tramtrack

DEVELOPMENTAL BIOLOGY

Embryonic

Maternally supplied TTK protein helps to establish the timing of the onset of zygotic expression of even-skipped and fushi tarazu, thereby preventing premature activation. Ectopic expression of the 69 kDa protein, but not of the 88 kDa form nearly abolishes the striped patterns of expression of both even-skipped and fushi tarazu. Therefore, ttk functions to establish the timing of the onset of zygotic expression of these two genes (Read, 1992a).

Initiation of ftz transcription is a gradual process, mediated by titration of TTK protein. When the local concentration of nuclei is experimentally altered, using a mutation that makes the migration of nuclei to the egg cortex uneven, ftz is activated only in nuclei that attain a high local density at the cortex (Pritchard, 1996).

The zygotic transcripts appear at stage 7 in the anterior mid-gut primordia and pole cells and later in the posterior mid-gut primordia. At stage 9, when FTZ stripes are no longer present, TTK mRNA, corresponding to the 69 kDa protein, forms a pattern of about 14 stripes in the ectoderm and mesoderm of the extended germ-band. These stripes later fuse, forming a 'tramtrack' pattern around the germ-band [Images], At stage 13, TTK mRNA is only found in the developing epidermis and gut (Brown, 1993). Gross defects in the larval cuticle resulting from misexpression of the 69 kDa protein suggests that this protein performs additional function in the patterning of ectoderm (Read, 1992b).

tramtrack acts downstream of Notch to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS (Guo, 1995). Both the loss of function and overexpression of ttk affect the fates of sensory organ precursor cells. numb is expressed in neurons, and is distributed to them asymetrically (Guo, 1995).

In Drosophila, cell-fate determination of all neuroectoderm-derived glial cells depends on the transcription factor Glial cells missing (Gcm), which serves as a binary switch between the neuronal and glial cell fates. Because the expression of Gcm is restricted to the early phase of glial development, other factors must be responsible for the terminal differentiation of glial cells. Expression of three transcription factors, Reversed polarity (Repo), Tramtrack p69 (Ttk69) and PointedP1 (PntP1), is induced by Gcm in glial cells. Repo is a paired-like homeodomain protein, expressed exclusively in glial cells, and is required for the migration and differentiation of embryonic glial cells. To understand how Repo functions in glial terminal differentiation, the mechanism of gene regulation by Repo was analyzed. Repo is shown to act as a transcriptional activator through the CAATTA motif in glial cells, and three genes are defined whose expression in vivo depends on Repo function. In different types of glial cells, Repo can act alone, or cooperate with either Ttk69 or PntP1 to regulate different target genes. Coordination of target gene expression by these three transcription factors may contribute to the diversity of glial cell types. In addition to promoting glial differentiation, it was found that Repo is also necessary to suppress neuronal development, cooperating with Ttk69. It is proposed that Repo plays a key role in both glial development and diversification (Yuasa, 2003).

Although ectopic Repo induces the appearance of many non-glial lacZ-expressing cells in the dorsal epidermis, cells within the CNS do not respond to ectopic Repo. In fact, even in the wild-type background, not all Repo-positive glia in the CNS expressed the ftz HDS reporter. This suggests that the mechanism by which Repo regulates transcription may be different in the CNS from the one for peripheral glia. One possible scenario is that the functions of Repo in the CNS require cooperation with one or more other factors, and that these interactions preclude Repo from acting through the CAATTA motif. Ttk69 and PntP1 are good candidates for such co-factors, because ttk and pointed are both required for the development of CNS glial cells. Although repo, ttk and pointed are expressed in overlapping subsets of CNS glial cells, their expression is mutually independent; Repo continues to be expressed in the ttk or pointed mutant background, and lacZ expression levels in enhancer-trap lines of ttk or pointed are unaffected in repo mutant embryos. Moreover, ectopic expression of Repo in the entire neuroectoderm does not increase the expression of pointed P1 mRNA or Ttk69, nor does ectopic expression of either Ttk69 or PntP1 affect Repo expression. All three genes are most probably regulated independently, downstream of the glial determinant Gcm (Yuasa, 2003).

Although glial specification by Gcm is well established, how the characteristics of individual glial cells are determined is poorly understood. Gcm expression is confined to the early stage of glial development, suggesting that Gcm itself does not participate in the terminal differentiation of glia. Moreover, Gcm also directs blood cell development; Gcm is expressed in macrophage precursors and ectopic expression of Gcm in crystal cell precursors causes the transformation of crystal cells to macrophages. These results clearly show that the expression of Gcm does not always lead to the determination and terminal differentiation of glia. In glial cells, Gcm induces the expression of three transcription factors, Repo, Ttk69, and PntP1, and the loss of these proteins causes abnormal glial development, although Gcm expression remains normal. Although gcm can direct repo expression in various contexts, repo is not expressed endogenously in blood cells, but is confined to Gcm-positive glial cells, lasting even after gcm expression has ceased. In repo mutant embryos, the migration, survival and terminal differentiation of glial cells are abnormal. This study shows that Repo activates gene expression in glia, and also demonstrates that Repo mediates the suppression of neuronal differentiation. These results suggest that Repo is the major factor that is necessary for glial development (Yuasa, 2003).

The synergistic effect of Repo and Ttk69 on M84 marker expression suggests a positive role of Ttk69 on glial differentiation. Since the major function of Ttk69 has been thought to be the inhibition of neuronal differentiation through transcriptional repression, the positive action of Ttk69 on glial gene expression could be an indirect effect through repressing transcription of a repressor for M84 expression. However, Ttk69 can activate transcription in yeast cells, suggesting that Ttk69 may also promote transcription, depending on the cellular context. Recent studies also implicate a role for Ttk69 in cell proliferation, through controlling the expression of cell cycle regulators. Overexpression of Ttk69 results in the inhibition of glial development, accompanied by the repression of the S-phase cyclin and glial proliferation. Since an increase in the number of cells that express M84 glial marker is observed upon co-expression of Ttk69 and Repo, the result cannot be accounted for by the ability of Ttk69 to inhibit glial cell cycle. Whereas ectopic expression of Ttk69 reduces the expression of the endogenous repo gene, the misexpression paradigm provides exogenous Repo through the GAL4/UAS control. Thus the existence of Repo might modify the activity of Ttk69, so that it plays a positive role on glial development (Yuasa, 2003 and references therein).

Glial fate determination involves not only the promotion of glial differentiation but also the suppression of neuronal properties. Because ectopic Gcm can induce neurogenesis in certain contexts, it is unlikely that Gcm directly represses neuronal differentiation. Ttk69 has been proposed to inhibit neuronal differentiation, mainly because of its loss-of-function phenotype in the sensory organ. Here, it has been shown that the co-expression of Repo and Ttk69 has a potent neuron-suppressing activity, and further demonstrated that the repo mutant permits neuronal differentiation even when Gcm is overexpressed. This strongly suggests that Repo functions not only to activate the transcription of glial genes, but also to prevent the neuronal differentiation of presumptive glial cells (Yuasa, 2003).

If glia and neuron represent two mutually exclusive cell states that must be chosen between early in development, it is somewhat strange that suppression of neuronal development should be carried out by proteins that are expressed throughout glial differentiation. The existence of continuous suppression of neuronal properties in glia suggests that cells within the nervous system may retain the potential to become neurons or glia throughout their cellular history. This idea is supported by the observation that Gcm is able to transform post-mitotic neurons into glia. Conversely, in the vertebrate nervous system, glial cells (astrocytes and oligodendrocyte-precursor) can respond to environmental signals and function as neural stem cells, generating neurons. The role of Repo and Ttk69 may be to suppress the ability of glia to respond to cues that would cause them to change into neurons or neural precursors (Yuasa, 2003).

Oogenesis

Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary identifies tramtrack

Cell migration contributes to normal development and homeostasis as well as to pathological processes such as inflammation and tumor metastasis. Previous genetic screens have revealed signaling pathways that govern follicle cell migrations in the Drosophila ovary, but few downstream targets of the critical transcriptional regulators have been identified. To characterize the gene expression profile of two migratory cell populations and identify Slbo targets, border cells and centripetal cells expressing the mouse CD8 antigen were purified and whole-genome microarray analysis was carried out. Genes predicted to control actin dynamics and the endocytic and secretory pathways were overrepresented in the migratory cell transcriptome. Mutations in five genes, including ttk, failed to complement previously isolated mutations that cause cell migration defects in mosaic clones. Functional analysis revealed a role for the Notch-activating protease Kuzbanian in border cell migration and identified Tie receptor tyrosine kinase as a guidance receptor for the border cells (Wang, 2006).

