proteins in directing metamorphic responses to ecdysone (Bayer, 1997).

Alleles of the Stubble-stubbloid locus at 89B9-10 act as dominant enhancers of broad alleles of the BR-C. Sb-sbd wild-type products are necessary for appendage elongation. Three other loci are implicated in imaginal disc morphogenesis based on their genetic interactions with both BR-C and/or Sb-sbd mutants. Enhancer of broad [E(br)] was identified as a dominant enhancer of the br1 allele of the BR-C and is a recessive lethal. Mapping of E(br) has led to the identification of two loci, blistered and l(2)B485, mutants of which interact with E(br) and the Sb-sbd locus. Blistered, but not l(2)B485, interacts strongly with the BR-C. Alleles of the blistered locus are viable and disrupt proper wing disc morphogenesis independent of genetic interactions. All three loci map within the 0.6-map unit interval between the genetic markers speck and Irregular facets and to the cytological region 60C5-6; 60E9-10 at the tip of chromosome 2R. Genetic evidence is consistent with the view that the BR-C regulates blistered (Gotwals, 1991).

The Broad-Complex (Br-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes that result from the ectopic expression of these zinc-fingers. The mechanism by which the Br-C affects chorion deposition has been investigated by using BrdU to follow the effects of Br-C misexpression on DNA replication and in situ hybridization to ovarian mRNA in order to evaluate chorion gene expression. Ectopic Br-C expression leads to prolonged endoreplication and to additional amplification of other genes, in addition to the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when Br-C is misexpressed. It is concluded that the target genes of the Br-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis (Tzolovsky, 1999).

BR-C mRNA is expressed in follicle cells in a dynamic pattern. Its expression is first detected in all follicle cells at stage 6. During stage 10A, all the columnar cells, except the dorsal anterior follicle cells, contain the BR-C transcript. However, only two groups of dorsal-lateral-anterior follicle cells express the BR-C mRNA during stage 10B, marking the dorsal appendage secreting cells. Z1 is the only zinc-finger isoform with levels of expression significantly high enough to be detected by in situ hybridization techniques during oogenesis. The distribution pattern of the BR-C protein appears to be similar to that of its mRNA during stages 6-8 of oogenesis, when all follicle cells stain. The protein is also detected in all columnar follicle cells except the dorsal anterior cells at stage 10, similar to the pattern of mRNA distribution. However, the follicle cells at the posterior pole appear to be stained at this stage; this differs from the mRNA distribution pattern. The late distribution pattern of Br-C protein and mRNA differs. A very strong signal is observed in two groups of the lateral-dorsal follicle cells at stages 11 and 12, but the posterior and ventral follicle cells are still stained. The signal in the posterior and ventral region disappears around late stage 13, leaving only the dorsal-appendage-associated follicle cells stained. The differences between the distribution patterns of the protein and mRNA presumably reflect the fact that the half-life of the protein is much longer than that of the mRNA. By the time late Br-C transcription occurs at the lateral-dorsal-anterior follicle cells, the protein translated from the early BR-C transcripts remains at the posterior and ventral side, while the mRNA has been degraded. Thus, the early and late protein distribution patterns overlap to form a gradient-like pattern at stages 11 and 12. The same reasoning could also be used to explain why the protein, but not the mRNA, is detected in the follicle cells at the posterior pole during stage 10 (Tzolovsky, 1999).

Genetically, the BR-C locus has three fully complementing functions: br (broad), rbp (reduced bristle number on palpus), and 2Bc, as well as a noncomplementing npr (nonpupariating) class. The Z1 isoform provides the full rbp⁺ function. Since the Z1 zinc-finger isoform is expressed during oogenesis and is detectable by in situ hybridization, it is predicted that the rbp functional domain will be required. To test this, female homozygous viable rbp¹ and rbp² mutants were dissected to examine the eggshell phenotype. It was found that the dorsal appendages were abnormal, being shorter and rougher than the wild type, and the eggshells were much more fragile. Shortening of the dorsal appendages was observed in rbp² homozygous mutants (Tzolovsky, 1999).

Ectopic BR-C expression appears to induce premature production of the chorion. The chorion is already present in stage-11 egg chamber. This could isolate the oocyte from the nurse cells and physically prevent dumping of the nurse cell components into the oocyte. This could result from an altered pattern of transcription and translation of the chorion genes, or from abnormalities in amplification of the chorion genes, or both. Therfore the study sought to determine whether the alterations in Br-C expression affect the timing or pattern of chorion gene amplification. Since the chorion is synthesized by most follicle cells, this function could be related to the earlier expression of the Br-C. To monitor amplification, the incorporation of BrdU in the follicle cell nuclei of wild-type ovaries and in ovaries misexpressing various isoforms of the Br-C was investigated. In wild-type ovaries, after eight mitotic cell divisions, the endoreplication phase of the follicle cell development of oogenesis begins (stage 6), and is completed by stage 10B; during this process the entire nucleus is labeled by BrdU. The endoreplication is asynchronous in wild-type and w¹¹¹⁸ (the host strain used to produce the transgenic lines) ovaries and occurs in both nurse cells and follicle cells. A continuous endoreplication is observed in the nurse-cell-associated follicle cells at stage 10B. This is followed by the chorion gene amplification phase when 4 spots of incorporation are seen per nucleus in the follicle cells overlying the oocyte. These 4 spots represent amplification of the two clusters of chorion genes. Two are always larger, presumably due to the higher level of amplification of the cluster on chromosome 3, as compared to the X-chromosome cluster. This amplification is first observed at the border between the oocyte and nurse cells and it soon spreads to the rest of the follicle cells. When the BR-C isoforms are misexpressed, there is prolonged and synchronized endoreplication until late stage 10B, followed by specific amplification of genes in each nucleus. These results are observed from 3.5 to 4.5 hr after heatshock. Extra spots of incorporated BrdU were also observed in the nuclei (Tzolovsky, 1999).

There are three possible explanations for this: either the heatshock could be responsible, the homologs of the chromosomes could have separated due to a defect in the cell cycle, or there could be amplification of DNA at additional sites in the genome. The host flies used to produce the transgenic lines, w¹¹¹⁸, were heatshocked and still showed 4 spots per nucleus, so heatshock itself is not responsible for the results. The number of spots per nucleus was counted and 6 or 12 spots were found in ~80% of the nuclei. Occasionally up to 28 spots were observed. If the cell cycle is affected, and the homologs have separated, one would expect to see many more than 28 spots per nucleus due to the polyploidy of the follicle cells. If the amplification sites vary one could not predict the numbers, and indeed the numbers may well be variable. This suggests that there are other sites in the genome induced to replicate by Br-C overexpression. It is concluded that the endoreplication of DNA and the amplification of the chorion genes depends on the Br-C encoded proteins or an unknown protein that is encoded by one of the downstream targets of the Br-C. This observation is consistent with the the fact that a mutation in the Br-C locus causes premature arrest of chorion gene amplification. In summary, a working model would be that the Br-C is activated by ecdysone in all follicle cells at stage 6 of oogenesis where its key function is the control of endoreplication, and then selective amplification. Later, when Br-C is turned off in all but the anterior-dorsal follicle cells that will secrete the appendages it has a second set of functions and is involved in the migration of cells and morphogenesis of the chorionic appendages. Recently this link between ecdysone, the Br-C, and morphogenesis has also been described for the progression of the furrow in the developing eye imaginal disc of Drosophila (Tzolovsky, 1999 and references therein).

Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. Isoform BRC-Z1 mediates the muscle attachment function of rbp⁺ and is expressed in both developing muscle fibers and their epidermal attachment sites. Two complementary studies have been carried out to determine the cellular site and mode of action of rbp⁺ during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. (1) Genetic mosaics, produced using the paternal loss method, reveal that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis is mutant, the vast majority of muscles detach or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis can support attachment of mutant muscles. (2) Ultrastructural analysis corroborates and extends these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, are reduced in number and show delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resemble the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, it is proposed that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle (Sandstrom, 1999).

BRC mutants of the rbp (reduced bristles on palps) complementation group have distinctive defects involving the most prominent thoracic muscles of adult Drosophila, the indirect flight muscles (IFM) and tergotrochanteral, or jump, muscle (TTM). The IFM consist of dorsal longitudinal muscles (DLM) and dorsoventral IFM (DVM), which are morphologically and biochemically specialized to produce the high contraction frequencies required for dipteran flight; TTM is a tubular muscle, resembling the other somatic muscles. rbp mutants have few intact DVM fibers, and these, along with the DLM and TTM, are often attached to incorrect epidermal muscle attachments (EMAs) (Sandstrom, 1999 and references therein).

IFM and TTM develop from myoblasts (called adepithelial cells because they are associated with the imaginal disc epithelium) that proliferates during larval and early pupal life. Early during metamorphosis, the myoblasts migrate, fuse with one another to form muscle primordia, and align between their respective EMAs. Subsequent maturation of the IFM includes stereotyped morphological changes that yield the differentiated muscles and elaborate myotendinous junctions (MTJs) of the adult fly. rbp⁺ (i.e. wild type) function is required for the IFM and TTM to establish and maintain attachment to epidermal tendon cells at correct EMA sites. In rbp mutants, thoracic muscle primordia make contact with EMAs during early pupal life. However, most incipient attachments of DVM with dorsal thoracic EMAs are unable to withstand the muscle shortening that occurs several hours later, resulting in muscle detachment, generally followed by complete degeneration. In addition, significant numbers of mutant muscles form junctions with inappropriate EMAs, e.g. DLM attaching to DVM sites. These defects can be completely rescued by expressing specific BRC isoforms (BRC-Z1 or -Z4, but not -Z2 or -Z3) from inducible transgenes at the beginning of pupal development. These data suggest that rbp mutations disrupt the interaction between developing epidermal tendon cells and muscles. BRC-Z1, the primary mediator of rbp⁺ function, is expressed in both thoracic epidermis and muscles of wild-type pupae. The rescuing transgenes are heat-shock-driven and, therefore, expressed in all tissues. In order to determine where rbp⁺ function is required for normal muscle attachment, mosaic animals whose thoracic muscles and epidermis are of different genotypes were examined. Developing MTJs of mutant and wild type were compared and subcellular defects were identified that resulted from reduced rbp⁺ function. Data from these two complementary approaches point to the dorsal EMA as the essential site of rbp⁺ action in controlling muscle attachment (Sandstrom, 1999).

It is proposed that transcription factor BRC-Z1, induced by 20E in the thoracic body wall, regulates a number of target genes whose products control specific features of tendon cell maturation. It is likely that intercellular signaling mechanisms are shared during embryonic and adult muscle attachment, just as neural cell fate determination is controlled similarly at both stages. In the embryo, muscle-independent expression of the transcription factor Stripe induces EMA specification and initital differentiation. During metamorphosis, Stripe is expressed in the EMAs of the IFM and TTM, initially in a muscle-independent manner. Viable stripe mutations disrupt IFM development. PS integrins, which play an essential role in muscle attachment by linking the extracellular matrix to the cytoskeleton, are expressed at developing EMAs in both embryos and pupae. Thus, studies of embryonic muscle attachment provide a useful framework for understanding mechanisms of BRC-Z1 action in the dorsal thoracic EMAs (Sandstrom, 1999 and references therein).

The muscle-independent phase of stripe expression, which in the embryo induces short stop (previously known as groovin or kakapo) and alien, is also rbp-independent in the pupal thorax. stripe expression in imaginal discs precedes the rise of BRC-Z1; expression in early pupal EMAs is normal in rbp mutants, consistent with normal tendon cell specification. It is therefore more likely that rbp⁺ function promotes a muscle-dependent phase of tendon cell differentiation. Embryonic muscle secretes the neuregulin-like molecule Vein, a signal received by the EGF receptor in the epidermis, resulting in expression of EMA-specific markers beta1 tubulin and the bHLH protein Delilah and further up-regulation of Stripe, Short stop and Alien. Hence, candidate target genes for BRC-Z1 in the pupal EMA include Egfr, delilah, beta1 tubulin, short stop, alien and stripe. The recent finding that Alien may be a co-repressor for the Ecdysone receptor (Dressel, 1999) suggests the additional possibility that BRC-Z1 overcomes Alien-mediated transcriptional repression, thereby unleashing the ecdysone cascade in pupal thoracic epidermis (Sandstrom, 1999 and references therein).

The mechanisms by which an organism becomes immune competent during its development are largely unknown. When infected by eggs of parasitic wasps, Drosophila larvae mount a complex cellular immune reaction in which specialized host blood cells, lamellocytes and crystal cells, are activated and recruited to build a capsule around the parasite egg to block its development. Parasitization by the wasp Leptopilina boulardi leads to a dramatic increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. Furthermore, a limited burst of mitosis follows shortly after infection, suggesting that both cell division and differentiation of lymph gland hemocytes are required for encapsulation. These changes, observed in the lymph glands of third-instar, but never of second-instar hosts, are almost always accompanied by dispersal of the anterior lobes themselves. To confirm a link between host development and immune competence, mutant hosts in which development is blocked during larval or late larval stages were infected. In genetic backgrounds where ecdysone levels are low (ecdysoneless) or ecdysone signaling is blocked (nonpupariating allele of the transcription factor broad), the encapsulation response is severely compromised. In the third-instar ecdysoneless hosts, postinfection mitotic amplification in the lymph glands is absent and there is a reduction in crystal cell maturation and postinfection circulating lamellocyte concentration. These results suggest that an ecdysone-activated pathway potentiates precursors of effector cell types to respond to parasitization by proliferation and differentiation. It is proposed that, by affecting a specific pool of hematopoietic precursors, this pathway thus confers immune capacity to third-instar larvae (Sorrentino, 2002).

Studies of Drosophila metamorphosis have been hampered by an inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, GFP was expressed in specific tissues of living prepupae and pupae and images of these animals were compiled into time-lapse movies. These studies reveal the dynamics and coordination of morphogenetic movements. Responses that have not been described previously include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp⁵ mutants of the Broad-Complex, and imaginal disc elongation and eversion is abolished in br⁵ mutants of the BR-C. Leg malformations associated with the crol³ mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals (Ward, 2003).

Broad complex, apoptosis and autophagic cell death

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms (Lee, 2001).

