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The evolution of abdominal reduction and the recent origin of distinct Abdominal-B transcript classes in Diptera

In insects, the Hox gene Abdominal-B (Abd-B) governs the development of the posterior-most segments, the number and fate of which differ within and between orders. A striking feature of insect evolution is a trend toward the reduction of posterior abdominal segments which is most pronounced in higher Diptera. In Drosophila melanogaster, two distinct Abd-B transcript classes and protein isoforms are expressed in non-overlapping domains and have discrete functions in patterning the posterior abdomen. It has been proposed that evolutionary changes in Abd-B structure and expression are responsible for the reduction of the dipteran abdomen. This study investigated the relationship between the evolution of the Abd-B gene and abdominal reduction by analyzing the structure and expression of homologs from four additional dipterans representing distinct clades within the order. The lower dipteran mosquito Anopheles gambiae expresses a single Abd-B transcript class, as do two species phylogenetically intermediate to mosquitoes and drosophilids. These results delimit the evolution of distinct functional Abd-B isoforms to within the dipteran radiation after the origin of the reduced abdominal morphology. Furthermore, the spatial distribution of Abd-B transcripts in non-drosophilid Diptera is identical to the combined domains of the two D. melanogaster Abd-B transcripts. Therefore, neither the structural evolution nor changes in the spatial regulation of Abd-B account for the derived abdomen of higher Diptera. The recent subfunctionalization of this Hox gene has occurred without any apparent morphological correlate. It is concluded that regulatory modifications to developmental programs downstream of or parallel to Abd-B are responsible for the evolutionary reduction of the higher dipteran postabdomen (Yodera, 2006).

This study investigated the relationship between the evolution of postabdominal morphology in higher Diptera and changes in the structure and expression of the Hox gene Abd-B. The evolution of Abd-B gene structure and distinct transcript classes occurred late within the higher dipteran radiation, after the evolutionary reduction of the postabdomen. Furthermore, it was found that the anterior expansion of Abd-B expression, compared with that of primitive insects, is an ancestral trait of Diptera. This may be ancestral to Holometabola since it was recently shown that the Abd-B homolog of Oncopeltus fasciatus (Hemiptera; a sister clade of Holometabola) is expressed as far anteriorly as A4 (Yodera, 2006).

Since neither Abd-B gene evolution nor regulation is correlated with the reduction of the higher dipteran adult postabdomen, but genetic evidence clearly indicates that Abd-Bm is required for this derived morphology, it is proposed that changes in the regulation of Abd-B target genes, as well as in parallel developmental pathways, must have occurred in order to promote the morphological evolution of the higher dipteran postabdomen. The major effector of the sex determination pathway, the transcription factor doublesex (dsx), plays a prominent role in patterning postabdominal morphology. dsx interacts genetically with Abd-B to pattern the sexually dimorphic postabdominal pigmentation of drosophilids. This interaction evolved within the drosophilid clade at least in part through evolution of the Bric-a-brac (bab) locus as a transcriptional target. The interaction between Abd-B and dsx in patterning the postabdomen is not restricted to epidermal pigmentation, as dsx is also necessary in D. melanogaster for the dimorphic development of the appropriate number and morphology of postabdominal segments (Yodera, 2006).

It is proposed that other downstream targets were acquired by the Abd-B/dsx regulatory network and promoted the morphological evolution of the higher dipteran postabdomen. In addition to genes that govern cell cycle regulation, other candidate target genes are those of the apoptotic pathway. Abd-B has previously been shown to function both as a positive and negative regulator of the proapoptotic gene reaper. The elucidation of Abd-B and dsx targets required for postabdominal morphology in D. melanogaster will be necessary to understand the evolution of postabdominal reduction in higher Diptera (Yodera, 2006).

The data indicate that compared with D. melanogaster, a similar degree of functional complexity, with regard to abdominal morphology, is achieved in another higher dipteran, Megaselia abdita, which appears to generate only a single class of Abd-B transcript. Therefore, the evolution of two distinct Abd-B transcript classes and protein isoforms, which occurred after the origin of higher Diptera, was not necessary to promote the derived postabdominal morphology of this clade (Yodera, 2006).

While no transcript homologous to D. melanogaster Abd-Br was identified in M. abdita, its Abd-B gene does encode a potential start codon at a homologous position to that of D. melanogaster Abd-Br. The possibility therefore remains that transcripts homologous to Abd-Br are expressed in M. abdita, but were not detected by the assay. If such transcripts are expressed in M. abdita, they do not encode a protein with comparable function to D. melanogaster Abd-Br as there is no evidence of transcriptional repression of the Abd-Bm homolog (Yodera, 2006).

What then was the evolutionary significance, if any, to the origin of structurally and spatially distinct Abd-B transcript classes and protein isoforms? One possibility is that the evolution of distinct Abd-B transcript classes was initially neutral and any functional differences between the two protein isoforms have evolved after their origin and are unrelated to segment reduction (Yodera, 2006).

The neutral origin of Abd-B transcript classes may be explained in light of recent insights into the evolution of alternate splicing. Exon duplication has been shown to be a major mechanism by which alternate splicing evolves. In a manner similar to gene duplication, exon duplication is thought to provide relief from functional constraints through the generation of redundant 'internal paralogs'; that is, separate but essentially functionally identical alternatively spliced transcripts (Yodera, 2006).

In D. melanogaster there is evidence that the derived organization of the Abd-B gene is the result of duplication and divergence of the m-specific exon. The three D. melanogaster Abd-Br transcripts contain two common 5' UTRs as well as additional transcript specific non-coding exons. It was previously recognized that the common Abd-Br non-coding exon, most proximal to translation initiation, shares significant sequence similarity to a portion of the Abd-Bm-specific coding exon. Conceptual translation of this Abd-Br non-coding exon yields a stretch of 29 amino acids 72% identical to the carboxy-most sequence of the Abd-Bm-specific N-terminus (approximately the same N-terminal residues that are conserved among the four Diptera examined in this work) (Yodera, 2006).

The D. melanogaster Abd-B transcript classes and the complementary expression of recently diverged paralogs fit the prediction of the Duplication-Degeneration-Complementation model (DDC) for the preservation of duplicate genes. The DDC model predicts that recently duplicated paralogs can become fixed following regulatory sequence evolution that results in complementary expression patterns equivalent to the sum of ancestral expression. In D. melanogaster, the transcriptional repression of Abd-Bm by Abd-Br accomplishes this subfunctionalization (Yodera, 2006).

What is most remarkable here is that a Hox gene has undergone a major reorganization, equivalent to gene duplication, in a relatively recent timeframe, yet no discernable morphological consequence is apparent. These findings delimit the evolution of separate dipteran Abd-B transcript classes to after the origin of the Cyclorrhaphan suborder, which arose an estimated 140 mya. By comparison, the primitive dipteran Hox3/zerknüllt gene, which has both zygotic and maternal functions, duplicated prior to the Cyclorrhaphan radiation, generating the zen and bicoid paralogs that evolved discrete zygotic and maternal functions, respectively (Yodera, 2006).

The uncoupling of ancestral functions into discrete paralogs (or transcripts classes in the case of Abd-B) is predicted to relax selective constraints and allow the paralogs to evolve new functions. Whether this has happened with the two Abd-B isoforms is not clear; however, the potential was undoubtedly increased as a result of subfunctionalization (Yodera, 2006).

Abdominal-B homologs in C. elegans

How do temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans? P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway; a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. lin-3 encodes a membrane-spanning protein with a single extracellular EGF domain that is similar in structure to members of the EGF family of growth factors. LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification: egl-5, an AbdominalB homolog, is a downstream target of the lin-3/let-23 pathway in specifying P12 fate, and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal (Jiang, 1998).