Gene expression profiling of migratory cells in the Drosophila ovary has allowed comparison of the global patterns of gene expression of developmentally regulated cell movements to that previously reported for invasive carcinoma cells. Of 30 genes that encode motility-associated proteins that were identified as upregulated in invasive breast carcinoma relative to the primary tumor, 23 have easily identified Drosophila homologs. Of these, 11 (48%) were identified as upregulated in migratory follicle cells in the current analysis. This seems noteworthy given that the cells derive from different organisms and different tissues. In contrast, only one of the cytoskeleton-associated, migratory cell-enriched genes was identified out of the top 419 genes upregulated in the adult Drosophila eye (Wang, 2006).

Finding a large number of genes that are differentially expressed in a microarray analysis can make it difficult to decide which individual genes merit additional, detailed study. One approach to limiting the number of genes in an analysis is to use stringent fold-change cutoffs. However, it is not clear that this is the best way to derive biologically meaningful information from large data sets. An alternative approach was used, employing a sensitive method to reveal a large number of differentially expressed genes and then separating the large data set into smaller sets by using gene ontology with GO Slim. This allowed discernment, in a relatively unbiased manner: genes that encode cytoskeletal proteins and proteins associated with the secretory and endosomal pathways were overrepresented in the migratory cell-enriched genes compared to the genome as a whole, providing a rationale for the selection of smaller, functionally related subsets of genes for further study (Wang, 2006).

The overrepresentation of cytoskeleton-associated gene products among the migratory cell-enriched genes is interesting to consider in light of the striking morphology of border cells during their migration. One, or occasionally two, cells at the front of the cluster extend a long dominant protrusion that can be up to 50 μm long. This may be a common morphology for cells migrating in vivo, since it has also been observed for cells of the rostral migratory stream and neural crest cells. It seems reasonable to propose that this extended morphology may require special regulation of the cytoskeleton: such regulation might differ in some respects from that of the broad, flat lamellae and ruffles formed by cells cultured on two-dimensional surfaces. For example, longer parallel bundles of F-actin are probably required to create and maintain long protrusions such as those observed in border cells (Wang, 2006).

Although the general idea that proteins associated with the cytoskeleton are important in migratory cells is not surprising, this analysis leads to generation of hypotheses regarding specific genes. For example, two proteins known to promote long, parallel actin bundles are among the migratory cell-enriched genes, including tropomyosin and fascin. Loss of function of fascin (encoded by the gene singed) does not result in a discernible border cell migration defect; however, this may be because of redundancy with tropomyosin. Similarly, loss of the filamin-like protein encoded by the cheerio locus causes a mild border cell migration defect. The microarray analysis reveals that another filamin-like protein (Jitterbug) is expressed at a higher level in the migratory follicle cell population. The microarray data therefore can guide the development of specific, testable hypotheses concerning possible gene redundancies (Wang, 2006).

In addition to proteins with well-characterized functions in actin dynamics, such as actin and actin-related proteins, a number of genes emerged from the microarray analysis that encode proteins with motifs or domains that suggest a specific role in regulating the actin cytoskeleton, but which have not yet been characterized at all. These include Rexin, a protein composed of three SH3 domains, and CG31352, which encodes a protein composed of three LIM domains and a motif resembling the villin headpiece. Mammalian homologs of these proteins exist but have not been characterized. It will therefore be of interest to determine if these genes and their products represent evolutionarily conserved, but previously unrecognized, contributors to cell motility (Wang, 2006).

Genes encoding proteins associated with the endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles, and endosomes were significantly overrepresented among the migratory cell-enriched genes compared to the genome as a whole. This observation suggests that border cells have a special need for dynamic trafficking of proteins to and from the cell surface. It has been proposed that dynamic cell-cell adhesion between border cells and nurse cells is required for the cells first to gain traction and then to translocate, and that this may involve high rates of turnover of membrane proteins such as E-cadherin. Moreover, it is clear that several receptor molecules such as Domeless, PVR, and EGFR are present at lower concentrations on the surfaces of the border cells as compared to other follicle cells. Therefore, it seems likely that there is a high rate of movement of these proteins onto and off of the plasma membrane. All of this traffic would likely require an upregulation of proteins functioning in the secretory and endocytic pathways. Consistent with this hypothesis, it has been shown that multivesicular bodies are markedly more prevalent in migrating border cells as compared to other follicle cells in the egg chamber (Wang, 2006).

Relatively little is known about the mechanisms governing centripetal cell migration. Border cells and centripetal cells take two different paths, but they arrive at the same place. Both cell types express Slbo and require E-cadherin, Rac, and myosin II for their respective movements. Thus, mechanical aspects of these two migrations may be similar. However, there are also differences in the migrations of these two cell types. Border cells completely exit the follicle cell epithelium during their migration down the center of the egg chamber. Centripetal cells, in contrast, stay connected to the outer follicle layer. In addition, the directions of the two migrations are quite different. Whereas border cells migrate posteriorly, centripetal cells migrate symmetrically toward the center, orthogonal to the path of border cell migration. Therefore, the cues that direct the two migrations must be different. Consistent with this, none of the known border cell guidance receptors is required for centripetal cell migration. In addition, the border cells and centripetal cells initiate migration at distinct times: the border cells complete their migration before the centripetal cells begin. The gene expression profile presented in this study provides a wealth of candidate genes to test for effects on centripetal cell migration and to flush out the similarities and differences between border cell and centripetal cell migration (Wang, 2006).

One goal on this study in determining the gene expression profile of border cells was to facilitate the molecular identification of genes corresponding to the mutations that cause border cell migration defects in mosaic clones. Five genes in the microarray lists, gliotactin, tramtrack, catsup, latheo, and zipper, were matched to mutant lines identified in mosaic screens. The next challenge will be to elucidate precisely how each of these genes contributes to border cell migration (Wang, 2006).

In addition to facilitating the identification of genes that cause cell migration defects in mosaic clones, the gene expression profile can identify genes that would be unlikely to be identified in such genetic screens. For example, all of the known guidance factors for border cell migration produce either no defect when mutated individually (ligands for the EGF receptor) or quite mild defects (PVF1). Their contributions become much more obvious when multiple mutations are combined. Therefore, it is a challenge to identify this class of proteins by using conventional forward genetics. In the gene expression profile reported here, an uncharacterized receptor tyrosine kinase was found to be expressed at higher levels in migratory cells and to be an Slbo target. Expression of a putatively dominant-negative form of this receptor exacerbated the migration defects associated with loss of PVR alone or loss of PVR and EGFR, implicating this receptor in guidance of border cell migration. Therefore, the expression profile has provided a source of candidate genes that would be difficult or impossible to identify by other methods (Wang, 2006).

Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells

The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1. In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).

Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).

In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).

The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).

Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).

Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).

Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).

These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).

What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).

An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

Effects of Mutation or Deletion

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. The first growth cones navigate toward the anterior most ventral unpaired median (VUM) cell and thus pioneer the prospective posterior commissure. Only when the posterior commissure is established, the anterior commissure forms. In later stages, midline glial cells, migrating toward the posterior, are required to separate anterior and posterior commissures into distinct axon bundles. The VUM neurons reside ventral to the posterior commissure and project in a characteristic axon-bundle to the anterior commissure. Migration of two midline glial cells occurs along these cell processes. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, a saturating ethylmethane sulfonate mutagenesis was conducted, screening for mutations that disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. Two gene functions are required for the initial formation of commissural tracts, in addition to the function of commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures (Hummel, 1999).

Subsequent analysis has defined four sequential steps involved in commissure development. Initially, single minded, jaywalker, Egf receptor and slit are involved in the first step in midline formation: the formation of the anlage of the CNS midline. Next the segment polarity genes hedgehog, engrailed, patched and wingless are involved in the specification of midline cell number. It is possible that midline and ectodermal pattern formations occur at the same time. In addition to the segment polarity genes other signaling mechanisms appear important. Notch, for example, is required to specify the different midline lineages. The third step in commissure formation consists of the formation of commissures. Once the midline cells have been specified, they guide commissural growth cones toward and across the midline. Here, the Netrins, frazzled, commissureless, weniger, schizo, roundabout and karussel play an essential role. The fourth step in commissure development involves the separation of the commissures. Contrary to midline specification and initial commissure formation, this process occurs relatively late during embryogenesis and thus a maternal contribution is not likely to rescue a mutant phenotype. In addition, the separation of commissures requires not only the differentiation of the midline glial cells but also the differentiation of the midline neurons as well as interactions of these two cell types for normal migration to occur. This might explain the large number of genes identified (Hummel, 1999).

