fork head


Targets of Activity

FKH binds specifically to the DNA target sequence of vertebrate HNF3 alpha-gamma (Kaufman, 1994).

Fork head regulates the central gap gene Kruppel in Malpighian tubules. Kruppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. The interactions of Kruppel with other terminal genes has been studied. The gap genes tailless and huckebein, which repress Kruppel in the central segmentation domain, activate Kruppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Kruppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Kruppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Kruppel in the Malpighian tubules differ strikingly from its role in segmentation (Gaul, 1990). Kr activity determines the neural fate of tip cells by acting as a direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. Fork head interacts with a 400 bp cis active element of Krüppel directing Kr expression in Malpighian tubules. There are two FKH binding sites. This element also contains functional binding sites for the restricted proneural bHLH factors (Hoch, 1998).

The molecular characterization is described of the paired-type homeobox gene D-Ptx1 of Drosophila, a close homolog of the mouse pituitary homeobox gene Ptx1 and the unc-30 gene of C. elegans, characterized by a lysine residue at position 9 of the third alpha-helix of the homeodomain. D-Ptx1 is expressed at various restricted locations throughout embryogenesis. Initial expression of D-Ptx1 in the posterior-most region of the blastoderm embryo is controlled by fork head activity in response to the activated Ras/Raf signaling pathway. During later stages of embryonic development. D-Ptx1 transcripts and protein accumulate in the posterior portion of the midgut, in the developing Malpighian tubules, in a subset of ventral somatic muscles, and in neural cells. Phenotypic analysis of gain-of-function and lack-of-function mutant embryos show that the D-Ptx1 gene is not involved in morphologically apparent differentiation processes. It is concluded that D-Ptx1 is more likely to control physiological cell functions than pattern formation during Drosophila embryogenesis (Vorbruggen, 1997).

Segment polarity genes are not activated by pair-rule genes in the head as they are in the trunk, but instead are activated by gap genes. The segment polarity genes hedgehog and wingless are two important targets of cnc and fork head, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labral region of the foregut, fated to give rise to the dorsal pharynx and fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsible for the maintenance of wg in the dorsal pharyngeal domain of wg expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995).

fork head is required for the activation of wingless, hedgehog and decapentaplegic in both the foregut and hindgut, considered to be ectodermal tissues. The larval hindgut can be divided into anterior intestine and the posterior intestine or rectum. The anterior rectum is composed of two different sub-regions, the small intestine and the large intestine. At the transition to the large intestine, there is a narrowing of tissues to form a constriction-like structure in the intestinal wall, and this structure is marked by a circular expression domain of crumbs. The attachment point of the Malpighian tubules marks the anterior boundary of the rectum.

wingless is expressed initially in the whole hindgut primordium, but becomes restricted to a ring in the small intestine anterior to the outgrowing Malpighian tubules, and to a ring in the posterior region of the rectum. hedgehog is also expressed in the hindgut primordium but becomes restricted to a ring of cells posterior to the outgrowing Malpighian tubules in the future small intestine of the hindgut. A second hh expression domain is located in the anterior portion of the rectum. These two expression domains are adjacent to the wg expression domains. dpp is expressed in the hindgut primordium and later on one side in the large intestine of the hindgut tube, in between the small intestine and the rectum. Thus the expression domains of wg, hh and dpp subdivide the hindgut tube into a central portion (the large intestine) where dpp is expressed, and two flanking regions (the small intestine and the rectum) where wg and hh are expressed. In fkh mutant embryos, the foregut, the midgut and the hindgut epithelia are disrupted, and fkh is required for the activation of each of these genes in the fore- and hindgut primordia. fkh is expressed in the entire foregut and hindgut, whereas wg, hh and dpp are expressed only in restricted domains. Since the expression of these genes appear not to be established through cross-regulatory interactions, there must be other factors which act to spatially regulate wg, hh and dpp expression along the hindgut (Hoch, 1996).

Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions, a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells, through the EGF-receptor acts to block fork head expression in preduct cells, thereby restricting gland identity to more dorsal cells. Fork head acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).

Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by fork head. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos. The trh gene product is necessary for invagination of all salivary duct cells and it is required for expression of downstream duct markers. Because eyg is also expressed in part of the salivary duct primordium, the relationship between trh and eyg was tested in the pathway for duct determination. In wild-type embryos, both trh and eyg expression in the salivary primordium begin early during stage 10. At this stage, eyg expression in trh-mutant embryos is indistinguishable from expression in wild-type embryos. Therefore, initiation of eyg expression in the salivary primordium is independent of trh. In early stage 12, however, eyg expression becomes dependent on trh. Although eyg is expressed strongly in the posterior preduct cells of wild-type embryos, this expression is completely absent in trh-mutant embryos. Therefore it is eyg maintenance, and not its initiation, that depends on trh. Whether trh expression depends on eyg was also tested and it was found that trh expression is unaffected in eyg null-mutant embryos (Jones, 1998).

fork head plays an important role in establishing the pregland/preduct border by dorsally limiting duct-specific gene expression. trh, like eyg, is also initially expressed throughout the gland primordium. In fkh-mutant embryos, trh transcript never disappears from the pregland cells (Isaac, 1996). Does fkh play a similar negative regulatory role in eyg transcription? When the wild-type eyg expression pattern is compared to that of fkh-mutant embryos, it becomes clear that fkh indeed negatively regulates eyg. eyg expression persists in gland precursors in fkh-mutant embryos. Thus, fkh represses expression of trh and eyg, both of whose expression disappears from the pregland cells at approximately the same time. eyg plays no role in the regulation of fkh expression (Jones, 1998).

Armed with the knowledge that (1) fkh is responsible for the exclusion of both trh and eyg from the pregland cells and (2) trh is necessary for maintenance of eyg expression in the duct cells, it is possible to ask whether fkh represses eyg in the pregland cells simply by repressing trh or if fkh downregulates trh and eyg independently. To address this question, embryos were generated that were doubly mutant for trh and fkh. If the reason for eyg disappearance from the pregland cells in wild-type embryos is disappearance of trh, then it would be predicted that eyg expression would not persist in trh;fkh-mutant embryos. eyg expression, however, does persist in pregland cells in trh;fkh-mutant embryos, suggesting that trh plays no role in eyg repression by fkh. Thus, after the initial establishment of the salivary primordium by Sex combs reduced, fork head excludes eyegone expression from the pregland cells so that eyegone's salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eyegone expression in the posterior preduct cells (Jones, 1998).

The Drosophila Fork head protein participates in salivary gland formation, since salivary glands are missing in fkh mutant embryos. The Fork head protein binds to an upstream regulatory region of the larval salivary gland glue protein gene Sgs3. Fork head interacts with the TGTTTGC box shown to be involved in tissue-specific Sgs3 expression. Fork head binding is prevented by the same single base substitutions that have been shown to interfere with the TGTTTGC element function in vivo. Furthermore, anti-Fork head antibody binds to >60 sites of polytene chromosomes, including the puffs of all Sgs genes. Fork head protein is detected in the nuclei of salivary glands of larvae at all examined stages. These data provide experimental evidence for the hypothesis that the protein encoded by fork head is required initially for salivary gland formation and is utilized subsequently in the control of larval genes specifically expressed in this organ (Mach, 1996).

A fundamental unresolved question in endocrinological research is how systemic signals like pulses of steroid hormones are converted into a variety of tissue- and stage-specific responses. The existence of three different Ecdysone receptor isoforms, which are differentially expressed in larval and imaginal tissues, may provide the first clue for the differential regulation of these responses. Sgs genes are salivary gland secretion protein genes, regulated in Drosophila by the molt cycle. Two regulatory elements were identified in the upstream region of the Drosophila Sgs-3 gene that are both able to bind the Ecdysone receptor/Ultraspiracle dimer (EcR/USP) and the product of the fork head gene. Interestingly, only one of the EcR/USP binding sites is able to recognize in vitro-translated EcR/USP, which provides evidence for the existence of different receptor forms having different DNA binding specificities. Deletions of the elements leads to a reduced accumulation of Sgs-3 mRNA without altering the temporal expression profile of the gene. The data are consistent with the hypothesis that the Ecdysone receptor directly contributes to the transcriptional activation of Sgs-3 by binding to at least one of the two elements. Since the Sgs-4 gene is also controlled by a functional EcR/USP binding site, a direct participation of EcR/USP in the formation of regulatory complexes may be of general importance for the hormonal control of Sgs genes (Lehmann, 1997).