In C. elegans there are twelve ventral cord precursor cells, P1-P12, numbered from anterior to posterior along the body axis. These cells divide postembryonically to generate cells of the ventral nervous system, as well as the vulva. P11/P12 are the most posterior pair of the ventral cord precursors. At hatching, the cells AB.plapappa (left side) and AB.prapappa (right side) are disposed laterally. In hermaphrodites, they start to migrate ventrally several hours after hatching and enter the ventral cord about 8-9 hours after hatching. The left cell migrates to the anterior and becomes P11; the right cell migrates to the posterior and becomes P12. Two hours later they each divide once. The anterior daughters, P11.a and P12.a are neuroblasts that will divide for three more rounds to generate several ventral cord neurons. These neurons are morphologically indistinguishable under Nomarski optics. The posterior daughter of P11, P11.p, does not divide but rather fuses with the large epidermal syncytium hyp7. P12.p divides once more about 1 hour prior to L1 molt to generate two cells: P12.pa, which becomes a unique epidermal cell (hyp12) and P12.pp, which undergoes cell death. P11.p and P12.pa can be distinguished by their different nuclear morphologies and positions observed with Nomarski optics. Prior to migration, both P11 and P12 cells are able to express the P12 fate: if only a single cell is present, it will adopt the P12- like fate. Therefore, P12 represents a primary fate, while P11 is a secondary fate (Jiang, 1998).

Mutants of several lin-3/let-23 pathway components have been reported to display defects in P11/P12 cell fate specification. Loss-of-function alleles of let-23 show a loss of the cell P12.pa with concommitant duplication of P11.p in the hermaphrodite tail. Lineage analysis in males indicates that this defect likely represents a transformation of P12 to P11 fate, since the anterior branch is also affected. Mutations at the lin-15 locus, which encodes negative regulators of the lin-3/let-23 pathway, have the opposite defect: P11 to P12 cell fate transformation. Other components, sem-5 and let-60, which encode a SH2/SH3 domain protein and a RAS protein, respectively, are also involved in P11/P12 cell fate specification (Jiang, 1998).

egl-5 might play a permissive role in P12 fate specification by setting up the competence of the cell to respond to the LIN-3 inductive signal, or egl-5 might be an instructive factor for P12 fate specification. To distinguish between these hypotheses, a test was performed of the effect of overexpression of EGL-5 on P11/P12 cell fate specification. Overexpression of EGL-5 in wild-type animals does lead to a P11 to P12 fate transformation. Could overexpression of EGL-5 suppress the P11/P12 defect of let-23 mutants? If egl-5 is a permissive factor for P12 fate, no rescue of the P11/P12 defect would be expected; whereas if egl-5 is an instructive factor, overexpression of EGL-5 should be able to rescue the P11/P12 defect caused by let-23 mutation, and may additionally result in a P11 to P12 cell fate transformation. Overexpression of EGL-5 suppresses the P11/P12 defect of let-23 mutants, as predicted by the instructive model. Therefore, egl-5 plays an active role in P12 fate specification and acts downstream of let-23. egl-5has been shown to be a downstream target of the lin-3/let-23 pathway for P12 fate specification (Jiang, 1998).

Next the interactions between lin-3 and lin-44 were tested by examining the P11/P12 defect in a strain defective in both genes. The strong synergy observed between lin-3 and lin-44 is consistent with the two signals acting in parallel. To confirm the interaction between lin-3 and lin-44, a test of synergy between lin-3 and lin-17 mutations was performed. lin-17, which encodes a putative seven-transmembrane protein similar to the Drosophila Frizzled protein, has been suggested to be a receptor for the LIN-44 protein. Similar synergistic interactions are found between lin-3 and lin-17 as have been found between lin-3 and lin-44. A synergistic interaction is also found between mutations of let-23, the EGF receptor for LIN-3 signal, and the Wnt signal LIN-44. These data support the hypothesis that both of the two signaling pathways, lin-3 and lin-44, are required for the P12 fate specification. How do these two signaling pathways function in concert to specify P12 fate? The favored model is that both pathways are required for proper P12 fate specification and that they act at different developmental times. Three sources of evidence support this model: (1) LIN-3 overexpression experiments indicate that LIN-3 signal is required in early L1 before P11/P12 enter the ventral cord to induce P12 fate. (2) lin-44 expression is turned on during embryogenesis, much earlier than the time of P11/P12 induction. (3) The unique effect of overexpression of LIN-3EGF during late embryogenesis in lin-44 mutants suggests that LIN-44 function may be important in the early phase of P12 fate specification. It is possible that the Wnt pathway regulates the competence of the cells to respond to the LIN-3 inductive signal. But the Wnt signal alone is not sufficient to promote P12 fate, since overexpression of LIN-44 in wild-type animals has no effect on P11/P12 cell fate specification (Jiang, 1998).

The following is a model for P12 neuroectoblast fate specification: in newly hatched larvae, LIN-44 signal acts via receptor LIN-17 to set up the competence of P11 and P12 cells to respond to the inductive signal and be able to express P12 fate. egl-5 expression is kept off in both P11 and P12 cells. Later, an inductive signal LIN-3 coming from the posterior region activates LET-23 receptor activity in the posterior cell of the P11/P12 pair. Activation of the lin-3/let-23 pathway turns on egl-5 expression, which specifies the posterior cell to take on P12 fate. lin-15 negatively regulates let-23 activity and prevents the anterior cell from becoming P12. Information from the Wnt signaling pathway may be integrated into the lin-3/let-23 EGF signaling pathway either at the level of LIN-3 signal, LET-23 receptor or egl-5 transcription. Thus the temporal and spatial co-ordination and interactions between the Wnt signal, EGF signal and HOM-C transcription factor are important for P12 fate specification (Jiang, 1998).

The Caenorhabditis elegans body axis, like that of other animals, is patterned by the action of Hox genes. In order to examine the function of one C. elegans Hox gene in depth, the postembryonic expression pattern of egl-5, the C. elegans member of the Abdominal-B Hox gene paralog group, was determined by means of whole-mount staining with a polyclonal antibody. A major site of egl-5 expression and function is in the epithelium joining the posterior digestive tract with the external epidermis. Patterning this region and its derived structures is a conserved function of Abd-B paralog group genes in other animals. Cells that initiate egl-5 expression during embryogenesis are clustered around the presumptive anus. Expression is initiated postembryonically in four additional mesodermal and ectodermal cell lineages or tissues. Once initiated in a lineage, egl-5 expression continues throughout development, suggesting that the action of egl-5 can be regarded as defining a positional cell identity. A variety of cross-regulatory interactions between egl-5 and the next more anterior Hox gene, mab-5, help define the expression domains of their respective gene products. In its expression in a localized body region, function as a marker of positional cell identity, and interactions with another Hox gene, egl-5 resembles the Hox genes of other animals. This suggests that C. elegans, in spite of its small cell number and reproducible cell lineages, may not differ greatly from other animals in the way it employs Hox genes for regional specification during development (Ferreira, 1999).

The C. elegans HSN motor neurons permit genetic analysis of neuronal development at single-cell resolution. The egl-5 Hox gene (Drosophila homolog: Abdominal-B), which patterns the posterior of the embryo, is required for both early (embryonic) and late (larval) development of the HSN. ham-2 encodes a zinc finger protein that acts downstream of egl-5 to direct HSN cell migration, an early differentiation event. The EGL-43 zinc finger protein, also required for HSN migration, is expressed in the HSN specifically during its migration. In an egl-5 mutant background, the HSN still expresses EGL-43, but expression is no longer down-regulated at the end of the cell's migration. A new role in early HSN differentiation has been found for UNC-86, a POU homeodomain transcription factor shown previously to act downstream of egl-5 in the regulation of late HSN differentiation. In an unc-86; ham-2 double mutant the HSNs are defective in EGL-43 down-regulation, an egl-5-like phenotype that is absent in either single mutant. Thus, in the HSN, a Hox gene, egl-5, regulates cell fate by activating the transcription of genes encoding the transcription factors HAM-2 and UNC-86, which in turn individually control some differentiation events and combinatorially affect others (Baum, 1999).