The analysis of mutations reveals two major phenotypic classes: the pointed and the tramtrack groups. pointed and tramtrack mediate different aspects of glial development. In pointed mutants no glial differentiation occurs, whereas ectopic pointed expression results in ectopic glial differentiation. tramtrack, in contrast, does not interfere with actual glial cell differentiation but appears to be required for the repression of neuronal differentiation in these cells. The pointed group consists of pointed itself, rhomboid, kastchen, klotzchen, kette, schmalspur, mochte gern, spitz, Star, cabrio and kubel. Mutations in eight other genes lead to an axon phenotype initially described for tramtrack. In tramtrack-type mutation (tramtrack, shroud, disembodied, spook, shade, shadow, phantom, and rippchen) commissures appear fused, but in contrast to pointed group mutations, connectives are not affected (Hummell, 1999).

The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons. Type I neurons are characterized by their single dendrite whose distal part is a modified cilium. In contrast, type II neurons possess several dendrites lacking ciliated structures. Type I neurons are associated with accessory cells (socket and shaft cells, known respectively as tormogen and trichogen cells) that form the non-neuronal part of the sense organ. Type II neurons are not associated with accessory cells. In Notch mutant embryos, the type I neurons are missing while type II neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication. It is proposed that a protoprecursor cell exists called p0, having both external sense organ and multidendritic cell potentiality. In the absence of Notch the two daughters of the protoprecursor will adopt the same fate, the multidendritic fate (Vervoort, 1997).

Both type I and type II neurons are absent in numb mutant embryos and also after the ubiquitous expression of tramtrack. This indicates that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates. Numb is thought to repress tramtrack, a gene that promotes non-neuronal verses neuronal fate. The analysis of string mutant embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate. A new mutant phenotype has been described, called X1. It prevents the acquisition of external sense cell fate. In these mutants, ASC-dependent neurons are converted into type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate (Vervoort, 1997).

A collection of transposable-element-induced mutations have been screened for those which are dominant modifiers of the extra R7 phenotype of a hypomorphic yan mutation. The members of one of the identified complementation groups correspond to disruptions of the tramtrack gene. As heterozygotes, ttk alleles increase the percentage of R7 cells in yan mutant eyes. Just as yan mutations increase ectopic R7 cell formation, homozygous ttk mutant eye clones also contain supernumerary R7 cells. However, in contrast to yan, the formation of these cells in ttk mutant eye tissue is not necessarily dependent on the activity of the sina gene. Furthermore, although yan mutations are dominant in interactions with mutations in the Ras1, Draf, Dsor1, and rolled genes to influence R7 cell development, ttk mutations only interact with yan and rl gene mutations to affect this signaling pathway. These data suggest that yan and ttk both function to repress inappropriate R7 cell development but that their mechanisms of action differ. In particular, TTK activity appears to be autonomously required to regulate a sina-independent mechanism of R7 determination (Lai, 1996).

Genetic interactions of yan with downstream components of the Sevenless pathway have been studied. A reduced activity of tramtrack results in enhancement of the mutant yan phenotype. ttk mutations produce extra R7 cells even in sina homozygotes while the yan mutation does not. This results indicates that TTK represses R7 induction downstream of the sites where YAN and SINA function (Yamamoto, 1996).

The Drosophila yan gene encodes an ETS domain nuclear protein with a transcription repressor activity that can be downregulated through phosphorylation by mitogen-activated protein kinase (MAPK). Before photoreceptor precursor cells commit to a particular cell fate, Yan is required to maintain them in an undifferentiated state. tramtrack (ttk) mutations have been identified that act as dominant enhancers of yan. ttk synergistically interacts with yan to inhibit the R7 photoreceptor cell fate. Since ttk products are nuclear proteins with zinc-finger DNA-binding motifs, yan and ttk represent two nuclear regulators essential for the control of cellular competence for neural differentiation. Reduction of either yan or ttk activity suppresses eye phenotypes of the kinase suppressor of ras (ksr) gene mutation, which is consistent with the involvement of yan and ttk in the Ras/MAPK pathway. Based on the fact that yan acts upstream of sina and ttk acts downstream of sina, it is expected that interaction between yan and ttk does not occur through direct protein associations. A more likely scenario is that yan controls expression of downstream genes that are critical for regulating ttk expression or function. A strong candidate target gene is phyllopod, which acts downstream of yan and upstream of sina (Lai, Z.-C., 1997).

Two classes of glial cells are found in the embryonic Drosophila CNS: midline glial cells and lateral glial cells. Midline glial development is triggered by EGF-receptor signaling, whereas lateral glial development is controlled by the glial cells missing (gcm) gene. Subsequent glial cell differentiation depends partly on pointed . tramtrack (ttk) is required for all CNS glia development. Mutant ttk embryos are characterized by an embryonic CNS axon pattern phenotype of fused segmental commissures, indicating a requirement of ttk during midline glial development. In ttk embryos, longitudinal axon tract formation is impaired and the connnectives appear thinner. This phenotype is indicative of a defect in the longitudinal glia (Giesen, 1997).

tramtrack encodes two zinc-finger proteins, one of which, ttkp69, is expressed in all non-neuronal CNS cells. ttk expression in the ventral cord is restricted to lateral and midline glial cells. All cells that express the glial marker Repo also express ttkp69. The transverse nerve exit glial cells (or DM cells) express ttkp69. In the CNS of stage 16 ttk mutants, there are about 20% less lateral glial cells than a wild-type CNS. In mutants, although the midline glial cells are initially present in normal number and position, they fail to perform their normal migration. Therefore ttk is required for normal glial development. The exit glial cells in mutant ttk embryos are slightly enlarged, but they are still able to ensheath both the segmental and intersegmental axon bundles. Like ttk, pointed is expressed in glial cells. However, unlike ttk, pointed is required for glial cell development. Ectopic ttkp69 expression in the neuroectoderm leads to a partial block of neuronal development as indicated by substantially reduced expression of the neuronal Elav antigen as well as other neuronal markers examined (Giesen, 1997).

Both Ttkp69 and pointed are downstream of gcm. gcm, however, is not expressed in midline glia, and ttkp69 as well as pointed expression in midline cells is normal in gcm mutants. pointed and ttkp69 are both expressed under the control of gcm in lateral glial cells; the expression of these genes appears to be independent of one another. Thus the two targets of gcm appear to act in parallel. Glial cell differentiation may depend on a dual process, requiring the activation of glial differentiation by pointed and the concomitant repression of neuronal development by tramtrack (Giesen, 1997).

Recently, double-stranded RNA (dsRNA) has been found to be a potent and specific inhibitor of gene activity in the nematode Caenorhabditis elegans (Fire, 1998). The potential of dsRNA to interfere with the function of genes in Drosophila, termed RNA inhibition or RNAi) has been investigated. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. dsRNA corresponding to four genes with previously defined functions was introduced. dsRNA is shown to potently and specifically inhibits the activities of wg, fushi tarazu (ftz), even-skipped (eve), and tramtrack (ttk). The reception mechanism of the morphogen Wingless was determined using dsRNA. Interference of the frizzled and Drosophila frizzled 2 genes together produces defects in embryonic patterning that mimic the loss of wingless function. Interference with the function of either gene alone has no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. These results demonstrate that dsRNA interference can be used to analyze many aspects of gene function (Kennerdell, 1998).

Target specificity of dsRNA interference was further assessed using the tramtrack (ttk) gene. ttk encodes two alternatively spliced proteins with different pairs of zinc fingers. Both proteins are together required for development of chordotonal (ch) organs of the embryonic nervous system. The number of ch neurons is greater in ttk mutant embryos. Compared with wild type, the number of neurons in each lateral cluster of five ch organs, termed lch5, is approximately doubled at the expense of other organ cell types. ttk is expressed in ch cells when the sensory organ precursors are determined at approximately stage 11-12. Since this corresponds to a time approximately 6 to 7 hr after dsRNA was normally injected into embryos, interference of ttk would also allow a test of whether dsRNA can persist for several hours. Persistence is an important issue because many endogenous RNAs are rapidly degraded in fly embryos. dsRNA was prepared corresponding to exons common in both ttk transcripts; embryos were injected with the RNA, and the lch5 organ was examined in each abdominal segment. All injected embryos exhibit lch5 organs with increased numbers of neurons. The potency of this effect is profound, with 90% (33 of 37) having all of their lch5 organs affected. In addition to the defects in the peripheral nervous system, almost all embryos fail to undergo dorsal closure and head involution, two defects observed in ttk mutants. A significant number of injected animals exhibit defects more profound than the previously characterized ttk null mutants. These defects include extensive hyperplasia of the nervous system reminiscent of mutants in the 'neurogenic' class of genes, which includes Notch. The dsRNA covers the highly conserved BTB/POZ domain and may have interfered with other BTB/POZ domain genes. Alternatively, the dsRNA may have interfered with both maternally supplied ttk transcripts and zygotic ttk activity. Since the described ttk mutants have only removed zygotic activity, it is possible that removal of both maternal and zygotic ttk activities would produce a neurogenic phenotype. Consistent with this possibility, ttk mutant cuticle phenotypes resemble Notch mutants (Kennerdell, 1998).

