In the Drosophila leg, activation of Notch leads to the establishment of the joints that subdivide the appendage into segments. Mutations in bowl result in phenotypes similar to Notch, causing fusion and truncations of tarsal segments (tarsomeres). Like its close relative Odd-skipped, Bowl is produced in response to Notch signalling at a subset of segment boundaries. However, despite the fact that bowl mutant clones result in fusion of tarsomeres, Bowl protein is only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere joints. One hypothesis to reconcile these data is that bowl has a role at an earlier stage in tarsal development. Therefore, the effects were investigated of bowl mutations on the expression of leg 'gap' genes that confer regional identity on the developing leg. Several of these genes have altered expression in bowl mutant cells. For example, bric-a-brac2 is normally expressed in the central part of the tarsus domain but expands into distal and proximal regions in bowl clones. Conversely, ectopic bowl leads to a reduction in bric-a-brac2, with a concomitant expansion of proximal (t1) and distal (t5) tarsomere fates. The bowl gene is therefore required for the elaboration of pattern in the tarsus and its effects suggest a progressive model for the determination of P/D identities. This mechanism might be important in the diversification of arthropod limbs, because it explains how segmented tarsomeres could have arisen from an ancestral limb with an unsegmented tarsus (de Celis Ibeas, 2003).

A second study has independently shown that the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. Since the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, it is suggested that they may be required in multiple developmental contexts to induce epithelial cellular changes (Hao, 2003).

Animal limbs develop as outgrowths from the main body axis that acquire proximal/distal (P/D) patterning to form a series of specialized skeletal structures. These structures are articulated and so one key consequence of P/D patterning is the establishment of joints between each skeletal element. In the Drosophila leg, the P/D axis is established through the combined activities of Wingless (Wg) and Decapentaplegic (Dpp), which intersect in the centre of the limb primordium. Wg and Dpp together induce the expression of Distal-less (Dll), a homeodomain protein required for the development of all distal leg structures, and Dachshund (Dac), a nuclear protein required for intermediate leg segments (femur and tibia). By the beginning of the third larval instar, the leg primordium is therefore subdivided into at least three regions. Subsequent patterning involves interactions between the factors expressed in these early territories. For example, several genes are required for the development of the tarsus, including rotund and bric-a-brac. Expression of these genes is promoted by Dll and restricted proximally by the combined activities of Dac and distally by a gradient of epidermal-growth-factor-receptor signalling. By the stage that a series of P/D regions have been established, further patterning appears to be independent of the initial inducers Wg and Dpp. However, it is not clear how these P/D regions are elaborated (for example, to give diversity to the distal tarsal structures) (de Celis Ibeas, 2003 and references therein).

A final stage in translating the P/D patterning into the definitive segmented structure of the insect adult leg is the formation of the inter-segmental joints. The leg consists of six true segments or podites (coxa, trochanter, femur, tibia, tarsus and pretarsus), which are independently moveable by muscles. In Drosophila, the tarsus is further subdivided into five tarsomeres (t1-t5), which have distinct characteristics but lack independent musculature. Development of both 'true' joints and inter-tarsomere joints requires Notch activity, shown by the loss of joints and fused segments in Notch mutant cells, and by the ectopic joints that are formed when extra sites of Notch activity are engineered. Consistent with its pivotal role in specifying joint development, Notch activity is detected at all segment/subsegment boundaries at the end of larval development, using transcription of the Enhancer of split target genes as a measure. However, expression of Notch ligands is first observed at a subset of locations at a much earlier stage shortly after the initial 'regional' domains of gene expression are established. There are two explanations for this. One is that the specification of joints occurs sequentially, with some joints being determined early and others (e.g., tarsomere joints) much later. Alternatively, Notch activity might have both earlier roles in P/D regionalization and patterning and later roles that build on these earlier events to establish the segmental boundaries and joints at the correct locations (de Celis Ibeas, 2003).

