Fushi tarazu

The orphan nuclear receptor alpha Ftz-F1, which is deposited in the egg during oogenesis, is an obligatory cofactor for Fushi tarazu. Mutation of Ftz-F1 causes a pair-rule phenotype even though the maternal alphaFTZ-F1 gene product is uniformly distributed through the embryo. Surprisingly, patterns of FTZ mRNA and protein expression in the alphaFTZ-F1 mutants are indistinguishable from wild type. In Ftz-F1 mutant embryos, as in ftz mutant embryos, Ftz-dependent engrailed stripes fail to be expressed, and wingless stripes expand. Thus alphaFtz-F1 is required for all Ftz activities tested except that for which it was first identified: regulation of the ftz promoter. Given this result, a test was made for direct interaction between Ftz protein and alphaFtz-F1. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. The conserved motif is independent of the Ftz homeodomain and is located in the central portion of the protein, flanked by prolines. Deletion of this motif disrupts all but one of the the Ftz activities described above, that is, it is still capable of broadening endogeneous ftz expression stripes. Thus removal of the alphaFtz-F1 interaction domain from the Ftz polypeptide results in the same loss of Ftz activities as removal of alphaFtz-F1. The Ftz-mediated repression of wingless requires both Paired and alphaFtz-F1. This interaction could involve either simultaneous or competitive interactions among the three proteins, as Prd also contacts residues 101-150 of Ftz. Paired may be a cofactor of Ftz or Ftz-F1 that is required for target genes that are repressed by Ftz, because Prd is required for Ftz-dependent wingless repression, but not for Ftz-dependent activation of engrailed or ftz auto-regulation (Guichet, 1997).

A native binding site for the homeodomain protein Fushi tarazu, the upstream regulatory region of the ftz gene itself, was used to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. The upstream regulatory region contains five Ftz binding sites and three Ftz-F1 binding sites. One Ftz-F1 site is adjacent to a medium-affinity Ftz binding site. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone would bind only high-affinity sites. By virtue of mutual binding to the same regulatory region, Ftz-f1 and Ftz are both implicated in Ftz autoregulation. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects, and engrailed transcription sites are not present in Ftz-f1 mutant embryos. The pair-rule phenotype is a result of abnormal ftz function, because ftz is expressed but fails to activate downstream genes (Yu, 1997).

Transcription of messenger RNA-encoding genes in vitro requires many protein factors. Transcription factor IID, possibly with the cooperation of TFIIA, binds to the TATA element of the promoter, forming a complex that can bind TFIIB followed by RNA polymerase II and other factors. One or more of these steps is thought to be facilitated by gene-specific transcriptional activation proteins; this seems to require TFIID-associated auxiliary factors and may involve direct contact between the activator and TFIID and/or TFIIB. In fact, the activity of the glutamine-rich fushi tarazu activation domain is mediated by interactions with TFIIB (Colgan, 1993).

The Engrailed homeodomain protein is an 'active' or dominant transcriptional repressor in cultured cells. In contrast, the Fushi tarazu homeodomain protein is an activator, both in cultured cells and in Drosophila embryos, where it activates several known target genes, including its own gene. This auto-activation has been shown to depend on targeting to a Fushi tarazu enhancer by the Fushi tarazu homeodomain. Fushi tarazu targeting and Engrailed active repression were combined in a chimeric regulator, EFE. When EFE is ubiquitously expressed, it overrides endogenous Fushi tarazu and causes a fushi tarazu mutant phenotype. Normal fushi tarazu target genes are affected as they are in fushi tarazu mutants. One such target gene is repressed by EFE even where fushi tarazu is not expressed, suggesting that the repression is active (John, 1995).

Comparison of the DNA targets of Bicoid, Fushi tarazu and Orthodenticle reveal the importance of the amino acid at position 50 of the homeodomain in discriminating between bases that lie adjacent to the TAAT core of homeodomain binding sites. FTZ has a preference for TAATG due to the presence of glutamine at position 50, while Bicoid prefers the consensus sequence TAATCC, specified by lysine at position 50. OTD also has a lysine at position 50 and the consensus sequence recognized is similar to that of BCD. Structural studies suggest that water-mediated hydrogen bonds and van der Walls contacts underlie the preferences for bases adjacent to the TAAT core (Wilson, 1996).

Protein-protein interactions are critical in homeodomain protein function. A homeodomain-deleted FTZ polypeptide (FTZ delta HD) is incapable of binding DNA in vitro, but can regulate endogenous ftz gene expression. FTZ delta HD can directly regulate ftz-dependent segmentation, suggesting that it can control target gene expression through interactions with other proteins. A likely candidate is the pair-rule protein Paired (PRD). FTZ delta HD bound directly to PRD in vitro and required PRD to repress wingless in vivo (Copeland, 1996).

The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. One of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala results in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescues the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. The T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein. Preliminary experiments with Ftz T263 mutants suggest that the phosphorylation state of T263 does not affect either the Ftz-Prd or the Ftz-Ftz-F1 interaction. Hence, a yet to be identified protein expressed in embryos is likely to be the relevant target (Dong, 1998).

In approximately half of all HOX proteins, residue 7 of the homeodomain (analogous to Ftz T263) is either a Thr or Ser residue, while position 5 is an Arg residue. This conserves the PKA recognition site, and suggests that each of these proteins probably shares the ability to be phosphorylated at this position. This includes the more divergent POU class homeodomain proteins Pit-1 and Oct-1, which like Ftz are phosphorylated efficiently by PKA, or a PKA-like kinase in vitro. It is also worth noting that some homeodomain proteins, such as En and Even-skipped (Eve), normally possess alanines at position 7. Hence, the substitution of Thr263 with Ala is a conservative one that is unlikely to exert its effect at the level of general homeodomain structure. This is consistent with the findings that this substitution has no apparent effect on DNA-binding activity, transactivating activity, protein stability or subcellular localization (Dong, 1998).

