BIOLOGICAL OVERVIEW

odd-skipped (odd) was first identified in the genetic screen of Nusslein-Volhard and Wieschaus (1980). In the trunk of odd-skipped mutant embryos, cuticle defects appear limited to alternate--odd-numbered--segments, hence, its name and designation as a pair-rule gene. In odd mutants, the posterior rows of the odd-numbered denticle rows are deleted, leaving the anterior regions intact. The deleted posterior rows are replaced with a mirror-image duplication of the remaining anterior rows. This duplication is attributed to an ectopic parasegmental boundary, which results from ectopic expression of engrailed (en) and wingless (wg). This suggests that Odd functions in the anterior regions of the odd-numbered segments to repress en and wg. Since odd encodes a zinc-finger protein, it presumably functions to regulate transcription of other segmentation genes.

Both Odd-skipped mRNA and Odd protein accumulate in a pair-rule pattern of seven stripes during cellular blastoderm (stage 5). The Odd pair-rule expression stripes reside in the even-numbered parasegments (corresponding to the odd-numbered segments). During gastrulation, Odd undergoes a transition from a pair-rule to a segment-polarity pattern of expression. This transition occurs during gastrulation (stages 6 and 7) by refinement of the pair-rule ("primary") stripes concomitant with activation of eight "secondary" stripes. Two of the secondary stripes lie anterior to the first primary stripe, while the remaining 6 fall between the primary stripes. By early germband extension (stage 8) [Images], Odd is expressed in evenly spaced stripes corresponding to the anterior margins of every segment (i.e. each Odd stripe is adjacent and posterior to an En stripe).

The Odd pair-rule stripes arise during cellularization in a highly dynamic manner; the proper expression of these stripes is critical for activation of the even-numbered En stripes. Extensive molecular and genetic studies indicate that Odd, Fushi-Tarazu (Ftz) and Even-skipped (Eve) interact with one another during cellularization to ensure activation of the even-numbered En stripes. According to the model, Ftz activates en while Odd represses en and Eve acts to restrict Odd activity, thereby allowing en to be activated in a narrow stripe within the Ftz domain. Initially, during mid-cellularization (stage 5) Odd, Eve and Ftz are all co-expressed in the domain of the future En stripe. By the end of cellularization, Eve and Odd clear out of this domain, leaving only Ftz in place to activate en. Odd protein remains in the posterior of the Ftz stripe, resulting in a phase-shift between the anterior margins of Ftz and Odd. The presence of Odd in the posterior domain of the Ftz stripe prevents activation of en in this domain. Removing Odd from the anterior margin of the Ftz stripe (establishing a Ftz/Odd phase-shift) is necessary for activation of en. In eve null embryos this phase-shift is not established and the even-numbered en stripes are not activated. In eve hypomorphs, there is a delay in the establishment of the Ftz/Odd phase-shift, and a corresponding delay in the activation of en (Ward, 1997 and Ward and Coulter, manuscript in prep.). Thus, genetic and molecular data are consistent with Eve functioning to restrict Odd activity within the Ftz stripe thereby allowing en to be activated in a narrow stripe. It is important to note that the Eve pair-rule stripes are responsible for establishing the Ftz/Odd phase-shift, rather than the later, Eve "secondary stripes" (Fujioka, 1995, Ward, 1997 and Ward and Coulter, manuscript in prep.).

While the expression of Ftz and Odd at the end of cellularization is consistent with Ftz activating en and Odd repressing en, the expression patterns do not define the mechanism by which Odd acts to prevent activation of en. Two hypotheses have been proposed to account for the correct positioning of a narrow En stripe within a broad Ftz stripe. In the Hierarchy model, en responds to a critical threshold level of Ftz activity within the Ftz stripe (Lawrence, 1989). In this model, Odd and Ftz are proposed to interact in a hierarchy, with Odd protein functioning to repress ftz, presumably at the transcriptional level (Mullen, 1995). A key feature of this model is that Odd represses en indirectly by reducing ftz levels. In the alternate, Combinatorial model, the presence of a negative regulator of en prevents en expression within portions of the Ftz stripe. According to this model, Odd is proposed to interact in a combinatorial (parallel) manner with Ftz. The distinguishing feature of this model is that Odd does not affect en by altering ftz levels; rather, Odd acts to prevent en activation within the Ftz stripe.