To investigate further the mechanisms involved in P/D limb development, genes were sought whose expression is dependent on Notch activity. Analysis of these genes could allow discovery whether they have roles in the initial P/D patterning as well as in the subsequent establishment of joints. The zinc-finger protein encoded by the gene bowl is detected at a subset of sites of Notch activity and its expression is dependent on Notch. The bowl gene is closely related to the segmentation gene odd-skipped, and is required for development of the embryonic hindgut. Analysis of bowl and odd-skipped function in the developing leg indicates that these genes are involved in the elaboration of pattern in the tarsus, leading to the proposal that Notch is important for patterning as well as for joint formation. The effects of Bowl on tarsal development suggest that P/D tarsal identities are determined progressively and might also explain how different numbers of tarsomeres could have arisen from an ancestral limb that is thought to have contained an unsegmented tarsus (de Celis Ibeas, 2003).

Thus the genes bowl and odd are involved in a novel aspect of this process that elaborates the pattern within the tarsus to generate the correct number and structural diversity of the tarsomeres. Mutations in bowl or odd cause cells at the proximal and distal positions in the tarsal region to acquire fates of more centrally placed cells, giving rise to truncated or fused tarsomeres. Conversely, ectopic Bowl leads to a transformation of central fates to more proximal or distal fates, again causing distortions and truncations of the tarsus. The changes in fate are manifest in the expression patterns of genes such as bab1 and bab2, which are normally present at the highest levels in t3/t4 tarsomeres and at lower levels in t2 and t5. Absence of bowl leads to elevated Bab2 levels in t2 or t5 and to expression in proximal regions (t1), where bab2 is normally silent. One notable feature of Bab1/Bab2 expression is that it is modulated into rings of higher and lower expression. This modulation is also partially lost in bowl mutant clones (and in Dac mutants), arguing that bowl is intimately associated with the elaboration of patterning (de Celis Ibeas, 2003).

Previous studies have shown that bab1/bab2 expression is promoted by Dll and that its proximal and distal limits are dependent on Dac proximally and on epidermal-growth-factor-receptor signalling distally. It is proposed that these activities not only define the initial domain of bab1/bab2 expression but also indirectly regulate the production of Bowl and Odd through their effects on Notch-ligand expression. Bowl is then necessary to fine tune bab2 expression so that its levels are low or absent in the extremities of the tarsus, allowing these to adopt t1 and t5 characteristics. If one of the factors responsible for positively regulating bab1/bab2 expression was present transiently, its decay would also contribute to the gradation in Bab2 expression and could explain why Bab2 is not turned on in the t1 cells that have lost Bowl at late stages (de Celis Ibeas, 2003).

The effects of Bowl and Odd on tarsal development were initially difficult to reconcile with their expression. In late stages of limb development (late L3/early pupal), the proteins are present only at sites of Notch activity outside the tarsus, not within the tarsus, even though the most obvious phenotypes are tarsomere fusions. All of the sites of expression are precursors for the 'true' joints (those with tendon attachments and direct muscle control), suggesting that Bowl/Odd could have a primary role in the establishment of joints and that the regulation of tarsal patterning has been acquired secondarily. It is proposed that effects on patterning occur because the proximal and distal parts of the tarsus are formed by cells that synthesize Bowl/Odd at an earlier stage and that the levels of Bowl/Odd determine the extent of tarsal gene expression. When the tarsus is first defined by the expression of bab, Bowl/Odd directly flank this domain. As the tarsus expands, Bowl and Odd are only retained at the boundary and are lost from the intervening cells; as a consequence, bab2 is derepressed. In this way, cells closest to the initial domain of Bab2 expression would contain Bowl/Odd for the least time and therefore have higher levels of Bab2 than those closer to the tibial boundary. A similar relationship between expression and phenotype has been seen with drumstick (drm; a gene related to bowl and odd that is required for hindgut morphogenesis). At late embryonic stages, drm expression is detected only in the most anterior cells of the small intestine, even though it influences cell behaviour along the whole length of the intestine. By tracing earlier phases of expression, it has been shown that drm is transiently expressed more broadly and gradually becomes restricted to the anterior hindgut boundary (Green, 2002), which is similar to what was observed for odd-lacZ expression in the leg. It is possible that these similarities in drm, odd and bowl regulation reflect a common underlying mechanism conserved between hindgut and leg morphogenesis (de Celis Ibeas, 2003).

