Mechanistic relationships between Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A signaling

Fragile X syndrome is caused by loss of the FMRP translational regulator. A current hypothesis proposes that FMRP functions downstream of mGluR signaling to regulate synaptic connections. Using the Drosophila disease model, relationships between dFMRP and the sole Drosophila mGluR (DmGluRA) were tested by assaying protein expression, behavior and neuron structure in brain and NMJ; in single mutants, double mutants and with an mGluR antagonist. At the protein level, dFMRP is upregulated in dmGluRA mutants, and DmGluRA is upregulated in dfmr1 mutants, demonstrating mutual negative feedback. Null dmGluRA mutants display defects in coordinated movement behavior, which are rescued by removing dFMRP expression. Null dfmr1 mutants display increased NMJ presynaptic structural complexity and elevated presynaptic vesicle pools, which are rescued by blocking mGluR signaling. Null dfmr1 brain neurons similarly display increased presynaptic architectural complexity, which is rescued by blocking mGluR signaling. These data show that DmGluRA and dFMRP convergently regulate presynaptic properties (Pan, 2008).

Protein Interactions

Chickadee/profilin interacts with Cappuccino in a two-hybrid screen for proteins that bind to Cappuccino. This, together with the similarity of mutant phenotypes, suggests that profilin and Cappuccino may interact during development (Manseau, 1996).



Chickadee is present throughout development, with a strong surge in expression in 12- to 24-hour embryos and a second less dramatic increase during pupal stages. Adult extracts show the lowest expression levels (Verheyen, 1994).

Drosophila fragile X mental retardation protein targets chickadee in the developmental regulation of activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, the Drosophila FraX model was used to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development (Tessier, 2008).

In the absence of dFMRP, elevated levels of total RNA/protein are evident during a restricted period of late pupal brain development, with the protein increase persisting into an early-use refinement period. These increases are transient and disappear in the mature brain thereby defining a limited developmental window of dFMRP function. The increase in protein is predicted as FMRP/dFMRP negatively regulates translation. The elevated RNA is more surprising. dFMRP/FMRP can both negatively and positively regulate mRNA stability, and, therefore, dFMRP may have a developmentally-restricted role primarily as a negative regulator of mRNA stability. Alternatively, the RNA increase may be caused by elevated transcription, via an uncharacterized direct or indirect transcriptional inhibition function of dFMRP. Because the increase in total protein/RNA is not biased towards selected dFMRP targets, these results suggest globally upregulated transcription/translation in the dfmr1 mutant brain during a restricted window of late maturation and early-use refinement (Tessier, 2008).

During brain development, dfmr1 mRNA and dFMRP protein levels tightly correlate with the above changes, but, surprisingly, dfmr1 mRNA levels inversely correlate with dFMRP protein levels in the mature brain. By 4 days after eclosion (AE), dfmr1 mRNA levels rise to levels nearly as high as those present during development, but dFMRP protein is maintained at a basal level in the mature brain. This change strongly suggests a distinct switch in dFMRP regulation, in which transcription and translation become uncoupled. Because dFMRP/FMRP represses the translation of its own mRNA, it is tempting to speculate that this negative-feedback mechanism specifically regulates dFMRP in the mature brain. FMRP modulates synaptic plasticity at maturity, as evidenced by decreased LTP and enhanced LTD in fmr1 knock-out (KO) mice. Consistent with such a mature function, elevated total protein levels are once again evident in the fully mature dfmr1-null brain. A similar increase in cerebral protein synthesis occurs in adult fmr1-KO mice. Together, these data suggest that a switch in dFMRP/FMRP regulation defines separate windows of function in development versus maturity (Tessier, 2008).

