Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Cloning and transcriptional analysis of the segmentation gene fushi tarazu of Drosophila

In the course of studying the Antennapedia (Antp) locus, we found that one of the 3' Antp exons has weak cross-homology to another gene affecting segmentation, fushi tarazu (ftz; meaning "not enough segments"), which is 30 kb to the left of Antp. Homozygous ftz- embryos die before hatching and lack alternate body segments. The reduced number of segments results from the fusion of the anterior portion of one segment with the posterior portion of the next segment. The ftz gene encodes a single 1.9 kb poly(A)+ RNA expressed exclusively from the early blastoderm to gastrula stages of embryonic development. The structure of the ftz gene has been analyzed by S1 nuclease mapping and by restriction mapping of a cDNA clone. The ftz gene consists of two exons, and it is the 3' exon that cross-hybridizes with the 3' exon of Antp. The role of ftz in cell determination is discussed.     

Drosophila fushi tarazu. a gene on the border of homeotic function

BACKGROUND: Hox genes specify cell fate and regional identity during animal development. These genes are present in evolutionarily conserved clusters thought to have arisen by gene duplication and divergence. Most members of the Drosophila Hox complex (HOM-C) have homeotic functions. However, a small number of HOM-C genes, such as the segmentation gene fushi tarazu (ftz), have nonhomeotic functions. If these genes arose from a homeotic ancestor, their functional properties must have changed significantly during the evolution of modern Drosophila. RESULTS: Here, we have asked how Drosophila ftz evolved from an ancestral homeotic gene to obtain a novel function in segmentation. We expressed Ftz proteins at various developmental stages to assess their potential to regulate segmentation and to generate homeotic transformations. Drosophila Ftz protein has lost the inherent ability to mediate homeosis and functions exclusively in segmentation pathways. In contrast, Ftz from the primitive insect Tribolium (Tc-Ftz) has retained homeotic potential, generating homeotic transformations in larvae and adults and retaining the ability to repress homothorax, a hallmark of homeotic genes. Similarly, Schistocerca Ftz (Sg-Ftz) caused homeotic transformations of antenna toward leg. Primitive Ftz orthologs have moderate segmentation potential, reflected by weak interactions with the segmentation-specific cofactor Ftz-F1. Thus, Ftz orthologs represent evolutionary intermediates that have weak segmentation potential but retain the ability to act as homeotic genes. CONCLUSIONS: ftz evolved from an ancestral homeotic gene as a result of changes in both regulation of expression and specific alterations in the protein-coding region. Studies of ftz orthologs from primitive insects have provided a "snap-shot" view of the progressive evolution of a Hox protein as it took on segmentation function and lost homeotic potential. We propose that the specialization of Drosophila Ftz for segmentation resulted from loss and gain of specific domains that mediate interactions with distinct cofactors.     

A screen for genes that interact with the Drosophila pair-rule segmentation gene fushi tarazu

The pair-rule gene fushi tarazu (ftz) of Drosophila is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, we conducted a screen to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, which define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, as Atro has been shown to function as a corepressor for Eve. In this study, we present evidence that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor.     

The pattern of cell death in fushi tarazu, a segmentation gene of Drosophila

The pair-rule mutant, fushi tarazu, causes deletion of alternate metameres. Here we show that there is cell death in the mutant which begins at the completion of germ band extension. We map the dying cells in the epidermis; they occur scattered all over those regions that, in the wild type, would form the even-numbered parasegments and are also found in posterior parts of the odd-numbered parasegments. In the affected zones, dying and dividing cells are intermingled; we suggest that cells from these zones may still give descendents that contribute to the larval cuticle. Cell death is not limited to those cells that would normally express ftz+, suggesting that it is some indirect consequence of the abnormal situation in the mutant embryo.     

Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila

Summary We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3 and 5 to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a housekeeping gene-rosy (ry), or Xdh. In contrast, DNA sequences 5 to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.     

Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis

The establishment of the segmental body pattern of Drosophila requires the coordinated functions of three classes of zygotically active genes early in development. We have examined the effects of mutations in these genes on the spatial expression of the fushi tarazu (ftz) pair-rule segmentation gene. Mutations in four gap loci and in three pair-rule loci dramatically affect the initial pattern of transverse stripes of ftz-containing nuclei. Five other pair-rule genes and several other loci that affect the larval cuticular pattern do not detectably affect ftz expression. No simple regulatory relationships can be deduced. Rather, expression of the ftz gene depends upon the interactions among the different segmentation genes active at each position along the anterior-posterior axis of the early embryo.     

Determinants of Drosophila fushi tarazu mRNA instability.