In the midline glia of the embryonic ventral nerve cord of Drosophila, differentiation as well as the subsequent regulation of cell number is under the control of EGF-receptor signaling. During pupal stages apoptosis of all midline glial cells is initiated by ecdysone signaling. In a genetic screen, mutations in disembodied, rippchen, spook, shade, shadow, shroud and tramtrack have been identified that all share a number of phenotypic traits, including defects in cuticle differentiation and nervous system development. Some of these genes were previously placed in the so-called 'Halloween-group' and have been shown to affect ecdysone synthesis during embryogenesis. The Halloween mutations not only affect glial differentiation but also lead to an increase in the number of midline glial cells, suggesting that during embryogenesis ecdysone signaling is required to adjust glial cell number similar to pupal stages. A P-element-induced mutation of shroud has been isolated: it controls the expression of ecdysone inducible genes. The P-element insertion occurs in one of the promoters of the Drosophila fos gene for which an as yet undescribed complex genomic organization is presented. The recently described kayak alleles affect only one of the six different Fos isoforms. This work for the first time links ecydsone signaling to Fos function and shows that during embryonic and pupal stages similar developmental mechanisms control midline glia survival (Giesen, 2003).

However, beside the overall phenotypic similarities displayed by mutants in the different genes differences were noted in the number of midline glial cells. In tramtrack and rippchen mutant embryos a reduction in the number of midline glial cells was observed. tramtrack is also a negative regulator of glial cell division. In the absence of tramtrack, additional glial cells can be detected in stage 14 embryos and only in older embryos a reduction in the number of glial cells can be observed. Interestingly, tramtrack and shroud appear to interact. In an effort to generate a tramtrack shroud double mutant it was noted that significantly fewer transheterozygous tramtrack/shroud flies eclosed. Thus, the BTB-Zn-finger proteins encoded by tramtrack may eventually be linked to Fos function (Giesen, 2003).

Initially, about six midline glial cells are generated in each abdominal neuromere. During the second half of embryogenesis the number of midline glial cells is reduced by apoptosis to three to four per neuromere. Only those glial cells that have formed extensive contacts to commissural axons are thought to survive. Indeed, when formation of commissural axons is impaired, as for example in the commissureless mutation, most midline glial cells die via apoptosis. Activation of Egfr by axon-derived Spitz and subsequent Ras/MAPK signaling in the midline glia is able to counteract apoptosis by inactivating the cell death protein Hid. Similar to the finding that the EGF receptor ligand Spitz regulates midline glia survival, the EGF-receptor ligand Vein, a Drosophila neuregulin homolog, provides trophic support for subsets of cortical glial cells. These findings parallel the survival promoting effects of neuregulin-1 on Schwann cells in the mammalian peripheral nervous system (Giesen, 2003).

Based on phenotypic similarities, tramtrack, disembodied, shroud, shadow, shade, spook and rippchen were classified as members of the tramtrack or Halloween-group. Mutations in the disembodied locus are characterized by defects in epidermal development, dorsal closure, head involution, midgut formation and CNS development. In particular, loss of disembodied function leads to an excess of midline glial cells that are not able to separate anterior and posterior commissures (Giesen, 2003).

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from each proneural cluster and then undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. phyllopod (phyl), known to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters (IIb cells are transformed into IIa cells), leading to the fate transformation of neuron and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development (Pi, 2001).

Genetic analyses show that phyl functions together with sina to promote SOP formation by antagonizing ttk activity. These results suggest that degradation of the Ttk protein is a major function of Phyl in the cell fate specification of SOP cells. Consistent with this idea, misexpression of ttk can inhibit the formation of SOP cells and suppress the ectopic bristle phenotype caused by misexpression of phyl. Several lines of evidence also indicate that Ttk functions as a repressor to inhibit SOP cell fate. (1) Ttk is expressed ubiquitously in the pupal notum except in SOP cells. (2) In embryos, overexpression of ttk inhibits the formation of es organs. (3) Injection of ttk dsRNA results in extensive increase of neurons in embryonic PNS, a phenotype observed in neurogenic mutants. All of these results suggest that ttk might play a negative role in the fate specification of SOP cells, and phyl promotes SOP fate specification by degrading Ttk (Pi, 2001).

Genetic analyses of phyl, sina and ttk are mostly consistent with the model that Phyl functions together with Sina to promote es organ development by degrading Ttk. In embryos, strong defects are detected only when both maternal and zygotic sina transcripts are removed, suggesting that maternally contributed sina transcript play an essential role in the development of embryonic es organs. Consistently, no genetic interaction between zygotic sina and phyl, and between zygotic sina and ttk was detected in embryonic PNS development. In adults, the bristle phenotypes in sina mutants are weaker than in phyl mutants. One possible reason is that the perdurance of sina gene products from maternal transcripts might supply activity for some adult bristles to develop normally. Another possibility is that phyl is able to down-regulate ttk activity in a sina-independent manner. In the Drosophila genome, a sequence (CG 13030) is located next to sina in the genome and encodes a putative protein with 50% identity and 70% similarity with Sina. It might be possible that sina functions redundantly with this gene in bristle development (Pi, 2001).

These studies of phyl/sina/ttk in es organ development and previous studies in photoreceptor differentiation indicate that the Drosophila eye and es organs depend on the same protein complex to specify their cell fate. In both cases, phyl mutations transform neural cells to non-neural cells. Both studies also show that phyl expression is tightly regulated by the upstream signaling pathways. The expression of phyl is activated by the Ras pathway in photoreceptor cells. In SOP cells, the transcription of phyl is likely activated by the proneural genes ac and sc, and is repressed by N signaling. Interestingly, it has been shown that the Egrf/Ras/Raf pathway acts antagonistically with the N pathway in SOP formation of adult macrochaetes and chordotonal organs. Whether these two pathways converge on phyl expression to regulate sensory organ formation remains to be examined (Pi, 2001).

tramtrack is required for dorsal appendage elongation

The Drosophila gene tramtrack encodes two transcriptional repressors, Ttk69 and Ttk88, which are required for normal embryogenesis and imaginal disc development. A novel female sterile allele of tramtrack called twin peaks (ttk^twk) has been characterized that, unlike other tramtrack alleles, has no effect on viability and produces no obvious morphological defects, except during oogenesis. Females homozygous for twin peaks produce small eggs with thin eggshells and short dorsal respiratory appendages. Complementation analyses, immunolocalization, and rescue data demonstrate that these defects are due to loss of Ttk69, which is expressed in the follicle cells and is required for normal chorion production and dorsal follicle-cell migration. Analyses of phenotypes produced by mutations in other loci that regulate eggshell synthesis suggest that the chorion production and follicle-cell migration defects are independent. Evidence suggests that twin peaks disrupts a promoter or promoters required for late-stage follicle-cell expression of Ttk69. It is hypothesized that loss of Ttk69 in all follicle cells disrupts chorion gene expression and lack of function in dorsal anterior follicle cells inhibits morphogenetic changes required for elongating the dorsal appendages (French, 2003).

Dorsal appendage morphogenesis in Drosophila oogenesis has been used as a model system for studying the relationship between patterning and morphogenesis. Each of the two dorsal respiratory appendages of the Drosophila egg chamber is formed by secretion of eggshell proteins into a tube of follicle cells. This tube is generated by cell shape changes and rearrangements within an epithelial sheet. Dorsal appendage formation is therefore similar to more complicated examples of organogenesis. In addition, the study of dorsal appendage formation provides several advantages that make it an excellent system for investigating the regulation of epithelial morphogenesis. For example, the signaling events that determine two populations of dorsal follicle cells are well understood. This understanding facilitates an ability to uncouple effects on patterning from morphogenesis. Further, powerful genetic tools, including mutations that disrupt dorsal appendage formation, have allowed for an unraveling of the genetic circuitry underlying the regulation of epithelial morphogenesis (French, 2003).

The Drosophila egg chamber contains 16 interconnected germline cells, consisting of 1 oocyte nourished by 15 highly polyploid nurse cells; these germline cells are surrounded by a monolayer of ~1000 somatic follicle cells. The follicle cells secrete the chorion that makes up the three layers of the eggshell: the vitelline envelope, the endochorion, and the exochorion. A subset of these follicle cells undergoes morphogenesis to generate the dorsal appendages, specialized structures that facilitate gas exchange in the developing embryo (French, 2003).