It was crucial to determine whether dFMRP function correlates with its developmental expression profile. A known dFMRP target is chickadee/profilin; dFMRP binds chickadee mRNA and negatively regulates its translation. Importantly, the dynamics of chickadee misregulation in the dfmr1-null brain indicate that the dFMRP functional requirement mirrors its developmental expression profile. Chickadee expression normally peaks during late-stage brain development (P4), and it is during this development window, and shortly following, that overexpression is manifested in the dfmr1-null brain. Generally, the increase in chickadee transcripts parallels the increase in protein, suggesting that dFMRP regulation may be at the level of the mRNA, for example, by affecting mRNA stability. dFMRP reportedly interacts with miRNA machinery to control mRNA levels of the sodium channel Pickpocket1. A similar mechanism for chickadee regulation would be consistent with the current results. Interestingly, the increase in Chickadee protein levels coincides with the period of use-dependent neural circuit refinement at eclosion. At least one dFMRP/FMRP target mRNA, futsch (MAP1B), is regulated specifically at postnatal day 10 in fmr1-KO mice. These new insights suggest it will be vital to ascertain the developmental expression of all putative FMRP targets in the context of these distinct windows of regulation in order to validate in vivo functions (Tessier, 2008).

During the peak period of dFMRP expression, there are two phases of dFMRP-dependent axon maturation. During late pupal development, dFMRP inhibits axon elongation, with dfmr1-null neurons exhibiting branches 25% longer than controls. This function is restricted to very late stages (P4), with no differences in branch length or number being observed earlier (P3). Immediately upon eclosion, dFMRP is required for use-dependent pruning, causing a decrease in both axon branch length and number. Pruning is most evident in the smallest presynaptic branches (<5 µm) and occurs quickly (hours) following the onset of adult activity. Targeted overexpression of dFMRP causes inverse defects in both phases of dFMRP requirement. Axon undergrowth is apparent early (P3) and axons fail to grow later (P4). Axon branches present in these neurons are short, filipodial-like structures, and, at eclosion, there is excessive pruning to result in ~30% fewer branches than in P4 and ~3 times fewer branches than in controls. Thus, both axonogenesis and axon branch pruning are bidirectionally modified by inverse changes in dFMRP expression (Tessier, 2008).

Blocking sensory input activity maintains dFMRP in its early development regulative state, with a correlative reduction in both dfmr1 mRNA and dFMRP protein. Both olfactory (Or83b) and phototransduction (ninaE) mutants similarly suppress dFMRP levels, indicating that these two primary modes of brain sensory input positively drive dFMRP expression. Similarly, mammalian FMRP expression is elevated following activity stimulation by both environmental enrichment and mGluR signaling activation. Blocking mGluR activity in Drosophila and mice can rescue some dfmr1 defects, including impaired learning and memory. From these similar findings, it is tempting to suggest that dFMRP/FMRP may function downstream of mGluR signaling activity, perhaps differentially in development versus maturity. Importantly, both Or83b and ninaE sensory mutants cause elevation of chickadee/profilin at the same time dFMRP is suppressed. This finding is consistent with activity-dependent regulation of dFMRP to regulate chickadee/profilin expression (Tessier, 2008).

This study shows that Drosophila neurons undergo activity-dependent pruning coincident with the onset of use. In the absence of dFMRP, pruning does not occur during the normal developmental window. Indeed, blocking sensory input activity leads to further increases in the axon branch number and length in dfmr1-null neurons. Moreover, at maturity, sensory stimulation following sensory deprivation does not induce pruning, probably because the dFMRP level has fallen too low. It is hypothesized that there is a threshold of dFMRP required for efficient activity-dependent pruning during the early-use period, which is normally defined by the window of high dFMRP expression. Reinstated sensory stimulation following sensory deprivation does cause a significant dFMRP-dependent increase in the number of long axon branches. These data are consistent with the need for high dFMRP expression to both limit axonal growth and mediate the early-use refinement of circuits. Importantly, it was confirmed, by using targeted expression of the exogenous light-gated channelrhodopsin-2 channel, that neuronal activation bidirectionally drives the pruning process. Light-driven activation of CHR2 channels induces pruning of the same small (<5 µm) axonal processes that aberrantly persist in the dfmr1-null brain. As predicted, the induced pruning process fails to occur in the absence of dFMRP (Tessier, 2008).