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (Ftz-f1) protein is a conserved member of the nuclear receptor superfamily, but has been thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. It is shown here that Ftz-f1 does in fact have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (Ftz). Ftz contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins (Schwartz, 2001).

Ftz-f1 deletion constructs were sequenced to ensure that no errors had been introduced during the course of PCR and cloning procedures. These analyses revealed a discrepancy between the sequences in this study and those of the published Ftz-f1 sequence. An 8 bp repeat beginning at position 3006 of the alphaFtz-f1 sequence was contained in the sequences obtained in this study. The predicted coding region of the revised sequence diverges at amino acid 1003 and encodes an additional 24 amino acids. This revised sequence contains an AF2 consensus that is highly similar to the sequences of vertebrate Ftz-f1 homologs. Confirmation of this revised sequence has recently been provided by the Drosophila genome sequencing project. The sequence of the Ftz-f1 protein of B. mori has also been reported to lack an AF2 motif. A brief examination of the DNA coding sequence reveals that the addition of a single nucleotide in an appropriate location would change the reading frame and yield an AF2 nearly identical to that of the Drosophila protein. Hence, a Bombyx Ftz-f1 cDNA was obtained and sequenced. An additional adenosine nucleotide was found that follows the thymidine at position 1659. The shifted reading frame generates a new C-terminus with 27 of the 34 predicted amino acids identical to the Drosophila sequence. An additional four residues are conservative substitutions. The C-terminal AF2s of the two insect proteins are identical at 15 of 16 positions. It is concluded that both insect proteins contain highly conserved AF2 motifs (Schwartz, 2001).

It is not yet known how Ftz and Ftz-f1 regulate their target genes. Interestingly, Ftz and Ftz-f1 are known to function as both transcriptional activators and repressors. Thus, Ftz may be able to act as both a coactivator and corepressor for Ftz-f1 function. Alternatively, these differential activities may depend on the actions of other cofactors, some of which may be target gene specific. The fact that Ftz-f1 utilizes Ftz as a coregulator of target genes, and that Ftz contains a nuclear receptor box, suggests the possibility that, like other nuclear receptor cofactors, Ftz may help recruit histone acetyltransferase or deacetylase complexes. Alternatively, Ftz may act via regulatory factors or complexes that are quite different from these. For example, Ftz target genes such as engrailed and homeotic genes of the Antennapedia and bithorax gene clusters are regulated by chromatin-organizing Polycomb and Trithorax group protein complexes. The Ftz-Ftz-f1 complex may play a role in recruiting these larger protein complexes (Schwartz, 2001).

Does Ftz-f1 have a ligand? There are several observations that argue both for and against the existence of a Ftz-f1 ligand. Motifs and residues required for ligand binding by other receptors are conserved in Ftz-f1. These include residues in the AF2 helix that, based on other receptor structures, are expected to contact the ligand-binding pocket. Ftz-f1 also contains a conserved residue in the third helix of the LBD (N840) that has been shown in other receptors to be a key ligand-contacting residue. Although a study with the Ftz-f1 homolog SF-1 suggests that its transcriptional activity could indeed be enhanced in the presence of certain oxysterols, subsequent studies have been unable to validate this finding. A more general observation in favor of a ligand is that while there are a large number of orphan nuclear receptors whose ligands have not been identified, there are currently no clear examples of receptors that are fully functional as activators in the absence of ligand (Schwartz, 2001 and references therein).

Observations that argue for the absence of a Ftz-f1 ligand include the avid binding of Ftz to Ftz-f1 observed in vitro in the absence of ligand. For most nuclear receptors with known ligands, the ligand is required to position the overlying AF2 in the proper orientation for coactivator binding. The ability of Ftz to bind in the absence of ligand suggests that coactivator binding to Ftz-f1 may be constitutive, and that temporal and spatial regulation of Ftz-f1 activity is controlled by the presence or absence of cofactors such as Ftz rather than by ligands. Alternatively, conditions in the cell may be more stringent, making the Ftz-Ftz-f1 interaction ligand dependent (Schwartz, 2001).

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. The murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. It is proposed that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, in contrast, influences Ftz-F1 activity by interacting with its AF-2 domain in a manner that mimics a nuclear receptor coactivator (Yussa, 2001).

The orphan receptor Ftz-F1 and homeodomain (HD) protein Ftz cooperate to promote the development of alternate body segments, presumably by activating expression of downstream target genes such as engrailed. Ftz and Ftz-F1 are each sequence specific DNA binding proteins with strong transcriptional activation domains. However, neither is apparently able to function in Drosophila embryos to regulate target gene expression in the absence of its partner, since mutation of either protein results in lethality accompanied by identical pair-rule defects. Each protein is necessary but neither Ftz nor Ftz-F1 alone is sufficient to select and activate target gene transcription in the embryo. Why do Ftz and Ftz-F1 require partners in vivo to regulate gene expression? By analogy to the function of beta ftz-f1 as a competence factor for the ecdysone response, alpha ftz-f1 can be seen as a competence factor for a 'pulse' of Ftz expression in seven stripes in the blastoderm embryo (Yussa, 2001).