Morphological studies of developing vertebrate embryos have resulted in the definition of three types of physiological cell death. The first type, widely known as apoptosis, is found in isolated dying cells that exhibit condensation of the nucleus and cytoplasm, followed by fragmentation and phagocytosis by cells that degrade their contents. The second type, known as autophagy, is observed when groups of associated cells or entire tissues are destroyed. These dying cells contain autophagic vacuoles in the cytoplasm that function in the degeneration of cell components. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. The third type, known as non-lysosomal cell death, is least common, and is characterized by swelling of cavities with membrane borders followed by degeneration without lysosomal activity. While autophagy fulfills the definition of programmed cell death, occurs during development of diverse organisms, and has been implicated in tumorigenesis, little is known about the molecular genetic mechanisms underlying this type of programmed cell death. The morphological characteristics that distinguish apoptosis and autophagy suggest that these cell deaths are regulated by independent mechanisms. Comparison of biochemical changes during lymphocyte apoptosis and insect intersegmental muscle autophagy also indicate that these physiological cell deaths occur by distinct mechanisms. However, recent studies of steroid-triggered cell death of Drosophila larval salivary glands suggest that these cells utilize genes that are part of the conserved apoptosis pathway, even though these cells exhibit characteristics of autophagy. Specifically, the caspase Dronc and homolog of ced4/Apaf-1 Apaf-1-related-killer (Ark), two components of the core apoptotic machinery, increase in transcription immediately prior to salivary gland cell death. Thus, characterization of the mechanisms governing the regulation of autophagy will identify how these cell deaths differ from those that occur by apoptosis (Lee, 2001 and references therein).

Larval salivary glands of Drosophila undergo rapid programmed cell death in response to ecdysone. This cell destruction can be detected using markers that are typically associated with apoptosis including nuclear staining by Acridine Orange, TUNEL to detect DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane. The changes in vacuolar structure that immediately precede the synchronous destruction of larval salivary gland cells are clearly more similar to autophagy than heterophagy (apoptosis). Large vacuoles increase in number in prepupal salivary glands, and rearrangement of the cytoskeleton and an increase in acid phosphatase activity are associated with these structures. Dynamic changes in salivary gland structure may reflect important biochemical changes during programmed cell death. Large Eosin-positive vacuoles appear to fragment, a distinct class of Eosin-negative vacuoles are formed that are closely associated with the plasma membrane, and vacuoles containing organelles are observed in the cytoplasm immediately preceding destruction of salivary glands. An increase in transcription of the caspase Dronc occurs at this stage, and inhibition of caspase activity blocks DNA fragmentation and partially prevents changes in vacuoles and plasma membranes, suggesting that these morphological changes may be attributed in part to the activity of enzymes typically associated with apoptosis (Lee, 2001 and references therein).

While morphological analyses of apoptosis and autophagy suggest different mechanisms for these forms of cell death, some genes that function in apoptosis also function during autophagy. Steroid-regulated genes impact distinct cellular changes in dying cells. Ecdysone impacts on the transcription of the cell death genes rpr, hid and diap2. This regulation is mediated by the ecdysone receptor, and a group of ecdysone-activated factors that include the BR-C, E74 and E93 genes. The function of the steroid-regulated BR-C, E74 and E93 genes in salivary gland cell death has been examined. E93 mutant salivary glands exhibit persistence of large vacuoles and plasma membranes, while these structures are destroyed in BR-C and E74A mutants. Two possible explanations exist for the differences in BR-C, E74A and E93 mutant salivary gland cell morphology. E93 mutant salivary glands could be arrested at an earlier stage of cell destruction that is similar to that of 12-hour wild-type cells, while BR-C and E74A mutants are arrested at a stage that is similar to 14.5-hour salivary gland cells. This model is supported by previous studies indicating that E93 function is required for proper regulation of BR-C and E74A transcription. Alternatively, E93 could function to regulate autophagy that results in destruction of vacuoles and plasma membranes, while BR-C and E74A do not function in the regulation of these cellular changes even though these genes are required for salivary gland cell death. The latter interpretation is intriguing when one considers that expression of E93 is sufficient to induce characteristics of apoptosis, and can induce the removal of cells even in the absence of the rpr, hid and grim cell death genes and nuclear apoptotic changes (Lee, 2001).

Several factors indicate that salivary gland autophagy is regulated by genes that also function in apoptosis. (1) Caspases function in salivary gland cell death. Expression of the baculovirus inhibitor of caspases, p35, inhibits destruction of this tissue. Furthermore, p35 expression prevents DNA fragmentation and partially inhibits morphological changes in vacuoles that are associated with autophagy, indicating that caspases are utilized during autophagy. Transcription of the Apaf1 homolog ark and the caspase, dronc increases immediately preceding salivary gland cell death, and this transcription is blocked in E93 mutants, further supporting that caspases function in salivary gland autophagy. (2) Transcription of the proapoptotic genes, rpr and hid increases immediately prior to salivary gland autophagy, and the transcription of these genes is blocked by mutations in steroid-regulated genes that are involved in this process. Ectopic expression of E93, a critical determinant of salivary gland autophagy, is sufficient to induce cell death with numerous characteristics of apoptosis. In addition, the association of Croquemort (Crq) expression with E93-induced removal of apoptotic cells and autophagy of salivary glands provides yet another link between these morphologically distinct forms of programmed cell death. Combined, these factors indicate that autophagy and apoptosis utilize at least some similar mechanisms (Lee, 2001).

The location and type of cell appears to be an important determinant for the type of programmed cell death that occurs in the context of animal development. Autophagy occurs when groups of cells or entire tissues die, while apoptosis occurs in isolated dying cells. These studies are consistent with these criteria; salivary gland destruction occurs by autophagy and requires E93 function, while ectopic induction of cell death by expression of E93 during embryogenesis has the characteristics of apoptosis. It is hypothesized that this is due to similarities between autophagy and apoptosis. Alternatively, autophagy and apoptosis may be mechanistically distinct, and the ability to induce ectopic cell death by expression of E93 is simply due to activating a death program in different cell types. This explanation is supported by data demonstrating that p35 inhibits salivary gland cell death, but that p35 is not capable of inhibiting E93-induced cell death in embryos. However, several possibilities exist to explain the disparity of these data. (1) Ectopic expression of E93 during embryogenesis may lead to higher than normal levels of this protein. In side-by-side comparisons with the proapoptotic genes rpr and hid, expression of E93 results in greater cell death and lethality. Thus, the strong killing potential of E93 may be sufficient to overcome inhibition of cell death by p35. (2) Other cell death genes are not inhibited by expression of p35, including cell death that is induced by ectopic expression of the caspase Dronc. (3) Inhibition of vacuolar changes by expression of p35 during salivary gland cell death is incomplete, even though DNA fragmentation is inhibited in this tissue. Thus, caspases may play a role in salivary gland cell death, and both p35 experiments and the transcription of dronc during salivary gland autophagy support this conclusion. However, it is possible that other proteolytic mechanisms act in concert with caspases in the bulk degradation of salivary gland cells (Lee, 2001).

It is concluded that Autophagy and apoptosis are morphologically distinct, suggesting that the mechanisms underlying the regulation of these forms of programmed cell death are different. Nearly all of the large polytenized larval cells die during Drosophila metamorphosis. The synchrony and volume of these cell deaths suggests that engulfment of each dying cell may be limited by the number of available phagocytes. One obvious distinction between autophagy and apoptosis is the location of the lysosomal machinery that degrades the dying cell. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. This distinction may account for much of the differences in the morphological appearance of these two forms of dying cells, but does not exclude the possibility that a single autophagic cell utilizes the mechanisms that exist in distinct apoptotic and phagocytic cells. The specific expression of Crq during autophagy supports this possibility, but genetic studies of crq function are needed to test this hypothesis. Future studies of autophagy, and its relationship to apoptosis, will illustrate the similarities and differences between these forms of programmed cell death (Lee, 2001).