Delayed pruning eventually occurs in dfmr1-null neurons to ultimately rescue the overbranching defect present in younger animals. A similar transient elongation of dendritic spines occurs in young postnatal Fmr1-KO mice, although a secondary overgrowth phenotype may appear months later in adult animals. In Drosophila, the delayed axon pruning in dfmr1-null neurons actually goes too far, resulting in reduced neuronal complexity in mature adult animals. The small presynaptic branches (<5 µm) are reduced 35% in dfmr1-null neurons compared with controls at 4 days. Because pruning normally occurs very rapidly (<3 hours after eclosion), coincident with initial use, it is likely that the pruning process is strictly controlled for that developmental time. By delaying pruning in the absence of dFMRP, it appears that other factors that buffer the extent of process elimination fail to provide adequate regulation of the mechanism. Indeed, this mitigation may be a function of dFMRP itself, as dFMRP levels drop drastically immediately following the normal pruning window. FMRP potentially regulates many proteins involved in a diverse set of functions. Understanding the developmental regulation of proteins that associate with FMRP and FMRP target mRNAs will be crucial to unraveling the underlying pruning mechanisms of activity-dependent neural circuit refinement (Tessier, 2008).


In wild-type ovaries, Chickadee/profilin is highly expressed in the cytoplasm of the somatic follicle cells as these cells migrate and enclose germline clusters to form egg chambers in the germarium. In these somatically derived cells, low levels of profilin are found associated with the plasma membrane. The high cytoplasmic expression in follicle cells decreases after stage 2, but the protein remains expressed in these cells throughout oogenesis. In the follicle cells covering the oocyte at stage 10, low levels of membrane associated profilin can be detected in addition to cytoplasmic profilin. Profilin is very abundantly expressed in a specialized set of follicle cells, called border cells, that migrate through the nurse cell cluster from the anterior of the egg chamber to the anterior edge of the developing oocyte. In the germarium, profilin is also expressed in the cytoplasm of the germline cells, however at a much lower level than in the somatic tissue. Profilin is detected in the nurse cell cytoplasm throughout oogenesis, with the highest level of expression at stage 10 (Verheyen, 1994).

Effects of Mutation or Deletion (part 1/2)

In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II (Zipper) filaments that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, which would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).

In wild-type testes, phalloidin staining reveals an F-actin-enriched contractile ring that encircles the spindle midzone (the bundle of interdigitated microtubules between the separating chromosome complements during anaphase and telophase) from late anaphase through telophase of both meiotic divisions. In contrast, in many meiotic divisions of chic mutants, no actin staining is visible at the cell equator. In most mutant ana-telophases with equatorial actin staining, only irregular patches of F-actin are observed. Chic protein is found in clear concentrations near the cell cortex, particularly in the equatorial zone. In chic mutants abnormal aggregations of F-actin are observed primarily in premeiotic mature spermatocytes at the S5 stage. These aggregates are enriched in alpha-spectrin and are almost invariably associated with ring canals, suggesting that these actin aggregates are in fact relatively undegraded remnants of the male fusome (see Drosophila Spectrin for more information on fusomes). It is suggested that in the absence of chic the disintegration of the fusome is partially blocked (Giansanti, 1998).

In addition to the absence of the contractile ring, the ana-telophases of all the chic mutants exhibit another obvious abnormality: a defect in central spindle morphology. In wild-type ana-telophases of both meiotic divisions, the two daughter nuclei are connected by a prominent bundle of interdigitating microtubules. In chic mutants, this central spindle structure is considerably less dense than in wild type, and microtubules show very little or no interdigitation. In wild type, the central spindle is already evident by mid-anaphase, before the actin ring constriction has caused substantial equatorial pinching. In the strongest chic mutants, these early stages of central spindle formation are never seen, indicating that the chic phenotype reflects a failure of central spindle assembly rather than a degradation of this structure in the absence of the actin ring. This result is surprising because the central spindle is a tubulin based cytoskeletal structure. There is substantial evidence that the central spindle is required for structuring of the actin based contractile ring but not similar evidence that the contractile ring is required for building the central spindle. Together, these observations indicate that chic mutations disrupt two major cytokinetic structures: the microtubule-based central spindle and the actomyosin contractile ring (Giansanti, 1998).

In wild-type primary spermatocytes during the prophase-prometaphase transition of the first meiotic division (stage M1), centrosomes migrate from a position just under the plasma membrane to the nuclear envelope, where they nucleate prominent asters. The two asters then separate and move around the periphery of the nuclear membrane, so as to establish a bipolar spindle. Similarly, during late telophase of the first meiotic division and the short interphase between meiosis I and meiosis II, asters separate and migrate to the opposite poles of secondary spermatocytes. Although the asters in chic mutants are improperly positioned by the start of prometaphase, relatively normal-looking bipolar spindles eventually form by late metaphase. It is remarkable that the function of these spindles, with respect to chromosome segregation, appears to be largely unimpaired. Only a very low frequency of onion-stage spermatids contain irregularly sized nuclei, such as micronuclei (Giansanti, 1998).