The fushi tarazu gene is essential for the establishment of the Drosophila embryonic body plan. When first expressed in early embryogenesis, fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, fushi tarazu mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of fushi tarazu mRNA is probably crucial for attaining this localized pattern of expression. mRNA stability in transgenic embryos was measured by a new method that does not use drugs or external interference. Experiments using hybrid genes that fuse fushi tarazu sequences to those of the stable ribosomal protein A1 mRNA provide evidence for at 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 identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other previously identified destabilization sequences suggests that FIE3 contains a novel RNA destabilization element.     

Localization of the fushi tarazu protein during Drosophila embryogenesis

The fushi tarazu (ftz) gene of Drosophila acts early in embryogenesis to regulate body segmentation. The localization of the ftz protein product in embryos was examined using indirect immunofluorescence microscopy. Antibodies were prepared against a β-galactosidase-ftz hybrid protein made in E. coli. The ftz protein was first detectable in blastoderm-stage embryos as seven stripes of nuclei encircling the embryos transversely. The stripes persist through the early events of gastrulation, but disappear before overt segmentation is visible. The ftz protein is expressed a second time in some nuclei of the developing nervous system. In contrast to the early pattern, at the later stage, ftz is expressed in each of fifteen metameric subunits of the embryo.     

Control of bithorax complex functions by the segmentation gene fushi tarazu of D. melanogaster

The properties of three dominant alleles of ftz are described. These alleles cause transformations of the first abdominal segment to the third and cause alternate segment pattern deletions that are out of phase with respect to those caused by ftz null alleles. To explain the effects of these mutations, a model is proposed in which ftz+ has two roles: to subdivide the body into parasegments and to activate appropriate bithorax complex functions in alternate parasegments. According to this model, the effects of the novel ftz alleles can be understood as arising from a slight widening of the blastoderm stripes of ftz expression.     

Stage-specific phosphorylation of the fushi tarazu protein during Drosophila development.

The regulatory protein encoded by the fushi tarazu (ftz) gene is expressed during three different stages of Drosophila embryogenesis in three different developing tissues. Previously, we demonstrated that ftz protein ectopically expressed throughout developing embryos under the control of an hsp70 heat shock promoter is heavily modified. Here we show that these negatively charged isoforms of the protein are the result of phosphorylation at as many as 16 sites. Phosphate groups could be removed in vitro by treatment with various phosphatases and could be added in vivo by incubating embryo-derived cells or nuclei in the presence of [32P]-orthophosphate. Phosphoamino acid analysis of immunoprecipitated ftz protein yielded both phosphoserines and phosphothreonines at a ratio of approximately 1:1. Interestingly we find that the endogenous ftz protein is also phosphorylated at multiple sites and that different subsets of the phosphoisoforms occur during different stages of development.     

Analysis of function of the pair-rule genes hairy, even-skipped and fushi tarazu in mosaic Drosophila embryos

We report the first attempt of its kind to study genetic interactions using young Drosophila embryos that are mosaic for wildtype and mutant cells. Using nuclear transplantation we make mosaic embryos in which a patch of cells lacks a particular segmentation gene, A. With antibodies, we than look at the expression of another gene that is known to be downstream of gene A, with respect to the cells in the patch. We have examples of patches of hairy cells (where we monitor the effect on fushi tarazu (ftz) expression), even-skipped (monitoring ftz) and ftz (monitoring engrailed and Ultrabithorax). Our main finding is that the dependence of engrailed expression on the ftz gene is strictly cell-autonomous. This result goes some way towards explaining the dependence of Ultrabithorax expression on ftz, a dependence we show to be locally cell-autonomous within parts of parasegments 6 and 8 but non autonomous within parasegment 7.     

Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5

The DNA-binding homeobox motif was first identified in several Drosophila homeotic genes but also in fushi tarazu, a gene found in the Hox cluster yet involved in segmentation, not anteroposterior patterning [1]. Homeotic transformations are not seen in insect ftz mutants, and insect ftz genes do not have Hox-like expression except within the nervous system [2] [3]. Insect ftz homeobox sequences link them to the Antp-class genes and Tribolium and Schistocerca orthologs have Antp-class YPWM motifs amino-terminal to the homeobox [2] [3]. Orthologs of ftz cloned from a centipede and an onychophoran [4] show that it predates the emergence of the arthropods, but the inability to pinpoint non-arthropodan orthologs suggested that ftz is the product of a Hox gene duplication in the arthropod ancestor [4] [5]. I have cloned ftz orthologs from a mite and a tardigrade, arthropod outgroups of the insects [6]. Mite ftz is expressed in a Hox-like pattern, confirming its ancestral role in anteroposterior patterning. Phylogenetic analyses indicate that arthropod ftz genes are orthologous to the Lox5 genes of lophotrochozoans (a group that includes molluscs) [7] and, possibly, with the Mab-5 genes of nematodes and Hox6 genes of deuterostomes and would therefore have been present in the triploblast ancestor.