The Drosophila gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. Sgs-1 was cloned from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences reveals that Sgs-1consists mainly of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 have been mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays reveal binding sites for the transcription factor fork head (Fkh) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. Fkh and SEBP3 are involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This could mean that the dramatic increase in Sgs RNA between early and late third instar is not due to an increase in the rate of transcription controlled by an enhancer element, but in fact is due to a protection of Sgs RNA against degradation, possibly by RNA secondary structure or by a specific factor(s) (Roth, 1999).

To test if the transcription factor Fkh, which controls the tissue-specific expression of Sgs-3 and -4, also binds to regions important for Sgs-1 expression, the Sgs-1 upstream region was scanned for sequences showing similarity to known Fkh binding sites. From 21 candidate sequences in the nt -1 to nt -1200 region containing the core motif TNNGTNA/T, four sequences were selected for binding studies. Only one of these sequences, which is deleted in the C-1021-del lines and mutated by base exchanges in the C-1021-mu lines, proved to be an in vitro Fkh target site. Surprisingly, a second putative Fkh target sequence, located between positions nt -980 and nt -1000, is not recognized by Fkh but is efficiently bound by SEBP3. SEBP3 is a factor that binds to two sites in the upstream region of the Sgs-4 gene that are required for full transcriptional activation of this gene. The SEBP3 binding site of Sgs-1 identified in this study shows a high similarity to the strong SEBP3 binding site of Sgs-4 located arround position nt -422. In particular, both binding sites contain an E-box, suggesting that SEBP3 belongs to the basic helix-loop-helix family of transcription factors. The detection of a SEBP3 binding site in the regulatory region of another Sgs gene, in addition to Sgs-4, suggests that SEBP3 might generally be required for the activation of Sgs genes (Roth, 1999).

The steroid hormone ecdysone controls multiple aspects of insect development, including larval molts and metamorphosis, and can induce specific genetic responses in different tissues. The regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile, have been identified by transgenic analysis. An ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the Fkh regulatory protein, which can potentially modulate the ecdysone genetic regulated response (Crispi, 2001).

For many ecdysone-regulated genes, the definition of the regulatory role that an ecdysone-responsive element can play in the determination of a specific genetic expression profile is often complicated by the dense organization of regulatory sequences responsible for the hormonal response and the developmental expression within short upstream regions acting as multifunctional regulatory domains. This type of organization, where interacting regulatory elements are usually tightly clustered or even superimposed, is common to many target genes in both invertebrate and vertebrate systems, and is often referred as a Hormone Response Unit (HRU). The results reported here reveal that the regulatory sequences directing the expression of the ecdysone-regulated ng-1 gene are organized quite differently. In fact, ng-EcRE deletion or sequence alterations results in a marked reduction of the amount of the ng-1::lacZ fused transcripts, but does not change substantially the developmental expression profile of the mutant transgenes. In addition, the cis-acting regulatory elements responsible for ng-1 tissue and stage-specific expression do not map in close association with the ng-EcRE, but are clustered within a distinct upstream regulatory region acting as a salivary gland enhancer in third instar larvae. Given that the ng-EcRE, in conjunction with its close flanking sequences, functions as an autonomous developmental enhancer, the results obtained here strongly imply that its activity can be widely modulated by additional regulatory elements, and suggest a critical role for context-dependent and combinatorial factor interactions in setting the specificity of its hormonal response. Within the ng-1 upstream enhancer, the cis-acting elements responsible for the temporal expression profile could not be distinguished from those involved in determining the tissue specificity. In fact, while the complete region directs a salivary-gland specific expression in third instar larvae, all the smaller subregions tested fail to activate the expression of the reporter gene. It is plausible that the tight linkage of protein binding sites within this regulatory region might cause this effect, given that, as determined by EMSA analysis, a set of at least five factors binds specifically at this regulatory domain. Thus, full-level activation of ng-1 may depend on a concerted action of EcR binding at the ng-EcRE and on a set of transcription factors, which includes the Drosophila Fkh protein, bound elsewhere, at the upstream control region. At the transcriptional level, the modulation of the ng-1 activity may be accomplished by the ng-EcRE in at least two different ways. (1) ng-EcRE/ecdysone-receptor complex can directly influence the binding or the activity of components of the transcription machinery; (2) receptor binding may alter chromatin structure locally, thus removing constraints imposed by chromatin conformation to the access of regulatory transcription factors at the upstream regulatory domain. Both hypotheses are consistent with the finding that the upstream regulatory region, although at a reduced level, is by itself able to properly activate the transcription of the reporter gene. However, given that the presence or functional integrity of the ng-EcRE, although relevant to the accumulation of ng-1 mRNA, is not strictly required either for the initiation or for the maintenance of ng-1 expression, it is also possible that ecdysone binding at the ng-EcRE might trigger alternative effects, such as modulation of the ng-1 mRNA half-life. Ecdysone-mediated regulation of the mRNA stability has already been described for the Sgs-3, Sgs-7 and Sgs-8 glue gene mRNAs, whose half-lives decline upon addition of ecdysone to cultured salivary glands. Thus, the possibility that ecdysone-mediated ng-1 regulation might occur, at least in part, at the post-transcriptional level cannot be excluded, and the peculiar location of the ng-EcRE within the ng-1 transcribed region makes this possibility even more intriguing. The recent finding that a ng-EcRE RNA probe is able to form multiple complexes when incubated with nuclear salivary gland extracts adds further support to this hypothesis (Crispi, 2001).