Analysis of Odd, Ftz, and En expression patterns in eve hypomorphs supports a model of combinatorial interactions between Ftz and Odd to define En stripes. In strong eve hypomorphs, ftz and odd are co-expressed at the anterior of each Ftz stripe for an extended period of time (up to 15 minutes post onset of gastrulation) prior to removal of Odd (i.e. establishment of the Ftz/Odd phase-shift). Despite the prolonged co-expression of Ftz and Odd, En is rapidly (within 5-10 minutes) activated subsequent to removal of Odd. This rapid activation of en strongly suggests that Ftz is present at a level sufficient to result in expression of en upon removal of Odd. Thus, the eve hypomorph data supports a model of combinatorial interactions between Ftz and Odd to define En stripes (Ward, 1997 and Ward, 2000a).

This essay courtesy of and copyright © 1997 Ellen J. Ward

The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation

Notch signaling controls formation of joints at leg segment borders and growth of the developing Drosophila leg. The odd-skipped gene family has been identified 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 brother of odd with entrails limited (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).

Morphogenesis of the Drosophila leg joint involves distinct changes in cell behavior, including apical constrictions of cells, changes in cell shape, and also likely changes in the adhesion between cells. Detailed studies of Drosophila joint morphogenesis have revealed that joints can be subdivided into different territories based on characteristic cell shape changes and gene expression profiles and their ultimate contribution to a particular structure of the adult leg joint. Although joint morphogenesis becomes evident during pupal stages of Drosophila development, specification of the distinct cell populations fated to participate in joint formation occurs during larval development. The Notch signaling pathway plays a fundamental role in specifying the formation of joints. Ultimately, however, it must be the target genes downstream of Notch signaling that define distinct joint territories (Hao, 2003).

The odd-skipped family is a key group of genes induced upon Notch activation that promotes morphological changes associated with joint formation during leg development. Expression of odd, sob, and drm is induced in cells responding to Notch activation; these cells lie distal to Notch ligand-expressing cells. bowl expression is also regulated by Notch. Ectopic expression of odd, as well as loss of bowl function, does not alter the expression of Notch ligands. Hence, the morphological changes induced by expression of these genes appear to be mediated downstream of Notch activity. Ectopic expression of odd, sob, and drm, like ectopic Notch activation, can cause alterations in the leg cuticle that resemble those that occur at joints, including deep creases within the cuticle and an absence of bristles. Importantly, their ectopic expression, like ectopic Notch activation, induces cells to form an invaginating furrow, while still remaining part of the disc epithelium. Interestingly, during normal leg development, mid-distal joint cells express odd; these are the same cells that will invaginate and ultimately fold under proximal joint cells. Moreover, the cells that invaginate because of their expression of odd, both in ectopic expression studies and in wild-type legs, accumulate high levels of apical filamentous actin. Further support for the idea that odd, sob, and drm control a specific aspect of cell morphogenesis, an invagination, as opposed to being more generally required for specifying joint fate, comes from the observation that ectopic odd expression does not induce the expression of other markers of joint fate, including nubbin, E(spl)mß, big brain, odd, and drm. This contrasts with the effect of Notch, which induces nubbin, E(spl)mß, big brain, odd, and drm expression. Moreover, FLP-out clones of Notch induce outgrowths of leg tissue, whereas FLP-out clones of odd, drm, and sob are not associated with leg outgrowths. Thus, while both ectopic Notch activation and ectopic odd or sob expression are capable of inducing an invagination into the disc epithelium, Notch activation must further organize additional aspects of joint formation and also leg growth. Taken together, the ectopic expression studies indicate that odd, sob, and drm are Notch target genes that mediate a subset of the activities of Notch during leg development, namely, they promote a cell morphological change, an epithelial invagination, which normally occurs during joint formation (Hao, 2003).

Interestingly, the involvement of the odd-skipped family of segmentation genes in promoting epithelial cellular changes may not be unique to the leg joint. odd-LacZ is expressed in the apodemes of the developing leg, which are tubes of invaginating cells that serve as muscle attachment. Thus, these genes may have a role in promoting the apodeme invagination as well. odd is also required for embryonic segmentation during which segmental borders are defined by intersegmental furrows; cells at the prospective segment boundary elongate and fold into the epidermis. While the relationship between odd expression and cell morphology during embryonic segmentation has not been elucidated, it is possible that one function of odd in segmentation is to orchestrate epithelial invaginations. Thus, the odd-skipped gene family may be required in multiple developmental contexts to induce epithelial cellular changes, such as promoting an invaginatiom. Since the odd-skipped family genes encode transcriptional regulators, it is hypothesized that they regulate the expression of genes involved in cytoskeletal architecture or cell adhesion (Hao, 2003).