Notch activation appears to be one key factor in promoting the accumulation of Bowl and Odd at the tarsal boundaries, but some data indicate that other factors are required and that the regulation might be indirect. (1) Bowl and Odd can only be induced at a subset of the locations where Notch is active, so Notch alone is not sufficient. (2) Although all Notch clones at the t5/tibia boundary result in a loss of Bowl protein, not all clones at the more proximal boundaries have a phenotype. Because the smaller clones tend to have the least effect on Bowl, Notch appears to initiate but not to maintain Bowl expression at these locations. (3) Although regulation of odd can be fully explained by its effects on transcription, Bowl might be subject to post-transcriptional regulation. When expression of bowl mRNA is driven through the leg (using GAL4 drivers), only low levels of Bowl protein are detected, at best, within the tarsus, suggesting that the translation or the stability of the protein are regulated. Candidates to participate in Odd and Bowl regulation include Spineless and Lines, a protein that acts antagonistically to Bowl and Drm in hindgut morphogenesis. Although the combined actions of Notch and these factors might explain the initial expression of Bowl and Odd, the mechanism that maintains their expression specifically at the boundaries of the tarsus is unclear. This aspect of regulation is crucial for the diversification of the tarsomeres and, if this model is correct, would be linked to proliferation. It is predicted that tarsal cells should show a bias in their patterns of proliferation, as is the case in more proximal regions of the leg, and that the progeny of Bowl-expressing cells should occupy the t1/t2 and t5 tarsal segments. It has not yet been possible to specifically monitor the proliferation pattern and fate of Bowl-expressing cells to test these predictions (de Celis Ibeas, 2003).

One extrapolation from the proposed model for tarsal development in Drosophila is that the basal or ancestral state consisted of a single tarsal segment, specified by uniform levels of Bab and directly flanked by sites of Bowl expression prefiguring the tarsal/tibial and tarsal/pretarsal joints. This is in agreement with the phylogenetic evidence, which points towards the ancestral arthropod limb having an unsegmented tarsus (as remains the case for many modern arthropods, including some insects). Furthermore, there is considerable variation in the extent of tarsal subdivision, with most insects having between two and five tarsomeres (some arachnids have further subdivisions). These differences in pattern could be explained by differences in either the duration or the rate of proliferation during the crucial phase when bowl/odd influence bab2 patterning. Although mutations in Notch or bowl/odd affect the extent of tarsal proliferation, as do mutations in spineless and bab2, none of these activities alone is sufficient to cause an increased length of the tarsus (although ectopic Notch activity can give ectopic outgrowth). Further investigation of how these factors combine to coordinate tarsal patterning and proliferation should help in the unraveling of the mechanism underlying the diversification of arthropod limb structure. Furthermore, as modifications of bab2 expression are correlated with diversification of pigmentation and trichome patterns in Drosophila species, the possibility that bab2 expression is intrinsic to diversification of tarsal patterning suggests that changes in the regulation of a single gene could contribute to the diversification of many different morphological traits (de Celis Ibeas, 2003).

GENE STRUCTURE

cDNA clone length - 3425 base pairs

Bases in 5' UTR - 344

Exons - 4

Bases in 3' UTR - 1381

PROTEIN STRUCTURE

Amino Acids - 744

Structural Domains

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. Two embryonically expressed odd-cognate genes, sob and bowel (bowl), have been molecularly characterized that encode proteins with highly conserved C2H2 zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although these results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl (Hart, 1996).

The most notable feature of the odd-skipped sequence is the presence of four tandem C2H2 zinc fingers in the carboxy-terminal third of the protein. Both sob and bowl encode C2H2 zinc fingers, and these show substantial homology with the amino acid and nucleotide sequences of odd. Unlike odd, the cognates encode five (rather than four) tandem zinc fingers, such that the first four amino-terminal fingers of each align with the Odd sequence. With the exceptions noted below, the genes show little if any significant homology beyond this region (Hart, 1996).