Apoptosis and autophagy are two forms of programmed cell death that play important roles in the removal of unneeded and abnormal cells during animal development. While these two forms of programmed cell death are morphologically distinct, recent studies indicate that apoptotic and autophagic cell death utilize some common regulatory mechanisms. To identify genes that are associated with apoptotic and autophagic cell death, changes in gene transcription were monitored by using microarrays representing nearly the entire Drosophila genome. Analyses of steroid-triggered autophagic cell death identified 932 gene transcripts that changed 5-fold or greater in RNA level. In contrast, radiation-activated apoptosis resulted in 34 gene transcripts that exhibited a similar magnitude of change. Analyses of these data enabled identification of genes that are common and unique to steroid- and radiation-induced cell death. Mutants that prevent autophagic cell death exhibit altered levels of gene transcription, including genes encoding caspases, non-caspase proteases, and proteins that are similar to yeast autophagy proteins. This study also identifies numerous novel genes as candidate cell death regulators and suggests new links between apoptosis and autophagic cell death (Lee, 2003).

The identification of genes that exhibit significant changes in RNA levels during steroid-triggered autophagic cell death and radiation-induced apoptosis prompted empirical analyses of transcription in mutants that block salivary gland cell death. Mutations in the ecdysone-regulated genes BR-C, E74A, and E93 prevent salivary gland programmed cell death and prevent proper transcription of the apoptosis genes rpr, W (hid), ark, Nc (dronc), and crq. The transcription of a subset of the newly identified genes was examined in BR-C, E74A, and E93 mutants by Northern blot hybridization because of their possible association with apoptosis and autophagy in dying salivary glands. Cohybridization of these Northern blots allows systematic investigation of how BR-C, E74A, and E93 might regulate transcription of genes that were identified with Genechips and provides a possible mechanism to explain steroid regulation of cell death (Lee, 2003).

The radiation-inducible genes CG10965, CG17323, CG7144, EG25E8.4, and CG5254 are induced in control dying salivary glands at head eversion, and this transcription is altered in mutants that prevent salivary gland cell death. CG10965 and CG17323 are not transcribed in salivary glands of BR-C mutants; they exhibit elevated levels of transcription in E74A mutants, and have reduced RNA levels in E93 mutants. CG7144 is transcribed at significantly reduced levels in BR-C mutants, is ectopically transcribed before the rise in ecdysone in salivary glands of E74A mutants, and may also be ectopically transcribed in E93 mutants. EG25E8.4 is not altered in BR-C and E74A mutants, but this RNA is significantly reduced in salivary glands of E93 mutants. CG5254 is not transcribed in BR-C mutants, had normal RNA levels in E74A mutants, and had reduced RNA levels in E93 mutants (Lee, 2003).

Several other categories of genes exhibit interesting patterns of regulation in BR-C, E74A, and E93 mutant salivary glands. The Bcl-2 family member buffy and the caspases Ice (drice) and dream (strica) are induced at head eversion in salivary glands of control animals, and they are altered to different extents in mutants. Similarly, the Drosophila genes that are most similar to the yeast autophagy genes apg2 (CG1241), apg4 (CG6194), apg5 (CG1643), apg7 (CG5489), and apg9 (CG3615) are induced just prior to cell death of wild-type salivary glands, and they are altered to varying extents in BR-C, E74A, and E93 mutants. It is particularly intriguing that E93 mutants have significantly decreased levels of CG6194, CG1643, and CG5489, since yeast with mutations in apg4, apg5, and apg7 are defective in autophagosome formation and size, and E93 mutants exhibit defects in vacuolar changes in dying salivary gland and midgut cells. In addition, the cysteine protease (CG5505), serine protease (CG3650), and metalloprotease (mmp1) all exhibit increases in RNA level immediately following the rise in ecdysone in dying wild-type salivary glands, and this change is accompanied by a decrease in the inhibitor of metalloproteases, timp. It is interesting that BR-C, E74A, and E93 mutations affect transcription of the non-caspase protease genes CG5505, CG3650, and mmp1, since caspase inhibitors do not completely block changes in dying salivary glands, and mutations in these ecdysone-regulated genes prevent degradation of salivary gland cells (Lee, 2003).

Drosophila salivary gland chromosomes were used to predict the first steroid-triggered transcription hierarchy based on chromosome puffing (chromatin decondensation). This study has identified several candidate genes in this signaling pathway based on correlative increases in transcription that are associated with chromosome puffs and with the proximity of binding sites of transcription factors in this pathway. Two putative puff genes, CG17309 (86E puff) and CG3132 (87A puff), increase following the rise in ecdysone titer and match the puffing patterns of these chromosome loci. CG17309 RNA is present before the rise in ecdysone in BR-C mutants, while it is reduced in salivary glands of E74A and E93 mutants. CG3132 appears to encode two transcription units that were either not detected or decreased in salivary glands of BR-C, E74A, and E93 mutants. The Smad anchor for receptor activation sara and the transcription regulator bun have increased RNA levels in dying salivary glands and have BR-C Z1 and E74A binding sites in the same region of the genome. sara is not induced in BR-C, E74A, and E93 mutant salivary glands. bun RNA was also not detected in BR-C and E93 mutant salivary glands, but it is expressed normally in E74A mutant salivary glands. These data provide a direct link between the ecdysone-regulated early genes and target genes (Lee, 2003).

It is concluded that developmental cues and genotoxic stress can both trigger programmed cell death. During steroid-triggered autophagic cell death in developing salivary glands, 932 gene transcripts were identified that either decreased or increased 5-fold or greater in RNA level. In contrast, radiation-activated apoptosis in embryos only identified 34 gene transcripts that exhibited a similar magnitude of change. The difference in the number of genes that were induced by these stimuli most likely reflects the presence of maternal RNAs for cell death genes that are deposited in embryos. Alternatively, the apoptotic machinery may exist in cells as proteins waiting to be posttranslationally activated following a death-inducing stimulus. Radiation-induced apoptosis in Drosophila embryos can be suppressed by treatment with cyclohexamide, suggesting that protein synthesis is necessary for activation of this cell death. In addition, studies of radiation-induced apoptosis have implicated p53, which is known to function as a regulator of transcription in this process. It is also possible that radiation-induced apoptosis is sufficiently asynchronous that it is difficult to detect changes in RNA levels in a very complex cell population. Comparative analyses of cell death microarray data has enabled the identification of a small group of genes that are induced by both ecdysone and radiation. While salivary gland autophagic cell death and radiation-induced apoptosis appear to be quite different, transcription of the common genes rpr, CG10965, CG17323, CG7144, EG25E8.4, and CG5254 is altered in mutants that prevent salivary gland cell death, further suggesting that these genes are important for this cell death. In addition, BR-C, E74A, and E93 mutants also impact transcription of numerous genes in salivary glands, including apoptosis regulators, non-caspase proteases and protease inhibitors, cell remodeling factors, and the genes that are similar to the yeast genes that function in protein degradation by autophagy. This study has identified numerous genes that exhibit interesting patterns of transcription during steroid- and radiation-induced programmed cell death, and future genetic studies will determine the importance of these genes in autophagy and apoptosis (Lee, 2003).

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes ßFTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death (Martin, 2004).