In Caenorhabditis elegans males, a row of epidermal precursor cells called seam cells generates a pattern of cuticular alae in anterior body regions and neural sensilla (called rays) in the posterior. The Abdominal-B homolog mab-5 is required for two posterior seam cells, V5 and V6, to generate rays. The V5 lineage generates one ray and the V6 lineage generates five rays. In mab-5 mutant males, V5 and V6 do not generate sensory ray lineages but instead generate lineages that lead to alae, cuticular ridges that extend along the two sides of the animal. Alae are normally generated by the V1-V4 cells only. Two independent regulatory pathways can activate mab-5 expression in the V cells. (1) The caudal homolog pal-1 turns on mab-5 in V6 during embryogenesis. (2) A Wnt signaling pathway is capable of activating mab-5 in the V cells during postembryonic development, however, during normal development Wnt signaling is inhibited by signals from neighboring V cells. The inhibition of this Wnt signaling pathway by lateral signals between the V cells limits the number of rays in the animal and also determines the position of the boundary between alae and rays (Hunter, 1999).

ceh-7 is a small Caenorhabditis elegans homeobox gene consisting of 84 amino acids that at present is not known to be closely related to any others. Examination of the expression pattern of ceh-7 using reporter constructs reveals that is expressed in a few cells of the male tail, which form a ring around the rectum. The most posterior member of the C. elegans Hox cluster, egl-5, an Abd-B homolog, is required for the proper development of several blast cells in the male tail. The expression of ceh-7 has been examined in mutant backgrounds of egl-5 and also mab-5, an Antp/Ubx/Abd-A homolog. Although ceh-7 is not expressed in egl-5 mutants, it is still expressed in mab-5 mutants. The late expression argues that ceh-7 is not involved in sex determination itself; rather, its role appears to be in the generation or differentiation of cell types specific to the male tail. Given the conserved nature of the Hox cluster, it appears possible that in vertebrates and flies, homeobox genes, perhaps even a ceh-7 homolog, might exist which control the development of sex-specific appendages (Kagoshima, 1999).

Specification of cell fate is key to understanding the development and function of a nervous system. In C. elegans, where cell lineages are reproducible and gene expression programs can be studied with single-cell resolution, it is possible to explore the progression of cell-state changes that lead to the generation of cells with individual identities. Studies were carried out to take advantage of the neurons of male sensory rays; these studies have addressed how neurons are programmed to adopt a particular neurotransmitter identity. There are nine similar bilateral pairs of rays extending out of the body on each side of the male tail. Each ray comprises two sensory neurons (denoted A-type and B-type) and a support cell surrounded by a hypodermal sheath. Each ray consists of similar cells and is generated by repetition of a stereotyped cell sublineage. Yet each ray also has individual characteristics. These include position within the genital specialization, morphology, neurotransmitter usage, expression of a sensory receptor, transcription factor expression profile and functional role in mating behavior. Further individual characteristics may encompass expression of other sensory receptors and modalities, axon pathfinding and synaptic targets. Thus, two developmental processes involved in the generation of each ray may be distinguished: a program of neurogenesis that results in the generation of three differentiated cell types and a pattern-formation process that assigns the characteristics that differ among the rays. The existence of two processes is supported by the identification both of genes required in all the rays and genes involved in specifying ray-specific properties (Lints, 1999).

Dopamine (DA) is expressed by R5A, R7A and R9A, the A-type sensory neurons present respectively in rays 5, 7 and 9. A TGFbeta-family signaling pathway and a Hox gene have been identified that are involved in directing the dopaminergic fate specifically to these three neurons. C. elegans genes encoding components of TGFbeta-like signaling cascades have been defined by genome sequence analysis and by genetic studies. A TGFbeta pathway involved in defining male ray morphology, which is referred to as the DBL-1 pathway, has been defined by mutations in six genes. The pathway ligand, encoded by a gene variously named dbl-1 and cet-1, is a member of the Vg1/Dpp/BMP subfamily of TGFbeta molecules, most closely resembling Nodal. sma-6 and daf-4 encode type I and type II receptors, respectively. sma-2, sma-3 and sma-4 encode SMAD proteins likely to act as downstream transducers. The DAF-4 receptor and the signal transduction pathway have been shown to act cell autonomously in specification of ray morphology. dbl-1 is expressed in several neurons within the tail, but the source important for ray morphology has not been identified (Lints, 1999).

Expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine were used to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway (the DBL-1 pathway), dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability (Lints, 1999).

One question of interest is whether pathways leading to the adoption of the neuronal cell fate are entirely independent of those that pattern individual neuronal properties. Several lines of evidence indicate that egl-5 acts in specification of the ray neuroblast fate, not only in ray 6, but also redundantly with mab-5 in other V-rays. egl-5 also acts in the specification of ray morphology and, as shown here, in the specification of DA expression. Since a single regulatory factor acts both to specify the neuronal cell fate and also the differentiated properties of individual neurons, this demonstrates that there are steps in common between the developmental pathways leading to expression of pan-neural genes and pathways leading to expression of neuron-specific genes. Similar multiple roles and times of action have been demonstrated for mab-5 in posterior hypodermal cell lineages and for the Hox gene lin-39 in vulva development. In its late action, EGL-5 could act directly on the cat-2 (tyrosine hydroxylase) promoter or alternatively on the promoter of another transcription factor at some level in the hierarchy upstream of cat-2. Additional intermediate transcription factors might include those of the LIM and POU families, which play widespread roles in C. elegans and in other organisms in specifying the properties of individual neurons. A transcription factor of the POU family regulates dopamine decarboxylase gene expression in Drosophila. Since EGL-5 is expressed in all branches of the ray sublineages of rays 3 to 6, additional factors must intervene to direct the action of EGL-5 in the dopaminergic pathway exclusively to the lineage branch leading to the A-type neuron. Likewise, turn-on of cat-2 appears to be delayed until the ray sublineage is completed. Whether lineage-branch-specific and timing cues are integrated by the promoter of the cat-2 gene itself, or by the promoters of intermediate transcription factors acting between egl-5 and cat-2, is an interesting question for future studies (Lints, 1999).

The lin-49 and lin-59 genes in C. elegans have been molecularly characterized, and their products have been found to be related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans, as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, it is proposed that lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes (Chamberlin, 2000).

A TGFbeta-like signal is required for spicule development in Caenorhabditis elegans males. This signal appears to originate in the male-specific musculature and is required for the migrations of cells within the proctodeum. The migrations of these cells form cellular molds, the spicule traces, in which the cuticle of the spicules is secreted, thus determining spicule morphology. Mutations in daf-4, sma-2, sma-3, and sma-4, which disrupt TGFbeta-like signaling, result in aberrant migrations and morphologically abnormal spicules. daf-4 codes for a type II TGFbeta-like receptor and the smas code for smad family proteins. daf-4, and hence the TGFbeta-like signal, is required prior to or during cell migrations. Therefore, the TGFbeta-like signal may act to prime the migrating cells or as a guidance cue. Mutations in lin-31 result in identical cell migration and spicule morphology defects. Thus, lin-31, which encodes a "winged helix" protein, may be a component of this TGFbeta-like signaling pathway. The TGFbeta-like signal required for spicule formation likely is coded for by the dbl-1 gene. Mutations in dbl-1 result in adult spicule, ray pattern, and body size defects. Spicule development in dbl-1 mutant males has not been analyzed. A possible source of the TGFbeta-like signal required for spicule development is the male-specific musculature. These muscles are derived from a single postembryonic myoblast, M. Expression of the TGFbeta-like signal in M cell descendants may require the egl-5 gene product, an Abdominal B homolog (Baird, 1999).