Studies on the function of odd, sob, and drm suggest that these genes may have a similar function during leg development. They share a common expression pattern at all stages of leg development, consistent also with their overlapping expression in the embryo. Importantly, ectopic expression of each gene is capable of inducing the same morphological changes in the adult cuticle and, for odd and sob, the same cellular changes in the leg disc epithelium. It is thus suggested that odd, sob, and drm act redundantly during leg segmentation. Hence, it is not surprising that, when only one of the genes is removed, no effect on leg development is observed. It will ultimately be of interest to determine the phenotype of leg tissue triply mutant for odd, sob, and drm (Hao, 2003).

bowl, in contrast, may have adopted functions that are independent of and/or not obscured by the other three members of the family. bowl expression appears largely distinct from the other three genes; its expression encompasses a broader domain that overlaps that of the other genes in proximal segments and tarsal segment 5, while bowl is the only odd-skipped family gene expressed in tarsal segments 1-4. The identical expression profile of odd, sob, and drm, yet distinct pattern of bowl, is also observed in other tissues. The observation that the odd-skipped family genes are expressed in overlapping domains in a number of different developmental contexts, yet are not always genetically redundant, suggests that their contribution to a particular morphogenetic process may depend on their relative expression levels or their interaction with other proteins in that particular tissue (Hao, 2003).

In fact, it has been shown that a physical interaction between one of the odd-skipped family members, Drm, and another transcriptional regulator, Lines, is important during hindgut morphogenesis. By interacting with Lines, Drm inhibits Lines activity in the embryonic hindgut, thereby allowing specification of the small intestine. Since the functionally significant Drm-Lines interaction was mapped to the first zinc-finger of Drm, it is conceivable that Odd, Sob, and Bowl may also interact with Lines in other developing tissues. Indeed, this does seem to be the case, since regulatory interactions amongst Drm, Bowl, and Lines operate during the patterning of the embryonic dorsal epidermis and the foregut. In these contexts, Lines inhibits Bowl, resulting in a particular cell type. The remaining cell types are controlled by Drm, which activates Bowl by causing inhibition of Lines (Hao, 2003).

The results are consistent with this molecular genetic circuit also functioning during Drosophila leg development. Clones of cells mutant for bowl are unable to participate in joint formation, resulting in melanotic protrusions from the leg cuticle in proximal segments and in a fusion of tarsal segments. The difference in the phenotype of bowl clones in proximal versus tarsal segments may be because proximal joints do not form in the same way as tarsal joints, although some of the changes in cell behavior are presumably conserved. Notably, the difference does not appear to be due to redundancy, because loss-of-function bowl mutations result in the fusion of the tibia and tarsal segment ta1, despite the fact that all four genes are expressed in the developing tibia. Thus, odd, sob, and drm are insufficient to induce tibia-tarsal 1 joint formation in the absence of bowl; the ability of these genes to induce morphogenesis might be dependent in some way on the expression of bowl. lines has also been shown to have a role during leg development. These results are all consistent with a molecular model in which Bowl and Lines interact to regulate joint formation during leg development, although it remains to be determined whether Lines inhibits Bowl function or whether a Bowl-Lines complex regulates the expression of genes effecting joint formation. It is further proposed that formation of proximal leg joints requires the additional contribution of Odd, Sob, and Drm, which act redundantly to relieve the repression of Bowl by Lines. In such a model, ptcGAL4-driven ectopic bowl expression would be insufficient to induce ectopic segmentation in the leg, as has been observed, since Lines would repress Bowl and hence render Bowl inactive. Also, drm bowl mutant clones would behave similar to bowl mutant clones, as has been observed; odd and/or sob would compensate for the loss of drm. While odd, sob, drm, and bowl may act together to regulate proximal leg segmentation, it appears that only bowl is essential to tarsal segmentation; odd, sob, and drm are not expressed in tarsal segments 1-4. This would suggest that within tarsal segments 1-4 an alternative mechanism regulates Bowl activity (Hao, 2003).