Studies of salivary glands indicate that caspases play an important role in their autophagic cell death. The caspase-encoding genes dronc and drice show an increase in their transcription following the rise in steroid that triggers salivary gland autophagic cell death. This increase in caspase transcription corresponds to the increase in active caspase protein levels and in the cleavage of substrates such as nuclear Lamins in dying salivary glands. Mutations in the steroid-regulated ßFTZ-F1, E93 and BR-C genes, which prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice expression, and have altered alpha-Tubulin, alpha-Spectrin and nuclear Lamin expression in salivary glands. Although E74A mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice levels and degraded nuclear Lamins. Although these data are consistent with the partially degraded morphology of E74A mutant salivary glands, it remains unclear what factor(s) E74A may regulate that are required for normal cell death. However, the data indicate that ßFTZ-F1, E93 and BR-C play a crucial role in determining caspase levels in dying salivary gland cells, and this is supported by the impact of these genes on the transcription of dronc. Significantly, inhibition of caspases by expression of either p35 or dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in nuclear Lamins and alpha-Tubulin, and death of salivary glands (Martin, 2004).

Broad regulates the autophagic death of midgut cells during development

Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Steroid regulation of changes have been characterized in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca, and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development (Lee, 2002).

The morphology of midguts was examined at the onset of metamorphosis to provide a framework for studies of genetic regulation of larval midgut cell death. Wild-type Canton S were staged in hours following puparium formation, fixed, embedded in paraffin, sectioned, and stained. New prepupae possess a larval esophagus, proventriculus, gastric caeca, and midgut structures, and exhibit no signs of larval cell death or adult midgut formation at this resolution. Two hours after puparium formation, the proventriculus, gastric caeca, and larval midgut are surrounded by an adult epithelium. In 4-h prepupae, the proventriculus and gastric caeca appear to compress toward the larval midgut, and the larval epithelium becomes convoluted, causing a large space in the larval lumen. Six hours after puparium formation, the proventriculus and gastric caeca can no longer be distinguished, and the larval midgut becomes further condensed. The larval midgut is extremely condensed 12 h after puparium formation, and the adult and larval epithelia have separated such that a defined adult lumen exists (Lee, 2002).

The death of larval midgut cells coincides with the increase in ecdysone that triggers puparium formation, and premature elevation of the ecdysone titer in third instar larvae is sufficient to ectopically induce cell death in larval midguts. In addition, mutations in the Ecdysone receptor and the ecdysone-regulated primary response gene BR-C prevent proper destruction of larval midguts. The role of the ecdysone-regulated primary response genes BR-C, E93, and E74 in larval midgut destruction was examined, since these genes regulate steroid-activated destruction of larval salivary glands (Lee, 2002).

To analyze the destruction of mutant larval midguts, animals were staged at pupal head eversion, fixed, embedded in paraffin, sectioned, and analyzed by light microscopy for defects in midgut structure. Head eversion was selected as the stage for analyses since this is 12 h after midgut destruction is initiated and the larval midguts of control animals are fully compressed at this time. BR-C (2Bc²) mutants have the strongest phenotype and always possess some remnants of the larval proventriculus and gastric caeca. While larval midgut destruction does not occur properly in BR-C mutants, the adult epithelium is formed and the midgut appears to be arrested at a stage that is similar to the midgut of wild-type animals 2-4 h following puparium formation (Lee, 2002).

The larval midgut shrinks dramatically during the first 6 h of pupariation when these cells are dying, suggesting that midgut shortening may be related to larval cell death. In order to quantify the relationship between the change in midgut length, cell death, and mutants that impact larval midgut destruction, the length of midguts was measured at puparium formation and head eversion. Wild-type Canton S midguts decrease from 7.80 to 1.60 mm in length, or 85%, during this interval. While BR-C, E93, E74A, and E74B mutant larval midguts all decrease in size between puparium formation and head eversion, this shrinking varies. BR-C mutant midguts decrease from 7.57 to 2.50 mm (65%) in length. E93 midguts decrease from 6.38 to 1.64 mm (74%) between puparium formation and head eversion. Midguts of E74A mutants decrease from 6.55 to 1.21 mm (81%), while E74B mutants change from 5.97 to 1.62 mm (73%) in length. Therefore, midgut shortening likely relies on the newly formed adult midgut epithelium, since midguts shorten in BR-C and E93 mutants that prevent proper destruction of larval cells (Lee, 2002).

DNA fragmentation accompanies the destruction of larval midguts. The TUNEL procedure was used to determine whether mutations in ecdysone-regulated genes prevent DNA fragmentation in larval midgut cells. Wild-type Canton S and mutant animals were staged at head eversion, fixed, embedded in paraffin, sectioned, and analyzed for the presence or absence of DNA fragmentation. Canton S possess compacted midguts and fragmented DNA at head eversion. While BR-C mutants have persistent larval structures, including gastric caeca, every larval midgut cell nucleus of these mutants appeared to be labeled, indicating that they possess fragmented DNA (Lee, 2002).

Similarly, the nuclei of E93, E74A, and E74B mutant midguts were all labeled following the TUNEL procedure. These data suggest that larval midgut cells do not die by apoptosis, since mutations in the BR-C and E93 genes prevent destruction of midgut cells, and the midgut cells of these mutants possess fragmented DNA (Lee, 2002).

Drosophila larval midguts exhibit markers of apoptosis immediately prior to destruction, including DNA fragmentation and nuclear staining by acridine orange, as well as increased transcription of the proapoptotic genes rpr and hid. While mutations in the BR-C and E93 genes prevent destruction of midgut cells, the midgut cells of these mutants possess fragmented DNA, suggesting that they do not die by apoptosis. Thus, transmission electron microscopy was used to analyze cell structure during cell death of midguts. Late third instar larval midguts possess microvilli facing the lumen, large nuclei with banded polytene chromosomes, and intact mitochondria in the cytoplasm. At this stage, very few indications of cell death exist, although small numbers of early stage autophagic vacuoles and swirls of rough endoplasmic reticulum are observed; this is one of the mechanisms by which autophagic vacuoles are formed. Larval midguts of new prepupae have microvilli facing the lumen, intact nuclei, and the cytoplasm has increased numbers of autophagic vacuoles and appears to possess more spaces than in late third instar larvae. Vacuoles that contain structures, including organelles such as mitochondria and crystalline inclusions, are abundant in the larval midguts of new prepupae and indicate that these cells die by autophagy. Two hours following puparium formation, the forming adult midgut is apparent, and the larval midgut cytoplasm possesses an increased number of vacuoles containing organelles, indicating that autophagy has progressed. Larval midguts of 4-h prepupae appear to exhibit an increase in the number of nuclei per area examined, which is likely due to the compression of this structure. Large numbers of crystalline inclusions were observed in the cytoplasm of larval midguts in 4-h prepupae. The proximity of nuclei increases and autophagic structures are abundant in larval midguts 6 h after puparium formation. Twelve hours after puparium formation, the cytoplasm of larval midguts appears more condensed since fewer spaces are observed, and numerous autophagic structures, including myelin-like membrane swirls, are detected. These data indicate that larval midguts die by autophagy and do not exhibit morphological characteristics of apoptosis (Lee, 2002).