Hox genes encode highly conserved transcription factors that control regional identities of cells and tissues along the developing anterior-posterior axis, probably in all bilaterian metazoans. However, in invertebrate embryos other than Drosophila, Hox gene functions remain largely unknown except by inference from sequence similarities and expression patterns. Recent genomic sequencing has shown that Caenorhabditis elegans has three Hox genes of the posterior paralog group. However, only one has been previously identified genetically, and it is not required for embryonic development. Identification of the remaining two posterior paralogs is described: the nob-1 gene and the neighboring php-3 gene. Elimination of nob-1 and php-3 functions causes gross embryonic defects in both posterior patterning and morphogenetic movements of the posterior hypodermis, as well as posterior-to-anterior cell fate transformations and lethality. The only other Hox gene essential for embryogenesis is the labial/Hox1 homolog ceh-13, required for more anterior patterning. Therefore, essential embryonic patterning in C. elegans requires only Hox genes of the anterior and posterior paralog groups, raising interesting questions about evolution of the medial-group genes (Auken, 2000).

nob-1 encodes a protein similar to AbdB and other members of the posterior paralog group. php-3 has considerably higher similarity than nob-1 to AbdB in the predicted homeodomain. (php-3 stands for posterior Hox paralog 3; 1 and 2 are egl-5 and nob-1, respectively). php-3 and nob-1 have similar gene structures and have 47% nucleotide identity throughout their coding regions I. There is as yet no basis for deciding whether the C. elegans mechanism is primitive or derived, that is, whether the common ancestor required only anterior and posterior group genes and only later co-opted medial group genes for embryonic roles, or whether it required medial group genes as well, which later became unnecessary for embryogenesis in the nematode line of descent. In Cnidarians, which occupy a basal position in animal phylogeny, only anterior-group and posterior-group Hox genes have been identified. (Auken, 2000).

sop-3 is required for the regulated expression of C. elegans Hox gene egl-5 in a postembryonic neuroectodermal cell lineage. Regulated expression of egl-5 in this cell lineage is necessary for development of the sensory rays of the male tail. sop-3 encodes a predicted novel protein of 1475 amino acids without clear homologs in other organisms. However, the sequence contains motifs consisting of homopolymeric runs of amino acids found in several other transcriptional regulators, some of which also act in Hox gene regulatory pathways. The genetic properties of sop-3 are very similar to those of sop-1, which encodes a component of the transcriptional Mediator complex, and mutations in the two genes are synthetic lethal. This suggests that SOP-3 may act at the level of the Mediator complex in regulating transcription initiation. In a sop-3 loss-of-function background, egl-5 is expressed ectopically in lineage branches that normally do not express this gene. Such expression is dependent on the Hox gene mab-5; expression is as also dependent on mab-5 in branches where egl-5 is normally expressed. Ectopic egl-5 expression is also dependent on the Wnt pathway. Thus, sop-3 contributes to the combinatorial control of egl-5 by blocking egl-5 activation by MAB-5 and the Wnt pathway in inappropriate lineage branches (Zhang, 2001).

SOP-3 characteristics exhibit a number of intriguing parallels with those of several known transcriptional regulators. This group of similar proteins includes the products of cap'n'collar isoform B, teashirt, lines, and mastermind of Drosophila, and lag-3 of C. elegans. All of these proteins have several or all of following characteristics: (1) they are nuclear proteins involved in regulation of transcription; (2) they contain homopolymeric runs of amino acids, usually involving Q, S, A, P and G; (3) they are involved in Hox gene regulatory pathways; and (4) they act by modulating the Wnt pathway. Strikingly, though they act in pathways involving well-conserved components, none of these proteins has clear orthologs in other organisms. cncB, tsh and lins, like sop-3, are all involved in Hox gene regulatory pathways, affecting the outcome of Hox gene action. CncB and Tsh have recognizable DNA-binding domains (a basic leucine zipper domain in CncB, a Zn-finger domain in Tsh) whereas SOP-3 and Lines do not. SOP-3, Tsh and Lin all affect the action of the Wnt pathway. Tsh modulates Wnt signaling by direct binding to the beta-catenin homolog Armadillo. Alone among these proteins, Lin does not contain homopolymeric runs of amino acids. LAG-3 and Mam differ from the other proteins in being involved in the LIN-12/Notch pathway rather than in Hox gene regulatory pathways, and their structures include a greater number and length of homopolymeric runs of Q residues (Zhang, 2001 and references therein).

LAG-3 provides an attractive model for the function of this putative family of transcriptional regulators. It has recently been shown to participate in a ternary complex between the ankyrin-repeat-containing intracellular domain of the LIN-12 receptor, which translocates to the nucleus upon signaling, and the target DNA-binding factor of this pathway, LAG-1. A reasonable premise for further investigation may be a possible similar role for SOP-3 as constituent of a multi-protein complex that includes the Tcf/LEF family member POP-1, beta-catenin homolog BAR-1, Groucho homolog UNC-37 and the Mediator component SOP-1. Why such a mode of action would allow for unusual evolutionary variability of protein sequence is not obvious, since the interactions involved are between evolutionarily conserved components. Possibly the mechanism allows for the function of proteins of mixed and variable functional domains that act in the nature of linker proteins. Such a mechanism might provide an important point of evolutionary flexibility that can lead to variation in the regulatory interactions involved in the combinatorial control of gene expression (Zhang, 2001).

Polycomb group (PcG)-mediated repression of C. elegans Hox genes has not been demonstrated, and genes homologous to components of one of the PcG complexes (PRC1) have not been identified in the C. elegans genome. A mechanism of general Hox gene repression exists in C. elegans, carried out in part by SOP-2, a protein related to, but not orthologous with, any PcG protein. sop-2 mutations lead to widespread ectopic expression of Hox genes and homeotic transformations. SOP-2 contains a SAM domain, a self-associating protein domain found in other repressors, including a core component of PRC1 and ETS transcription factors. Phylogenetic analysis indicates that this domain is more closely related to those of the ETS family than to those of PcG proteins. The results suggest that global repression of Hox genes has been taken over by a different branch of the SAM domain family during the evolution of nematodes (Zhang, 2003).

As seen in fly or mouse PcG mutants, in sop-2 mutants, Hox genes are not expressed in every cell, and each Hox gene is ectopically expressed at a different level. For instance, mab-5 is not expressed in the tail region, and the ectopic expression domains of mab-5 and egl-5 appear to be much broader than those of ceh-13 and lin-39. The simplest explanation for these gene-specific patterns is that, for each gene, transcription factors that will activate expression in the absence of sop-2 gene function are present or active in only a subset of cells. Alternatively, sop-2 repression may be redundant with other repressive mechanisms in some tissues. Crossregulation between Hox genes may also contribute to the Hox gene expression patterns observed in sop-2 mutants, as the data suggest for lin-39 and egl-5 (Zhang, 2003).

sop-2 also appears to play a role in regulating the expression of nonhomeotic genes, since sop-2 mutants have pleiotropic effects not known to be associated with Hox gene misexpression, including abnormalities in body size, sex determination, and vulva development. PcG mutants in other organisms also cause defects that may be caused by inappropriate expression of nonhomeotic genes. For instance, M33 (Pc homolog) mutant mice have slow gonad growth that leads to male to female sex reversal, and loss of function of mel-18, mph-1/rae28, bmi-1 (Pc, ph, and Psc homologs, respectively), and M33 result in cell proliferation defects. Thus, regulation of these distinct pathways may be inherent properties of some PcG genes (Zhang, 2003).

Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. A gene has been identified, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis (Alper, 2001).

Much of the C. elegans epidermal layer, the hypodermis, is composed of several multinucleate cells (syncytia) that are formed by the fusion of mononucleate cells throughout embryonic and postembryonic development. One such syncytium, hyp7, extends over most of the length of the worm and contains 133 nuclei, close to 15% of all somatic nuclei in the worm. How is the fusion of all these cells coordinately regulated to allow formation of hyp7? To understand how the hyp7 syncytium is generated, the regulation of the fusion decision of one group of cells called the Pn.p cells that line the ventral surface of the worm during the first larval stage (L1) has been studied. Pn.p cell fusion is regulated by two genes of the C. elegans Hox gene cluster. The Hox cluster consists of six genes: ceh-13, lin-39 and mab-5, homologs of Drosophila labial, Sex combs reduced and Antennapedia, respectively, and egl-5, php-3 and nob-1, three Abdominal-B homologs. In C. elegans, as in other organisms, the Hox genes regulate the choice of cell fates along the AP body axis. However, the simple Hox gene expression pattern in C. elegans is insufficient to explain the complex Pn.p cell fusion pattern. This is due to the sex-specific, post-translational regulation of two Hox genes, lin-39 and mab-5. In hermaphrodites, MAB-5 is inactive and only LIN-39 influences Pn.p cell fusion fate. In males, both LIN-39 and MAB-5 are active, but the two proteins interact in an unusual way to control cell fusion. It is quite likely that in most species, Hox proteins interact with each other and with other factors to generate more complexity than their expression patterns alone would allow (Alper, 2001).

