apontic

Transcriptional Regulation

tracheae defective (tdf), now more properly termed apontic, is required for the formation of the tracheal system during Drosophila embryogenesis. It encodes a putative bZIP transcription factor (Tdf). Antibodies directed against Tdf detect a nuclear protein in all tracheal cells before invagination and throughout tracheal system morphogenesis. Examination of tdf mutants reveals that tdf activity is not necessary for determining tracheal cell identity but for subsequent morphogenetic cell movements. tdf activity is under the control of trachealess, the key regulator gene for tracheal development. In contrast, tdf activity is not dependent on and does not interfere with the fibroblast growth factor-(FGF) and Decapentaplegic- (Dp) mediated signaling that directs guided tracheal cell migration. These results suggest that lack of tdf activity affects tracheal cell migration in general rather than specific aspects of cell migration. tdf activity involves both maternal and zygotic components; its requirement and hence, its effects, are not limited to tracheal system formation. The complex spatiotemporal patterns of Tdf expression in the embryo suggests that defects in tdf mutants may be the result of impaired cell migration. Therefore, it is proposed that the bZIP protein Tdf functions as a co-regulator of target genes that provide cells with the ability to migrate (Eulenberg, 1997).

The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some downstream genes but not others. While tracheal expression of tkv depends on vvl, it appears to be independent of trh. The opposite appears to be the case for two other tracheal genes, apontic/tracheal defective (tdf) and pebbled (peb) [also known as hindsight (hnt)], which code for two putative transcription factors. Both genes appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes seem to be common targets of vvl and trh but others seem to depend only on one of them (Boube, 2000).

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hnt^peb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt³⁰⁸-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

Protein Interactions

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the Oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the Oskar mRNA. To begin to understand how Bruno acts in translational repression, a yeast two-hybrid screen was performed to identify Bruno-interacting proteins. One interactor proves to be the product of the apontic gene (Lie, 1999).

Apt is an RNA binding protein. Remarkably, the regions of the OSK 3' UTR bound by Apt, the AB and C regions, are precisely those bound by Bru. A test of Apt binding was performed to determine if Bru and Apt have the same RNA binding specificity: a series of RNAs was used to map the Bru binding sites, called BREs, within the OSK C region. Three of these RNAs retain the BREs and are bound by Bru, while a fourth RNA, CDelta4, lacks the BREs and fails to bind Bru. Apt binds all four RNAs, including CDelta4, indicating that Apt can bind to sites other than BREs (Lie, 1999).

Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. Mutants in apt are zygotic lethal, and some alleles also cause arrested oogenesis. Several different apt alleles were used for all analyses, since the genetics of apt are complex and different alleles have different effects. Testing for a genetic interaction between the aret (bruno) and apt mutants, dosages of both genes were reduced to see if this would provide a distinct phenotype. Females heterozygous for aret, heterozygous for any of the five apt alleles, or transheterozygous for both aret and an apt allele, were crossed to wild-type males, and the progeny embryos were then examined for cuticular defects. Females transheterozygous for aret and apt produce a fraction of embryos with head defects. Head defects can result from ectopic or excessive posterior body patterning activity, because this activity interferes with expression of the anterior body patterning morphogen, Bicoid. Consequently, the observed head defects could be explained if both Bru and Apt contribute to repression of OSK mRNA translation. Given this interaction, Apontic is likely to act together with Bruno in translational repression of Oskar mRNA (Lie, 1999).

Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the Oskar mRNA 3' untranslated region. A sequence shared by all of the bound RNAs could not be identified. Thus, despite its ability to efficiently discriminate between different parts of the OSK mRNA, Apt appears to be relatively promiscuous in its binding and may recognize many sites or perhaps a structural feature common to many RNAs (Lie, 1999).

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. Drosophila has been used to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, Drosophila Mbf1 was found to form a ternary complex including Mbf1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (Tdf)/Apontic. A Drosophila mutant has been isolated. This mutant lacks the mbf1 gene; no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system (Liu, 2003).

