Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.     

Pattern formation in the Drosophila embryo: allocation of cells to parasegments by even-skipped and fushi tarazu

The first sign of metamerization in the Drosophila embryo is the striped expression of pair-rule genes such as fushi tarazu (ftz) and even-skipped (eve). Here we describe, at cellular resolution, the development of ftz and eve protein stripes in staged Drosophila embryos. They appear gradually, during the syncytial blastoderm stage and soon become asymmetric, the anterior margins of the stripes being sharply demarcated while the posterior borders are undefined. By the beginning of germ band elongation, the eve and ftz stripes have narrowed and become very intense at their anterior margins. The development of these stripes in hairy-, runt-, eve-, ftz- and engrailed- embryos is illustrated. In eve- embryos, the ftz stripes remain symmetric and lack sharp borders. Our results support the hypothesis (Lawrence et al. Nature 328, 440-442, 1987) that individual cells are allocated to parasegments with respect to the anterior margins of the eve and ftz stripes.     

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.     

Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene

The distributions of the products of the homeotic genes Sex combs reduced (Scr) and Ultrabithorax (Ubx) and of the segmentation genes, fushi tarazu (ftz), even skipped (eve) and engrailed (en) have been monitored in polyhomeotic (ph) mutant embryos. None of the genes monitored show abnormal expression at the blastoderm stage in the absence of zygotic ph expression. Both Scr and Ubx are ectopically expressed in the epidermis of ph embryos, confirming the earlier proposal, based on genetic analysis, that ph+ acts as a negative regulator of Antennapedia (ANT-C) and bithorax (BX-C) complex genes. At the shortened germ band stage, en is also ectopically expressed, mainly in the anterior region of each segment. In contrast to these effects in the epidermis, the expression of en, Ubx, Scr and ftz is largely or completely suppressed in the central nervous system, whereas eve becomes ectopically expressed in most neurones.     

Autocatalytic ftz activation and metameric instability induced by ectopic ftz expression

Inappropriate expression of the Drosophila pair-rule gene, fushi tarazu (ftz), causes cuticular pattern deletions apparently complementary to those in ftz larvae. We show that the two patterns actually originate similarly, in both cases affecting the even-numbered parasegmental boundaries. The reciprocal cuticular patterns derive from differing patterns of selector gene expression (homoeotic transformations). The primary effect of ectopic ftz activity is to broaden ftz domains by autocatalytic activation of endogenous ftz expression in an additional anterior cell. This activates engrailed (en) and represses wingless (wg) expression, consistent with their proposed combinatorial control by ftz (and other pair-rule genes) to define parasegmental primordia. We propose that the anterior margin of each ftz stripe is normally defined by the posterior even-skipped (eve) boundary.     

Analysis of a fushi tarazu autoregulatory element: multiple sequence elements contribute to enhancer activity.

Regulatory sequences or factors involved in the regulation of target genes of Drosophila homeodomain proteins are largely unknown. Here, we identify sequence elements that are involved in the function of the fushi tarazu (ftz) autoregulatory element AE, a direct in vivo target of the homeodomain protein ftz. A systematic deletion analysis of AE in transgenic embryos defines multiple elements that are redundantly involved in enhancer activity. Sequences juxtaposed to ftz binding sites are not strictly required for enhancer function. Several sequence motifs are conserved in other developmentally regulated genes of Drosophila melanogaster and in the AE homologue of Drosophila virilis. The D. virilis AE is functional in D. melanogaster. The sequence motifs identified here are candidate elements contributing to the target specificity of the homeodomain protein ftz.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

Expression of a homologue of the fushi tarazu (ftz) gene in a cirripede crustacean

In Metazoa, Hox genes control the identity of the body parts along the anteroposterior axis. In addition to this homeotic function, these genes are characterized by two conserved features: They are clustered in the genome, and they contain a particular sequence, the homeobox, encoding a DNA-binding domain. Analysis of Hox homeobox sequences suggests that the Hox cluster emerged early in Metazoa and then underwent gene duplication events. In arthropods, the Hox cluster contains eight genes with a homeotic function and two other Hox-like genes, zerknullt (zen)/Hox3 and fushi tarazu (ftz). In insects, these two genes have lost their homeotic function but have acquired new functions in embryogenesis. In contrast, in chelicerates, these genes are expressed in a Hox-like pattern, which suggests that they have conserved their ancestral homeotic function. We describe here the characterization of Diva, the homologue of ftz in the cirripede crustacean Sacculina carcini. Diva is located in the Hox cluster, in the same position as the ftz genes of insects, and is not expressed in a Hox-like pattern. Instead, it is expressed exclusively in the central nervous system. Such a neurogenic expression of ftz has been also described in insects. This study, which provides the first information about the Hoxcluster in Crustacea, reveals that it may not be much smaller than the insect cluster. Study of the Diva expression pattern suggests that the arthropod ftz gene has lost its ancestral homeotic function after the divergence of the Crustacea/Hexapoda clade from other arthropod clades. In contrast, the function of ftz during neurogenesis is well conserved in insects and crustaceans.     

Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results.