Understanding how these interactions modulate Hox protein activity is therefore necessary to understand fully how an animal body plan is laid out. At the end of the first larval stage, some of the 12 Pn.p cells fuse with the hyp7 syncytium in a sex-specific pattern. In hermaphrodites, anterior (P1.p and P2.p) and posterior P(9-11).p cells fuse with the hyp7 syncytium while the six central cells P(3-8).p remain unfused. These six unfused cells, the vulval precursor cells, remain competent to develop further, and some of these cells generate the hermaphrodite vulva later in development. The Pn.p cell fusion pattern is different in males, with P1.p, P2.p, P7.p and P8.p fusing with hyp7 and P(3-6).p and P(9-11).p remaining unfused. The posterior unfused cells generate male-specific copulatory structures later in development (Alper, 2001).

Two Hox genes, lin-39 and mab-5, are known to influence Pn.p cell fusion. lin-39 is expressed in P(3-8).p in both hermaphrodites and males. In hermaphrodites, lin-39 prevents fusion of those Pn.p cells in which it is expressed and therefore P(3-8).p remain unfused. Thus, in a lin-39 mutant, all hermaphrodite Pn.p cells fuse with the hyp7 syncytium and are unable to generate a vulva. The regulation of Pn.p cell fusion in males is more complex because both lin-39 and mab-5 can affect the fusion decision. mab-5 is expressed in P(7-11).p in both sexes, but only functions in males. Acting alone, either Hox gene is able to prevent fusion of those cells within which it is expressed: P(3-6).p for lin-39 and P(9- 11).p for mab-5. However, when cells express both Hox genes (P7.p and P8.p), those cells fuse with hyp7, much like cells that contain neither Hox gene (P1.p and P2.p). The ability of these two Hox genes to negate each other's effects in males occurs post-translationally; that is, LIN-39 and MAB-5 proteins can somehow inhibit each other's activity when both proteins are present in the same cell. Moreover, the relative levels of the two proteins do not matter because the two proteins are still capable of inhibiting each other when one of the Hox genes is strongly overexpressed. This result argues against a model in which the two Hox proteins simply sequester each other and, as a consequence, titrate each other's activity. Instead, something else appears to be limiting in this cell fate decision. One possibility is that both proteins bind to regulatory sites in the same target gene, which in turn encodes a protein that directly affects cell fusion. In this model, the binding of either protein alone influences the activity of the fusion gene, whereas the binding of both Hox proteins together does not (Alper, 2001).

In summary, Hox protein activity is regulated in two key ways to control the Pn.p cell fusion decision. (1) MAB-5 is present in the same cells in both sexes but only functions in male Pn.p cells. Thus, something keeps MAB-5 inactive in the hermaphrodite Pn.p cells. (2) Both Hox proteins can interact to inhibit each other when present in the same Pn.p cell in males. To identify genes that affect Pn.p cell fusion by regulating Hox protein activity, mutations were isolated that alter the Pn.p cell fusion pattern. One such mutation, ref-1(mu220) (REgulator of Fusion-1) prevents fusion of posterior Pn.p cells in hermaphrodites, largely, but not completely, by affecting the sex-specific activity of MAB-5. ref-1 mutants also exhibit a defect in the specification of the fate of a hypodermal cell located on the lateral surface of the worm in this same posterior body region as well as other defects in the anterior part of the worm. ref-1 has been cloned and it encodes a transcription factor with two basic helix-loop-helix (bHLH) domains, both of which are distantly related to the hairy/Enhancer of split [E(spl)] subfamily of such proteins (Alper, 2001).

During larval development in C. elegans, some of the cells of the ventral epidermis, the Pn.p cells, fuse with the growing epidermal syncytium hyp7. The pattern of these cell fusions is regulated in a complex, sexually dimorphic manner. It is essential that some Pn.p cells remain unfused in order for some sex-specific mating structures to be generated. The pattern of Pn.p cell fusion is regulated combinatorially by two genes of the C. elegans Hox gene cluster: lin-39 and mab-5. Some of the complexity in the Pn.p cell fusion pattern arises because these two Hox proteins can regulate each other’s activities. A zinc-finger transcription factor, REF-2, is described that is required for the Pn.p cells to be generated and to remain unfused. ref-2 encodes a zinc-finger transcription factor of the odd-paired (opa)/Zic family. REF-2 functions with the Hox proteins to prevent Pn.p cell fusion. ref-2 may also be a transcriptional target of the Hox proteins (Alber, 2002).

Hox genes encode evolutionarily conserved transcription factors involved in morphological specification along the anteroposterior body axis of animals. The two most striking features of Hox genes are colinearity and the strong sequence conservation. Among all animals studied so far, the nematode Caenorhabditis elegans contains one of the most divergent Hox clusters. The core cluster contains only four members, which in part deviate from the colinearity rule. In addition, orthologous and paralogous nematode Hox sequences diverge substantially. Given these nematode-specific features, it was asked how these Hox proteins evolved and how they provide functional specificity. The role of MAB-5 during ray formation was investigated and an in vivo assay was established using Cel-mab-5 regulatory elements to express orthologous, paralogous and chimeric cDNAs in a Cel-mab-5 mutant background. The MAB-5 ortholog from Pristionchus pacificus, but not the C. elegans paralogous Hox proteins can rescue Cel-mab-5. Experiments with chimeric, truncated and mutagenized Hox proteins suggest the specificity to be conferred by the N-terminal arm and helix I, but not helix II of the homeodomain (Gutierrez, 2003).

In summary, this work provides the first detailed analysis of the functional specificity of nematode Hox genes by studying orthologous, paralogous and chimeric proteins for their role in ray formation. Besides the limited sequence conservation of nematode Hox proteins, the N-terminal arm and helix I of MAB-5 are sufficient to induce ray formation when provided in an otherwise LIN-39 protein. Thus, although the homeodomain is the most highly conserved part of nematode Hox proteins, it is this part of the protein that confers most of the functional specificity. At the same time, similar studies with LIN-39 during vulva formation suggest the importance of regions other than the homeodomain in providing functional specificity. Although still in their infancy, these studies support the view that there is no common mechanism in providing specificity to nematode Hox proteins (Gutierrez, 2003).

Axin, APC, and the kinase GSK3ß are part of a destruction complex that regulates the stability of the Wnt pathway effector ß-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3ß, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/ß-catenin and POP-1/Tcf. How BAR-1 is regulated by the EGL-20 pathway has been investigated. First, a negative regulator of the EGL-20 pathway, pry-1, has been characterized. pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. These results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. PRY-1 interacts with BAR-1, SGG-1, and APR-1 and overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that PRY-1 can functionally interact with vertebrate destruction complex components. Finally, SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. It is concluded that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/ß-catenin signaling in C. elegans (Korswagen, 2002).

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; a subset also express egl-5. The following observations are reported: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. It is concluded that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes; that sem-4 indirectly modulates mec-3 expression through its repression of egl-5, and that this negative regulation is required for proper determination of neuronal fates. It is suggested that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes (Toker, 2003).

Hox genes appear to be targets not only of sem-4 but also of other sal genes. Drosophila sal might negatively regulate Sex combs reduced (Scr) and other Drosophila Hox genes. Loss of sal function in Drosophila BX-C minus embryos produces some limited ectopic expression of the Hox gene Scr. Mutations in sal enhance the phenotypes of Polycomb group (PcG) mutants. These genes are known to be negative regulators of Hox genes. Loss of sal function affects AP patterning in Drosophila. Mutations in sal incompletely transform both head and tail structures into trunk-like structures: sal activity has been shown to promote head development. Hox genes in mammals might also be targets of sal family genes. Patients with TBS, which is caused by mutations in SALL1, display characteristic features of syndromes associated with mutations in HOX genes (Toker, 2003).