Each finger is a 28-residue repeat that matches the canonical consensus for the C2H2 zinc finger class. A comparison of the zinc finger amino acid sequences of the three Drosophila genes indicates that the Sob and Bowl proteins are most similar. Besides having a fifth, C-terminal finger that is not present in the Odd protein, the two cognates show greater similarity among the first four fingers as well. Over this region, Sob and Bowl are 97.3% identical while sharing 86.6% and 87.5% identity with Odd, respectively. Substitutions among the three sequences occur at 15 different positions within these four fingers. At 12 of these, Sob and Bowl share the same residue, and at 2 of the 3 positions where Sob and Bowl do differ, the Odd sequence diverges from both cognates (Hart, 1996).

The conservation between Sob and Bowl is notably lower in the fifth (C-terminal) finger, where they share 85% identity, such that the zinc fingers of the two cognates are 95% identical overall. Interestingly, a similar trend toward increasing divergence in more C-terminal fingers is apparent when the first four fingers of all three proteins are compared. While substitutions are relatively rare among the first two (N-terminal) zinc fingers, they increase in frequency in the third and fourth fingers. A C-terminal bias is also apparent in the divergence between the C. elegans gene B0280.4, the strongest homologue identified in a BLAST search, and the first three fingers of Odd. Beyond the zinc fingers, homology searches indicate no extensive regions of amino acid sequence conservation among the three cognates. A small region of homology occurs at the C-terminal ends of the Odd and Bowl proteins, which terminate with the sequences GFTIDEIMSR and GFSIEDIMRR, respectively. (This region of the Sob sequence is clearly distinct. The relative position of the zinc fingers varies among the three proteins, falling near the C-terminal end of Odd (residues 215-326 out of 392 total) and Sob (residues 389-528 of 577), but in the amino terminal half of Bowl (residues 233-372 of 744) (Hart, 1996).

Drm is a member of the Drosophila odd-skipped (odd) family of zinc finger encoding genes that includes odd, sister of odd and bowl (sob), and bowel (bowl). These genes map close to each other, suggesting that the family has arisen by relatively recent duplication. Like bowl and sob (but not odd), drm contains a splice donor site within the R74 codon of the second zinc finger. Interestingly, this splice site has been conserved evolutionarily, since it is also present in both the mouse and human odd-skipped related (Osr) genes Osr1 and Osr2 (Green, 2002).

The Drm protein contains two zinc finger motifs (compared to four in Odd and five in both Sob and Bowl). The zinc fingers in Odd, Sob, and Bowl conform to the canonical C₂H₂ structure (C-X₂-C-X₁₂-H-X₃-H) that is most commonly associated with a DNA-binding function, but in some cases, can have protein-binding capability. In Drm, the first zinc finger conforms to the canonical C₂H₂ sequence and has a high degree of similarity (~95%) to the first finger of the other Odd family members. The second zinc finger of Drm is divergent; the primary sequence conforms to the canonical C₂H₂ sequence up to the H73 residue, but the second His residue is replaced by a Cys, with H-X₄-C spacing between the latter two zinc-coordinating residues. This residue spacing is found in other C₂HC fingers with demonstrated protein-binding activity. Computer modeling with respect to the known structure of the Drosophila U-shaped (Ush) C₂HC zinc finger shows that the Drm C₂HC finger is theoretically capable of folding around a zinc ligand. Another distinguishing feature of Drm is the divergent linker region between its zinc fingers. The most common linker, found in over 50% of known C₂H₂ fingers, consists of five residues with the consensus sequence TG(E/Q)(K/R)P. The Odd, Sob and Bowl linkers all have the conserved sequence TDERP, whereas the Drm linker (KSPEIT) is different both in sequence and length. Since its C₂H₂ and C₂HC zinc fingers are, in principle, capable of either DNA or protein binding, Drm may function by either or both of these mechanisms (Green, 2002).

date revised: 25 July 2005

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