By examining expression of arc in different mutant embryos, it was determined that transcription factors known to be required for patterning and maintenance of various developing epithelia control arc expression in those domains. tll and hkb, which are required to pattern the posterior 15% of the embryo, control arc expression in the posterior midgut primordium. fkh, which appears to act as a maintenance, or permissive, transcription factor, is required for expression of arc throughout the gut. byn, which is required for hindgut development and specifies its central domain (the large intestine), controls expression of arc in the elongating hindgut. Kr and cut, required for evagination and extension of the Malpighian tubule buds control expression of arc in the tubule primordia (Liu, 2000).

The Drosophila salivary gland is a simple tubular organ derived from a contiguous epithelial primordium, which is established by the activities of the homeodomain-containing proteins Sex combs reduced (Scr), Extradenticle (Exd), and Homothorax (Hth). EGF signaling along the ventral midline specifies the salivary duct fate for cells in the center of the primordium, while cells farther away from the source of EGF signal adopt a secretory cell fate. EGF signaling works, at least in part, by repressing expression of secretory cell genes in the duct primordium, including fork head (fkh), which encodes a winged-helix transcription factor. Fkh, in turn, represses trachealess (trh), a duct-specific gene initially expressed throughout the salivary gland primordium. trh encodes a basic helix-loop-helix PAS-domain containing transcription factor that has been proposed to specify the salivary duct fate. In conflict with this is the idea that trh specifies salivary duct fate: three genes, dead ringer (dri), Serrate (Ser), and trh itself, are expressed in the duct independently of trh. Expression of all three duct genes is repressed in the secretory cells by Fkh. Ser in the duct cells signals to the adjacent secretory cells to specify a third cell type, the imaginal ring cells. Thus, localized EGF- and Notch-signaling transform a uniform epithelial sheet into three distinct cell types. In addition, Ser directs formation of actin rings in the salivary duct (Haberman, 2003).

trh is initially expressed throughout the salivary gland, in both duct and secretory cell primordia, but becomes restricted to the duct cells by fkh. It has been suggested that Fkh acts through repression of trh to limit expression of all duct genes to only the ventral preduct portion of the salivary gland primordium. Since it has been shown that expression of at least three genes is trh-independent, it is unclear how their expression is limited to the duct. Whether or not expression of the trh-independent duct genes is affected by Fkh was tested. Since salivary gland cells undergo apoptosis in fkh mutants, the experiments were performed in the background of the H99 deficiency, which blocks apoptosis by removing the apoptosis-activating genes hid, grim, and reaper. As in fkh mutants alone, all salivary gland cells remain on the surface of the embryo in fkh H99 embryos. In these embryos, secretory cells express the secretory marker Pasilla (PS) and Trh is expressed in all salivary gland cells. Similarly, expression of both Dri and Ser expanded into the secretory cells of fkh H99 embryos, suggesting that fkh is required to prevent secretory cell expression of multiple duct genes independently. Expression of all three genes is also observed throughout the salivary gland primordium of fkh mutants without the H99 deficiency, demonstrating that the observed expression profiles are not affected by the H99 deficiency. Also, expression of all of these genes is unchanged in H99 homozygous embryos, further indicating that the changes in gene expression are due to fkh (Haberman, 2003).