Drosophila and mammalian studies have suggested that sal genes might function as PcG genes. Mutations of Drosophila sal cause limited ectopic expression of the Hox genes Ubx and Scr, and sal mutations enhance mutations in the PcG genes polyhomeotic and Polycomb-like. Human SALL1 localizes to chromocenters in mammalian cells and mouse sall1 interacts with components of chromatin remodeling complexes. One additional speculation is that Drosophila sal might bind to a 138 bp silencing sequence in the Polycomb response element in Abd-B, the egl-5 ortholog. Two sites have been identified that match the SEM-4 binding sequence in this Drosophila silencing element (Toker, 2003).

Polycomb group (PcG) chromatin proteins regulate homeotic genes in both animals and plants. In Drosophila and vertebrates, PcG proteins form complexes and maintain early patterns of Hox gene repression, ensuring fidelity of developmental patterning. PcG proteins in C. elegans form a complex and mediate transcriptional silencing in the germline, but no role for the C. elegans PcG homologs in somatic Hox gene regulation has been demonstrated. Surprisingly, it is found that the PcG homologs MES-2 [E(Z)] and MES-6 (ESC), along with MES-3, a protein without known homologs, do repress Hox expression in C. elegans. mes mutations cause anteroposterior transformations and disrupt Hox-dependent neuroblast migration. Thus, as in Drosophila, vertebrates, and plants, C. elegans PcG proteins regulate key developmental patterning genes to establish positional identity (Ross, 2003).

The three mes genes act upstream of the Hox genes mab-5 and egl-5 during V ray differentiation, and loss of mes activity can restore normal ray development and mating ability to males mutant in the mab-5 activator pal-1. Males lacking mes activity display anterior expansions of tail structures and ectopic expression of the Hox reporter egl-5::gfp and the Hox target lin-32::gfp. This regulation is not restricted to the male tail: mes-2, -3, and -6 also repress lin-39::lacZ expression in the midbody and head and mab-5 activity in a migrating neuroblast. Consistent with a general somatic regulay function, MES protein expression is widespread in larvae, particularly males. These findings suggest that the regulatory relationship between PcG chromatin proteins and thtore Hox genes has been conserved in nematodes (Ross, 2003).

Presented here is an analysis of cis-regulatory elements in the C. elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. Phylogenetic footprinting was used to efficiently identify cis-regulatory elements and these have been characterized with gfp reporters and tissue-specific rescue experiments. The complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. This analysis has allowed the implication of several new regulatory factors significant to the control of egl-5 expression (Teng, 2004).

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, a C. elegans lineage was studied that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. It has been shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. This study examined tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, the T-box protein TBX-2 was identified as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage (Singhvi, 2008).

Abdominal-B homologs in other invertebrates

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes has suggested that homologs with related functions are present in diploblastic animals, in particular, in Hydra. Phylogenetic analyses show that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. See Phylogenetic relationships among 200 Antp-class genes. Among the 13 non-Hox families, 9 reported here have diploblastic homologs: Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh/Hex, Not, and Dlx. Among the Hox/paraHox, poriferan sequences are not found, and the cnidarian sequences form at least five distinct cnox families. Cnox-1 shows some affinity to paralogous group (PG) 1; this group includes Drosophila Labial. Cnox-2 is related to Drosophila Intermediate neuroblast defective. Cnox-3 and 5 show some affinity to PG9-10; this group includes Drosophila AbominalB. Cnox-4 has no counterparts in Drosophila or vertebrates. Intermediate Hox/paraHox genes (PG 3 to 8 and lox) do not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 are not found chromosomally linked within a 150-kb range and display specific expression patterns in the adult head. During regeneration, cnox-1 is expressed as an early gene whatever the polarity, whereas cnox-2 is up-regulated later during head but not foot regeneration. Finally, cnox-3 expression is reestablished in the adult head once the head is fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods (Gauchat, 2000)

The expression patterns of Hox genes have not previously been comprehensively analyzed in a myriapod. The expression patterns are presented of the ten Hox genes in a centipede, Lithobius atkinsoni, and these results are compared to those from studies in other arthropods. Three major findings are reported. (1) It has been found 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 (spiders) and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. (2) Two 'extra' Hox genes were found 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. (3) It was 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 (Hughes, 2002).

The expression of the Hox genes corresponds roughly with the tagmatic divisions in the centipede. The expression of the genes lab, pb, Hox3 and Dfd is confined to the head, while the trunk is apparently under the control of Antp, Ubx, abd-A and Abd-B. Interestingly, the maxilliped segment has expression of three genes that extend both into the head (Scr and ftz) and into the trunk (Antp). The maxilliped segment is thought to be homologous to the first trunk or thoracic segment of other mandibulate arthropods. The appendages of this segment in the centipede, however, have been highly modified. While their leg-like structure is still evident, they develop to become short and broad fangs, complete with a poison gland. Thus, the first legs of the centipede are modified to become more mouthpart-like, and are used for prey capture and manipulation. This mixed head/trunk identity of the segment seems to be reflected in the Hox code found there. While the segment itself has only a 'trunk' Hox gene (Antp), the appendages have expression of Antp as well as the 'head' genes Scr and ftz, which are also expressed in the maxillary II segment. It remains to be determined how these genes contribute to the development of the centipede fangs. It would also be interesting to know whether the evolution of this novel appendage is correlated with a shift in the expression of these genes. Further studies of Hox expression in other myriapods such as a millipede, or functional studies in the centipede, would be very interesting regarding these issues (Hughes, 2002).

Expression of genes along the centipede trunk is, like the morphology of the trunk, fairly homonomous. Antennapedia extends along the whole trunk in early stages, and later retracts to cover legs one through four. It is not clear whether this later, more restricted domain imparts any developmental difference to these segments, as none is evident morphologically. It is intriguing to note that this restriction to the anteriormost segments of the trunk is reminiscent of a similar restriction of Antp expression in the pleon of malacostracan crustaceans and the thorax of insects. Perhaps the domain of Antp expression was restricted to the anterior portion of the trunk in the myriapod-like mandibulate ancestor, but was only exploited fully in the specialized differentiation of the crustaceans and insects. In the centipede, Ubx and abd-A expression patterns are similarly expressed along the trunk, although Ubx expression fades from the extreme posterior segments. Expression of Abd-B is strongest in the telson, but faint expression extends over the mid-region of leg segments two to seven. Since the genes Ubx, abd-A and Abd-B are likely to have similar roles in patterning the trunks of all mandibulates, it is suggested that the myriapods have developed their unique body plan largely by expanding the number of segments under the control of the 'trunk' genes. This is a similar scenario to that provided by recent findings that snakes seem to have created an elongated body by increasing the numbers of somites under the control of thoracic Hox genes (Hughes, 2002).

Antibodies were used to examine the expression patterns of Antennapedia (Antp), Ultrabithorax (Ubx), Ubx and abdominal-A combined (Ubx/abd-A), and Distalless (Dll) in the embryos of the moth Manduca sexta. The spatial and temporal pattern of Antp expression in Manduca is correlated with the anterior migration of two patches of epithelium that include the anterior-most tracheal pits, and with the development of functional spiracles. Ubx expression shows an intricate pattern that suggests complex regulation during development. Throughout Manduca embryogenesis, the expression of Ubx/Abd-A and Dll is similar to that reported for other insects. However, there is no apparent reduction in Ubx/Abd-A expression in the Manduca abdominal proleg primordium that expresses Dll. The expression of these four proteins was also examined in embryos of the Manduca homozygous homeotic mutant Octopod (Octo). The Octo mutation results in the transformation of A1 and A2 in the anterior direction, with homeotic legs appearing on A1 and occasionally A2. These results suggest that in Octo animals there is a reduction in the level of Ubx protein expression throughout its domain (Zheng, 1999).

