Cofactor-interaction motifs and the cooption of a homeotic Hox protein into the segmentation pathway of Drosophila melanogaster

Some Drosophila Hox-complex members, including the segmentation gene fushi tarazu (Dm-ftz), have nonhomeotic functions. Characteristic expression in other arthropods supports an ancestral homeotic role for ftz, indicating that ftz function changed during arthropod evolution. Dm-Ftz segmentation function depends on interaction with ftz-F1 via an LXXLL motif and homeodomain N-terminal arm. Hox proteins interact with the cofactor Extradenticle (Exd) via their YPWM motif. Previously, we found that Dm-ftz mediates segmentation but not homeosis, whereas orthologs from grasshopper (Sg-ftz) and beetle (Tc-Ftz), both containing a YPWM motif, have homeotic function. Tc-Ftz, which unlike Sg-Ftz contains an LXXLL motif, displays stronger segmentation function than Sg-Ftz. Cofactor-interaction motifs were mutated in Dm-Ftz and Tc-Ftz and effects were evaluated in Drosophila to assess how these motifs contributed to Ftz evolution. Addition of YPWM to Dm-Ftz confers weak homeotic function, which is increased by simultaneous LXXLL mutation. LXXLL is required for strong segmentation function, which is unimpeded by the YPWM, suggesting that acquisition of LXXLL specialized Ftz for segmentation. Strengthening the Ftz/Ftz-F1 interaction led to degeneration of the YPWM and loss of homeotic activity. Thus, small changes in protein sequence can result in a qualitative switch in function during evolution.     

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.     

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.     

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.     

fushi tarazu: a Hox gene changes its role

The Hox genes play a role in anteroposterior axis specification of bilaterian animals that has been conserved for more than 600 million years. However, some of these genes have occasionally changed their roles in evolution. For example, the insect gene fushi tarazu (ftz), although localised in the Hox cluster, no longer acts as a Hox gene, but is involved in segmentation and nervous system development. Recent data of Mouchel-Vielh et al., and Hughes and Kaufman on ftz homologues in a crustacean and a myriapod, respectively, shed new light onto the evolution of this gene.     

Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede

The diversity of the arthropod body plan has long been a fascinating subject of study. A flurry of recent research has analyzed Hox gene expression in various arthropod groups, with hopes of gaining insight into the mechanisms that underlie their evolution. The Hox genes have been analyzed in insects, crustaceans and chelicerates. However, the expression patterns of the Hox genes have not yet been comprehensively analyzed in a myriapod. We present the expression patterns of the ten Hox genes in a centipede, Lithobius atkinsoni, and compare our results to those from studies in other arthropods. We have three major findings. First, we find that Hox gene expression is remarkably dynamic across the arthropods. The expression patterns of the Hox genes in the centipede are in many cases intermediate between those of the chelicerates and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. Second, we found two 'extra' Hox genes in the centipede compared with those in DROSOPHILA: Based on its pattern of expression, Hox3 appears to have a typical Hox-like role in the centipede, suggesting that the novel functions of the Hox3 homologs zen and bicoid were adopted somewhere in the crustacean-insect clade. In the centipede, the expression of the gene fushi tarazu suggests that it has both a Hox-like role (as in the mite), as well as a role in segmentation (as in insects). This suggests that this dramatic change in function was achieved via a multifunctional intermediate, a condition maintained in the centipede. Last, we found that Hox expression correlates with tagmatic boundaries, consistent with the theory that changes in Hox genes had a major role in evolution of the arthropod body plan.     

Regulation and function of the Drosophila segmentation gene fushi tarazu

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.     

Colinear and segmental expression of amphioxus Hox genes.