Larval midgut cells possess vacuoles that contain cytosolic structures, such as mitochondria, indicating that these cells die by autophagy. Thus, whether mutations in the BR-C, E93, E74A, and E74B genes prevent the destruction of the cytoplasm was tested. The midgut cells of BR-C mutants exhibit variable cytoplasmic staining: some cells are extremely osmophylic, while others are not stained as dark. BR-C mutant midgut cells contain intact mitochondria and do not exhibit obvious alterations in cytosolic structures from midguts of third instar larvae other than containing large spaces. In contrast, E93 mutant midguts possess numerous cells that contain swollen mitochondria, and many of these organelles rupture. Not all E93 mutant midgut cells completely lack autophagic structures, however, since some mitochondria are enclosed by membranes. The midguts of E74A and E74B mutants contain intact mitochondria that are observed in autophagic vacuoles. While BR-C mutants exhibit defects in the destruction of gross larval structures and E93 mutants exhibit defects in the destruction of cytosolic midgut structures (such as mitochondria), no similar defects were observed in either E74A or E74B mutant midguts, which possess numerous normal autophagic structures (Lee, 2002).

Expression of the caspase inhibitor p35 prevents midgut cell death). Since caspases are generally considered proteases that regulate apoptosis, it was necessary to determine whether caspases and other cell death regulators are transcribed in midguts that die by autophagy. While it is known that rpr and hid are induced in dying midguts, it is not known whether other candidate cell death regulators are induced in these cells. Therefore, developmental Northern blots were prepared from wildtype midguts at stages preceding and during cell death (Lee, 2002).

Transcription of rpr, hid, ark, dronc, and crq increases in wild-type animals following the late larval pulse of ecdysone that triggers larval midgut cell death. Since mutations in the BR-C and E93 genes prevent proper destruction of larval midguts, Northern blots were prepared from midguts of these mutants at stages preceding and during cell death. BR-C 2Bc² mutants have altered transcription of rpr, hid, and crq, but do not impact the transcription of ark and dronc. In contrast, E93 mutants possess altered transcription of dronc, but do not change the transcript levels of the other cell death genes known to be expressed in dying midguts. Although midguts die by autophagy, they transcribe core apoptosis regulators during this cell death, and mutants that prevent autophagy alter transcription of apoptosis genes (Lee, 2002).

Studies of ecdysone-triggered destruction of Drosophila larval midguts and salivary glands illustrate many similarities in these dying cells. However, several important differences exist between ecdysone-regulated midgut and salivary gland programmed cell death. Consider that these two tissues are triggered to die by independent pulses of ecdysone. While the nuclear receptor ßFTZ-F1 is responsible for specifying ecdysone induction of BR-C, E74A, and E93 immediately prior to larval salivary gland programmed cell death, the factor(s) that specify the timing of the cell death response in larval midguts 12 h earlier remain unclear. BR-C and E93 appear to be critical regulators of midgut cell death, but it is unclear how the ecdysone receptor complex activates these genes in midguts. ßFTZ-F1 is not expressed in midguts prior to ecdysone-induced cell death of this tissue, so other factors must be responsible for induction of BR-C and E93 in midguts. One possibility is that the hormone receptor complex activates BR-C and E93 independently of a factor such as ßFTZ-F1. Alternatively, another nuclear receptor, or possibly an unrelated transcription regulator, may regulate BR-C and E93. Future genetic studies and analyses of the BR-C and E93 promoters will define the mechanism for the stage-specific induction of cell death by ecdysone in larval midguts (Lee, 2002).

The distributed association of future adult cells within the epithelium of larval midguts is another important difference between ecdysone-regulated midgut and salivary gland programmed cell death. The close association of larval and adult midgut cells may be one of the reasons why larval midgut exhibits a less synchronized cell death than salivary glands. Both salivary glands and midguts require the function of the E93 and BR-C genes. However, mutations in these genes appear to result in different effects in salivary glands and midguts; BR-C appears to play a more important role in midguts. While both salivary glands and midguts express the cell death genes rpr, hid, ark, dronc, and crq, the impact of mutations in BR-C and E93 are very different in the midgut than in salivary glands. BR-C affects transcription of rpr, hid, and crq, but E93 mutants only affect dronc transcription in midguts. In contrast, mutations in E93 prevent proper transcription of all of these cell death genes in dying salivary glands. Clearly, many more genes may be involved in the complicated autophagic cell death of midguts. While several similarities and differences have been identified between salivary gland and midgut death, future analyses are needed to clarify the mechanism by which the steroid ecdysone triggers midgut programmed cell death (Lee, 2002).

Broad complex and eye development

The progression of the morphogenetic furrow in the developing Drosophila eye is an early metamorphic, ecdysteroid-dependent event. Although Ecdysone receptor-encoded nuclear receptor isoforms are the only known ecdysteroid receptors, it has been shown that the Ecdysone receptor gene is not required for furrow function. DHR78, which encodes another candidate ecdysteroid receptor, is also not required. In contrast, zinc finger-containing isoforms encoded by the early ecdysone response gene Broad-complex regulate furrow progression and photoreceptor specification. br-encoded Broad-complex subfunctions are required for furrow progression and proper R8 specification, and are antagonized by other subfunctions of Broad-complex. There is a switch from Broad complex Z2 to Z1 zinc-finger isoform expression at the furrow that requires Z2 expression and responds to Hedgehog signals. These results suggest that a novel hormone transduction hierarchy involving an uncharacterized receptor operates in the eye disc (Brennan, 2001).

Despite the demonstrated lack of requirement for known or candidate ecdysteroid receptors in transducing the hormone requirement for furrow function, previous evidence suggested that the early ecdysone response gene Broad-complex plays a role in this process. Broad-complex proteins are expressed at the furrow in an ecdysoneless-dependent manner, and discs null for all BR-C function display defects in furrow progression and ommatidial organization. The expression pattern in the eye disc of the different zinc finger-containing BR-C isoforms was characterized using antibody and mRNA probes. An antibody that recognizes all isoforms ('core antibody') stains cells both in and flanking the furrow, with a peak of expression just posterior to the furrow. BR-C is highly expressed in the nuclei of cells in the peripodial membrane. BR-C proteins appear to be expressed in cells until they differentiate; immediately posterior to the furrow, Elav-expressing differentiating photoreceptors express BR-C, whereas more posteriorly, expression is stronger in cone cells. Staining with an antibody that detects only Z1-containing isoforms of BR-C is restricted to posterior to the furrow, and a Z3-specific antibody stains the entire disc at very low levels, with slight upregulation in photoreceptors posterior to the furrow. Although no Z2-specific antibody is available, it was reasoned that the staining anterior to the furrow seen with the core antibody might be represented by Z2-containing isoforms. This was confirmed by in situ hybridization using probes specific for the Z1 and Z2 zinc fingers. Z2 mRNA is detected anterior to the furrow, followed by Z1 posterior to the furrow. A switch in BR-C isoforms expression from Z2 forms to Z1 forms around the time of pupariation in imaginal discs has been described. These results show that this switch from larval to prepupal forms is precocious and asynchronous in the eye disc, and is associated with the morphogenetic furrow as it traverses the disc. Although levels of BR-C expression increase dramatically during the last few hours of the third instar, corresponding to the late larval ecdysone pulse, BR-C expression still remains highest near the furrow (Brennan, 2001).