At stage 10 of oogenesis, the oocyte occupies the posterior half of the egg chamber, the nurse cells the anterior half, and the oocyte nucleus is positioned at the dorsal anterior corner of the oocyte. The majority of follicle cells forms a columnar layer over the oocyte, while a few follicle cells are stretched out over the nurse cells. During stage 10B, those follicle cells closest to the nurse cell/oocyte boundary begin to migrate centripetally, between nurse cells and oocyte. The centripetal cells secrete the operculum (a thin layer of chorion that functions as an escape hatch for the larva), the collar (a hinge on which the operculum swings), and the micropyle, a coneshaped structure through which the sperm enters (French, 2003).

Shortly after centripetal migration (stage 10B), the nurse cells rapidly transfer their contents into the oocyte (stage 11) then begin to degenerate and undergo apoptosis (stages 12-14). At the same time, two groups of approximately 65-80 anterior, dorsal follicle cells, one on each side of the dorsal midline of the egg chamber, migrate over the nurse cells, laying down the chorion of the two dorsal appendages. Extensive studies have defined the signaling events that determine two populations of dorsal follicle cells. Dorsal follicle-cell fate determination begins when transcripts encoding the TGFalpha-like signaling molecule Gurken (Grk) become localized in a cap above the oocyte nucleus. Grk signals via the epidermal growth factor receptor homolog (Egfr) to the follicle cells, activating a signal transduction cascade involving the Ras/Raf/MAPK pathway. This initial signaling event defines a set of dorsal anterior follicle cells and induces a second signaling cascade involving three additional Egfr ligands. This second cascade amplifies and refines the initial Grk signal, leading to the definition of two separate populations of dorsal follicle cells. These events are required for the production of two separate dorsal appendages. Disruptions of this process result in dorsalization or ventralization of the follicular epithelium and the eggshell. Partial ventralization generally results in failure to determine two separate populations of cells, leading to the production of a single dorsal appendage at the dorsal midline. Complete ventralization results in the absence of dorsal cell fates and the concomitant loss of dorsal appendages (French, 2003).

Information along the anterior-posterior axis also contributes to cell-fate determination within the dorsal appendage primordia. The BMP2/4 homolog encoded by dpp is expressed in the stretch cells and a single row of centripetally migrating cells. This morphogen radiates posteriorly and alters columnar cell fates. High levels of Dpp repress dorsal identities and specify operculum; moderate levels synergize with Grk to define dorsal, while low levels of Dpp are insufficient to allow cells to respond to Egfr signaling. Thus, loss-of-function mutations generate short, often paddleless appendages, while overexpression either expands the operculum at the expense of appendage material or creates multiple, often antler-shaped dorsal structures. The subsequent events underlying dorsal appendage morphogenesis are only beginning to be understood. Analyses of cultured wild-type egg chambers have revealed several phases of dorsal appendage morphogenesis. From stages 10B to 12, two groups of dorsal anterior follicle cells move out from the follicular epithelium to form short tubes. Each tube extends forward over the nurse cells, secreting chorion proteins that make up the cylindrical stalk of the dorsal appendage. Cells at the anterior end of the tube change shape to produce the flattened paddle of the distal dorsal appendage. Finally, upon oviposition, the entire follicular epithelium sloughs off, leaving behind the chorionic structures (French, 2003).

The most striking defect is a failure of the ttk^twk mutant to complete dorsal anterior follicle-cell migration, leading to the production of short, nublike dorsal appendages. Female flies homozygous for twk also produce small, round eggs with a weak eggshell. This chorion defect causes the eggs to be fragile and easily ruptured, and is likely due to a severe reduction in transcript levels of at least one gene in the third-chromosome cluster of chorion genes, as well as a moderate reduction of transcription of the X-chromosome chorion-gene Cp36 (French, 2003).

While many known mutations affect chorion synthesis, nearly all reduce chorion gene amplification. The proteins that regulate transcription of the chorion genes are not known. Two transcription factors that bind to the promoter of the chorion gene Cp15 have been isolated, but the functional significance of this binding has yet to be investigated. In twk egg chambers, chorion gene amplification is normal; nevertheless, these egg chambers produce extremely weak eggshells due to reduced mRNA from at least two of the major chorion genes. Ttk69 is consequently the only known protein required for the transcription of any of the major chorion genes (French, 2003).

The effect on chorion gene transcription could arise in two ways. Since Ttk69 is a transcription factor, it is tempting to speculate that the chorion genes are direct targets for transcriptional regulation by Ttk69. Alternatively, regulation of the chorion genes by Ttk69 may be indirect, a result of Ttk69 regulating factors upstream of chorion gene transcription. This second hypothesis is favored for two reasons. (1) The hypothesis that Ttk69 directly regulates the chorion genes requires that Ttk69 function as a transcriptional activator. Currently, however, the only demonstrated function for Ttk69 is as a repressor of transcription. (2) From analyses of BDGP sequences, no matches to the Ttk69 consensus-binding sequence were found within 1 kb of any of the major chorion genes. Thus, it is more likely that Ttk69 regulates factors upstream of the chorion genes rather than the chorion genes themselves (French, 2003).

Intriguingly, the effect of the twk mutation on chorion gene expression is not uniform. While a reduction in the amount of at least two of the major chorion transcripts is seen, the genes in the third-chromosome cluster are affected to a much greater degree than the X-chromosomal gene Cp36. This result is interesting in light of the exquisite temporal regulation of the individual chorion genes: although the genes are organized into clusters, each gene is expressed during a precise and unique window in time. Thus, further analysis of twk may shed light on the individual regulation of the chorion genes. In addition to the defects in expression of the major chorion genes, an apparent vitelline envelope defect is seen in twk eggs. It is therefore possible that Ttk69 is required for production of vitelline envelope proteins. Alternatively, this phenotype may reflect a requirement for one or more of the major chorion proteins in stabilizing the vitelline envelope. Indirect evidence supports this latter hypothesis: until stage 12, Cp36, the earliest of the major chorion proteins, accumulates predominantly in the vitelline envelope, and dec2 mutant egg chambers lacking Cp36 display the same vitelline envelope defect exhibited by twk egg chambers. Thus, interactions between chorion layers may be required for structural integrity of the eggshell (French, 2003).

In wild-type ovaries, the dorsal anterior follicle cells undergo a characteristic series of cell shape changes and movements leading to the production of a tube of cells that secrete the chorionic appendages. These events initiate normally in twk ovaries but arrest soon after the beginning of tube extension. Although Ttk69 is present throughout oogenesis, the loss-of-function phenotype is not evident until stage 12. This delay may be due to early transcription from exon 1b, which is not disrupted in twk ovaries, combined with perdurance of the protein until stage 12. Alternatively, tube formation, which occurs normally in twk ovaries, and tube extension, which is defective, may be distinct processes under separate genetic regulation. Indeed, mutations in kayak (Fos), hemipterous (JNKK), Jun related antigen (Jra, Jun), and puckered (JNK phosphatase) all lead to the production of short dorsal appendages, in some instances very similar to twk. These investigations demonstrate that proper regulation of the Jun kinase pathway is required for normal tube extension and support the hypothesis that the initiation of morphogenesis is a process distinct from tube extension. The possibility that ttk might regulate the Jun kinase pathway in follicle cells, or vice versa is considered. twk mutations do not alter the expression of Jun. Furthermore, strong alleles of basket (JNK) and Jra (Drosophila Jun) do not dominantly enhance the twk dorsal appendage phenotype. These genes may act in separate pathways, both of which regulate tube extension. Alternatively, Ttk69 might act downstream of the JNK pathway and loss-of-function resulting from twk mutations is so severe that defects in other pathway members produce no discernable change in dorsal appendage morphology (French, 2003).

The finding that twk is an allele of ttk is somewhat unexpected for several reasons. Of the more than 50 published alleles of ttk, nearly all are lethal; the rest display reduced viability. The lethality associated with mutations in ttk is attributed to several factors, including defects in the developing nervous system, failure of dorsal closure, and other early embryonic defects. A completely viable but female sterile allele of ttk, ttk^twk, reveals an unexplored function for Ttk69 during oogenesis (French, 2003).

