chickadee

Transcriptional Regulation

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. The genome-wide transcriptional response to activation of the JNK pathway has been analyzed in the Drosophila embryo by serial analysis of gene expression (SAGE) and loci encoding cell adhesion molecules and cytoskeletal regulators were identified as JNK responsive genes. The role in embryogenesis of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly (Jasper, 2001).

With SAGE, virtually every transcript in a sample RNA population can be identified and quantitated by generating specific 14 bp sequence tags from a defined position. Concatemers of such tags are then sequenced, and the frequency with which a given tag is detected represents a direct measure of the abundance of the corresponding mRNA (Jasper, 2001).

SAGE in Drosophila has become particularly powerful with the availability of the Drosophila genomic sequence. Due to the comparatively small size of the fly genome (1.2 x 108 bases of euchromatin), the 14 bases of a SAGE tag (2.7 x 108 possibilities) are typically sufficient to locate the respective transcript in the genome without having to rely on further information (Jasper, 2001).

The dorsal closure process takes place between embryonic stages 13 and 16, corresponding to 10-16 hr of development at 25°C. To capture the relevant changes of gene expression involved in setting up and executing dorsal closure, SAGE libraries were generated from 4-16 hr old Drosophila embryos in which the JNK pathway was either repressed by the ubiquitous expression of a dominant-negative form of Basket (Bsk^DN) or ubiquitously activated due to expression of a constitutively active form of Hemipterous (Hep^act). A library from wild-type embryos was prepared as a reference. The expression of the transgenes was dependent on Gal4 that was expressed ubiquitously under the control of the armadillo promoter starting at around 4.5 hr after egg laying. The effect of the Bsk^DN and Hep^act molecules on the transcription of target genes was therefore limited to the period of embryogenesis relevant for dorsal closure (Jasper, 2001).

Among the upregulated genes identified, several were known from previous studies to interact genetically or biochemically with components of the JNK pathway or to be required for embryonic dorsal closure. However, dpp and puc, until now the only known JNK-responsive genes, were not among them. Despite a significant upregulation of both dpp and puc in Hep^act-expressing embryos as detected by RT-PCR, significant numbers of the corresponding tags were not obtained in the SAGE experiment. The absence of dpp and puc in this analysis is due to the low expression levels of these regulatory transcripts. To generate statistically relevant SAGE data for such rare messages, greater numbers of tags will have to be sequenced (Jasper, 2001).

Consistent with the proposed role of JNK signaling in reorganization of the actin cytoskeleton, this analysis identified several cytoskeletal regulators as JNK target genes. One example is the Drosophila homolog of the profilin gene, chickadee, which is strongly upregulated in Hep^act-expressing embryos. Although mutants for chic have been described as defective in a number of actin-dependent processes, a role in dorsal closure has not yet been reported. Mutants lacking chic function were examined. In addition to pleiotropic defects observed in cuticle preparations, about 30% of these embryos secreted a very thin cuticle with obvious dorsal closure defects. The lateral epidermis failed to stretch normally during the closure process, confirming that the defects detected were not secondary effects caused by the weak cuticle (Jasper, 2001).

To further investigate the proposed role of chic downstream of JNK signaling in dorsal closure, whether chic and hep mutations interact genetically was examined. When female flies homozygous for the X-linked hypomorphic mutation hep¹ are crossed to wild-type males, the male offspring die with mild dorsal closure defects: only 30% of these embryos are completely open, whereas in the remaining 70%, the segments a5-a8 close normally. In contrast, when hep¹ homozygous females are crossed to chic²²¹ heterozygous males, the dorsal open phenotype is enhanced and the number of completely open embryos in the offspring is increased to around 65%. Thus, the gene dose of chic becomes critical in embryos with compromised hep function. In an independent experiment, it was found that in chic heterozygous embryos the phenotypic effects of Hep^act expression are suppressed. Together with the molecular data, these genetic interactions suggest that chic is required downstream of hep for normal dorsal closure (Jasper, 2001).

To understand the cellular function of Hep-induced chic expression in the embryo in more detail, the actin cytoskeleton in the relevant genotypes was examined. Hep^act-expressing embryos display ectopic foci of actin polymerization among the lateral epithelial cells and show increased actin polymerization in leading edge cells, resulting in a stronger actin cable compared to wild-type embryos. In contrast, the leading edge of embryos lacking JNK activity shows a less prominent actin cable overall, which is occasionally disrupted. Significantly, the same phenotype can be observed in chic mutant embryos, consistent with Chic and Hep acting in the same pathway (Jasper, 2001).