Bar is a dominant mutation causing premature arrest of the furrow, which results in the deep anterior nick in the adult eye. Since Bar has the dominant effect of stopping the furrow early, one might expect loss-of-function mutations at other loci that normally act to promote furrow progression to be genetic enhancers of Bar and loss-of-function mutations in genes that normally antagonize the furrow to act as genetic suppressors of Bar. Thus, genetic interactions between BR-C sub loci and Bar were examined. Mutants defective for different BR-C subfunctions display unexpected heterogeneity in their genetic interactions with Bar, suggesting that the role of the BR-C in the regulation of furrow function might be complex. BR-C has several recessive lethal complementation groups that correspond to mutations that remove the function of all or individual zinc finger-containing isoforms subgroups. npr1 mutations lack all function, whereas rbp, br and 2Bc mutant groups correspond to the loss of Z1-, Z2-, and Z3-containing isoforms, respectively. Both npr1/Bar and br/Bar eyes are significantly smaller than +/Bar, indicating a dominant enhancement of the Bar furrow-stop phenotype, consistent with the earlier reports that the BR-C is required for furrow progression. However, br/Bar eyes are smaller than npr1/Bar, suggesting that the BR-C might encode isoforms that act antagonistically during furrow progression, so that the effect of losing isoforms that positively regulate furrow progression is more severe than losing all isoforms. This idea is supported by the observation that rbp/Bar and 2Bc/Bar eyes are larger than +/Bar, suppressing the phenotype, and possibly representing furrow-antagonistic functions of rbp- or br-encoded BR-C isoforms (Brennan, 2001).

Hemizygous males of all BR-C mutant groups survived through the third instar: the eye discs of these males displayed defects consistent with the genetic interactions with Bar. npr1/Y discs show ommatidial disorganization, and signs of furrow failure, including mature ommatidial clusters at the furrow. br/Y discs show a much more dramatic failure of furrow progression, as well as ommatidial disorganization. rbp/Y and 2Bc/Y discs do not show any defects. While rbp does interact strongly with Bar it shows no eye disc defect alone -- it may be that rbp is a redundant function (Brennan, 2001).

Disc clones lacking individual BR-C subfunctions were generated to further characterize the mutant phenotypes. Both npr1 and br clones displayed defects in ommatidial organization, including the wrong number of photoreceptors in clusters. rbp and 2Bc clones appeared normal. To investigate the stages at which BR-C function is required for normal third instar eye development, mosaic clones spanning the morphogenetic furrow were examined. In contrast to br clones, which are frequently associated with some slowing of furrow progression, none of over fifteen npr1 furrow-spanning clones was associated with any retardation of the furrow. This is consistent with the hemiygous disc phenotypes that show a more severe furrow effect in br than npr1 mutants. Neither npr1 nor br clones at the posterior margin of the disc show any visible defects in furrow initiation; this is consistent with the lack of requirement for ecdysoneless function for furrow initiation. Both npr1 and br clones are associated with ommatidial disarray, and are indistinguishable in this regard. Consistent with this, both npr1 and br clones are defective in the specification of the founding R8 photoreceptors, as shown by Atonal expression. atonal is the proneural gene for photoreceptor cells, and is first expressed in a broad equivalence group of cells anterior to the furrow, and subsequently restricted to expression in the R8 founder photoreceptors. A complex network of inductive and inhibitory signals mainly involving the Notch pathway specifies the R8 cells and ensures their correct spacing. In both npr1 and br clones, clusters of three Atonal-positive R8 cells are seen. This suggests a failure in the lateral inhibition mechanisms that pattern these founder cells. The cell cycle is influenced by steroid hormones, including the insect ecdysteroids. Cell cycle control in the Drosophila eye disc is essential for the orderly specification of the retinal array; the synchronous cell cycle arrest in G1 in the furrow is necessary for proper cell-cell communication (Brennan, 2001).

Dpp, produced from cells in the furrow, is thought to regulate cell cycle synchronization in cells anterior to the furrow. Cyclin B is expressed in cells at the G2/M transition. Normally, Cyclin B is expressed in unsynchronized cells far anterior to the furrow, turned off just anterior to (as well as within) the furrow and reactivated in a tight band of cells posterior to the furrow. Cyclin B expression was assessed in br clones. In a long narrow br clone that extends across the furrow, there is a delay in cessation of Cyclin B expression anterior to the furrow. However, close examination reveals that this is a non-autonomous defect; the greatest delay occurs just adjacent to the clone, in a region that is directly anterior to the section of the mutant clone that crosses the furrow. It is suggested that the delay in cell cycle synchronization anterior to the furrow is a secondary defect resulting from delays in events at the furrow, possibly including delayed production of Dpp (Brennan, 2001).

br mutations lack the function of Z2 isoforms of the Broad-complex, which are expressed both in and anterior to the furrow in the eye disc. This corresponds to the zone in which the earliest defects in clones are noted: defects in R8 photoreceptor patterning. To confirm the hypothesis that lack of Z2 isoform expression anterior to the furrow is the cause of the defects in br and npr1 clones, expression of various BR-C isoforms was assessed in clones. As expected, in npr1 clones, expression of all BR-C proteins is eliminated. More surprising was the finding that br clones are unreactive to an antibody that recognizes all BR-C proteins. This suggests that the expression of Z2 isoforms anterior to the furrow is required for the subsequent expression of Z1 isoforms posterior to the furrow. Evidence of such positive autoregulation between Z2 and Z1 expression has been described previously. rbp clones also lack expression of Z1 isoforms, yet have no defects in retinal specification, suggesting that the defects in br clones are due to the lack of Z2 expression, and not to the lack of Z1. Although 2Bc clones do show Z3 protein, this protein is presumably nonfunctional, because a 2Bc¹ allele has been shown to be a genetic null (Brennan, 2001).

BR-C expression in the eye disc is dependent on ecdysoneless function, suggesting that it is regulated by ecdysteroid titer. While BR-C expression in the eye disc is unlikely to be regulated by EcR, Usp may play a role. BR-C and usp disc clone phenotypes are opposite in that the former shows some furrow retardation while the latter shows a slight acceleration. Possible Usp repression of BR-C, or Usp regulation of BR-C isoform ratio could explain these differences. BR-C and usp eye clones also resemble each other in that both show ommatidial disarray. This similarity underscores the importance of orderly progression of the furrow for subsequent events, and may reflect an essential hormonal input that regulates timing of furrow progression. Local events appear to regulate BR-C expression at a number of levels. Two regulatory inputs into the switch from Z2 to Z1 expression at the furrow have been suggested: (1) the lack of Z1 expression in br clones suggests that prior expression of Z2 isoforms anterior to the furrow is required for subsequent expression of Z1 isoforms; (2) the ectopic Z1 expression seen surrounding a PKA clone suggests that Hh-dependent signaling at the furrow also promotes the switch to Z1 isoforms. Cessation of BR-C expression in eye disc cells correlates with cell-fate determination; photoreceptors diminish expression levels earlier than cone cells, and failure to differentiate in smo;Mad cells is associated with prolonged expression of BR-C proteins. BR-C proteins are thought to confer competence to respond to ecdysteroid signals; in the asynchronously developing eye disc, it may be essential to spatially restrict such competence (Brennan, 2001).

How might BR-C proteins exert their effects on eye development? Although ecdysteroids regulate ommatidial patterning at many stages of eye development in Manduca, evidence suggests that the critical stage at which early eye development in Drosophila is regulated by Broad-complex is just anterior to and in the furrow. This corresponds to the site of Z2 expression and the zone at which the earliest defects are detected, in R8 specification. No transcriptional targets of BR-C have been identified in the eye; the aberrant specification of R8 founders suggests that targets may include elements of the Notch signaling pathway. BR-C regulation of such targets is likely to be complex, with different isoforms acting antagonistically (Brennan, 2001).

Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH-PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often fail when MET is absent. Patterns of BRC protein accumulation during metamorphosis reveals essentially no difference between wild-type and Met-null individuals. JH agonist treatment does not block accumulation of BRC proteins. It is proposed that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC (Wilson, 2005).