Why does Ftz-F1 fail to activate Ftz-F1/Ftz (F1F) targets in the absence of Ftz protein? Ftz-F1 proteins are strong transcriptional activators in a variety of cell systems. In Drosophila, ftz-f1 is maternally deposited and Ftz-F1 is found in all somatic nuclei before Ftz is expressed in seven stripes. Yet, Ftz-F1 does not detectably activate transcription in the absence of Ftz either temporally (before Ftz is expressed zygotically) or spatially (in regions of the embryo outside the seven Ftz stripes). Thus the presence of Ftz functions as an apparent 'on' versus 'off' switch to enable Ftz-F1 to activate transcription. Two mechanisms are proposed that may contribute Ftz-F1's requirement for Ftz to activate transcription. (1) Ftz-F1 might require Ftz for stable DNA binding. In vitro, Ftz-F1 activates transcription of the ftz proximal enhancer (323-fPE) and F1F reporter constructs more strongly than does Ftz, consistent with the in vitro DNA binding properties of the two proteins. In fact, the order of magnitude of synergy of transcriptional activation of Ftz-F1 compared to Ftz-F1 + Ftz (5-10 fold) is similar to the enhancement of DNA binding conferred by Ftz on Ftz-F1. While this mechanism likely contributes to Ftz-F1 activation, the enhancement seen in vitro may not translate into the apparent 'on' vs. 'off' state of Ftz-F1 in Ftz-expressing vs. Ftz-non-expressing cells in vivo (Yussa, 2001). Therefore, (2) a model in which Ftz-F1 is actively repressed in the absence of Ftz, even if it is bound to cognate DNA target sites, is favored. Ftz-F1 is present in all somatic nuclei before Ftz is expressed and may bind cognate DNA sequences, but remains quiescent until it is activated by interaction with Ftz protein. It is proposed that interaction of Ftz with Ftz-F1 through its LXXLL motif displaces corepressor molecules, allowing productive transcription complexes to form. The candidates for repressor molecules that keep Ftz-F1 in an 'off' state are the corepresssors that inhibit activity of other nuclear receptor family members. The corepressors identified in Drosophila that are expressed throughout the blastoderm embryo include Alien, which interacts with Ftz-F1 in GST-pulldown assays and SMRTER. A corepressor might bind directly to Ftz-F1. It is also possible that another partner of Ftz-F1 recruits a corepressor to the complex, as has been shown for the murine ortholog of Ftz-F1 (SF-1) which is regulated by interaction with the nuclear receptor Dax-1 that recruits corepressors and inhibits SF-1 activity (Yussa, 2001).

Why does Ftz require Ftz-F1 for target gene regulation in vivo? Ftz and Ftz-F1 bind cooperatively to DNA to select specific target sites that are composite binding sites for the two proteins. The binding of Ftz and Ftz-F1 to composite sites is stabilized by protein-protein interactions mediated by at least two regions of each partner protein. The composite nature of the binding site raises the selectivity of Ftz binding by requiring a heterodimeric site for productive interaction. The affinity of Ftz for composite sites is dramatically increased by Ftz-F1. Ftz protein can bind to monomeric 'ATTA' core sites in vitro and it appears to associate with a wide array of sites in the genome, as determined by UV cross-linking experiments. However, it is proposed that in vivo, binding of Ftz to monomeric 'ATTA' sites is transient, during the time that Ftz scans the genome for cognate DNA binding sites. This relatively unstable binding does not allow Ftz to effectively activate transcription on its own, consistent with the finding that concatamerized Ftz binding sites do not mediate a Ftz-dependent pattern of gene expression in vivo. Ftz binds productively to composite heterodimeric sites, where it is stabilized by protein-protein interactions with Ftz-F1. This notion is also consistent with reports that fusion of Ftz to a strong VP16 activator does not alter target gene regulation in vivo. Thus, specificity of gene regulation by Ftz protein is achieved at the level of DNA binding/target site selection, as a result of interaction with DNA binding cofactors such as Ftz-F1 (Yussa, 2001).

Future studies are required to elucidate the detailed mechanism whereby transcription is activated by the Ftz/ Ftz-F1 complex. One candidate mechanism is direct contact of TFIIB by Ftz and/or Ftz-F1. Ftz was shown to directly contact TFIIB, activating transcription via a C-terminal region that does not appear to be involved in contacting Ftz-F1. Similarly, SF-1 interacts with TFIIB via the Ftz-F1 box and adjacent proline-rich region, neither of which appear to be necessary for interaction with Ftz. Thus, these regions of the proteins could be available in the DNA bound ternary complex to directly contact the basal transcription machinery (Yussa, 2001).

Does Ftz interaction obviate a requirement for a Ftz-F1 coactivator? The novel possibility is suggested that Ftz substitutes for coactivator function for Drosophila Ftz-F1. For mammalian SF-1, standard coactivator interactions have been demonstrated in vitro and in cell culture, suggesting that SRC and CBP/p300 family proteins are partners of SF-1. Drosophila CBP has been well characterized and one p160/SRC-type coactivator, Taiman, was recently identified. Like Ftz, Drosophila CBP has one LXXLL motif while Tai has four such motifs, as is typical for mammalian coactivators. However, it is unlikely that interaction with either of these coactivators is sufficient to activate Ftz-F1 since both dCBP and Tai are expressed ubiquitously in the embryo, including the cells where Ftz-F1 is apparently inactive. One interesting possibility is that dCBP acts as a corepressor in the context of Ftz-F1, as it has been shown to do for TCF. Thus dCBP might silence Ftz-F1 by interacting with its AF-2 domain via an LXXLL motif. This interaction could be displaced by Ftz because of an intrinsically higher affinity of its LXXLL motif as compared to that of dCBP. Note that peptides with variations of the LXXLL motif have different affinities for AF-2 domains of nuclear receptors. Alternatively, Ftz may interact preferentially with Ftz-F1 because of the additional protein-protein and protein-DNA interactions that bring high levels of Ftz protein in close proximity to Ftz-F1, driving the interaction of its LXXLL motif with Ftz-F1 (Yussa, 2001).

In addition to its role in DNA binding, the Ftz HD is involved in direct protein-protein interactions with its cofactor Ftz-F1. These findings underscore the importance of the HD, which is absolutely required for the wild type function of Ftz, as it is for other Hox proteins. Some years ago, a 'HD-independent' activity of Ftz was described. These studies made use of a protein carrying a deletion within the HD (DHD) that removes helix 2 and portions of helices 1 and 3. It was shown that this protein is able to cause 'anti-ftz' phenotypes that result from mis-expression of Ftz with a heat inducible promoter and it was suggested that this protein can rescue ftz-dependent cuticle when similarly overexpressed. In contrast to these results, Ftz DHD was unable to rescue ftz mutants when the protein was expressed under control of endogenous ftz regulatory elements. In addition, even subtle mutations within this region of the HD abolish rescue potential. Finally, FtzDHD was unable to rescue any cuticular defects associated with ftz mutations when expressed using native ftz regulatory elements. Thus it is likely that under conditions of overexpression, interactions with Ftz-F1 through the AF-2/LXXLL domains of the proteins can to some extent overcome the endogenous requirement for the HD by positioning Ftz on the DNA of some target elements, allowing for gene activation (Yussa, 2001).