GENE STRUCTURE

Bases in 5' UTR - 189

Bases in 3' UTR - 591

PROTEIN STRUCTURE

Amino Acids - 392

Structural Domains

The ODD protein is highly basic and contains four tandem Cys-Cys/His-His zinc finger repeats in the C-terminal half of the molecule, consistent with a presumed function for ODD as a DNA binding protein and transcriptional regulator. There is a polyserine run and three polyglutamate runs corresponding to an OPA sequence (Coulter, 1990).

There are two embryonically expressed odd-skipped-cognate genes, sob and bowel, that encode proteins with highly conserved C2H2 zinc fingers. While the Sob and Bowel 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. The closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns: in contrast, the less-tightly linked bowel gene is expressed in a distinctly different pattern at the termini of the early embryo. odd appears to be strongest at the cellular blastoderm stage when it is expressed in seven stripes. Later, when the segment polarity-like pattern develops, the intensity of the stripes diminishes. The sob transcripts show the reverse, gaining in intensity at gastrulation. Bowel accumulates in a strong cap at the posterior pole, a relative weak and nonuniform domain at the anterior pole, and a broad transverse stripe that lies just anterior to the presumptive cephalic furrow. At early gastrulation, the terminal expression of bowel is supplement by low intensity transverse stripes in the trunk primordium (Hart, 1996).

EVOLUTIONARY HOMOLOGS

Odd-skipped family in Drosophila

The terminal genes of Drosophila specify non-segmented regions of the larval body that are derived from the anterior and posterior regions of the early embryo. Terminal class genes include both maternal-effect loci (typified by the receptor tyrosine kinase Torso) that encode components of a signal transduction cascade and zygotic genes (e.g. Tailless and Huckebein) that are transcribed at the poles of the embryo in response to the local activation of the pathway. A zygotic gene, bowel (FlyBase name: brother of odd with entrails limited or bowl), has been characterized that is a zinc finger homolog of the pair-rule segmentation gene odd-skipped. bowel transcripts are initially expressed at both poles of the blastoderm embryo and in a single cephalic stripe. This pattern depends upon Torso and Tailless activity, but is not affected in huckebein mutants. Five mutations that affect the Bowel protein were isolated and sequenced, including a nonsense mutation upstream of the zinc fingers and a missense mutation in a putative zinc-chelating residue. bowel mutants die as late embryos with defects in terminal derivatives including the hindgut and proventriculus. These results indicate that the developmental roles of odd-skipped and bowel have diverged substantially, and that bowel represents a new member of the terminal hierarchy that acts downstream of tailless and mediates a subset of tailless functions in the posterior of the embryo (Wang, 1996).

The Drosophila embryonic hindgut is a robust system for the study of patterning and morphogenesis of epithelial organs. In a period of about 10 h, and in the absence of significant cell division or apoptosis, the hindgut epithelium undergoes morphogenesis by changes in cell shape and size and by cell rearrangement. The epithelium concomitantly becomes surrounded by visceral mesoderm and is characterized by distinct gene expression patterns that forecast the development of three morphological subdomains: small intestine, large intestine, and rectum. At least three genes encoding putative transcriptional regulators, drumstick (drm), bowl, and lines (lin), are required to establish normal hindgut morphology. drm and bowl are two genes closely related to odd-skipped and are also closely linked to odd. The defect in hindgut elongation in drm, bowl, and lin mutants is due, in large part, to the requirement of these genes in the process of cell rearrangement. Further, drm, bowl, and lin are required for patterning of the hindgut, i.e., for correct expression in the prospective small intestine, large intestine, and rectum of genes encoding cell signals (wingless, hedgehog, unpaired, Serrate, dpp) and transcription factors (engrailed, dead ringer). The close association of both cell rearrangement and patterning defects in all three mutants suggest that proper patterning of the hindgut into small intestine and large intestine is likely required for its correct morphogenesis (Iwaki, 2001).

Focusing on hindgut elongation that occurs after stage 10, neither apoptosis nor cell proliferation contribute significantly to the process. Thus, hindgut morphogenesis occurs normally in the apoptosis-deficient DfH99 mutant, and the only cell proliferation occurring in the hindgut after stage 10 is in a small domain at the anterior of the small intestine. The morphogenesis of the hindgut after stage 10, in particular its elongation and narrowing, must therefore be driven by changes in cell size, shape, and rearrangement (Iwaki, 2001).