The P element in twk is inserted into a 5' exon that was previously uncharacterized. Examination of EST data provided by the BDGP as well as computational analysis indicate that at least two promoters for the ttk gene may exist in this region, and that loss of transcription from one or both of these potential promoters leads to an oogenesis specific defect. Neither of these potential promoters is specific to oogenesis; furthermore, twk disrupts transcription from this upstream region not only in oogenesis, but also in adult tissues. Nevertheless, transcription beginning in this exon is absolutely required for columnar follicle cell expression of ttk late in oogenesis. Presumably, alternative promoters can provide Ttk69 function in other tissues (French, 2003).

Ttk69 has been most extensively characterized in the developing embryonic nervous system, where it serves primarily as a bimodal cell fate switch, repressing transcription of genes required for determination of neuronal cell fate. In twk ovaries, however, dorsal anterior follicle cell fate is correctly determined. The expression patterns of Broad, which responds to both Grk and Dpp signaling, and Jun, which is regulated by the JNK pathway, are completely normal in twk egg chambers. Thus, ttk must function in dorsal anterior follicle cells after initial cell fate determination (French, 2003).

What, then, is the function of ttk in dorsal appendage morphogenesis? Assuming that Ttk69 functions as a transcriptional repressor in the ovary as it does in other tissues, it can be speculated that a gene or genes must be repressed to allow tube extension to proceed. One simple hypothesis is that cell rearrangements or shape changes are necessary to allow part of the follicular epithelium to reorganize and extend forward over the nurse cells. Cell rearrangements might require the down-regulation of adhesion between cells, while shape changes might require altered levels of cytoskeletal proteins or apical/basal polarity determinants. Thus, it is tempting to speculate that one function of ttk in dorsal appendage formation is the repression of genes encoding regulators of cell morphology or adhesion (French, 2003).

Control in time and space: Tramtrack69 cooperates with Notch and Ecdysone to repress ectopic fate and shape changes during Drosophila egg chamber maturation

Organ morphogenesis requires cooperation between cells, which determine their course of action based upon location within a tissue. Just as important, cells must synchronize their activities, which requires awareness of developmental time. To understand how cells coordinate behaviors in time and space, Drosophila egg chamber development was analyzed. The transcription factor Tramtrack69 (TTK69) was found to control the fates and shapes of all columnar follicle cells by integrating temporal and spatial information, restricting characteristic changes in morphology and expression that occur at stage 10B to appropriate domains. TTK69 is required again later in oogenesis: it controls the volume of the dorsal-appendage (DA) tubes by promoting apical re-expansion and lateral shortening of DA-forming follicle cells. TTK69 and Notch were shown to compete to repress each other's expression, and a local Ecdysone signal is required to shift the balance in favor of TTK69. It is hypothesized that TTK69 then cooperates with spatially restricted co-factors to define appropriate responses to a globally available (but as yet unidentified) temporal signal that initiates the S10B transformations (Boyle, 2009).

This paper has demonstrated that TTK69 plays a central role in regulating the behavior of follicle cells during mid-to-late oogenesis. At S10 and again at S12, TTK69 integrates temporal and spatial information to coordinate the behaviors of subpopulations of follicle cells (Boyle, 2009).

Elsewhere in development, TTK69 acts as a determinant of cell fate. Best studied is its role in binary cell-fate decisions downstream of N-mediated lateral inhibition, such as in asymmetric sensory organ precursor (SOP) cell division, where N becomes active in one of two daughter cells, leading to TTK69 expression and repression of neural determinants. TTK69 also acts downstream of N to promote endocycle entry at S6, and in the transition from endocycle to chorion gene amplification at S10. Recent studies demonstrate a role for TTK69 in morphogenesis. ttk^twk, an allele of ttk69 that does not disrupt patterning, prevents DA elongation, and TTK69 is required for proper cell shape and fate during tracheal morphogenesis (Boyle, 2009).

This study advances understanding of TTK69 significantly by reporting four novel mechanisms of TTK69 action. (1) TTK69 controls DA tube volume by inducing apical expansion and lateral shortening, independent of basal extension. (2) Loss of TTK69 in mid-oogenesis causes cells to adopt inappropriate fates without inducing a binary cell-fate switch. (3) A surprising, mutually repressive relationship was found between TTK69 and N that is modulated by Ecdysone receptor activity. (4) TTK coordinates the response to temporal and spatial signals and thereby ensures the fidelity of egg chamber maturation (Boyle, 2009).

Although apical constriction has been well studied, less is known about apical re-expansion and the mechanisms that determine final apical size. Surprisingly, ttk^twk reveals independent control of apical and basal surfaces. Although DA-forming cells elongate, the tube extends only when roof-cell apices expand (Boyle, 2009).

The ttk^1e11 allele deletes a portion of the TTK69 zinc finger. Although long regarded as null, The possibility is considered that some truncated protein, expressed even at undetectable levels, might cause gain-of-function effects. Several lines of evidence support the argument against this possibility. First, heterozygous cells, the majority in the ttk^1e11 mosaics, show no phenotype; thus, any gain-of-function effect would be recessive, a very rare occurrence. Furthermore, N-CA phenocopies ttk^1e11 and causes TTK69 downregulation, independently demonstrating that TTK69 reduction produces the ttk^1e11 phenotypes (Boyle, 2009).

ttk^1e11 caused dramatic shape and fate transformations in all columnar follicle cells, leading to cells with highly constricted apices, elevated E-cadherin, and altered Broad (BR) levels. These changes did not result from constitutive activation of BR or cell-shape determinants, as S10A and earlier egg chambers did not exhibit any of these phenotypes. Thus, TTK69 regulates diverse processes at S10B. Unlike its role in SOP cell division, TTK69 does not switch cells from one type to another. Rather, ttk^1e11 cells take on aspects of many S10B follicle cell subtypes, not all of which are normally exhibited by a single cell (e.g. high basal E-cadherin and apical constriction) (Boyle, 2009).

Ecdysone signaling was required to flip the bistable relationship between N and TTK69 toward TTK69 at S10. Inactivating Ecdysone receptor via a dominant-negative construct largely mirrored ttk^1e11. Additional phenotypes, such as failure to generate stretch cells, were probably due to earlier requirements for EcR function, processes that do not involve TTK69. Interestingly, TTK69 levels were sometimes elevated in the cytoplasm and excluded from the nuclei of EcR-DN-expressing cells, indicating that Ecdysone may promote nuclear localization of TTK69. This mechanism could provide more rapid and reversible control over TTK69 activity than transcriptional regulation. The mutually repressive relationship between TTK69 and N could accelerate this change, once triggered (Boyle, 2009).

How does Ecdysone signaling produce this change? Autocrine signaling within the germline could induce downregulation of Delta, thereby reducing N activity. At the same time, Ecdysone signaling to the follicle cells could stabilize TTK69 in the nucleus, modulating expression of target genes that control N degradation. Alternatively, as activated N can block Ecdysone signaling at S10B, decreased Delta activity could allow Ecdysone receptor function in the follicle cells, indirectly activating TTK69. Regardless of the exact mechanism, these relationships would act as a positive feedback loop to ensure a rapid and reliable switch between the two states (Boyle, 2009).

The transition from S10A to S10B is marked by the adoption of specific follicle-cell fates in positions specified by EGF and DPP signaling. Strikingly, many of the observed phenotypes appeared at S10B but not S10A, revealing roles for TTK69, N and EcR in regulating temporal maturation to S10B. Thus, fundamental differences exist between S10A and S10B follicle cells. It is hypothesized that a signal experienced by all follicle cells induces this change. Importantly this signal cannot be a combination of EGF and DPP, as these signals are spatially restricted to the dorsal anterior at this stage, and ttk^1e11, UAS-N-CA and UAS-EcR-DN have equivalent effects in all columnar follicle cells (Boyle, 2009).

What is this signal? Probably, several factors contribute. Ecdysone itself is an interesting candidate. While ttk^1e11 mutant and N-CA-expressing cells were apically constricted after S10B, EcR-DN-expressing cells more closely resembled S10A cells (smaller overall with smaller nuclei). This distinction could indicate that Ecdysone is required to progress beyond S10A. EcR-DN-expressing cells, however, do not perfectly resemble S10A cells. Differences could be due to dominant effects of EcR-DN complexes, which could strongly repress EcR targets rather than simply failing to activate them. Another potential input at this time could be prostaglandin signaling, which induces nurse-cell dumping. Finally, the egg chamber grows during this period and overall egg chamber nutrition could determine the timing of the S10A-S10B transition. Insulin signaling is required earlier for vitellogenesis and may continue to monitor growth during this period (Boyle, 2009).

