Expression patterns of the rogue Hox genes Hox3/zen and fushi tarazu in the apterygote insect Thermobia domestica

Many embryonic patterning genes are remarkably conserved between vertebrates and invertebrates, and the Hox genes are paradigmatic examples of this conservation. Yet even Hox genes can change dramatically in evolution. Two genes in particular--Hox3 and fushi tarazu--lost their ancestral roles as homeotic genes and play very different developmental roles in the fruit fly Drosophila melanogaster. The Drosophila Hox3 homologs zerknullt and bicoid act in extraembryonic tissues and in establishment of the anteroposterior axis, respectively, whereas fushi tarazu acts in segmentation and neurogenesis. It would be valuable to know what mechanisms allowed Hox3 and ftz to abandon their ancestral roles as homeotic genes and take on new roles. To explore the evolutionary transition of these genes, we analyzed their expression in a primitive insect, the firebrat Thermobia domestica. The expression patterns seem to represent a stage of evolution intermediate between the ancestral state seen in basal arthropods and the derived expression patterns in Drosophila. These expression data help us to narrow the period in which the gene transitions took place. Hox3 appears to have evolved directly into zen within the insects, whereas ftz seems to have adopted the expression patterns of a segmentation and neurogenesis gene earlier in the mandibulate arthropods.     

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.     

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.     

Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.     

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.     

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.     

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.     

Hox Gene Loss during Dynamic Evolution of the Nematode Cluster

HOX GENES ARE IMPORTANT: their central role in anterior-posterior patterning provides a framework for molecular comparison of animal body plan evolution. The nematode Caenorhabditis elegans stands out as having a greatly reduced Hox gene complement. To address this, orthologs of C. elegans Hox genes were identified in six species from across the Nematoda, and they show that rapid homeodomain sequence evolution is a general feature of nematode Hox genes. Some nematodes express additional Hox genes belonging to orthology groups that are absent from C. elegans but present in other bilaterian animals. Analysis of the genomic environment of a newly identified Brugia malayi Hox6-8 ortholog (Bm-ant-1) revealed that it lay downstream of the Bm-egl-5 Hox gene and that their homeodomain exons are alternately cis spliced to the same 5' exon. This organization may represent an intermediate state in Hox gene loss via redundancy. The Hox clusters of nematodes are the product of a dynamic mix of gene loss and rapid sequence evolution, with the most derived state observed in the model C. elegans.     

Hox gene duplication in fish

The study of Hox gene clusters continues to serve as a paradigm for those interested in vertebrate genome evolution. Recent exciting discoveries about Hox gene composition in fishes challenges conventional views about vertebrate Hox gene evolution, and has initiated lively debates concerning the evolutionary events making the divergence of the major vertebrate lineages. Comparative analyses indicate that Hox cluster duplications occurred in early vertebrate evolution, and again within the order Cypriniformes of teleost fish. Loss of Hox genes was more widespread than duplication during fish evolution.     

Selection on coding regions determined Hox7 genes evolution

The important role of Hox genes in determining the regionalization of the body plan of the vertebrates makes them invaluable candidates for evolutionary analyses regarding functional and morphological innovation. Gene duplication and gene loss led to a variable number of Hox genes in different vertebrate lineages. The evolutionary forces determining the conservation or loss of Hox genes are poorly understood. In this study, we show that variable selective pressures acted on Hox7 genes in different evolutionary lineages, with episodes of positive selection occurring after gene duplications. Tests for functional divergence in paralogs detected significant differentiation in a region known to modulate HOX7 protein activity. Our results show that both positive and negative selection on coding regions are influencing Hox7 genes evolution.     

Is the evolution of Cnox-2 Hox/ParaHox genes "multicolored" and "polygenealogical?"

Understanding the evolution of metazoan bauplans is linked to understanding the evolution of Hox and ParaHox genes. At the base of metazoan radiation we see in both cases a quite confusing picture yet. Here Cnox-2 is one of the best studied diploblast Hox genes. Homologs of this gene are known from Placozoa and several Cnidaria. In those cases where full length gene sequences, or at least full length homeobox sequences, are available the relationship to Hox genes from triploblastic animals as well as the classification to Hox or ParaHox genes can be controversially discussed. The existing data on possible gene functions also reveal a quite heterogeneous picture. It seems conceivable that part of the "multicolored" picture relates to a "polygenealogical" origin of the Cnox-2 gene.     

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.     

Characterization of Hox genes in the bichir,Polypterus palmas

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred.We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.     

Hox gene evolution in nematodes: novelty conserved

The conserved homeobox (Hox) gene cluster is neither conserved nor clustered in the nematode Caenorhabditis elegans. Instead, C. elegans has a reduced and dispersed gene complement that is the result the loss of Hox genes in stages throughout its evolutionary history. The roles of Hox genes in patterning the nematode body axis are also divergent, although there are tantalising remnants of ancient regulatory systems. Hox patterning also differs greatly between C. elegans and a second 'model' nematode, Pristionchus pacificus. The pattern of Hox gene evolution may be indicative of the move to deterministic developmental modes in nematodes.     

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

Hox, ParaHox, ProtoHox: facts and guesses

The Hox gene cluster has captivated the imagination of evolutionary and developmental biologists worldwide. In this review, the origin of the Hox and ParaHox gene clusters by duplication of a ProtoHox gene cluster, and the changes in their gene numbers in major Metazoan Transitions are reviewed critically. Re-evaluation of existing data and recent findings in Cnidarians, Acoels, and critical stages of vertebrate evolution suggest alternative scenarios for the origin, structure, and changes in Hox gene numbers in relevant events of Metazoan evolution. I discuss opposing views and propose that (i) the ProtoHox cluster had only two genes, and not four as commonly believed: a corollary is that the origin of Bilaterians was coincident with the invention of new Hox and ParaHox gene classes, which may have facilitated such a transition; (ii) the ProtoHox cluster duplication was a cis duplication event, rather than a trans duplication event, as previously suggested, and (iii) the ancestral vertebrate cluster possessed 14 Hox genes, and not the 13 generally assumed. These hypotheses could be verified or refuted in the near future, but they may help critical discussion of the evolution of the Hox/ParaHox family in the metazoan kingdom.