After the cessation of the postblastoderm mitoses, an endoreplication cycle increases the size of the cells of the large intestine (but not small intestine or rectum). Inhibition of this endoreplication by different genetic manipulations results in a shorter large intestine with a smaller cell size, but roughly normal diameter. Endoreplication thus appears to be required to bring the large intestine to its full length, but not to play a critical role in reducing hindgut diameter. There is a change in cell shape, from columnar to cuboidal, as the hindgut elongates; such a change increases epithelial surface area and thus could contribute to hindgut elongation, but not to a reduction in its diameter (Iwaki, 2001).

The threefold elongation of the hindgut is accompanied by a three- to four-fold reduction of circumferential cell number, but not by appreciable cell proliferation or apoptosis. The major process driving this stereotypic elongation and narrowing must therefore be cell rearrangement. Elongation by cell rearrangement is a morphogenetic process of broad significance: it has been shown to drive gastrulation and embryonic axis elongation, and elongation of various tissues, throughout the bilateria. To date, few molecules required for this process have been identified. Elongation by cell rearrangement of the Drosophila germband, ovarian terminal filaments, and stigmatophore requires the Evenskipped homeodomain, Bric a brac BTB, and Grain GATA proteins, respectively, while that of the C. elegans dorsal epidermis requires the DIE-1 zinc finger protein. The genetic pathways in which these presumed transcriptional regulators function have not yet been determined. Only the Xenopus Brachyury transcription factor has been shown to affect cell rearrangement by controlling expression of a specific target, Wnt11, which acts via the planar cell polarity pathway to orient cell intercalation. A fuller understanding of the molecular basis of oriented cell rearrangement clearly depends on the identification of additional required genes and genetic pathways (Iwaki, 2001).

Since the hindguts of their mutant embryos are shorter and wider than normal, drm, bowl, and lin have been identified as possible regulators of the cell rearrangement that drives hindgut elongation. Analysis of hindgut morphology and gene expression patterns in mutants indicates that drm, bowl, and lin function in hindgut development after the primordium has already been established and internalized by gastrulation. No massive apoptosis in the hindgut (as seen in fkh, cad, and byn) is observed in drm, bowl, or lin hindguts. The number of cells in the hindgut epithelium of drm, bowl, or lin mutants is within 20% of wild type, demonstrating that cell proliferation is roughly normal in these mutants. The byn and fkh genes are expressed normally throughout drm, bowl, and lin hindguts, and otp is expressed throughout drm and bowl hindguts. The hindgut visceral mesoderm, on the basis of its expression of Connectin, appears to be established normally in drm, bowl, and lin mutants. Taken together, these results indicate that early events in hindgut development, namely the establishment and maintenance of the primordium (including initiation of gene activity, and cell proliferation throughout the primordium), its internalization during gastrulation, and its investment with visceral mesoderm, all occur more or less normally in drm, bowl, and lin mutants. The shorter overall length, and the two- to three-fold greater circumferential cell number seen in drm, bowl, and lin hindguts, must therefore be a result of a failure to complete the cell rearrangement that elongates and narrows the wild-type hindgut (Iwaki, 2001).

Patterning of the Drosophila hindgut serves as a microcosm of the complex anteroposterior and dorsoventral patterning that takes place during vertebrate gut development. In the Drosophila hindgut, patterning along the anteroposterior axis gives rise to the small intestine, large intestine, and rectum; patterning along the dorsoventral axis gives rise to the large intestine ventral and large intestine dorsal domains, and the boundary cells. Previous studies described gene expression patterns in the different domains of the developing Drosophila hindgut (as well as the requirement of fkh for these expression patterns), but did not identify any genetic activity that distinguished among or specified the different domains (Iwaki, 2001).

This study shows that drm, bowl, and lin are required for the gene expression patterns that distinguish these three domains: lin is required for expression characteristic of large intestine (dpp, dri, and en) and rectum (Ser, hh, and wg); drm and bowl are required for expression characteristic of small intestine (hh and upd). By both morphological criteria (cell shape, presence or absence of boundary cells) and gene expression patterns (expanded expression of genes expressed in the small intestine), lin hindguts appear to consist of a greatly expanded small intestine and to lack the large intestine and rectum. In contrast, both morphological and gene expression characteristics of drm and bowl hindguts indicate that they lack most or all of the small intestine, and consist only of large intestine (which remains unelongated) and rectum. A model consistent with these data is that lin functions in the hindgut to repress small intestine fate and to promote large intestine and rectum fate, while establishment of the small intestine requires the activity of drm and bowl. The requirement for drm (but not bowl) for wg expression at the most anterior of the hindgut could be explained if the domain of bowl function in the small intestine does not extend to the most anterior of the hindgut (consistent with the expression of bowl). Since they have opposite effects on Ser expression, bowl and drm may function in different ways, possibly in different pathways, to promote small intestine fate (Iwaki, 2001).