A cDNA encoding Drosophila Mbf1 was cloned from a larval CNS library. The predicted protein of 145 amino acids has 44%, 64% and 83% identity to MBF1 from yeast, human and silkmoth, respectively. MBF1 consists of two structural domains: a well-structured C-terminal half that binds the general transcription factor TBP; and a flexible N-terminal half that participates in binding to various activators. The region conserved in Drosophila Mbf1 includes both of these functional domains. Expression of Drosophila mbf1 cDNA partially rescued the yeast mbf1 mutant phenotype upon amino acid starvation, indicating that the ability to bind partner transcription factors is also conserved between yeast and Drosophila. In situ hybridization has revealed that a large amount of maternal mbf1 mRNA is deposited to the egg. Likewise, MBF1 protein is present in preblastoderm embryos and is later expressed in many tissues, including the CNS and the trachea. Widespread expression of MBF1 is also seen in post-embryonic stages, with particularly high levels in the larval salivary glands, gonads and adult gonads (Liu, 2003).

The relationship between Drosophila Mbf1 and Tdf is similar to that between yeast MBF1 and its partner transcription factor GCN4. Just as yeast MBF1 contacts GCN4 through its bZIP domain, Drosophila Mbf1 binds the bZIP domain of Tdf. Moreover, the lack of GCN4-dependent activation in yeast mbf1 mutant can be partially restored by expressing Drosophila Mbf1. The sequence and functional conservation between yeast and Drosophila Mbf1 indicates that the interaction with bZIP proteins is a conserved feature of the bridging factor MBF1 (Liu, 2003).

Genetic studies of mbf1 in yeast and Drosophila suggest that MBF1-associated transcription factors have two pathways for activation. In addition to the MBF1-mediated recruitment of TBP via its bZIP domain, GCN4 also recruits the SAGA complex with its N-terminal activation domain and effects transcription through chromatin modification. Likewise, Drosophila Tdf has a region similar to the glutamine-rich transactivation domain and may employ an activation pathway independent of recruiting TBP through Mbf1. Such a pathway may account for the residual expression of the TDS-lacZ reporter gene in the absence of Mbf1. Although Yeast MBF1 is essential for GCN4-dependent transcription of its target gene HIS3, low level of Tdf-dependent transcription of the TDS-lacZ gene can still occur in the absence of MBF1. This suggests that the relative importance of the two pathways is different between GCN4 and Tdf. The DNA-binding domain of Drosophila FTZ-F1 carries a basic region homologous to those in bZIP proteins and binds Mbf1 through this region. However, loss of mbf1 showed no effect on FTZ-F1-dependent transcription in vivo, suggesting that the activation by FTZ-F1 relied solely on the pathway through its transactivation domain. In an in vitro transcription system, the transactivation domain does not seem to be functional because FTZ622 polypeptide bearing only the DNA-binding domain of FTZ-F1 shows the same transcriptional activity as the intact FTZ-F1. This may explain the difference in the MBF1 requirement between FTZ-F1-dependent transcription in vivo and in vitro (Liu, 2003).

It is possible that the role of Mbf1 becomes more critical under certain circumstances, when rapid induction of gene expression is demanded by environmental conditions. The expression of the TDS-lacZ reporter in mbf1^- background varies considerably from embryo to embryo, suggesting that certain conditions that are uncontrolled in these experiments may render transcription particularly dependent on the Mbf1-mediated pathway. In the natural environment, there are many stimuli that alter the gene expression profile: UV radiation, poison agents, nutrient starvation and so on. Therefore, direct recruitment of TBP by Mbf1 may become essential for rapid activation of transcription under such conditions. In agreement with this idea, the yeast mbf1 disruptant is viable under normal culture conditions, but sensitive to amino acid starvation (Liu, 2003).

Studies on MBF1 homologs also support the idea that MBF1 may function when gene expression is required in response to developmental or environmental signals. Rat MBF1 has been isolated as a calmodulin-associated peptide 19 (CAP-19) and human MBF1 has been identified as endothelial differentiation-related factor 1 (EDF1). EDF1/MBF1 is downregulated when endothelial cells are induced to differentiate. Interestingly, EDF1/MBF1 binds to calmodulin in the cytoplasm under low Ca²⁺ conditions but the two proteins dissociate when intracellular Ca²⁺ is high. The released EDF1/MBF1 is then phosphorylated and shuttled into the nucleus, where it binds TBP. Nuclear translocation of MBF1 has also been observed at a specific stage of molting in the silkworm B. mori. Considering the Ca²⁺ elevation upon exposure to the molting hormone ecdysteroid , these data raise an intriguing possibility that MBF1 is involved in Ca²⁺-induced gene activation. Although in this study the developmental roles of MBF1 were studied only in association with Tdf function, Drosophila Mbf1 may also be involved in other biological processes, such as stress response, homeostasis and longevity (Liu, 2003).