A role for Fkh as a master regulator of secretory cell fates has been rejected by multiple groups. A model is proposed where the salivary gland fate and the distinction between duct and secretory fate within the primordium is initiated by the coordinate system provided by the early patterning genes. Scr/Exd/Hth in the absence of Dpp- and EGF-signaling specifies the secretory cell fate, and Scr/Exd/Hth in the absence of Dpp-signaling and in the presence of EGF-signaling specifies the salivary duct fate. As a consequence of this combinatorial system for cell fate specification, multiple different genes are activated in the different salivary gland cell types. It is the combined activities of these downstream genes that make secretory cells different from duct cells. Moreover, since Scr and hth expression disappears from the salivary gland quite early, the downstream target genes must maintain as well as elaborate on these cell fate decisions (Haberman, 2003).

fkh has many roles in secretory cell development. Fkh prevents secretory cell apoptosis, mediates apical constriction during invagination, regulates its own expression, maintains expression of dCrebA, and regulates expression of the ecdysone-stimulated glue genes sgs3 and sgs4. Fkh has been found to represses expression of all tested duct genes in the secretory cells. In fkh mutants, trh, Ser, and dri are expressed throughout the salivary gland primordium in both duct and secretory cells. It is unclear whether Fkh directly regulates duct gene expression or regulates expression through some currently unidentified upstream activator(s). The 4-kb Ser salivary duct enhancer used in these studies contains several potential Fkh binding sites, indicating that Fkh repression of Ser could be direct. Fkh repression of duct gene expression suggests a role for Fkh in reinforcing the secretory cell fate. Fkh is required to maintain the distinction between duct and secretory primordia that is initially established by EGF-signaling. First, EGF-signaling initiates the distinctions between duct and secretory cells by blocking expression of secretory-specific genes in the duct primordium. Then, the genes whose duct expression is blocked by EGF-signaling, specifically fkh, maintain this distinction by repressing duct gene expression and maintaining their own expression, thus sharpening the boundaries between duct and secretory primordia by interpreting the gradient of EGF-signaling into a binary cell fate decision (Haberman, 2003).

The gradient of EGF signal from the ventral midline initiates early differences in duct versus secretory cell populations. The boundary then becomes more firmly established by Fkh. It is proposed that the salivary gland imaginal ring cells are then specified at the boundary between the duct and secretory cells in the salivary gland primordium. While the assay for imaginal ring specification analyzed salivary glands two days after embryogenesis, two lines of evidence suggest that imaginal ring specification occurs during embryogenesis. Ser is expressed in the salivary duct cells beginning at embryonic stage 11, when the duct cells and the adjacent secretory cells are still on the surface of the embryo. Notch, the receptor for Ser, is transiently upregulated in the secretory cells at stage 11. Thus, at this stage, the gland has high-level expression of ligand in the duct primordia and high-level expression of the receptor in adjacent secretory cells and, therefore, this is when signaling is likely to occur. Furthermore, the salivary ducts of Ser mutants have abnormal distal ends that can be observed in late stage embryos, indicating that a defect in salivary gland formation has already occurred. While it was not possible to assay for imaginal ring formation in the embryo due to a lack of markers for imaginal ring cells, the evidence suggests that Ser acts during embryogenesis to specify the imaginal ring. Nonetheless, the possibility cannot be ruled out that Ser specifies the imaginal ring at later embryonic stages when the salivary gland has internalized and when the salivary gland cells are in their final relative positions (Haberman, 2003).

Apoptosis in developing Drosophila embryos is rare and confined to specific groups of cells. How do salivary glands of Drosophila embryos avoid apoptosis? senseless (sens), a Zn-finger transcription factor, is expressed in the salivary primordium and later in the differentiated salivary glands. The regulation of sens expression in the salivary placodes is more complex than observed in the embryonic PNS. sens expression is initiated in the salivary placodes by fork head (fkh), a winged helix transcription factor. The expression of sens is maintained in the salivary glands by fkh and by daughterless (da), a bHLH family member. salivary gland-expressed bHLH (sage), a salivary-specific bHLH protein has been identified as a new heterodimeric partner for da protein in the salivary glands. In addition, the data suggest that sage RNAi embryos have a phenotype similar to sens and that sage is necessary to maintain expression of sens in the embryonic salivary glands. Furthermore, in the salivary glands, sens acts as an anti-apoptotic protein by repressing reaper and possibly hid (Chandrasekaran, 2003).