Given the highly conserved nature of the HD, the finding that it is involved in the Ftz/Ftz-F1 interaction led the authors to ask if other Hox proteins could interact with Ftz-F1. Preliminary results indicate that a number of Hox proteins coordinately activate transcription in conjunction with Ftz-F1 in cells. Experiments are underway to determine whether these interactions -- which are less potent than Ftz -- are strong enough to support interactions between Ftz-F1 and Hox proteins during Drosophila development. Such interactions would have been masked in previous genetic studies by the fact that the earliest function of Ftz-F1 in segmentation results in a phenotype which precludes analysis of Ftz-F1 function at slightly later stages when Hox genes such as Ubx, Antp and Scr are active. An additional question is whether conserved Hox proteins are partners of mammalian Ftz-F1 proteins. Co-expression of and interaction between Hox proteins and Ftz-F1 have not been investigated in mammals. Several partners of SF-1 have been characterized, including the nuclear receptor Dax-1, the zinc finger transcription factor WT-1, and the Bcd-family HD protein Ptx-1. For Ptx-1, the interaction domain with SF-1 maps outside of the HD. However, interactions between other nuclear receptors and the HDs of other proteins have been reported. Since ftz-f1 but not ftz genes are found in the vertebrate lineage, an intriguing possibility consistent with the results discussed above is that vertebrate Ftz-F1 proteins interact with HDs of Hox proteins that are conserved throughout evolution, to execute unique functions as nuclear receptor/Hox protein heterodimers (Yussa, 2001).

Orphan receptors for whom cognate ligands have not yet been identified form a large subclass within the nuclear receptor superfamily. To address one aspect of how they might regulate transcription, the mode of interaction between the Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product Fushi tarazu (FTZ) was investigated. Strong interaction between these two factors was detected by use of the mammalian one- and two-hybrid interaction assays without addition of ligand. This interaction requires the AF-2 core and putative ligand-binding domain of FTZ-F1 and the LXXLL motif of FTZ. The requirement of these elements has been further confirmed by examination of their target gene expression in Drosophila embryos and observation of a cuticle phenotype in transgenic fly lines that express mutated factors (Suzuki, 2001).

In Drosophila cultured cells, FTZ is required for FTZ-F1 activation of a FTZ-F1 reporter gene. These results reveal a resemblance in the mode of interaction between FTZ-F1 and FTZ and that of nuclear receptor-coactivator and indicate that direct interaction is required for regulation of gene expression by FTZ-F1. Thus, it is proposed that FTZ may represent a category of LXXLL motif-dependent coactivators for nuclear receptors (Suzuki, 2001).

The general structure of the LBD of nuclear receptor superfamily members is composed mainly of 12 helices. Interaction with ligand induces allosteric changes in conformation, especially in the configuration of helix 12 at the C terminus of the LBD, leading to transcriptional activation or repression. Helix 12 is often referred to as the AF-2 core (or AF-2 activation domain, tauc or tau4) that serves in some receptors as a conserved domain essential for ligand-dependent transcriptional activation. Transcriptional coactivators such as CBP/p300, TRAP220, and p160 family factors, SRC-1/NcoA-1, TIF2/GRIP1/NcoA-2, and p/CIP/ACTR/AIB1, have been shown to mediate activating signals through binding to nuclear receptors in a ligand-dependent manner. For this receptor-coactivator interaction, conserved sequences containing a short signature motif of LXXLL (where L is leucine and X can be any amino acid) have been implicated. The conserved leucines in these so-called LXXLL motifs, or NR boxes, appear indispensable for interaction with nuclear receptors. In nuclear receptors, the importance of helices 3, 5, and 12 (AF-2 core) in the LBD has been demonstrated, and computational modeling studies have predicted that helices 3, 5, and an appropriately realigned helix 12 form an interacting surface for the LXXLL. The majority of nuclear receptors, however, are 'orphans', for which cognate ligands have not yet been identified and the molecular mechanisms of their transcriptional regulation remain unclear. From an evolutionary aspect, the extension of the structural conservation to domains including the LBD strongly suggests a functional significance, raising a naive question. Despite large-scale ligand screenings that have been undertaken by many groups, why are there still so many 'orphans' remaining? One answer may be that the structural conservation in the LBD implicates an importance for interactions with various intracellular factors other than small lipophilic molecules (Suzuki, 2001).

Ubiquitous expression of FTZ in early embryos under control of the heat shock promoter broadens even-numbered engrailed (en) stripes, represses alternate wg stripes, and results in a so-called anti-ftz cuticle phenotype, in which roughly reciprocal segments are missing compared with the ftz larval cuticle phenotype. However, ectopic en induction or wg repression was not observed by expressing a construct (mutFTZ) containing substitutions in the tandem leucines, indicating that the LXXLL motif in FTZ is necessary for the ectopic expression of en and the repression of wg. FTZ-dependent en induction and wg repression were also observed by expression of both FTZ-F1 and FTZ under control of the heat shock promoter in the ftz-f1 mutant embryo but not when FTZ-F1DeltaAF2C was used instead of FTZ-F1. An anti-ftz cuticle phenotype was produced by forced expression of wild-type FTZ but not by expression of mutFTZ. In ftz-f1 mutant embryos, anti-ftz phenotypes were obtained when FTZ-F1 and FTZ were coexpressed under heat shock control but not upon replacement of FTZ-F1 with FTZ-F1DeltaAF2C. These observations indicate that interaction through the LXXLL motif in FTZ and AF-2 core in FTZ-F1 is necessary for producing an anti-ftz phenotype and further support the results of the one- and two-hybrid assay (Suzuki, 2001).