How does TTK69 integrate this temporal signal with the spatial pattern to achieve appropriate responses at the correct time and place? ttk^1e11 cells adopt aspects of multiple cell types. In wild type, each cell performs a subset of behaviors, repressing inappropriate responses in a ttk69-dependent fashion. For example, roof cells need TTK69 to prevent BR downregulation, whereas main body cells need TTK69 to prevent apical constriction. How does TTK69 repress different processes in different cells? Spatial information from DPP and EGF must contribute to TTK69 activity. TTK69 expression, however, although dynamic and variable, has no consistent spatial pattern and is required in all columnar follicle cells (Boyle, 2009).

A conceptual solution is to imagine TTK69 working in concert with two co-factors that are spatially restricted by EGF and DPP signaling: one co-factor is expressed in a U\M pattern, all follicle cells except the T using the system described previously (see Yakoby, 2008), and one is present in a U\R pattern (all follicle cells except the roof). When combined with TTK69, the U\M factor would repress basal E-cadherin, N expression and the early clearance of BR that normally occurs in the T. In combination with TTK69, the U\R factor would repress apical constriction and BR upregulation, which normally occur in the roof. Removal of TTK69 causes a failure to repress all of these behaviors, leading to the observed phenotypes, with moderate BR levels as a result of both up- and downregulation. These co-factors could take the form of BTB transcription factors that dimerize with TTK69. Alternatively, they could modify TTK69 by phosphorylation or mono-ubiquitylation or affect expression of other genes that alter the response to TTK69 function (Boyle, 2009).

In conclusion, this study has identified several novel functions for TTK69. At S10, presumably in collaboration with region-specific interacting proteins, it facilitates spatially correct responses to a uniform temporal signal; its function at S12 coordinates a return to a cuboidal cell shape. The regulatory network by which TTK69, N and Ecdysone receptor control the progression of egg chambers from mid- to late oogenesis could serve as a simple model to explain how a bistable system flips its state to regulate the temporal progression of development (Boyle, 2009).

Division of labor: Subsets of dorsal-appendage-forming cells control the shape of the entire tube

The function of an organ relies on its form, which in turn depends on the individual shapes of the cells that create it and the interactions between them. Despite remarkable progress in the field of developmental biology, how cells collaborate to make a tissue remains an unsolved mystery. To investigate the mechanisms that determine organ structure, this work studied the cells that form the dorsal appendages (DAs) of the Drosophila eggshell. These cells consist of two differentially patterned subtypes: roof cells, which form the outward-facing roof of the lumen, and floor cells, which dive underneath the roof cells to seal off the floor of the tube. This paper presents three lines of evidence that reveal a further stratification of the DA-forming epithelium. Laser ablation of only a few cells in the anterior of the region causes a disproportionately severe shortening of the appendage. Genetic alteration through the twin peaks allele of tramtrack⁶⁹ (ttk^twk), a female-sterile mutation that leads to severely shortened DAs, causes no such shortening when removed from a majority of the DA-forming cells, but rather, produces short appendages only when removed from cells in the very anterior of the tube-forming tissue. Additionally it was shown that heterotrimeric G-protein function is required for DA morphogenesis. Like TTK⁶⁹, Gβ13F is not required in all DA-forming follicle cells but only in the floor and leading roof cells. The different phenotypes that result from removal of Gβ13F from each region demonstrate a striking division of function between different DA-forming cells. Gβ mutant floor cells are unable to control the width of the appendage while Gβ mutant leading roof cells fail to direct the elongation of the appendage and the convergent-extension of the roof-cell population (Boyle, 2010).

Much of the study of developmental biology has focused on understanding the mechanisms by which cells become selected to perform various roles in the formation of structures. Classically, this patterning involves changes in gene expression that often presage further changes in form and behavior. For example, during oogenesis in Drosophila, a uniform epithelium of follicle cells surrounds a cluster of 16 germline-derived cells composed of 15 nurse cells and one oocyte. By stage 10 (S10, of 14 stages) signaling between these cell types has established a polarized microtubule network within the oocyte and has also defined distinct domains within the follicular epithelium. Midway through S10 (at the onset of S10B), the follicle cells over the oocyte appear indistinguishable from each other at a morphological level, but have already become patterned into subregions destined for different functions. Most cells will secrete the main body of the eggshell while others will synthesize specialized structures involved in fertilization, gas exchange, or hatching (Boyle, 2010).

This study has investigated the link between the patterning and morphogenesis of the follicle cells that make the dorsal appendages (DAs), protrusions of chorion that aid in making oxygen accessible to the embryo. Once the cells destined to participate in DA formation have been determined—a process that culminates in S10B—these cells rearrange to form a tube during S11 and elongate that tube through S12 and S13, reaching the final product at S14. Notably, mitosis in follicle cells stops during S6, so this morphogenesis is achieved solely through cell shape change and movement. This cessation of cell division also places an upper limit on the time at which the genetic mosaics discussed in this paper were generated; thus, the expression of the gene removed will have ceased at least 13 h before the initiation of tube formation. The exception to this general rule involves the tramtrack^twk (ttk^twk) allele, in which a P element inserted into an upstream promoter disrupts expression only late in oogenesis. Since one of the proximal promoters (1b) is still functional early in oogenesis, TTK69 expression persists until S10B, making the ttk^twk allele useful in avoiding earlier tramtrack requirements (Boyle, 2010).

The patterning of the DA-forming cells starts with signaling by the EGF and DPP pathways, which leads to changes in the level at which follicle cells express the transcription factor Broad (BR). BR is initially expressed at a uniform, low level throughout the epithelium, but at S10B the cells destined to form the roof of the DA lumen express elevated BR. The single row of cells destined to dive underneath the roof to seal off the floor of the lumen (as well as adjacent cells that contribute to the anterior face of the eggshell) cease expressing BR altogether. Collectively the roof and floor cells form the DA primordium (Boyle, 2010).

While much about the mechanisms by which these cell types are patterned have been discovered, comparatively little is known about their relative functions in the process of DA morphogenesis. What forces do the roof and floor cells generate? Does anterior migration of the floor cells pull the roof cells forward or vice versa? Does convergent extension of the roof cells pull the floor, thereby determining the width of the tube, or does the inward migration of the floor drive convergent extension in the roof cells? Beyond even the roof vs. floor distinction, cells in different regions of the DA-forming epithelium must perform distinct tasks. For instance, among the roof cells lining the anterior of the primordium, the cells on the lateral side must swing toward the posterior in a hinge-closing movement while those at the hinge point must migrate anteriorly over the squamous follicle cells that lie over the nurse cells (the stretch cells) in order to elongate the tube. If cells form these different locations were removed or immobilized, how would the rest of the structure react? Two primary hypotheses are considered. First, the remaining cells may re-organize to take over the role of the missing cells no matter which cells are affected. Second, genetically altering or laser ablating cells at certain positions may cause the unrecoverable failure of a subset of the movements required for proper DA formation. Due to their obvious patterning differences, it was predicted that roof cells would not be able to assume the role of the affected floor cells. The possiblity was also considered that the roof and floor consisted of additional subregions, and it was asked exactly which subset of behaviors would be impacted when disrupting cells at various positions within the DA primordium (Boyle, 2010).

This paper addresses these questions using laser cell ablation and genetic mosaics. It was found that the DA primordium can be split not only into the two known regions (roof and floor) but that the roof population itself consists of two regions, which are referred to as the 'leading roof', those roof cells adjacent to the floor cells, and the 'trailing roof', the remainder of the roof-cell population. Of particular interest is the area surrounding the hinge point, as these cells lead the anterior migration. Ablation of a few cells near the hinge causes disproportionate defects in DA elongation, while ablation of cells in the posterior of the DA primordium causes only very minor elongation defects. Further, mosaics were generated of ttk^twk, a mutation in an upstream regulatory region that affects expression of the transcription factor TTK69 during S10B and later, and which causes a severe DA elongation defect. ttk^twk does not cause DA defects when removed from the majority of DA-forming cells, but only when clones occur in the anterior of the structure. Finally a role was demonstrated for heterotrimeric G-protein signaling in DA morphogenesis, and it was shown that Gβ13F is required for distinct behaviors in the floor and the leading roof, yet is dispensable from the remainder of the follicle cells (Boyle, 2010).

This study presents the strongest evidence to date that cells from distinct regions of the DA primordium are responsible for specific shape changes affecting the entire structure. Cells from the anterior 1/3 of the DA-forming region are responsible for elongation of the whole tube while the remaining cells are dispensable. Leading roof cells control convergent extension of the whole population, and floor cells control the lateral width of the lumen. Thus, it was shown that the DA-forming epithelium is more finely stratified than previously appreciated, both in terms of the roles that cells play in morphogenesis and the genes that they require to perform those roles. In doing so, it was demonstrated that heterotrimeric G-protein signaling is required for DA morphogenesis. A subset of the roof cells, defined as the leading roof, requires TTK69 and Gβ13F for DA elongation, yet those genes are not required in trailing roof cells. These results suggest that the leading roof cells, rather than the roof population as a whole, are responsible for driving DA elongation. Similarly, the different DA shapes that result when removing Gβ13F from the floor cells versus the roof suggest that the floor, and not the roof, controls lumen width (Boyle, 2010).