Lesions in twinstar (tsr), a gene encoding a Drosophila cofilin (an actin severing and depolymerizing protein), cause a syndrome of phenotypic effects that have both similarities and differences to those described above for chic mutations. In tsr, as in chic spermatocytes, centrosome separation and migration are defective, abnormal accumulations of F-actin are apparent, and cytokinesis often fails after one or both meiotic divisions. tsr and chic mutant phenotypes, however, can be easily distinguished on the basis of several criteria. In tsr mutants, the central spindle is normal, and the contractile ring still forms (though in misshapen and enlarged form), in contrast to the situation for chic. The types of F-actin aggregates formed in tsr spermatocytes are much different from those in chic. It is concluded that tsr and chic mutations differ in how they interfere with meiosis (Giansanti, 1998).

To further investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).

The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. It is of interest that these figures also show severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin , preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells. (Giansanti, 1998).

In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).

The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).

Anillin is a 190 kDa actin-binding protein that concentrates in the leading edges of furrow canals during Drosophila cellularization and in the cleavage furrow of both somatic and meiotic cells. Anillin behavior was examined during D. melanogaster spermatogenesis, and a focus was placed on the relationships between this protein and the F-actin enriched structures. In meiotic anaphases, anillin concentrates in a narrow band around the cell equator. Cytological analysis of wild-type meiosis and examination of mutants defective in contractile ring assembly (Chickadee and KLP3A), reveals that the formation of the anillin cortical band occurs before assembly of the F-actin based contractile ring, and does not require the assembly of the contractile ring. However, once the acto-myosin ring is assembled, the anillin band precisely colocalizes with this cytokinetic structure, accompanying its contraction throughout anaphase and telophase. In Chickadee and KLP3A mutant ana-telophases, the cortical anillin band fails to constrict, indicating that its contraction is normally driven by the cytokinetic ring. These findings, coupled with the analysis of anillin behavior in twinstar mutants, suggests a model for the role of anillin during cytokinesis. During anaphase anillin would concentrate in the cleavage furrow before the assembly of the contractile ring, binding the equatorial cortex, perhaps through its carboxy-terminal pleckstrin homology (PH) domain. Anillin would then interact with the actin filaments of the acto-myosin ring through its actin-binding domain, anchoring the contractile ring to the plasma membrane throughout cytokinesis (Giansanti, 1999).

Genomic deletions of the chickadee locus result in a late embryonic lethal phenotype indicating that profilin is essential in flies. Viable alleles of chickadee with defects in oogenesis, spermatogenesis and bristle formation provide insight into profilin function in a variety of cell types. Defects in oogenesis include the failure to assemble nurse cell actin filament bundles in addition to abnormal regulation of mitosis, binucleate cells and stalled cell migration. Malformed bristles are a result of aberrant actin assembly (Verheyen, 1994).

To clone metazoan genes encoding regulators of cell shape, a functional assay has been developed for proteins that affect the morphology of the fission yeast S. pombe. A Drosophila cDNA library was constructed in an inducible expression vector and transformed into S. pombe. When expression of the Drosophila sequences is induced, aberrant cell shapes are found in 0.2% of the transformed colonies. Four severe phenotypes representing defects in cytokinesis and/or cell shape maintenance were examined further. Each display drastic and specific reorganizations of the actin cytoskeleton. Three of the cDNAs responsible for these defects appear to encode cytoskeletal components: the actin binding proteins profilin and cofilin/actin depolymerizing factor and a membrane-cytoskeleton linker of the ezrin/merlin family. These results demonstrate that a yeast phenotypic screen efficiently identifies conserved genes from more complex organisms and sheds light on their potential in vivo functions (Edwards, 1994).

Actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine is able to slow prehair extension. At higher doses, a complete blockage of hair development is seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton results in the development of multiple prehairs along the apical cell periphery. The multiple hair cells are a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development, since treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton. Several other mutant genotypes produce branched or split bristles or hairs. For example, mutations in singed, chickadee and capping protein produce bristles and/or hairs that are split, bent or stunted in ways that partially resemble cytochalasin D treatment. However, the phenotypes associated with these mutations do not resemble those seen with CD treatment as closely as the phenotype associated with crinkled (e.g. there is not hair splitting in sn mutants). The recent finding that mutations in the small G-protein rho result in an inturned-like phenotype and that the expression of a dominant negative form of rac also results in multiple hair cell phenotype is interesting with regard to the interaction of the actin and microtubule cytoskeletons. Small G-proteins of the rho and rac families are thought to interact with the actin cytoskeleton, yet they produce a wing hair phenotype that is similar to what is seen with the disruption of the microtubule cytoskeleton. This could be due to both the small G-proteins and the micotubule cytoskeleton being required for localizing a common component or activity to the vicinity of the distal vertex, or to the small G-proteins affecting the structure of the microtubule cytoskeleton, or to the microtubular cytoskeleton functioning in the localization of the small G-proteins or, alternatively, these two classes of proteins could be functioning in parallel pathways that function independently to restrict prehair initiation to the distal region of the cell. The observation that the expression of a dominant negative form of rac1 causes a disruption of the microtubule array suggests the possibility that the phenotypes associate with G-protein loss could be due to their disrupting the structure/function of the microtubule cytoskeleton and not to their being part of the frizzled signaling/signal transduction pathway (Turner, 1998).

The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee disrupts this nurse cell cytoplasm transport. The nurse cells from chickadee mutant egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. It is suggested that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells (Cooley, 1992).

Nurse cells are cleared from the Drosophila egg chamber by apoptosis. DNA fragmentation begins in nurse cells at stage 12, following the completion of cytoplasm transfer from the nurse cells to the oocyte. During stage 13, nurse cells increasingly contain highly fragmented DNA and disappear from the egg chamber concomitantly with the formation of apoptotic vesicles containing highly fragmented nuclear material. In mutant egg chambers that fail to complete cytoplasm transport from the nurse cells (dumpless chambers), DNA fragmentation is markedly delayed and begins during stage 13, when the majority of cytoplasm is lost from the nurse cells. These data suggest the presence of cytoplasmic factors in nurse cells that inhibit the initiation of DNA fragmentation. The dumpless mutants studied include cheerio and kelch, which both have aberrant ring canal morphology that does not permit cytoplasm to pass easily from the nurse cells to the oocytes. The chickadee, singed and quail gene products are necessary for the proper formation of cytoplasmic actin filament bundles that form in nurse cells at stage 10B, just prior to the onset of cytoplasmic transport. reeper and hid are expressed in nurse cells beginning at stage 9 and continuing throughout stage 13. The grim transcript is not expressed as strongly as rpr or hid. The negative regulators DIAP1 and DIAP2 are also transcribed during oogenesis. However, germline clones homozygous for the deficiency Df(3)H99, which deletes rpr, hid and grim, undergo oogenesis in a manner morphologically indistinguishable from wild type, indicating that genes within this region are not necessary for apoptosis in nurse cells (Foley, 1998).

Profilin is required for posterior patterning of the Drosophila oocyte. Disrupting the actin cytoskeleton with cytochalasin D induces microtubule bundling and microtubule-based cytoplasmic streaming within the oocyte, similar to that which occurs prematurely in cappuccino and spire mutant oocytes. After examining a number of mutants that affect the actin cytoskeleton, it was found that chickadee, which encodes the actin-binding protein, profilin, shares this phenotype. In addition to the microtubule misregulation, mutants in chickadee resemble cappuccino in that they fail to localize Staufen and Oskar mRNAs to the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee (Manseau, 1996).

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis: chic is an enhancer of Ras1

The synthesis of dorsal eggshell structures in Drosophila requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. Advantage of this process was taken to identify genes that link patterning and morphogenesis; lethal mutations on the second chromosome were screened for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. These genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61ß, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway: Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a 'neurospecific' receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype (Schnorr, 2001).