The function of lin as both an activator and repressor of gene activity in the developing hindgut is consistent with molecular and genetic characterization of its function in other embryonic tissues. In the developing dorsal epidermis, lin is required for transcriptional regulation (both activation and repression) of targets downstream of wg signaling. In the developing posterior spiracles, lin is required for the activation by Abd-B of its transcriptional targets. lin encodes a novel protein that is expressed globally throughout the embryo, including the developing hindgut. When ectopically expressed, Lin protein is detected in nuclei of cells signaled by Wg. The early expression of wg throughout the hindgut primordium, starting at the blastoderm stage and continuing to stage 10, might, analogous to its effect in the dorsal epidermis, activate Lin. This might be required for Lin to carry out its function, demonstrated here, of promoting expression of genes characteristic of large intestine identity (otp, dpp, en, and dri), and repressing expression of genes characteristic of small intestine identity (hh, upd, and Ser) (Iwaki, 2001).

It has been shown by genetic analysis that bowl and drm function to establish the small intestine. bowl encodes a zinc finger protein related to Odd-skipped and is expressed strongly in the hindgut primordium starting at the blastoderm stage and continuing through stage 11. Although the Bowl protein has not been shown to be nuclear or to bind DNA, the fact that it encodes five tandem zinc fingers suggests that it is a transcription factor. Thus, Bowl might function in the hindgut as an activator or coactivator of transcription of genes characteristic of small intestine fate. Finally, drm encodes a zinc finger protein related to Bowl and Odd-skipped and is expressed during stage 10 in the anterior of the developing hindgut, consistent with its required role in establishing the small intestine. drm, like bowl, is required for gene expression characteristic of small intestine fate. The drm protein may, like Bowl, function as a transcriptional regulator in the small intestine primordium (Iwaki, 2001).

Thus drm, bowl, and lin are required for both patterning and cell rearrangement of the hindgut. At least one other putative transcriptional regulator expressed in the hindgut has similar properties: Dichaete encodes a Sox protein required for en, hh, and dpp expression in and elongation of the hindgut. The question therefore arises whether any of the genes expressed in different hindgut domains are mediators of the required role of drm, bowl, lin, or Dichaete in hindgut morphogenesis (Iwaki, 2001).

The phenotypes described for wg, hh, dpp, dri, Ser, and en do not suggest a role for these genes in hindgut elongation by cell rearrangement. Mutations in Ser, dri, and en do not appear to affect overall hindgut morphology. The hindgut in wg mutants is extremely small, suggesting that the critical function of wg in hindgut development is in establishing and maintaining the primordium, but not in elongation. dpp mutant hindguts are shorter, consistent with the role of dpp in endoreplication in the large intestine; nevertheless, dpp hindguts have a roughly normal diameter. In hh mutant embryos, the rectum degenerates and hindgut length is reduced, but the overall morphology, particularly the narrowing of the large intestine, appears normal (Iwaki, 2001).

Only in upd embryos is a defect in both elongation and narrowing of the hindgut observed; significantly, upd is expressed only in the small intestine, the same domain that is largely missing from drm and bowl mutants. Shorter and wider hindguts are seen in younger upd embryos, but the majority of hindguts in mature upd embryos appear normal. Thus, while upd may at least partially mediate drm and bowl function in the hindgut, there must be other targets of these genes that are required for cell rearrangement in the hindgut (Iwaki, 2001).