Actin-based filopodia that extend dorsally from wild-type leading edge cells have been proposed to mediate the movement and proper alignment of the lateral epidermal sheets during dorsal closure. In hep-deficient embryos, these structures do not form. chic mutants share this phenotype and show an almost complete lack of these filopodia, indicating that this defect is a consequence of insufficient profilin expression in JNK pathway mutants (Jasper, 2001).

Protein Interactions

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Ena can bind directly to Chickadee, the Drosophila homolog of profilin. Furthermore, Ena and profilin are colocalized in spreading cultured cells. The proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo (Ahern-Djamali, 1999).

To identify target proteins for the C-terminal 243 amino acids of Ena, a yeast two-hybrid screen was performed. The C-terminal 243 amino acids of Ena, which include the consensus binding site for profilin and a proline-rich consensus site for binding the Abl SH3 domain, were fused to the DNA binding domain of the yeast transcription factor GAL4 and used to screen a Drosophila third-instar larval library whose inserts were fused to the activation domain of GAL4. The separately expressed domains are unable to activate transcription of the reporter genes HIS3 and LacZ unless a protein-protein interaction takes place. Of 20.5 million clones screened, 9 interacted with Ena as assessed by expression of both the HIS and LacZ reporter genes. One of these clones carried a cDNA encoding full-length Chickadee. The interaction is specific, because a construct with the Ena N-terminal domain fused to the DNA binding domain of GAL4 does not interact with the same isolated Chickadee clone. Of the seven remaining clones, two were partial Ena cDNAs and the other five are unique sequences that are yet to be described (Ahern-Djamali, 1999).

The region of the Ena protein used as bait in the yeast two-hybrid screen contains several matches to a putative profilin binding site. To test whether these sequences are important for mediating the interaction with Chickadee, DNA encoding Ena amino acids 440-490 that contain these putative binding sites, and DNA encoding Ena amino acids 490-684 were fused to the DNA binding domain of the yeast transcription factor GAL4. Yeast were cotransformed with each of these constructs, and the chickadee cDNA was fused to the activation domain of GAL4 and tested for activation of transcription of the reporter genes HIS3 and LacZ. An interaction is detected when chickadee is cotransformed with Ena amino acids 440-490 and not Ena amino acids 490-684, suggesting that this interaction is mediated by proline-rich sequences in Ena. Ena and Chickadee have also been shown to interact in vitro (Ahern-Djamali, 1999).

Profilin has been shown to be localized to cortical microfilament webs and leading lamellae of spreading or locomoting cells. In Drosophila, profilin is expressed ubiquitously throughout development, as for example, the high levels of profilin in the ventral nerve cord of stage 16 embryos. The Ena protein is localized to actin stress fibers and focal adhesions in cultured cells and is localized to the axonal tracts of the developing Drosophila embryonic central nervous system, although the small size of these cells makes higher-resolution localization difficult. Because Ena and Chickadee interact in vitro and are expressed in the nervous system of Drosophila embryos, it was speculated that these two proteins might interact in vivo in regions of dynamic actin remodeling. The subcellular distribution of transfected Drosophila Ena and endogenous profilin were compared in cultures of spreading Ptk2 cells. Ena and profilin colocalize to the periphery of the spreading cells. The colocalization, together with the biochemical interactions, suggests that Ena and profilin associate in vivo (Ahern-Djamali, 1999).

The proline-rich region of Ena contains multiple consensus binding sites for the SH3 domain in addition to the profilin binding sequences. It has been shown with a filter binding assay that Ena binds the Abl and Src SH3 domains in vitro. The SH3 binding specificity of Ena was examined further by using a solution binding assay. Ena was expressed in Drosophila S2 cells, and the transfected cell lysates were incubated with a series of GST-SH3 fusions. Ena binds specifically to the Drosophila and murine Abl-SH3 domains and the murine src SH3 domain. Ena also bind to the C-terminal but not to the N-terminal SH3 domain of Drk. The two Ena peptides most closely matching the Abl consensus binding motif partially and specifically block Ena binding to the Abl SH3 domain, although they do not block Src SH3 domain binding. Peptides derived from Ena proline-rich sequences most closely matching the optimal sequences for Src or Drk SH3 binding do not compete for Ena binding with any of the SH3 domains tested (Ahern-Djamali, 1999).