Met is essential for the manifestation of the toxic and morphogenetic effects of JH/JHA in Drosophila (Wilson, 1986; Riddiford, 1991; Wilson, 1996; Restifo, 1998). Met mutants are resistant to these effects of methoprene (Wilson, 1986). MET can bind JH III with specificity and nanomolar affinity (Shemshedini, 1990; Miura, 2005), suggesting that it is a component of a JH receptor. Met encodes a bHLH-PAS transcriptional regulator family member (Ashok, 1998) and MET can activate a reporter gene in transfected Drosophila S-2 cells (Miura, 2005; Wilson, 2005 and references therein).

Evidence was found for interaction between Met and BR-C as reflected by synergistically reduced viability and oogenesis seen in double mutants. Consistent results were seen with different combinations of Met and br or Met and rbp alleles, indicating that the interactions are not allele-specific in either direction. Met interacts with both the weak viable alleles br¹ and rpb² as well as the severe alleles br⁵ and rbp¹ during pupal development. Each of the weak alleles possesses sufficiently functional gene product to permit completion of pupal development; but this amount is insufficient when MET is absent or defective. The more severe rbp¹ homogygotes are pupal-lethal, but only at late metamorphosis, in the pharate adult stage. Lethality was shifted in rbp¹ Met²⁷ pupae to prepupal/early pupal development, suggesting that MET absence causes the rbp¹ product to be inadequate during these earlier stages in pupal development. Homozygotes of br⁵ and 2Bc die in the early and late prepupal stage, respectively, and the double mutants with Met²⁷ show a similar phenotype, demonstrating that the interaction cannot shift lethality to an earlier stage, late third-instar larvae. These observations are consistent with the interaction between BR-C and Met beginning in prepupal or early pupal development. While the Met-BR-C interaction is interpreted as enhancing the lethality of br and rbp mutations, it is also possible that Met becomes an essential gene when BR-C function is reduced, or that the interaction is mutual, such that both mutations become more severe in phenotype when they are present together (Wilson, 2005).

Genetic interaction became strikingly evident when complementation failures between mutant alleles from different BR-C complementation groups occurred in the presence of Met²⁷. Without MET, developing animals may be less able to make use of the partial functional redundancy among BRC isoforms. The interaction between mutant alleles of BR-C and Met is also evident in the adult stage when oogenesis is examined. Both the rate of oviposition and the paucity of vitellogenic oocytes in ovaries of br¹ Met²⁷ and rpb² Met²⁷ females reflects almost complete failure of oogenesis, with only a few eggs oviposited during the lifetime of the female (Wilson, 2005).

Previous studies have also detected BR-C interaction with other genes. Double mutants ofBR-C with another primary response gene, E74, show interaction for some but not all of the phenotypic characters. In addition to interactions among transcription regulators of the ecdysone cascade, br alleles interact with genes involved in imaginal disc morphogenesis, including those encoding an atypical serine protease, Stubble-stubboid, non-muscle myosin II heavy chain (Zipper), the Drosophila serum response factor transcription factor (Blistered), the small GTPase Rho1, cytoplasmic tropomyosin and 22 others. Although BR-C expression and function overlap the JH/JHA-sensitive period, methoprene treatment does not block BRC expression in either wild-type or Met null mutants. Furthermore, the methoprene phenocopy, which excludes complementation group-specific defects (e.g., larval salivary gland persistence, which is rbp-restricted), is not consistent with methoprene simply reducing BRC expression. It is proposed that JH application results in abnormal function of BRC proteins, thus phenocopying certain characteristics common to all BR-C mutants. Therefore, the link between BR-C mutant phenotypes and JH-induced defects could be abnormal regulation of target genes, resulting in the phenotypic characteristics observed. Several possibilities have been suggested to explain methoprene pathology and BR-C phenocopy, including BRC interaction with an unidentified partner, perhaps MET (Restifo 1998). It is believed that the Met-BR-C genetic interaction can be explained best by this hypothesized protein-protein interaction between MET and BRC to regulate one or more target genes. Supporting this hypothesis are the following findings: first, both proteins are located in the nucleus, so there is no compartmental barrier to interaction. Second, both proteins appear to be transcription factors: BRC isoforms bind specific DNA sequences and regulate transcription. BR-C mutants have misexpressed secondary-response and other target genes. MET is a member of the bHLH-PAS family of transcription factors (Ashok, 1998) and was recently shown to act as one (Miura, 2005). Third, both are found at common times during development, such as prepupae and during vitellogenic oocyte development. Finally, PAS domains in bHLH-PAS proteins are thought to promote protein-protein interaction, either with other PAS proteins or as coactivators with nuclear receptor proteins, and the BTB/POZ domain of BRC has been implicated in proteinprotein interaction (Wilson, 2005 and reference therein).

In Met²⁷ mutants, BRC protein accumulation profiles are normal. Since metamorphosis is not derailed in Met²⁷ pupae, then BRC⁺ function in these pupae does not seem to be adversely affected. The fly may be protected from absence of MET by functional redundancy (Wilson, 1998). A candidate for the redundant substitute is the PAS gene germ cell expressed (gce), a gene with high (~70% amino acid identity) homology to Met that could substitute for MET to rescue larval and/or pupal development. However, this substitute does not appear to be satisfactory if BR-C is mutant. When a gce mutant becomes available, its phenotype could help evaluate this hypothesis (Wilson, 2005).

How does the application of exogenous JH act to phenocopy BR-C? It is clear that the action of these compounds occurs through MET, probably acting as a JH receptor component. JH is present during larval development when it presumably acts to prevent premature metamorphosis resulting from each wave of 20E secretion that triggers a molt. This fail-safe mechanism may occur by JH binding by and conformational change of MET, resulting in regulation of genes necessary for molting or perhaps simply blocking expression of metamorphic genes. Studies with Drosophila S-2 cells have implicated the transcription factor E75A in promoting JH regulation of larval development. At metamorphosis, when little or no JH is present, BR-C is expressed, and it is proposed that BRC dimerizes with the non-liganded MET protein to regulate a different set of target genes, promoting the initiation of metamorphosis. If exogenous JH is present during this time, it binds to MET and results in a more larval conformation, resulting in inappropriate binding to BRC and leading to a change in target gene expression patterns consequently seen as defects characteristic of BR-C mutants (Wilson, 2005).

Other work has implicated BR-C in the action of the JH agonist pyriproxyfen during metamorphic disruption. Application of this compound to white prepupae results in re-expression of BRC-Z1 in the abdomen during late pupal development, which in turn causes abnormal development of abdominal epidermis, including bristle disturbances. Those findings differ from those with methoprene in two significant ways. First, a lethal dose of methoprene causes a mild enhancement and prolongation of BRC protein accumulation in young pupae, but no re-expression at later times. Second, the modest effect of methoprene on BRC protein profiles cannot mediate the developmental effects of this JHA because the same mild persistence of BRC is seen in Met²⁷ mutants, that are protected against methoprene-induced defects. It is not clear what underlies the difference in response of BR-C to methoprene and pyriproxyfen. It is noted that pyriproxyfen is a more powerful JH agonist than methoprene (Riddiford, 1991), but qualitative differences in the actions of the two compounds may exist as well (Wilson, 2005).

In summary, the results provide genetic evidence that supports other studies implicating BR-C as a focal point for interaction of JH and 20E signaling pathways, and they suggest that BRC and MET interact to regulate expression of one or more effector genes involved in metamorphic development (Wilson, 2005).

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.