Thus, results using embryos strongly suggest that FTZ-F1 activates engrailed in vivo through the AF-2-LXXLL-dependent direct interaction. In early fly embryos, FTZ-F1 seems to function as an activator for engrailed only in regions where FTZ is also present despite the uniform expression of FTZ-F1. Such situations mimic that of the requirement for a ligand by a nuclear receptor in controlling its function and specificity in gene expression. The characteristic cooperation of FTZ-F1 and FTZ provides a novel example of transcriptional regulation by a nuclear receptor, which may be an alternative pathway to the conventional one using lipophilic ligands. From an evolutionary aspect, it has been proposed that the ancestral nuclear receptor had no ligand and the ability to bind a ligand was acquired by a subset of descendent receptors later in evolution. It has also been presumed that FTZ-F1 is one of the most ancient receptors based on its distribution among species. It is believed that transcriptional activation by FTZ-F1 through binding to FTZ might represent a primitive style of regulation by nuclear receptors before the acquisition of ligand-binding ability. The existence is presumbed of yet unidentified corresponding factors for other orphan receptors as well as for ligand-responsive receptors, which may form a new group of nuclear receptor coactivators and play critical roles for development and metabolism (Suzuki, 2001).

Drosophila mediator complex is used by Fushi-tarazu

To decipher the mechanistic roles of Mediator proteins in regulating developmental specific gene expression and compare them to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates promoter-specific activation signals to the basal transcription machinery (Park, 2001).

Previous studies in yeast and human cells have suggested that transcriptional activator proteins interact with Mediator complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes with the VP16 protein were examined. After incubation of nuclear extracts with an excess of GST fusion protein beads containing either wild-type or mutant (Delta456FP442) VP16 activation domain, the supernatants were analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating with GST-VP16 but not with GST-VP16^{Delta456FP442}. However, the amounts of dGCN5, dTAF_II40, dTAF_II250, and dTBP in the extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most strongly bound to and most readily recruited to the activation domain (Park, 2001).

In addition to the model VP16 activator derived from herpesvirus, dMediator interacts with Drosophila transcriptional activators Dorsal and heat shock factor (dHSF). When dMediator complex was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein G-agarose beads through anti-dSOH1 Ab and the binding of diverse ³⁵S-labeled Drosophila transcription factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).

To evaluate the requirement of dMediator for activated transcription in response to the Drosophila activator proteins that interact with dMediator, the ability of dMediator-deficient nuclear extracts to support transcriptional activation by the Dorsal and Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level of transcriptional activation is reduced significantly (five- and three-fold activations, respectively) in nuclear extract that has been depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by the sequence-specific transcription factor Even-skipped (Park, 2001).

dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer elements. However, the requirement of TAFs, or at least dTAF_II250, in activated transcription appears to be redundant in the in vitro transcription system used and affected by such factors as the core promoter organization or nucleosomal structure of transcriptional templates. Several TAF components in the TFIID complex indeed have biochemical activities and structural motifs adequate for the recognition of specialized settings of transcription templates. For example, certain TAFs recognize the Inr and DPE sequences located in many Drosophila core promoters and increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAF_II250, which has a HAT catalytic activity and also possesses a histone octamer-like module comprising the histone H2B-, H3-, and H4-like TAFs. Although not experimentally demonstrated, these TAFs may have some roles in the transcriptional regulation of nucleosomal templates (Park, 2001).

The sequence-specific transcription factors which interact physically with dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and Fushi-tarazu. These factors contain different types of activation domains (acidic and glutamine-rich domains). Most of these transcription factors have been shown to activate transcription either constitutively or inducibly. It is noteworthy that dHSF interacts with and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170), suggests that some function of Mediator is required for HSF-mediated transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is conserved between yeast and Drosophila, transcriptional activation by HSF might utilize the conserved Rgr1 components of the Mediator complexes (Park, 2001).

Although some human Mediator complexes appear to have a negative effect on activated transcription, dMediator does not exhibit such an activity in an in vitro transcription system reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is more plausible that the dMediator-Krüppel interaction observed is a part of the mechanism for transcriptional activation rather than transcriptional repression. Taken together with the fact that dMediator is dispensable for basal transcription, the lack of defect of the dMediator-depleted nuclear extracts on transcriptional repression by Even-skipped protein suggests that dMediator is required mainly for the mediation of transcriptional activation signals to the basal transcription machinery. Very recently, developmental roles of certain dMediator proteins found in the Drosophila genome database have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).

Like yeast Mediator, dMediator bind with the CTD repeats of Drosophila Pol II. This implies that though dMediator was purified separately from Pol II, these two complexes indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS. Such interactions may be involved in the regulation of Pol II preinitiation complex assembly. Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator. Also, TFIIE interacts with the Mediator protein Gal11. Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).

dMediator contains the protein kinase component Cdk8, which can phosphorylate serine residues in the CTD. This catalytic kinase subunit seems responsible, at least in part, for the Pol II phosphorylation by dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II repeats, suggesting the presence of a functional interaction between these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).

Post-transcriptional regulation

When first expressed in early embryogenesis, Fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, FTZ mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of FTZ mRNA is probably crucial for attaining this localized pattern of expression. There are least two destabilizing elements in the Fushi tarazu mRNA, one located within the 5' one-third of the mRNA and the other near the 3' end (termed FIE3 for ftz instability element 3'). The FIE3 lies within a 201-nucleotide sequence just upstream of the polyadenylation signal and can act autonomously to destabilize a heterologous mRNA. Further deletion constructs have identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other destabilization sequences suggests that FIE3 contains a novel RNA destabilization element (Riedl, 1996).

A class of pre-mRNAs that are efficiently processed (spliced) in HeLa extracts depleted for U1 snRNP (delta U1 extracts) without the addition of excess SR proteins contrasts with other pre-mRNAs that can be spliced only when high concentrations of SR splicing factors are added. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila Fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon either U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element is found adjacent to the branch site. In the other, two elements flanking the 5' splice site are critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon (Crispino, 1996).