Laser ablation of the leading DA-forming cells severely disrupted tube elongation. Although dying cells could physically block more posterior cells, similar phenotypes occurred when disrupting leading cells by generating loss-of-function clones. Mosaic analysis also has potential drawbacks, including removal of gene function at earlier developmental periods (except when using specific alleles such as ttk^twk). Large clones are generated earlier in development, however, and such clones did not produce an effect unless specific cells were targeted, those same cells that exhibited a disproportionate effect upon laser ablation. Thus, these two distinct but complementary approaches reveal the importance of spatial position during DA morphogenesis (Boyle, 2010).

Clonal analysis of ttk^twk demonstrated that TTK69 is required during tube elongation, S12-S13, only in the anterior of the DA-forming region. This finding is particularly surprising for two reasons. One, earlier in oogenesis, at S10, TTK69 is required in all columnar follicle cells to pattern the epithelium; loss of function at that time produces cell shape changes in any cell that is mutated. Two, morphological analysis of ttk^twk cells revealed highly elongated yet apically constricted cell shapes throughout the DA-roof population. So how could removal of TTK69 (or Gβ13F) from the leading cells disrupt cell behaviors in other DA regions where these genes are still expressed? Two primary hypotheses are considered: TTK69, G proteins, or any such factor could be required specifically in those leading cells to relay a signal to the more posterior DA cells, which then respond by changing their shape and producing a correctly shaped DA. An alternative hypothesis is that these factors are required only to shape the leading roof and/or floor cells, which then pull or otherwise physically constrain the remainder of the cells into proper shape (Boyle, 2010).

Since the leading roof and floor cells outline the DA, moving them into proper position may be sufficient to shape the appendage if the remaining cells simply fill in the space in a lowest-energy fashion. It is also possible that TTK69 and Gβ13F act in all DA cells, but in a way that is only relevant to the leading roof and floor cells due to geographical constraints. For example, TTK69 and Gβ13F could regulate expression or activity of a heterophilic cell-cell adhesion molecule responsible for adhesion between roof cells and another cell type. If the function of this hypothetical adhesion protein were to diminish in some cells, it would not matter as long as those cells never come in contact with the cells expressing the protein's ligand. As the trailing roof cells contact only other roof cells along lateral surfaces (contacting the DA lumen at apical surfaces and extracellular matrix at basal surfaces), all would be well as long as proper function occurred in the leading roof that does contact other cell types (Boyle, 2010).

What role do G proteins play in DA morphogenesis and what molecular mechanisms underlie their genetic interaction with TTK69? The failure of Gβ13F^δ–96A mutant cells to cross the nurse-cell/oocyte boundary is suggestive. G-protein-coupled receptor signaling could be required in the DA-forming cells to receive a signal sent from the stretch -- or possibly some other anterior -- cells, a signal that attracts or orients them. Alternatively, the failure in convergent extension when Gβ13F is removed from the roof cells indicates that G proteins might be required to establish planar cell polarity at the boundary between roof and floor cells. This planar polarity could be required in turn not only for convergent extension of the roof, but also to orient the direction of roof-cell migration. Similar defects in the planar orientation of the floor cells could explain their disorganization and the consequent widening of the lumen (Boyle, 2010).

That Gγ1 is a suppressor of the ttk^twk phenotype suggests that TTK69 and heterotrimeric G-protein signaling downstream of the Gβγ subunit may be opposed in function, but the various G-protein mutants that were observed did not result in longer DAs, but in shorter ones. This result need not be inconsistent from a molecular perspective, however. In order for DA-forming cells to migrate toward the anterior of the egg chamber -- and in particular to cross over the nurse-cell/oocyte boundary -- they will require both the assembly and disassembly of adhesion complexes with their substrate, the stretch cells. If TTK69 and Gβ13F affect this expression in opposite directions, it would explain their opposition from a molecular perspective while still being required in the leading cells for anterior elongation of the DA tube (Boyle, 2010).

This hypothesis also explains the ttk^twk and Gβ13F phenotypes. ttk^twk results in highly elongated DA-roof cells stretched out along the path of their migration, consistent with an inability to disassemble adhesion complexes with the substrate along their migration path. By contrast, Gβ13F^δ1–96A mutant cells reach up to, but do not cross over, the stretch-cell-covered nurse cells, consistent with an inability to form such contacts (Boyle, 2010).

This study reveals that the boundaries of subpopulations are especially likely to behave in special ways due to their unique access to adjacent cell types. Parallels in other systems lead to the suggestion that this result is likely to be broadly applicable beyond DA morphogenesis. For instance, during Drosophila tracheal morphogenesis, a small number of cells at the tip of the branch directs the convergence of the cells that follow them. Similarly, during wound healing, cells along the leading edges form an actin purse-string and extend filopodia toward each other to facilitate the rapid closure of the gap. Like the leading roof cells, these cells at the edges are not fundamentally different from the cells that follow behind (both leading and trailing cells being, ultimately, a part of the wounded structure), yet their location causes them to perform an important function upon which all the cells behind them depend (Boyle, 2010).

In conclusion, this study has increased the resolution with which the process of DA morphogenesis can be understood, jointly in terms of the spatial organization of the cells that form the DA tubes, in terms of the functions of subpopulations in driving DA morphogenesis, and in terms of identifying new pathways that regulate tube elongation. Such a reductionist approach has proven highly valuable when studying other systems, such as gastrulation, where the development of modern understanding was dependent on breaking the complex movements into specific components such as convergent extension and spreading. Using the simpler system of DA morphogenesis, these intricate processes cn be understood at a higher level of detail, thereby providing a model system that facilitates understanding morphogenesis from its most basic molecular components to the overarching macroscopic events (Boyle, 2010).

Tramtrack is genetically upstream of genes controlling tracheal tube size in Drosophila

The Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. Multiple roles of Ttk have been discovered in the development of the tracheal system on the morphogenetic level. This study sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. Gene expression changes were profiled after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes (Rotstein, 2011).

The microarray results confirm previous observations and provide new data for the different Ttk tracheal requirements. For instance, the transcription factor Esg, which plays a pivotal role in fusion cell identity specification is lost when Ttk is over-expressed, but still present in Ttk loss-of-function conditions. The microarray data confirm this regulation, and in addition identifies other genes already shown to directly or indirectly modulate fusion fate as Ttk targets, like hdc, CG15252, or pnt. Similarly, polychaetoid (pyd), which has been identified as a Ttk target in in situ hybridisation analysis, is differentially expressed in the microarray conditions (it should be noted however that pyd is not formally a candidate due to inconsistencies between microarray replicates; in fact only splice variant pyd-RE shows a response), explaining in part the requirement of Ttk in tracheal cell intercalation. In addition, it is tempting to speculate about other candidate targets to mediate this function of Ttk in intercalation, like canoe for instance, which has been recently shown to act with pyd during embryogenesis (Rotstein, 2011).

The microarray analysis pointed to a regulation of the Notch signalling pathway or its activity by Ttk, likely acting as a negative regulator. In contrast, it has previously been observed that Ttk acts as a downstream effector of N activity in the specification of different tracheal identitites. Indeed, it was shown that Ttk levels depend on N activity in such a way that when N is active, Ttk levels are high, whereas when N is not active, Ttk levels are low. Thus, lower levels of Ttk were observed in tracheal fusion cells due to the inactivity of N there. Therefore, Ttk acts as a target of N in fusion cell determination. Now, the results of the microarray add an extra level of complexity to the Ttk-N interaction. The observation that in turn Ttk also transcriptionally regulates several N pathway components suggests that Ttk is involved in a feedback mechanism that could play a pivotal role in biasing or amplifying N signalling outcome (Rotstein, 2011).

Interactions between Ttk and N have been observed in different developmental contexts, emphasising the importance of such regulations. Several examples illustrate the regulation, either positive or negative, of Ttk expression by N activity. In addition, a recent report provides evidence of a regulation of N activity by Ttk and proposes a mutually repressive relationship between N and Ttk which would also involve Ecdysone signalling. The results are consistent with many of these observations, indicating that they could represent general molecular mechanisms of morphogenesis. Thus, tracheal cell specification could serve as an ideal scenario to investigate the intricate, and often contradictory, interactions between N and Ttk and the complexity of N signaling (Rotstein, 2011).

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date revised: 17 August 2020

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