Several lines of evidence suggest that Drosophila Mbf1 has partners other than Tdf. Mbf1 is expressed in a wide spatiotemporal pattern, including tissues and stages where Tdf is absent. Although Tdf is not expressed in the salivary gland, immunolocalization of Mbf1 on salivary gland chromosome revealed a large number of loci associated with Mbf1. Furthermore, FLAG-tagged Mbf1 pulled down many proteins besides Tdf. Although mbf1-null mutants are viable under laboratory conditions, tdf becomes haploinsufficient in mbf1^- genetic background, clearly indicating the importance of Mbf1 in the expression of the genomic information. This finding opens a way to identify new partners of Mbf1 through genetic screening for loci that exhibit dominant phenotypes in the absence of Mbf1. Characterization of Mbf1 partners will contribute to the knowledge of how co-activators mediate specific biological events (Liu, 2003).

DEVELOPMENTAL BIOLOGY

Maternal and Embryonic

See the embryonic expression pattern of apt at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

apontic expression occurs in both the somatic follicle cells and the germline nurse cells and oocyte. APT transcripts are detected as early as stage 2A at low levels in the germarium and at higher levels in the follicle cells. The amount of APT mRNA in the soma decreases during the remainder of oogenesis, while the level in the germline increases. APT mRNA becomes concentrated in the oocyte and also accumulates in the nurse cells at about stage 6. APT transcripts continue to be found in both the oocyte and nurse cells throughout oogenesis. To determine when and where Apt protein is expressed during oogenesis, antisera directed against a recombinant Apt protein were prepared and used for protein detection by confocal microscopy of whole-mount ovaries. Apt protein appears in both the germline and somatic cells of the ovary throughout all stages of oogenesis. In the germline, Apt protein is present in both cytoplasm and nuclei. Within the nurse cells the protein is more concentrated in the cytoplasm, while in the oocyte more protein is found in the nucleus. The protein, however, is not localized to any subdomain within the cytoplasm of either the nurse cells or the oocyte. Although Apt protein is not strictly nuclear or cytoplasmic in cells of the female germline, the protein is highly concentrated in nuclei of the ovarian follicle cells and in post-cellularization-stage embryos. The developmental differences in subcellular location suggest that Apt may have functions, perhaps different, in both nuclei and cytoplasm (Lie, 1999).

Nuclear proteins expressed from maternal mRNAs are sometimes present at high levels in the cytoplasm of early embryos. Examples include the Bicoid, Caudal and Hunchback proteins, which appear in both nuclei and cytoplasm shortly after egg laying. As nuclear divisions progress and the density of nuclei increases, nuclear localization of these proteins remains strong while the fraction of protein in the cytoplasm diminishes. Thus there appears to be no early impediment to nuclear localization, simply a paucity of nuclei. In contrast, the subcellular distribution of Apt protein appears to be actively controlled in early development. Apt protein was monitored in early embryos. Even after migration of nuclei to the surface of the embryo, Apt protein remains evenly distributed between nuclei and cytoplasm, unlike any of the examples described above. This unusual persistence of Apt protein in the cytoplasm suggests the existence of a mechanism to control its distribution, reinforcing the notion of roles for Apt in both cytoplasm and nuclei (Lie, 1999).