One class of localized transcripts is encoded by the zygotic Drosophila pair-rule genes [including fushi tarazu and hairy (h)], which are required to establish reiterated (segmental) embryonic pattern. Pair-rule transcripts are transcribed in the syncytial blastoderm embryo as seven distinct transverse stripes along the anterior/posterior axis. At this stage, a monolayer of nuclei at the embryonic surface subdivides the cortical cytoplasm ('periplasm') into apical and basal compartments. Most zygotic transcripts do not localize to a specific periplasmic compartment in blastoderm embryos, but pair-rule transcripts accumulate exclusively in the apical periplasm. The function of this localization is not yet established, although it may serve to restrict protein diffusion within the syncytial embryo. Several lines of evidence have argued against pair-rule transcripts localizing by cytoplasmic transport. (1) Pair-rule mRNAs are not detectable in the basal cytoplasm, even after extensive overstaining or stabilization of transcripts. (2) Pair-rule transcripts are extremely unstable (t1/2 6.5 min), so they are more likely to localize directly and rapidly. Stabilizing the transcripts does not prevent localization, showing that their selective accumulation is not due to selective degradation of basal transcripts. These observations have led to the proposal that Drosophila pair-rule transcripts localize directly (i.e., by selective [vectorial] export through one side of the nuclear envelope) (Francis-Lang, 1996). (3) Further evidence for this model comes from experiments examining aneuploid blastoderm embryos, in which pair-rule transcripts localize apically to displaced internalized nuclei (Francis-Lang, 1996): this indicates linkage between the nucleus and sites of transcript localization. Evidence connecting transcription and localization comes from studies showing that bcd transcripts localize apically only in cells where they are being synthesized. bcd transcripts are made in nurse cells, before being transported into the adjacent oocyte where they localize anteriorly. Maternal bcd transcripts localize apically to nuclei in nurse cells but not in the mature egg. Zygotic transcripts with a bcd 3'-untranslated region (3'UTR) localize apically in blastoderm embryos. Together, these results indicate that the bcd apical localization signal only operates when coupled with synthesis (Lall, 1999 and references).

Previous studies have shown that the 3'UTRs of pair-rule transcripts are necessary and sufficient to target transcripts apically (Davis, 1991). In the case of ftz, the localization signal resides within a 1.3 kb region of the ftz gene, which includes the ftz 3'UTR (Davis, 1991). This signal has been defined more precisely using germline transformation and it has been found that hybrid lacZ-ftz 3'UTR transcripts with 205 bp of ftz 3' genomic sequence are apically localized (LTf2). Transcripts that lack the last 53 bases of the 3'UTR fail to localize apically, showing that the ftz 3'UTR is necessary and sufficient to target a heterologous transcript apically (Lall, 1999).

Maternal Drosophila and Xenopus transcripts that localize via cytoplasmic mechanisms also have localization signals in the 3'UTR. Thus, endogenous pair-rule mRNAs might localize similarly, despite previous indirect evidence for a nuclear mechanism. This possibility was examined directly by testing whether transcripts injected into the blastoderm cytoplasm can localize specifically: if nuclear events are essential for apical accumulation of pair-rule transcripts, ftz mRNAs that have not been exposed to such an environment should not localize. To check localization, visualized transcripts that had been labeled with fluorescent tags were visualized directly. Capped, polyadenylated transcripts incorporating aminoallyl-UTP were synthesized in vitro and chemically labeled with either fluorescein (FITC) or Rhodamine (Rh). FITC-labeled ftz and Rh-labeled ftzDelta3' transcripts were injected into the basal periplasm during nuclear cleavage cycles 13 or 14, when endogenous pair-rule transcripts are restricted to the apical periplasm. Localization of the RNAs was examined by confocal microscopy 0-30 min after injection. Initial experiments have shown that both transcripts are short lived, in accord with in vivo measurements of ftz transcript half-life. Thus, the transcripts do not persist long enough to test for selective localization. To overcome this problem, cycloheximide was coinjected. This has previously been shown to stabilize endogenous pair-rule transcripts without affecting their localization. Under these conditions, injected ftz and ftzDelta3' transcripts are still readily detectable 30 min or more after injection. No evidence of selective localization of the full-length ftz transcripts is found. In essentially all embryos (>99%; n > 400), full-length ftz transcripts fail to accumulate selectively in the apical periplasm; both injected transcripts diffuse out from the site of injection and colocalize for at least 30 min. Thus, injected ftz transcripts are unable to mimic the apical localization shown by endogenous pair-rule and reporter transcripts, indicating that purely cytoplasmic mechanisms are insufficient to account for pair-rule transcript localization (Lall, 1999).

Naked pair-rule transcripts could be unable to localize either because localization depends on vectorial nuclear export, or because transcripts need prior exposure to a nuclear environment for apical targeting (e.g., to recruit nuclear proteins that are subsequently required for localization in the cytoplasm). To distinguish between these alternatives, a test was performed to see whether nuclear proteins might promote cytoplasmic localization of pair-rule transcripts. FITC-ftz and Rh-ftzDelta3' were exposed to Drosophila embryonic nuclear extracts and coinjected the 'preincubated transcripts' with cycloheximide into the basal periplasm of cycle 14 blastoderm embryos. Strikingly, preincubated FITC-ftz transcripts specifically accumulate in the apical periplasm within 10 min of injection, whereas ftzDelta3' transcripts, lacking the 3'UTR, remain unlocalized. Nuclear extract is much more active than control proteins in promoting apical transcript localization. Together, these results indicate that nuclear extracts include specific factors required to localize pair-rule transcripts in blastoderm embryos (Lall, 1999).

Preincubated ftz transcripts localize in apical caps above the nuclei, thereby differing slightly from endogenous transcripts that localize as a continuous stripe domain. A further difference is that the accumulations of preincubated transcripts appear more particulate than those of endogenous transcripts, although this may reflect differences in expression levels or detection methods. In any case, the efficiency of localization is high, and little residual transcript remains at the site of injection. Thus, nuclear extracts include factors that specifically promote apical localization of preincubated ftz transcripts. Endogenous ftz transcripts are never observed in the basal periplasm, indicating that localization by a cytoplasmic mechanism should be extremely rapid. Efficient localization occurs in all embryos 4-5 min after injection, and 50% of embryos display apical localized transcripts 2-2.5 min after injection. Thus, preincubated transcripts also localize rapidly (Lall, 1999).