Insects show a dramatic diversity in the number and segmental distribution of abdominal appendages. For example, among Lepidoptera larvae, the number of abdominal appendages varies from none to seven pairs. The number of appendages also varies during the course of embryonic development. During early stages of embryogenesis of Pieris rapae and Bombyx mori ventral appendages are present on all abdominal segments. Later some of these appendages regress, leaving prolegs on A3-A6 and on the terminal segment. In Drosophila both Ubx and Abd-A act to repress Dll and the development of abdominal appendages. However, such repression is absent in the crustacean, indicating that the repressive function of Ubx and Abd-A evolved in insects. The presence of homeotic legs in Octo Manduca demonstrates that Manduca Ubx also represses appendage development. However, in the beetle Tribolium and the grasshopper Schistocerca an A1 appendage, the pleuropodia, develops despite high level of Ubx in A1 appendage primordium. Thus the repressive function of Ubx on A1 appendage development evolved late in insect evolution, in the Diptera/lepidoptera lineage. These findings indicate that the repressive function of Abd-A evolved even later than that of Ubx. The expression of Dll and the emergence of prolegs in A3-A6 apparently initiated in the presence of strong Abd-A expression, suggests that in Manduca the repressive function of Abd-A on A3-A6 appendages has not yet evolved. This conclusion for Manduca Abd-A differs from that which has been suggested for the butterfly Precis, where Abd-A is believed to suppress appendages development. In order to develop prolegs both Ubx and Abd-A are locally repressed in the proleg primordia (Zheng, 1999).

In an abdominal segment the A1 appendage developed in Octo is a thoracic leg instead of an abdominal proleg. This suggests that the level of Ubx/Abd-A protein determines the type of appendage that develops in the abdominal region. This observation is consistent with the following hypothesis: high levels of Ubx and/or Abd-A protein(s) act to suppress leg development, while moderate levels of expression, as occur in T3 of wild-type and A1 and A2 of Octo animals, is a permissive environment for thoracic limb development. The expression of Abd-A in the near absence of Ubx protein directs the development of abdominal prolegs, as in A3-A6. Based on this hypothesis, the type of A2 homeotic appendage in Octo should be variable and depend on the level of Ubx expression on A2. If the expression of Ubx in A2 of Octo is above a certain threshold level, thoracic legs should develop on A2. However, if the expression is lower, the A2 appendages should develop as abdominal prolegs. Consistent with this is the finding that in Octo animals, the identity of the A2 homeotic appendage is variable. Some A2 homeotic limbs have characteristics of thoracic legs, while the others possess the characteristics typical of the abdominal prolegs. This model does not explain the absence of prolegs in A7, A8 and their presence in the terminal segment. However, it is quite possible that a proper combination of Abd-A and Abd-B dictates the pattern of appendage development in these more posterior segments (Zheng, 1999).

During the embryogenesis of Drosophila, the homeotic genes are required to specify proper cell fates along the anterior-posterior axis of the embryo. Partial cDNAs of homologs of the Drosophila homeotic gene teashirt and five of the homeotic-complex (HOM-C) genes were cloned from the thysanuran insect, Thermobia domestica (the firebrat), and these genes were assayed for their embryonic expression patterns. The HOM-C genes examined were labial, Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B. Since the expression pattern of these HOM-C genes is largely conserved among insects and since Thermobia is a member of a phylogenetically basal order of insects, the ancestral expression patterns of these genes in insects could be inferred. The expression patterns of the Thermobia HOM-C genes were compared with their expression in Drosophila and other insects; the potential roles these genes may have played in insect evolution are discussed. Interestingly, the teashirt homolog shows greater variability between Thermobia and Drosophila than any of the HOM-C genes. In particular, teashirt is not expressed strongly in the Thermobia abdomen, unlike Drosophila teashirt. It is proposed that teashirt expression has expanded posteriorly in Drosophila and contributed to a homogenization of the Drosophila larval thorax and abdomen (Peterson, 1999).

The earliest domain of Abd-B expression and much of its dynamic pattern are similar among Thermobia, Drosophila, and Schistocerca. The pattern of Abd-B in Thermobia and Schistocerca are nearly identical and likely reflect the ancestral expression pattern. The differences between Drosophila and the Thermobia/Schistocerca pattern reflect the differences in the number of abdominal segments, the modification of the posterior abdominal segments in Drosophila and implicate changes in the Abd-B expression pattern with the evolution of these differences. As with other HOM-C genes, firebrat and grasshopper Abd-B expression patterns are essentially equivalent. As in firebrats, grasshopper Abd-B first appears in the caudal region posterior to the tenth abdominal segment. It is never expressed in any part of the cercal appendages. Expression begins before all abdominal segments have appeared and then extends anteriorly through pA8 at a lower level of expression after all abdominal segments have been established. The anterior expansion of Abd-B expression occurs well after the posterior abdominal segments have been formed. Thus, like Ubx in the thoracic segments, Abd-B is restricted from more anterior segments until they have passed the period of early differentiation (when early cell fates are established). Therefore, Abd-B and Ubx in their anterior domains probably modify the development of specific cells as they develop into cuticle and various specialized structures. Thus, the onset of Abd-B expression in pA8-aA10 (PS14-15) well after abd-A expression probably allows abdominal cell fate specification of these parasegments to occur, after which Abd-B may have a role in promoting certain secondary fates (Peterson, 1999).

Abd-B is required for the proper development of the Drosophila terminalia and has a role in modifying the development of more anterior segments. In Drosophila, it is known that these two roles are not only temporally separable but are genetically separable (r and m alleles), as they result from the activity of two related but different protein products of Abd-B. The Abd-B R ('regulatory') protein is expressed posterior to the eighth and last abdominal segment (PS14-15). One effect of this expression is to eliminate segmentation in this region of the embryo. A distinct A9 segment develops only in Abd-B minus embryos, wherein abd-A expression moves into the posterior segments. In contrast,the Abd-B M protein is expressed in anterior segments after abd-A expression has been established. In aA8 (posterior PS13), the M protein is expressed at high enough levels to repress abd-A expression, but in pA4-pA7, abd-A and Abd-B m are coexpressed and work together to specify segmental fates that are moderately different from fates in more anterior abdominal segments (Peterson, 1999).

It is unknown whether r and m homologs exist in firebrats or grasshoppers. If they do, the probes or antibody (grasshopper) used on these species would have detected both transcripts/proteins to reveal a composite pattern. It has been concluded that there is no homolog to the M protein (or at least no 'm' function) in grasshoppers, citing the fact that the R protein and grasshopper Abd-B have the same anterior expression limit (pA8). It is suggested here that both r and m functions may exist in grasshoppers and firebrats and that an anterior expansion of Abd-B expression could explain the evolution of the derived Drosophila terminus. (1) The M protein is thought to repress abd-A expression at high levels. This is consistent with the retraction of abd-A expression from A10, where high levels of Abd-B accumulate in firebrats and grasshoppers. (2) The R protein prevents segmentation and the early Abd-B expression in firebrats and grasshoppers occurs in an unsegmented region of the embryo. If m and r activity are both ancestral, then a simple expansion of Abd-B expression during the evolution of Drosophila would lead to a shortened abdomen (a larger unsegmented region) due to r function and a more anterior role for Abd-B in abdominal patterning (pA4 in Drosophila versus pA8 in the non-drosophilids) due to m function. (3) Also, the stable posterior border of abd-A expression would shift anteriorly (A7 in Drosophila versus A10). All of these differences are observed and support the notion that both r and m function are ancestral. While these functions might not be separated into different proteins, they may be distinguishable by timing of expression. The separation of these functions into separate transcripts and proteins may have facilitated the anterior expansion of Abd-B expression coincident with the shortening of the Drosophila embryo (Peterson, 1999).