Transcripts from apt are expressed in a complex and dynamic pattern that includes, but is not limited to, the head regions affected in apt mutants. At the syncytial blastoderm stage maternal apt transcripts are distributed throughout the embryo. At cellular blastoderm, apt transcripts can be detected at both poles of the embryos. During germ band elongation most of the posterior expression disappears and a segmentally repeated pattern of expression arises in the trunk. apt transcripts in the trunk region are detected in cells along the ventral midline, in dorsal cells abutting the amnioserosa, and in the tracheal placodes (cells that will invaginate to form the respiratory tree). In the head of stage 9-10 embryos, apt is expressed in the dorsal part of the acron, the entire clypeolabrum and the ventral gnathal region. Transcripts can also be detected in the anterior-lateral regions of the mandibular, maxillary and labial lobes. During stage 11, apt transcripts accumulate at high levels in the dorsal ridge, and at this stage, transcript levels gradually disappear from the vicinity of the tracheal pits. As the amnioserosa is absorbed and dorsal closure ensues, dorsal mesodermal cells arranged in a single row on either side of the amnioserosa accumulate apt transcripts; later, these cells, still expressing APT transcripts, will contribute to the dorsal vessel. The patterned expression of apt also persists to late stages of embryogenesis in head epidermis and CNS (Gallon, 1997).

APT mRNA is maternally supplied. After gastrulation, at the beginning of germ band extension (stage 8), apt is uniformly expressed in the mesoderm and extends into the hindgut primordium. At stage 10, expression in the mesoderm ceases, except for the mesectodermal cells of the ventral midline. Later, during stage 11, apt expression is observed in the tracheal pits, the CNS, and the head region, as well as prominently in the heart progenitor cells. Expression in the heart precursors persists during the process of heart tube formation and in the differentiated heart during embryogenesis (Su, 1999).

Effects of Mutation or Deletion

Only a few genes have been identified that participate in the developmental pathways that modulate homeotic (HOX) protein specificity or mediate HOX morphogenetic function. To identify more HOX pathway genes, a screen was carried out for mutations in loci on the Drosophila second chromosome; a number of genes in this region interact with the homeotic gene Deformed. Genetic and molecular tests on the eight genes isolated in the screen place them in three general categories. (1) Two genes appear to encode trithorax group functions, i.e. they are general activators of Hox gene expression or function. (2) Four genes encode abundant, widely expressed proteins that may be required to mediate Dfd morphogenetic functions in certain tissues, including two genes for collagen IV protein variants. (3) two of the genes are required for the development of a subset of embryonic Dfd-dependent structures, while leaving many other segmental structures intact. One of these two was cloned and characterized. The cloned gene, apontic (apt), is required for the elaboration of dorsal and ventral head structures. The apt transcript pattern is normal in Dfd and Scr mutants, and the Dfd and Scr transcript patterns are normal in apt mutants. It is proposed that apt acts in parallel to, or as a cofactor with, HOX proteins to regulate homeotic targets in the ventral gnathal region (Gellon, 1997).

apontic is required for the formation of some but not all Deformed- and Sex combs reduced-dependent ventral gnathal structures. The lateral arms of the H-piece are missing and the lateralgraten are shortened in Dfd mutants; the hypostomal sclerites and dorsal pouch are missing in Scr mutants. Other Dfd- and Scr-dependent structures are intact in apt mutants, such as the ectostomal sclerites (Dfd-dependent), mouth hooks (Dfd-dependent) and cross bar of the H-piece (Scr-dependent). Thus, the apt phenotype suggests that apt might be contributing to the diversification of Dfd and Scr function in a specific cell population within each selector's domain. Such overlap in phenotypes could occur in principle by three different mechanisms: (1) apt could mediate Dfd and Scr functions by regulating their transcription in a particular region (i.e. apt acts upstream); (2) apt could be a target of Dfd and Scr in a discrete population of cells (apt acts downstream), or (3) apt could act in conjunction with Dfd and Scr (or with Dfd and Scr targets) to produce a distinct biological effect in a subpopulation of cells (apt acts in parallel). Whole mount in situ hybridizations of apt mutant embryos with DFD and SCR mRNA probes were performed their and patterns of transcription were found to be indistinguishable from wild type. Therefore, apt is not required to establish or maintain Dfd and Scr transcription. Conversely, apt transcription was examined in Dfd and Scr mutants by in situ hybridization and no changes were detected. Thus, neither Dfd nor Scr is required to establish or maintain apt transcription. It is concluded that apt acts in parallel with Dfd and Scr proteins to produce ventral gnathal structures (Gellon, 1997).