Localization of preincubated ftz is dependent on microtubules but independent of microfilaments. Early cycle 14 embryos were injected with 2 µg/ml of colcemid and 10 min later, injected with preincubated FITC-ftz transcript mixture. Localization is almost completely disrupted: the behavior of FITC-ftz resembles that of coinjected Rh-ftzDelta3'. This inhibition of localization indicates that pair-rule transcript localization depends on an intact microtubule cytoskeleton. By contrast, preincubated FITC-ftz transcripts still localize apically in embryos that have been coinjected with Cytochalasin B, which disrupts actin-dependent processes such as anchoring of nuclei to the cortex and, indeed, causes displacement of nuclei into the basal periplasm. Thus, processes disrupted by Cytochalasin B are not required for apical localization of preincubated FITC-ftz transcripts (Lall, 1999).

Nuclear factors promoting cytoplasmic transcript targeting are evolutionarily conserved. A human nuclear extract from TIG-3 cells (human fetal lung fibroblasts) can also promote pair-rule transcript localization. Preexposed FITC-ftz transcripts specifically localize apically (21/21 at 50 ng/µl protein), indicating that human nuclear extracts indeed promote pair-rule transcript localization. This degree of activity is higher than that of an equivalent Drosophila extract, although the proportion of localized transcripts within each embryo appears lower with human extracts. In any case, an activity that promotes pair-rule transcript localization is conserved between flies and humans (Lall, 1999).

For several reasons, the possibility was considered that the nuclear factors facilitating localization are hnRNP proteins: (1) hnRNP's are well conserved between flies and humans; (2) the nuclear factors that facilitate localization appear to function in the cytoplasm and therefore must shuttle between nucleus and cytoplasm, as do hnRNP's; (3) the Drosophila Squid (Sqd) hnRNP protein, a homolog of the mammalian hnRNP-A/B proteins, is required for gurken transcripts to localize during oogenesis, and (4) hnRNP-A2 has been shown to bind to a 3'UTR sequence required for the localization of MBP transcripts in rat oligodendrocytes (Lall, 1999 and references).

To test whether Sqd protein promotes cytoplasmic transport, transcripts were preincubated with each of the three Sqd protein isoforms and injected into blastoderm embryos. All three Sqd isoform fusion proteins are active in promoting apical localization. Thus, preexposure to any of the Sqd isoforms leads to localization of labeled ftz but not ftzDelta3' transcripts. Association of ftz transcripts with Sqd is very rapid, being essentially complete within the 2 min period required to establish injections. A test was also performed to see whether the ability of hnRNP proteins to promote transcript localization has been evolutionarily conserved. ftz transcripts preincubated with each of the human hnRNP-A1, -A2 and -B proteins localize apically, showing that the activity of this class of A/B hnRNP proteins is conserved between Drosophila and humans (Lall, 1999).

Immunostaining of early embryos shows that Sqd is indeed present in blastoderm nuclei, as expected if Sqd is the major in vivo localizing activity. Unfortunately, a direct test for whether Sqd is required to localize pair-rule transcripts could not be made because strong sqd mutant eggs are not fertile. However, it was determined whether Sqd selectively recognizes the ftz 3'UTR by examining protein extracts from ovaries, the major source of Sqd in blastoderm embryos. Proteins that bind to ftz-3'UTR transcripts were labeled by UV cross-linking to ³²P-labeled transcripts and visualized following gel electrophoresis. ftz-3'UTR transcripts label a predominant 42 kDa protein in Drosophila ovary extracts, the same size as Sqd and as the activity labeled by the grk-3'UTR. The 42 kDa protein is indeed Sqd, being immunoprecipitated by anti-Sqd antibodies and only weakly labeled by a control nanos-3'UTR transcript. Thus, Sqd binds specifically to the ftz-3'UTR and represents a major such activity in oocyte extracts (Lall, 1999).

The ability of human hnRNP proteins to promote localization and the uniform expression of Sqd within blastoderm nuclei argue against a vectorial export mechanism. Localization of preincubated pair-rule transcripts requires an intact microtubule cytoskeleton and is likely to be mediated by microtubule-dependent motors. In wild-type blastoderm embryos, each nucleus is indeed capped by an apical bundle of microtubules that could serve as a framework for transcript transport. Apical transport along these microtubules would require transport by a dynein-like motor. However, pair-rule transcripts can localize elsewhere in aneuploid blastoderm embryos. In 3L- embryos, some nuclei become internalized and lack associated apical microtubules (Francis-Lang, 1996); nevertheless, transcripts accumulate adjacent and apical to these nuclei, raising the possibility that pair-rule transcripts run along a different, minority class of microtubules. After transport, pair-rule transcripts are anchored to the cytoskeleton, as shown by their lack of diffusion in blastoderm embryos. However, the transcripts still localize apically to nuclei displaced from the periphery of the embryo following Cytochalasin B treatment, indicating that transcript attachment sites differ from those of nuclei and are perhaps not microfilament based. In the latter case, they would differ from those already implicated in transcript localization (Lall, 1999 and references).

Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the same machinery and RNA signals drive specific accumulation of maternal RNAs in the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos; interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor complex that drives transcript localization in a variety of tissues (Bullock, 2001).

Asymmetric RNA localization is evident during zygotic development, especially in the unicellular syncytial blastoderm embryo. At this stage, several transcripts including those of the pair-rule and wingless (wg) segmentation genes lie exclusively apically of the layer of several thousand peripheral nuclei. Localization of these transcripts seems to be mediated by signals within their 3' untranslated regions (UTRs), and to be driven on microtubules by the minus-end-directed molecular motor, dynein. The linkers and other factors that provide the cargo specificity are unknown. Nor is it clear if transcript localization in blastoderm embryos relates to that in other types of cells (Bullock, 2001).