Though the head is the most morphologically derived of the tagmata in the Drosophila embryo, the embryonic thorax is also highly modified relative to other insects. Most striking is the complete absence of larval thoracic limbs. Larval locomotion is performed by means of ventral denticles present in belts on the thoracic and abdominal segments. The modification of the drosophilid larval thorax has caused thoracic and abdominal segments to become more similar. In essence, the Drosophila larva could be thought of as having only two tagmata, a head and a trunk, instead of three. Consistent with this idea, some aspects of homeotic gene expression, such as the register of expression and domains of function of Drosophila Antp, are more similar between thorax and abdomen than between thorax and head. In contrast to this, in other insects the gnathocephalon and thorax show greater similarity. It is suggested that posterior expansion of tsh may be a causal factor in the trunk homogenization of Drosophila embryos. At the same time, the differences in expression of Drosophila Scr, Antp and Ubx are related to a greater distinction of the morphology of the thoracic segments relative to one another (Peterson, 1999).

Changes in the expression of the Hox genes Ultrabithorax and Abdominal in different crustaceans correlate well with the modification of their anterior thoracic limbs into feeding appendages (maxillipeds). In branchiopod crustaceans (such as Artemia), which do not have maxillipeds, Ubx and abdA are expressed throughout the thoracic region. In peracarids, the first, and sometimes second, of the eight thoracic segments bear limbs that have acquired several characteristics of feeding appendages. The modification of these segments correlates with the repression of Ubx and abdA in these segments. Uniform early expression becomes modulated within individual metameres during later development. Decapods are generally described as having three pairs of maxillipeds and five pairs of walking limbs in their thorax. In Periclimenes Ubx and abdA expression is excluded from the first three thoracic parasegments and limbs, is weaker in T4, and stronger in more posterior segments. In Homarus, only the T1 and T2 limbs appear to be distinctly reduced at hatching. Ubx and abdA staining is absent from the first two thoracic parasegments and strong in T3 and more posterior segments. Thus, the anterior boundary of embryonic expression of Ubx and abdA in Homarus appears to be shifted backwards by two metameric units corresponding to the morphological transition in thoracic limbs seen at hatching. It is suggested that spatially modulated distribution of Ubx and abdA expression and temporal changes in the expression of Hox genes are responsible for different decisions on regional identity. In some limbs identity could be determined as a mosaic, with some parts of a segment retaining a thoracic identity and others becoming homeotically transformed to a gnathal fate (Averof, 1997).

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body, consisting of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. The expression patterns was studied of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologs of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. It is concluded that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. It is argued that these segments constitute a unique body region that bears no obvious homology to previously characterized domains of Hox gene activity (Copf, 2003).

The Strongylocentrotus purpuratus genome contains a single ten-gene Hox complex >0.5 megabase in length. This complex was isolated on overlapping bacterial artificial chromosome and P1 artificial chromosome genomic recombinants by using probes for individual genes and by genomic walking. Echinoderm Hox genes of paralog groups (PG) 1 and 2 are reported. The cluster includes genes representing all paralog groups of vertebrate Hox clusters, except that there is a single gene of the PG4-5 types and only three genes of the PG9-12 types. The echinoderm Hox gene cluster is essentially similar to those of the bilaterally organized chordates, despite the radically altered pentameral body plans of these animals (Martinez, 1999).

The Hox cluster of the sea urchin Strongylocentrous purpuratus contains ten genes in a 500 kb span of the genome. Only two of these genes are expressed during embryogenesis, while all of eight genes tested are expressed during development of the adult body plan in the larval stage. Reported is the spatial expression during larval development of the five 'posterior' genes of the cluster SpHox7, SpHox8, SpHox9/10, SpHox11/13a and SpHox11/13b. The five genes exhibit a dynamic, largely mesodermal program of expression. Only SpHox7 displays extensive expression within the pentameral rudiment itself. A spatially sequential and colinear arrangement of expression domains is found in the somatocoels, the paired posterior mesodermal structures that will become the adult perivisceral coeloms. No such sequential expression pattern is observed in endodermal, epidermal or neural tissues of either the larva or the presumptive juvenile sea urchin. The spatial expression patterns of the Hox genes illuminates the evolutionary process by which the pentameral echinoderm body plan emerged from a bilateral ancestor (Arenas-Mena, 2000).

The common ancestor of echinoderms and hemichordates was almost certainly a bilaterally organized animal in which the posterior pair of coeloms terminated near the anus. All of the posterior Hox genes are in fact expressed in the somatocoels. The original A/P orientation of the somatocoels that can safely be inferred for the bilateral common ancestor of echinoderms and hemichordates can still be seen in hemichordates. In the evolutionary process leading to S. purpuratus this axis was evidently altered by a 90° shift in the digestive tract and the associated somatocoelar structures, so that what was originally at the tail of the animal is now on one side (this presumes, or rather predicts, that in hemichordates the sequence of homologous Hox gene expression patterns will run directly along the A/P body axis). The definitive morphological change in the evolution of the echinoderm body plan is coelomic stacking such that viewed from the oral surface of the pentaradial adult body plan, the left somatocoel comes to lie beneath (actually interdigitated within) the hydrocoel of the rudiment; the right somatocoel comes to lie beneath the left. The roughly circular shape the somatocoels assume as they surround the digestive tract late in development, including its stubby intestine, facilitates this arrangement: were the gut removed, the coeloms would resemble a stack of coins. The morphological stacking transformation would be much more different to conceive were the gut and somatocoels elongate linear structures. However, the price that had to be paid is clear: formation of the adult body plan requires formation of a new terminal hind gut and anus, since the original one is in the wrong place. Indeed, in echinoids, neither the larval hindgut and anus nor the esophagus survives metamorphosis. These are replaced in the juvenile during the period between metamorphosis and the resumption of feeding. It is not yet known whether the posterior Hox genes are reactivated during this process. The difference between the right and left somatocoelar Hox gene expression patterns, both quantitative and qualitative, can be regarded as molecular correlates of the developmental process of coelomic stacking. Thus, the left somatocoel, which is in contact with the rudiment and forms parts of the oral region of the animal (e.g. its dental sacs), expresses the Hox genes differently than does the right somatocoel. The right somatocoel contributes to the development of some of the structures of the lateral and anal surfaces of the adult, and correlated with its different developmental role is a different and less intense pattern of posterior Hox gene expression. Unfortunately, very little is known about the developmental biology of adult structures in the late larva, e.g. the specific contribution of the coeloms to the mesenteries, the gonad rudiment or the complex and regionally specific endoskeletal plates and spines. If the Hox gene expression patterns of the right somatocoel indeed play a role in setting up spatial progenitor fields in which subsequent morphogenetic events are to occur, these would be among the likely body parts for which they might be required (Arenas-Mena, 2000).

In animals, region specific morphological characters along the anteroposterior axis are controlled by a number of developmental genes, including Hox genes encoding homeodomain transcription factors. Although Hox genes have been regarded to play a key role in the evolution of morphological diversity, as well as in the establishment of the body plan, little is known about the function of Hox genes in invertebrates, except for in insects and nematodes. The present study addresses the role of Hox genes in body patterning during the larval development of the ascidian Ciona intestinalis conducting knockdown experiments of the seven Hox genes expressed during embryogenesis. Experimental results have demonstrated that Ci-Hox12 plays an important role in tail development through the maintenance of expression of Ci-Fgf8/17/18 and Ci-Wnt5 in the tail tip epidermis. Additionally, it has been shown that Ci-Hox10 is involved in the development of GABAergic neurons in the dorsal visceral ganglion. Surprisingly, knockdown of Ci-Hox1, Ci-Hox2, Ci-Hox3, Ci-Hox4 and Ci-Hox5 did not give rise to any consistent morphological defects in the larvae. Furthermore, expression of neuronal marker genes was not affected in larvae injected with MOs against Ci-Hox1, Ci-Hox3 or Ci-Hox5. In conclusion, it is suggested that the contribution of Hox genes to the larval development of the ascidian C. intestinalis might be limited, despite the fact that Ci-Hox10 and Ci-Hox12 play important roles in neuronal and tail development (Ikuta, 2010).

Table of contents

Abdominal-B: Biological Overview | Promoter Structure | Transcriptional Regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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