It is concluded that, if correct patterning of the hindgut is a prerequisite for its elongation by cell rearrangement, either all the targets of drm, bowl, and lin that are the essential components of the necessary patterning have not been identified, or the genes presently identified have overlapping or redundant function. Consistent with the idea that cell rearrangement in the Drosophila hindgut requires its correct patterning, convergent extension during vertebrate gastrulation has been shown to depend on patterning of cell fates along the dorsoventral axis of the embryo. It is, of course, possible that hindgut patterning and cell rearrangement, although closely associated both temporally and in the drm, bowl, and lin mutant phenotypes, do not have a necessary relationship to each other. A number of genes are known that, without affecting patterning, control cell rearrangement by directly affecting morphogenetic movements. This is a property of the Drosophila GATA transcription factor-encoding grain in stigmatophore elongation and of the zebrafish trilobite locus in body axis elongation. Thus, drm, bowl, lin, and Dichaete, in addition to patterning the hindgut, might be regulating other genes that independently control cell rearrangement. Nonetheless, the relationship between patterning of both small intestine and large intestine, on the one hand, and cell rearrangement, on the other hand, is striking. drm and bowl hindguts have a substantial cohort of large intestine cells, yet fail to complete cell rearrangement, presumably due to absence of the small intestine. lin hindguts have an excess of small intestine cells and also fail to complete cell rearrangement, presumably due to absence of the large intestine. The connection between hindgut patterning and cell rearrangement observed in drm, bowl, and lin mutants supports the idea that interaction between two correctly patterned anteroposterior subdomains, the small and large intestine, is a requirement for cell rearrangement in the hindgut tubule (Iwaki, 2001).

Elongation of the Drosophila embryonic hindgut epithelium occurs by a process of oriented cell rearrangement requiring the genes drumstick (drm) and lines (lin). The elongating hindgut becomes subdivided into domains -- small intestine, large intestine and rectum -- each characterized by a specific pattern of gene expression dependent upon normal drm and lin function. drm encodes an 81 amino acid (10 kDa) zinc finger protein that is a member of the Odd-skipped family. drm expression is localized to the developing midgut-hindgut junction and is required to establish the small intestine, while lin is broadly expressed throughout the gut primordium and represses small intestine fate. lin is epistatic to drm, suggesting a model in which localized expression of drm blocks lin activity, thereby allowing small intestine fate to be established. Further supporting this model, ectopic expression of Drm throughout the hindgut produces a lin phenotype. Biochemical and genetic data indicate that the first conserved zinc finger of Drm is essential for its function. A pathway has thus been defined in which a spatially localized zinc finger protein antagonizes a globally expressed protein, thereby leading to specification of a domain (the small intestine) necessary for oriented cell rearrangement (Green, 2002).

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).

Odd-skipped homologs in other arthropods

The number of leg-bearing segments in centipedes varies extensively, between 15 and 191, and yet it is always odd. This suggests that segment generation in centipedes involves a stage with double segment periodicity and that evolutionary variation in segment number reflects the generation of these double segmental units. However, previous studies have revealed no trace of this. The expression of two genes, an odd-skipped related gene (odr1) and a caudal homolog, is described that serve as markers for early steps of segment formation in the geophilomorph centipede, Strigamia maritima. Dynamic expression of odr1 around the proctodaeum resolves into a series of concentric rings, revealing a pattern of double segment periodicity in overtly unsegmented tissue. Initially, the expression of the caudal homolog mirrors this double segment periodicity, but shortly before engrailed expression and overt segmentation, the intercalation of additional stripes generates a repeat with single segment periodicity. These results provide the first clues about the causality of the unique and fascinating "all-odd" pattern of variation in centipede segment numbers and have implications for the evolution of the mechanisms of arthropod segmentation (Chipman, 2004).

These observations invite comparison with the process of segment generation in Drosophila. There, a pattern of double segment periodicity is first generated and then subdivided to yield the final single segment repeat. However, the generation of the 'pair-rule' pattern in Drosophila shows few if any similarities with the early stages of segmentation in Strigamia . Drosophila subdivides the entire body axis into unique domains by activating 'gap genes' under the influence of maternal gradients and then uses the complex promoters of the pair-rule genes to compute a repetitive pattern of gene activity from this underlying aperiodic pattern. The generation of this pattern is almost static with respect to the forming cells of the blastoderm. In Strigamia, the initial patterns of odr1 expression are not static with respect to the underlying cells. It is suggested that the patterns of odr1 gene expression are oscillations of cell state, coordinated as waves that move across the population of cells in the blastodisc, sharpening to encompass fewer cells and stabilizing to double segment periodicity. Thus, despite the fact that odd-skipped is one of the genes expressed in a pair-rule pattern during Drosophila segmentation, it is thought likely that the processes that generate this pair-rule pattern are different in the two species (Chipman, 2004).