In an effort to isolate genes required for heart development and to further the understanding of cardiac specification at the molecular level, PlacZ enhancer trap lines were screened for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, is particularly interesting because of its early pattern of expression in cardiac precursor cells, an expression pattern dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. A gene was isolated and characterized in the vicinity of B2-2-15 that exhibits an identical expression pattern to that of the reporter gene of the enhancer trap. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles: segments of heart cells (those of myocardial and pericardial identity) are often missing. These abnormalities become obvious shortly before the assembly of the heart precursor cells at the dorsal midline. The defects in heart tube formation are seen with three markers: (1) Evenskipped, which is present in a subset of pericardial cells (EPC); (2) Mef2, which marks the cardial cells of the heart, and (3) Zfh-1, which is primarily present in the non-EPC pericardial cells. No obvious defects are observed in somatic and visceral muscle patterning, suggesting a specific requirement for apt in heart formation, as opposed to other mesodermal derivatives. Since the initial cardiac mesoderm seems to form normally in these mutants, it seems likely that apt is primarily required for a late differentiation step, such as the correct assembly of the heart tube. This would be consistent with a cell autonomous function of apt in the developing heart (Su, 1999).

apt mutant embryos or larvae develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. The heart rate of wildtype or apt heterozgous animals varies over a large range (28-146 beats per minute). The overall average heart rate is about 80 beats per minute. When moving, the apt homozygous mutant embryos and larvae have heart rates, on average, only about a quarter the heart rate in wildtype. The heart rate and cardiac defects may be the cause of death for apt mutants during late embryonic or early larval stages (Su, 1999).

Earlier genetic analyses of apt have concentrated on the zygotic phenotype. To define more completely the role of apt in the female germline, females were created with apt minus germline clones using the FLP/DFS method. Ovaries containing germline clones were dissected, stained with DAPI to highlight nuclei, and examined for phenotype. Different apt mutants display dramatically different ovarian phenotypes. One allele is indistinguishable from wild type, because females with germline clones of this allele have phenotypically wild-type ovaries and lay eggs that develop into fertile adults. Females with germline clones of another allele also have phenotypically wild-type ovaries, but a small fraction of the eggs laid develop into embryos with head defects. In contrast, ovaries from females with germline clones from two other mutants have phenotypes that are similar to one another and severe: development is arrested in early oogenesis, and the oocyte fails to differentiate, with all nuclei becoming polyploid. In addition, some of the egg chambers have an abnormal number of nuclei. It is concluded that apt is necessary for oogenesis and that loss of apt activity leads to a developmental arrest during oogenesis. Just as for arrest (bruno) mutants, the developmental arrest occurs too early to allow the ovaries to be examined for defects in OSK mRNA translation (Lie, 1999).

Boube, M., Llimargas, M. and Casanova, J. (2000). Cross-regulatory interactions among tracheal genes support a co-operative model for the induction of tracheal fates in the Drosophila embryo. Mech. Dev. 271-278.

Eulenberg, K. G. and Schuh, R. (1997). The tracheae defective gene encodes a bZIP protein that controls tracheal cell movement during Drosophila embryogenesis. EMBO J. 16(23): 7156-7165.

Gellon, G., et al. (1997). A genetic screen for modifiers of Deformed homeotic function identifies novel genes required for head development. Development 124(17): 3321-3331.

Gunkel, N., Yano, T., Markussen, F. H., Olsen, L. C. and Ephrussi, A. (1998). Localization-dependent translation requires a functional interaction between the 5' and 3' ends of Oskar mRNA. Genes Dev. 12: 1652-64.

Lie, Y. S. and Macdonald, P. M. (1999). Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation. Development 126: 1129-1138.

Liu, Q.-X., et al. (2003). Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems. Development 130: 719-728. 12506002

Su, M. T., et al. (1999). The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart. Mech. Dev. 80(2): 125-32.

Wilk, R., Pickup, A. T., Hamilton, J. K., Reed, B. H. and Lipshitz, H. D. (2004). Dose-sensitive autosomal modifiers identify candidate genes for tissue autonomous and tissue nonautonomous regulation by the Drosophila nuclear zinc-finger protein, Hindsight. Genetics 168(1): 281-300. 15454543

date revised: 30 June 2005

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