The cephalochordate amphioxus has a single Hox gene cluster. Here we describe the genomic organization of four adjacent amphioxus genes, AmphiHox-1 to AmphiHox-4, together with analysis of their spatiotemporal expression patterns. We demonstrate that these genes obey temporal colinearity and that three of the genes also obey spatial colinearity in the developing neural tube. AmphiHox-1, AmphiHox-3, and AmphiHox-4 show segmental modulation of their expression levels, a two-segment phasing of spatial colinearity, and, at least for AmphiHox-4, asymmetrical expression. AmphiHox-2 is unlike other amphioxus Hox genes: it does not obey spatial colinearity and it has no positional expression in the neural tube. AmphiHox-2 is expressed in the preoral pit of larvae, from which the homologue of the anterior pituitary develops. We suggest that the ancestral role of chordate Hox genes was primarily in the neural tube and that chordate Hox genes can functionally diverge in a manner analogous to that of Drosophila ftz or zen.     

Evolution: hox genes and the cellared wine principle

Two Drosophila Hox genes involved in segmentation, fushi tarazu and bicoid, appear to have acquired these roles by functional divergence from classical homeotic genes. Recent results indicate how genes with critical functions in development can evolve completely different functions among species.     

Hox genes and the evolution of the arthropod body plan

In recent years researchers have analyzed the expression patterns of the Hox genes in a multitude of arthropod species, with the hope of understanding the mechanisms at work in the evolution of the arthropod body plan. Now, with Hox expression data representing all four major groups of arthropods (chelicerates, myriapods, crustaceans, and insects), it seems appropriate to summarize the results and take stock of what has been learned. In this review we summarize the expression and functional data regarding the 10 arthropod Hox genes: labial proboscipedia, Hox3/zen, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-A, and Abdominal-B. In addition, we discuss mechanisms of developmental evolutionary change thought to be important for the emergence of novel morphological features within the arthropods.     

ftz evolution: findings, hypotheses and speculations (response to DOI 10.1002/bies.201100019)

In a recent paper, Merabet and Hudry discuss models explaining the functional evolution of fushi tarazu (ftz) from an ancestral homeotic to a pair-rule segmentation gene in Drosophila. As most of the experimental work underlying these models comes from our research, we wish to reply to Merabet and Hudry providing an explanation of the experimental approaches and logical framework underlying them. We review experimental data that support our hypotheses and discuss misconceptions in the literature. We emphasize that the change in ftz function required changes in both expression pattern and protein function and review the evidence that these functional changes involved a switch in cofactor-interaction motifs during arthropod radiations. While agreeing with Merabet and Hudry that protein context likely contributes to Ftz function, we argue that until supporting evidence for alternative mechanisms is obtained, the role of cofactor-interaction motifs in driving a functional switch remains compelling.     

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.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes.

We have generated several transgenic Drosophila strains containing different mouse Hox genes under heat shock control and studied how their generalized expression affects Drosophila larval patterns. We find that they have spatially restricted effects which correlate with their genetic order and expression pattern in the mouse; as they are expressed more posteriorly in the mouse, they have more extensive effects in Drosophila. The generalized expressions of Hoxd-8 and d-9 modify Drosophila anterior head segment(s), but have no effect in the rest of the body. Hoxd-10 expression affects head and thorax, but not the abdomen. Finally, Hoxd-11 alters head, thorax not the abdomen. Finally, Hoxd-11 alters head, thorax and abdomen. The developmental effect of the Hox genes consists of a homeotic transformation of the affected segment(s), which exhibit a 'ground' pattern similar to that obtained in the absence of homeotic information, suggesting that Hox genes are able to inactivate Drosophila homeotic genes, but do not specify a pattern of their own. A partial exception is Hoxd-11 which, even though it has a general suppressing effect, can also activate the resident Abdominal-B and empty spiracles genes in ectopic positions. Our results strongly suggest a general conservation of the functional hierarchy of homeotic genes that correlates with genetic order and expression patterns.     

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.     

Chelicerate Hox genes and the homology of arthropod segments

Genes of the homeotic complex (HOM-C) in insects and vertebrates are required for the specification of segments along the antero-posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM-C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia ( pb ) and Deformed ( Dfd   ). In addition, we have cloned larger cDNA fragments of pb, zerknullt ( zen / Hox3 ) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co-linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD-A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.