A main class of Ras1 Enhancers consists of mutations that disrupt bona fide cytoskeletal regulatory genes. These Enhancers may interact in the pathway in a variety of ways (Schnorr, 2001). chickadee (chic) is the Drosophila homolog of Profilin, a conserved actin binding protein. The chic gene produces both constitutive and ovary-specific transcripts through alternative promoters. Mutations that disrupt the ovary-specific transcript result in sterile females that produce dumpless eggs; at a low frequency, these eggs exhibit fused dorsal appendages. In these mutants, cytoplasmic actin cables in the nurse cells are missing. As a result, the rapid transfer of cytoplasm that occurs in late-stage 10 and 11 is disrupted when the nurse cell nuclei drift into the ring canals and impede further cytoplasmic flow (Schnorr, 2001).

The cause of the D/V patterning defect exemplified by the fused dorsal appendages is more obscure. The female sterile alleles do not disrupt follicle cell expression of chic, suggesting the defect is linked to chic function in the germ line. In situ hybridization studies, however, do not reveal a significant change in the level or degree of localization of grk mRNA, which encodes the TGFalpha-like D/V morphogen. These results suggest that the defect is more subtle than a gross perturbation of the cytoskeleton. Mutations disrupting either the ovary transcript or both the ovary and constitutive transcript of chic enhance a weak Ras1 eggshell phenotype. One explanation may involve the interaction of Chic, the actin cytoskeleton, and the microtubule-based cytoplasmic streaming thought to facilitate the uniform distribution of molecules in the oocyte during nurse cell cytoplasmic transfer. Premature streaming may inhibit the localization or function of molecules involved in translation or secretion of the Grk morphogen. Alternatively, Chic might be necessary to form an actin-based anchoring network critical for tethering Grk protein (Schnorr, 2001).

Although the data suggest that Chic must have a germ-line function, these data do not rule out an additional function in the follicle cells. During egg formation, follicle cells undergo shape changes and migrations while secreting the eggshell. These functions require the reorganization and directed movement of the follicle cell cytoskeleton, possibly effected in part by the Chic protein (Schnorr, 2001).

Actin reorganization is necessary for salivary gland invagination

Epithelial invagination is necessary for formation of many tubular organs, one of which is the Drosophila embryonic salivary gland. Actin reorganization and control of endocycle entry are crucial for normal invagination of the salivary placodes. Embryos mutant for Tec29 (Flybase designation - Btk29A), the Drosophila Tec family tyrosine kinase, show delayed invagination of the salivary placodes. This invagination delay is partly the result of an accumulation of G-actin in the salivary placodes, indicating that Tec29 is necessary for maintaining the equilibrium between monomeric actin (G-actin) and filamentous actin (F-actin) during invagination of the salivary placodes. Furthermore, normal invagination of the salivary placodes appears to require the proper timing of the endocycle in these cells; Tec29 must delay DNA endoreplication in the salivary placode cells until they have invaginated into the embryo. Taken together, these results show that Tec29 regulates both the actin cytoskeleton and the cell cycle to facilitate the morphogenesis of the embryonic salivary glands. It is suggested that apical constriction of the actin cytoskeleton may provide a temporal cue ensuring that endoreplication does not begin until the cells have finished invagination (Chandrasekaran, 2005).

Salivary placode cells must constrict apically and move their nuclei basally in order to invaginate into the embryo. Therefore, the process of salivary gland invagination is expected to require actin-myosin contractility. Tec29 is responsible for regulating the actin reorganization in the salivary placodes prior to invagination. Lack of Tec29 in the salivary placodes results in a shift of actin in the salivary placodes from F-actin to more G-actin, leading to incomplete invagination of the salivary placodes cells. This change in the balance between F-actin and G-actin is observed only on the apical surfaces of these cells, suggesting that the delayed invagination is due to aberrant apical constriction of the salivary placodes cells in Tec29 embryos. Increasing actin depolymerization further in Tec29 mutants by a mutation in chic increases the salivary invagination delay, whereas promoting actin polymerization in Tec29 mutants by mutating twinstar partially rescues the invagination delay. Thus, these results provide the first evidence that actin reorganization is necessary for salivary gland invagination (Chandrasekaran, 2005).

Continued: chickadee Effects of mutation part 2/2

chickadee: Biological Overview | Evolutionary Homologs | Regulation | References

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