There is a rapid apical localization of fluorescently labelled fushi tarazu ( ftz) pair-rule transcripts injected into the basal cytoplasm of the cycle 14 blastoderm embryo. Although these experiments indicated a requirement for nuclear proteins fluorescein, labelling compromizes the structure of the transcripts, and pair-rule [even-skipped, hairy (h), ftz, paired and runt] and wg transcripts labelled with several other fluorochromes localize apically within 5-8 min without the need for exogenous protein. Indeed, injected unlabelled transcripts also localize apically. The protein-free assay retains specificity for apical transport, since transcripts that are normally unlocalized [Krüppel (Kr), huckebein] or enriched in the basal cytoplasm (string) are not transported apically and instead diffuse away from the site of injection (Bullock, 2001).

Blastoderm localization signals can drive transcript transport during oogenesis. This view is supported by more detailed analysis of maternally expressed pair-rule transcripts. The injection assay reveals a minimum region between positions 1,374 and 1,579 in ftz that is necessary and sufficient for localization in blastoderm embryos. A similar region of ftz seems to be required for localization of transcripts into the oocyte. Furthermore, h and runt transcripts, driven maternally by the Hsp70 promoter, also accumulate specifically in the oocyte and later reside at its anterior cortex, whereas Kr or truncated h transcripts lacking most of the 3' UTR fail to localize either in blastoderm embryos or during oogenesis (Bullock, 2001).

Whether Egl and BicD are present in early embryos was examined. Both proteins are supplied maternally to the embryo. They are noticeably enriched apical to the nuclei at blastoderm stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the proteins is present in the basal cytoplasm (Bullock, 2001).

Whether endogenous Egl and BicD can associate with injected localizing transcripts, as might be expected if they are components of the RNA localization machinery, was tested. Injection of h transcripts leads to marked enrichment of Egl and BicD protein levels at the sites of RNA localization. Similar results are found on injection of the other tested maternal and zygotic localizing transcripts ( ftz, bcd, grk, K10, nos, osk and w). Both proteins accumulate basally at the site of injection within 1-2 min. Protein recruitment is not inhibited in embryos preincubated with colcemid, showing that it is not dependent on intact microtubules. Thus, the proteins are recruited locally before transport and are transported together apically with transcripts (Bullock, 2001).

Whether BicD and Egl are required for apical localization in blastoderm embryos was examined. Strong BicD alleles block oogenesis early, and weaker mutant mothers that lay fertilized eggs (BicD^HA40/BicD^R26 and BicD^H3/BicD^R26) retain sufficient BicD activity for a normal apical distribution of endogenous pair-rule transcripts. However, the reduced BicD activity in these embryos no longer supports efficient transport of injected transcripts: 62% of BicD^HA40 /BicD^R26 and 73% of BicD^H3/BicD^R26 embryos show no or weak localization 5-8 min after injection, compared with 10% of wild-type embryos. Moreover, an antibody against BicD blocks RNA transport. Preinjection into the basal cytoplasm of anti-BicD antibody 4C2 strongly inhibits the localization of injected h, ftz, grk and stg-K10TLS transcripts in 70%-75% of embryos. The microtubule cytoskeleton is not obviously affected by the brief (~20 min) antibody treatment, indicating that the effects on RNA transport are probably direct. Injection of anti-BicD antibody prevents apical localization of endogenous pair-rule transcripts, also leading to anteroposterior smearing of their distribution. Thus, apical transcript localization seems to be important in restricting the range of activity of pair-rule genes, and allowing their combinatorial control of Drosophila segmentation (Bullock, 2001).

Injecting blastoderm embryos with anti-Egl also inhibits apical localization of both exogenous and endogenous pair-rule transcripts, without overtly disrupting the microtubule network. Moreover, its effect is more potent in embryos from mothers containing only a single copy of the egl gene, indicating that the antibody disrupts RNA localization by inhibiting the activity of Egl. Egl and BicD are probably also involved in transporting other cargoes. The arrangement of peripheral nuclei is disrupted after injection of antibodies to either of the two proteins, consistent with data showing a requirement for BicD in nuclear migration in eye imaginal disc cells. Embryos injected with either antibody undergo abnormal morphogenesis, which is also indicative of Egl and BicD transporting additional cargoes (Bullock, 2001).

These results indicate that Egl and BicD are principal elements of a complex that transports RNA in blastoderm embryos. Egl and BicD appear to be present as pools of excess cytoplasmic protein that associate selectively with localizing transcripts and are transported together apically. Protein recruitment occurs before transport and does not require microtubule integrity; rather, transport depends on Egl and BicD activity. Egl and BicD probably act directly to mediate RNA transport associated with establishment and maintenance of the oocyte. Thus, mutant transcripts that are defective in export from nurse cells into the oocyte fail to recruit Egl or BicD in blastoderm embryos. grk transcripts are also recognized by the Egl-BicD-microtubule transport pathway, which is consistent with the hypothesis that nurse cells are a source of these transcripts for the early oocyte and that they do not derive exclusively from the oocyte nucleus (Bullock, 2001).

Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of protein/RNA co-transport. The complex may have an additional role in anchoring transcripts at their destination. Alternatively, maintenance of localized transcripts might not depend on an independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).

Dhc, Egl and BicD have markedly similar distributions during oogenesis and in blastoderm embryos, and seem to function together in specifying oocyte identity. It is proposed that an Egl/BicD complex links specific RNAs to dynein and the microtubules. The same machinery may operate elsewhere in Drosophila. For example, inscuteable transcripts, which localize asymmetrically in neuroblasts, also localize apically when injected into blastoderm embryos. Indeed, germline transcripts localize apically when expressed in follicle cells. Egl and BicD homologs have been identified in Caenorhabditis elegans and mammals, and might comprise part of an evolutionarily conserved cytoskeletal system for transporting transcripts and other cargoes (Bullock, 2001).

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