Although da and sage are necessary for maintaining sens expression, initiation of sens in the salivary placodes did not depend on either of these genes. Since sens expression in the salivary placodes initiates at stage 11, later than primary Scr target genes, it was thought sens might be indirectly activated by Scr through one of these primary targets. As expected, sens expression was found to be absent in Scr mutant embryos. sens expression is unchanged in embryos mutant for several Scr-regulated early transcription factors such as huckebein, trachealess and eyegone. However, fkh mutant embryos show a complete absence of sens expression in the salivary placodes and never express sens at the later stages. The expression of sens in the PNS is unaffected in these mutants. da and sage RNAs were unchanged at stages 10 and 11 in fkh mutants, indicating that the lack of sens is not due to the effects on sage or da expression. There was a slight reduction in sage RNA at stage 12, which may be due to the positive feedback loop between sens and sage in the salivary placodes. Thus, sens expression in the salivary placodes is initiated by fkh and is maintained at high levels throughout embryogenesis by da and sage (Chandrasekaran, 2003).

Thus, the regulation of sens in the salivary glands is more complicated than in the proneural tissues. sens expression in the salivary glands can be divided into two parts: initiation and maintenance. sens is initiated in the salivary placodes in response to fkh, one of the initial set of salivary genes that are directly activated by Scr at the beginning of stage 10 (4.3 hours AEL). sens expression begins about an hour later and may be directly regulated by fkh. There are FKH binding sites present at the 3' end of sens and a fragment carrying these sites is sufficient to recapitulate the expression in the salivary glands (Chandrasekaran, 2003).

Since sens is a fkh target and because both sens and fkh embryos show extensive salivary apoptosis, it was thought that apoptosis in fkh mutants might be caused by lack of sens. Because rescuing cell death in fkh mutants does not rescue normal morphogenesis, it was suggested that sens normally protects salivary cells from cell death, and other fkh target genes direct the cell movements and shape changes needed to form the salivary gland. However, the apoptosis of the salivary placodes in fkh mutants could not be rescued by ubiquitous expression of sens. There are two explanations for this result. The first possibility is that sens was not overexpressed at high enough levels to overcome cell death. However, this is likely not to be the case because the same arm-GAL4:UAS-sens combination was used to rescue the sens phenotype. Furthermore, arm-Gal4:UAS-P35 rescues cell death in sens mutants. Thus, the second possibility is favored, that loss of fkh leads to multiple proapoptotic changes, only one of which is the failure to activate sens (Chandrasekaran, 2003).

Although Fkh can initiate expression of sens in the salivary placodes, both Da and Sage are required for high level sens expression at later stages. Da is also known to control the expression of sens in the PNS. There, it partners with the proteins of the Achaete-Scute Complex or with Atonal to regulate sens expression. For sens regulation in the salivary primordium, a new Da partner, Sage, which belongs to the bHLH proteins of the Mesp family, has been identified. These results are the first to demonstrate the ability of Mesp family members to heterodimerize with Da. It is shown, using RNAi, that absence of sage leads to a decrease in the size of the glands and a reduction in levels of Sens. In turn, Sens appears to positively regulate the levels of sage mRNA in the salivary glands. The existence of this positive feedback loop leads to the question of which protein, Sage or Sens, is the true antagonist of apoptosis in the salivary glands. The presence of sage mRNA in sens mutants sheds some light on this issue. In sens mutants, high levels of Rpr-11-lacZ are induced at stage 12, in the salivary placodes. At this stage, sens mutant embryos still express sage and da mRNA in the placodes at normal levels. Reduction in sage mRNA is not observed until stages 13-14, by which time the salivary glands of sens mutants are already reduced in size. These results indicate that sens, not sage, is necessary to maintain the survival of the salivary gland cells (Chandrasekaran, 2003).

A similar circuit controls the regulation of expression of Gfi1, the vertebrate ortholog of sens, in the inner ear cells of mice. The bHLH protein Math1, termed Atoh1, a homolog of atonal, is necessary to maintain Gfi1 mRNA, but not for its initiation in the inner ear cells. It would be interesting to examine if fkh family members are involved in this case to initiate the Gfi1 expression. However, the feedback regulation of sens onto sage or proneural genes is not observed between Gfi1 and Math1 (Chandrasekaran, 2003).

Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4αSG1 and PH4αSG2

Forkhead (Fkh) is required to block salivary gland apoptosis, internalize salivary gland precursors, prevent expression of duct genes in secretory cells and maintain expression of CrebA, which is required for elevated secretory function. This study characterized two new Fkh-dependent genes: PH4αSG1 and PH4αSG2. In vitro DNA-binding studies and in vivo expression assays show that that Fkh cooperates with the salivary gland-specific bHLH protein Sage to directly regulate expression of PH4αSG2, as well as sage itself, and to indirectly regulate expression of PH4αSG1. PH4αSG1 and PH4αSG2 encode α-subunits of resident ER enzymes that hydroxylate prolines in collagen and other secreted proteins. Salivary gland secretions are altered in embryos missing function of PH4αSG1 and PH4αSG2; secretory content is reduced and shows increased electron density by TEM. Interestingly, the altered secretory content results in regions of tube dilation and constriction, with intermittent tube closure. The regulation studies and phenotypic characterization of PH4αSG1 and PH4αSG2 link Fkh, which initiates tube formation, to the maintenance of an open and uniformly sized secretory tube (Abrams, 2006).

The diverse activities of Fkh support a major role for this protein in controlling many of the tissue specific functions of the salivary gland. Indeed, the long list would support a model wherein Fkh could be viewed as an organ-specifying gene, much like the role proposed for its C. elegans homologue, PHA-4, in pharynx development (Gaudet, 2002). Nonetheless, these studies reveal notable differences in the roles of the two genes in organ formation. PHA-4 is proposed to directly regulate expression of all pharynx-specific genes (Gaudet, 2002), whereas Fkh is required for the expression of only about one-third of the salivary gland-specific genes tested so far (>200 genes). Moreover, many downstream genes are indirect targets of Fkh. For example, although Fkh is required for high-level expression of 34 secretory pathway component genes, its role in their activation is largely indirect through maintenance of CrebA, which is more directly involved in the expression of these genes (Abrams, 2005). As observed with the secretory pathway genes, more than half of the Fkh targets require Fkh only for maintenance, not initiation, suggesting that regulation is mediated by Fkh-dependent downstream transcription factors. Even with SG1 and SG2, which absolutely require Fkh for all stages of expression, only SG2 appears to be regulated directly by Fkh. Fkh regulation of SG1 appears to be through an intermediate, currently unidentified transcription factor, even though the SG1 enhancer contains two sites that bind Fkh protein in vitro. Indeed, detailed enhancer analyses have revealed only a small number of direct transcriptional targets of Fkh, including both sage and CrebA, which encode transcription factor genes that, in turn, either function downstream of or in parallel with Fkh to regulate gene expression. Thus, although Fkh functions as a key regulator of salivary gland development, it does so in collaboration with other early expressed transcription factors (Abrams, 2006).

When this analysis of Fkh regulation of SG1 and SG2 was undertaken, every potential direct binding site was sought within the SG1 and SG2 enhancers using data from three different studies, that revealed a seven bp core consensus Fkh-binding site. Although four such consensus sites were found within each enhancer, not all of the sites were bound by Fkh protein in vitro. Three sites showed strong binding, two sites showed moderate or weak (non-specific) binding and three sites were not bound at all. Subsequently, Takiya (2003) reported Fkh DNA binding to be strongly influenced by negative cooperativity among neighboring bases. The binding data from this study are consistent with those findings. The three strong Fkh-binding sites in the SG1 and SG2 enhancers matched the optimal sequences for binding determined by Takiya (2003) in all positions. The moderate Fkh-binding site had a C in position 11, which was shown to reduce binding affinity. The weak Fkh-binding site had an inhibitory T9A10 dinucleotide, and the three sites that did not bind Fkh at all each had an unfavorable A7T8 dinucleotide as well as nucleotides unfavorable for binding at positions 9 and/or 11. The demonstration that the Fkh-binding sites within both the SG2 and sage enhancers are required for their full level salivary gland expression indicates a direct correlation between in vitro studies and site occupancy in vivo; sites that function in vivo are bound by the Fkh protein in vitro. These studies of SG1 regulation, however, indicate that not all sites bound by Fkh protein in vitro are necessary in vivo, as SG1 reporter gene expression was unaltered when the Fkh sites were disrupted (Abrams, 2006).