To determine whether the proline motifs identified in the peptide binding experiment as Abl SH3 binding sites are sufficient to mediate Abl SH3 binding, site-directed mutagenesis was employed to change eight prolines to alanine, thereby eliminating many of the PXXP motifs present in the sites. Serial two-fold dilutions of transfected cell lysates containing either the mutant Ena protein (Ena8 P to A) or wild-type Ena were tested for solution binding to the Abl SH3 domain. At higher concentrations of protein, it is difficult to detect an effect of the proline-to-alanine mutations on binding. However, at lower concentrations of the Ena proteins, binding of the mutant protein is markedly reduced when compared with the wild-type Ena protein. To examine the in vivo effect of the proline-to-alanine mutations on Ena function, transgenes expressing wild-type Ena and the Ena8 P to A mutant proteins were tested for their ability to rescue ena mutant lethality. The ena8 P to A transgene rescues the embryonic lethality associated with loss-of-function mutations in ena as well as the wild-type ena transgene. The ena8 P to A-rescued flies are phenotypically normal and have viability and fertility comparable to wild-type ena-rescued flies. Thus, the proline-to-alanine mutations present in Ena8 P to A are not sufficient to disrupt an essential function of the Ena protein (Ahern-Djamali, 1999).

Interestingly, there is some overlap in the binding sites for the Abl SH3 domain and some of the putative binding sites for P to A profilin. Because mutation of the prolines in these overlapping regions reduces binding to the Abl SH3 domain, it was hypothesized that these mutations might also disrupt binding to Chickadee. The mutant ena8 P to A cDNA was subcloned in the pGex expression vector, and the resulting mutant Ena fusion protein, GST-Ena8 P to A, was compared with wild-type GST-Ena in solution binding assays for its ability to pull down Chickadee from serial dilutions of lysates prepared from adult Drosophila. The GST-Ena8 P to A fusion protein and wild-type GST-Ena pull down approximately equivalent amounts of Chickadee, suggesting that different amino acids may be important for Ena binding to profilin and the Abl-SH3 domain, despite the overlap observed in some of their putative binding sites. It is worth noting that no SH3 domain-bearing proteins were isolated in the yeast two-hybrid screen that identified profilin as a binding partner for Ena. Perhaps the proline-rich sequences present in the Ena bait are not the most important for binding to SH3 domains. Alternatively, the conditions in the yeast two-hybrid screen may not favor detection of an interaction between an SH3 domain and proline-rich sequences. Another possibility is that Ena's interaction with the Abl SH3 domain may be less physiologically relevant than its interaction with Chickadee. Indeed, mutations that disrupt binding to the Abl SH3 domain in vitro have no effect in vivo when they are expressed from a heterologous promoter. It will be important to identify critical amino acids for the interaction between chickadee and Ena and to examine whether these mutations have any in vivo effects (Ahern-Djamali, 1999).

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).

Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. A robust and specific genetic interaction is found between between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors (Wills, 2002).

Genetic experiments suggest that Abl interacts with a number of actin regulatory proteins to control cytoskeletal assembly. Given the functional redundancy observed between CAP and Profilin in yeast, it was thought that Capt and Profilin might participate in some form of protein complex regulated by the Abl kinase. S2 cells were transfected with full-length Drosophila Abl (dAbl), Drosophila Src64 (dSrc), or the truncated mammalian v-Abl and then Capt immunoprecipitations were assayed with anti-Profilin (Chic) and anti-actin antibodies. No significant binding of Capt and Profilin were seen in cells transfected with dSrc or v-Abl or in untransfected controls where endogenous dAbl is expressed at very low levels. However, an association of Capt with Profilin and with actin was observed when dAbl was elevated, suggesting a model where Abl, Capt, and Profilin function together in a cytoskeletal protein complex (Wills, 2002).

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Exportin 6 (Exp6) has been identified as a novel family member from higher eukaryotes; it mediates nuclear export of profilin-actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin-actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. No indications was found for a major and direct role for CRM1 in actin export from mammalian or insect nuclei (Stuven, 2003).