Functional analysis of SG1 and SG2 suggests a role for apical secretion in the maintenance of uniform open salivary gland tubes. This finding supports studies in the Drosophila trachea, demonstrating the importance of apical secretions during tracheal remodeling showed that two apical proteins, Piopio (Pio) and Dumpy (Dp), are required during secondary branch formation. In this process, cells that are arrayed side-by-side in a multicellular tube rearrange to an end-to-end configuration to form unicellular tubes, while maintaining tube integrity with uniform lumenal space. In pio or dp mutants, tracheal cells detach from the main tracheal artery just as they complete their rearrangements to form unicellular tubes. It has been suggested that this occurs because the formation of the autocellular junctions of the unicellular tubes continues to completion instead of stopping at the point where the cells contact their proximal neighbors. It was further suggested that Pio and Dp contribute to an apical ECM that prevents reduction in lumen diameter as the secondary branches form. A role for an apical ECM in maintaining tube diameter has also been discovered in the multicellular tubes of the dorsal trunk. Genetic or pharmacological disruptions in chitin synthesis lead to regions of tube constriction and dilation. The existence has been demonstrated of a transient chitin network in the tracheal lumen that ensures that as the different segments of the dorsal trunk fuse to form a continuous tube, uniform tube diameter is maintained (Abrams, 2006 and references therein).

These studies demonstrate a correlation between apical ECM volume/morphology and lumen size uniformity, even in tubes not undergoing extensive cell rearrangements. The phenotypes of SG1/SG2-deficient salivary glands suggest that the apical ECM has both a barrier function that prevents cells from contacting one another and closing the tube, as well as a scaffolding function that prevents lumenal dilation. Similar defects were observed with mutations in pasilla (ps), which encodes a splicing factor homologous to the mammalian proteins Nova1 and Nova2 (Seshaiah, 2001). At the TEM level, ps mutants exhibit a decrease in secreted lumenal content and a reduction in the number and size of apical secretory granules. Although, as a splicing factor, Pasilla must be acting indirectly to affect secretion levels, its phenotype demonstrates a direct correlation between secretory volume and lumen size uniformity, a correlation supported by the defects in SG1/SG2-deficient glands. As SG1 and SG2 encode enzymes that could modify proteins in apical secretions, their role in this process is likely to be more direct (Abrams, 2006).

The apical matrix of wild-type glands forms a fibrillar network structure that is not apparent in salivary glands of embryos missing SG1 and SG2. This phenotype suggests that protein modification by the SG1 and SG2 prolyl-4-hydroxylases (PH4s) alters the character of secreted apical proteins to allow them to form fibrillar structures that maintain an expanded network of ECM. A role for prolyl hydroxylation in the formation of fibrillar collagen has been known for decades. Although canonical collagens are not expressed in the Drosophila salivary gland, a large number of uncharacterized genes encoding secreted proteins that contain the Pro-X-Gly repeats exist, which could be substrates for SG1/SG2 prolyl hydroxylation. Collagens are the major protein components of the ECM, where they serve key structural roles as exemplified by mutations in the human genes that lead to fragile bones, bone and joint deformities, as well as fragile skin and blood vessels. A 'structural' role for a collagen-related protein(s) in the apical matrix of salivary glands is consistent with these observations. Interestingly, formation of collagen fibrils occurs post-secretion, where enzymes outside the cell remove the propeptides from procollagen to allow fibrillar collagen formation. Similarly, the fibrillar nature of the lumenal secretions of the wild-type Drosophila salivary gland is not visible in the subapical secretory vesicles, suggesting that the fibrillar structures found in the apical matrix also form post-secretion. It is proposed that the denser apical matrix with reduced volume is the basis for the defects observed in SG1/SG2-deficient salivary glands. In areas where there is little to no apical content, the opposing sides of the tubes meet to either close or form very small lumena lined with small apical surfaces and closely arrayed adherens junctions. The similarity of the SG1/SG2 deficiency phenotypes to those seen with mutations affecting the Drosophila trachea suggests the potential for shared mechanisms for maintaining lumen size uniformity in epithelial tubes (Abrams, 2006).

fork head: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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