Interestingly, odd-skipped family members are downstream targets of the Notch signaling pathway during Drosophila limb segmentation. Recently, it has been shown that the Notch ligand Delta and its target hairy are expressed in a striped pattern during early development and segmentation in the embryo of the spider Cupiennius salei. It has been suggested that Notch signaling in the spider is generating a reiterated pattern through a mechanism analogous to that shown for vertebrate segmentation. These two observations, taken together, suggest the possibility that the odd-skipped family in Strigamia, and possibly in other arthropods, is modulated through a Notch-Delta-mediated oscillator to generate the first serially repeated pattern that begins the segmentation process (Chipman, 2004).

A separate and unresolved issue is whether there are similarities between the process that resolves the pair-rule repeat of Drosophila into a single segment pattern and the process whereby secondary caudal stripes intercalate between primary stripes to generate the single segment repeat in Strigamia. The possibility that such 'frequency doubling' processes may be widespread among the arthropods is supported by the observation of analogous phenomena in chelicerates and short germ insects: in the mite Tetranychus urticae, expression of the paired gene in the prosoma is initially at double segment intervals, with secondary stripes intercalating between them to generate the single segment repeat. In the growing abdomen of the grasshopper Schistocerca americana, paired gene expression also shows a transition from double to single segment periodicity, though in this case the process is one of stripe splitting rather than intercalation. However, in other cases, either no such pattern has been described or the periodicity of gene expression is not yet clear. Of particular relevance in this context is a recent study of segmentation gene expression in the lithobiomorph centipede, Lithobius atkinsoni (only distantly related to Strigamia). The expression of even-skipped in the posterior of the Lithobius germ band shows broad rings around the proctodeum that could reflect dynamic expression, resolving into stripes. However, there is as yet no evidence of subsequent frequency doubling (Chipman, 2004).

These results provide a possible explanation for the observation that, in nature, centipede segment number varies in two-segment increments. It is proposed that variation in segment number among centipedes is caused by variation in the number of cycles of a primary segmentation oscillator, each cycle of which generates two segments. The anteroposterior range of this process may well extend beyond the trunk to include the poison claw and parts of the head and genital regions. Therefore, the occurrence of odd rather than even numbers of leg-bearing segments is not incompatible with this explanation (Chipman, 2004).

While pair-rule patterning has been observed in most insects examined, the orthologs of Drosophila pair-rule genes have shown divergent roles in insect segmentation. In the beetle Tribolium castaneum, while odd-skipped (Tc-odd) was expressed as a series of pair-rule stripes, RNAi-mediated knockdown of Tc-odd (Tc-odd(RNAi)) resulted in severely truncated, almost asegmental phenotypes rather than the classical pair-rule phenotypes observed in germbands and larval cuticles. However, considering that most segments arise later in germband stages of Tribolium development, the roles of Tc-odd in segmentation of growing germbands could not be analyzed properly in the truncated Tc-odd(RNAi) germbands. This study investigated the segmentation function of Tc-odd in germband stages of Tribolium development by analyzing Tc-odd(RNAi) embryos that resumed germband extension. In the larval cuticles of Tc-odd(RNAi) embryos, normal mandibular and maxillary and loss of the labial segments were consistent in the head, whereas a broad range of segmentation defects including loss or fusion of thoracic and/or abdominal segments was observed in the trunk. Interestingly, a group of Tc-odd(RNAi) germbands showed pair-rule-like defects in the segmental stripes of the segment-polarity genes, engrailed, hedgehog, or wingless, in the abdominal regions. While the pair-rule genes even-skipped, runt, odd, and paired were misregulated in the growing Tc-odd(RNAi) germbands, paired expression required for odd-numbered segment formation was largely abolished, which might cause the pair-rule-like defects. Taken together, these findings suggest that Tc-odd can function as a pair-rule gene in the germband stages of Tribolium development (Jeon, 2020).

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).

In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).

The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).

The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).

These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).

The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).

Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).

The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).

It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).

The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).

Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn’s role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).

The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).

These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).

This study provides a genetic model of adult mouthpart development in Tribolium castaneum based on 13 genes. While previous studies have examined patterning in species with derived mouthpart morphologies, T. castaneum retains the pleisomorphic, mandibulate state of insect mouthparts. These results demonstrate the conservation of many gene functions in the maxilla and labium, relative to the legs, thus supporting the interpretation of novel gene functions in groups with derived mouthpart morphology as indicative of their specialized morphogenetic roles in those species. Mandibulate mouthparts such as those of T. castaneum include medial maxillary and labial endites, and the current data are consistent with hypotheses of reiteration in the PD axis and specification by Notch signaling, but rule out a direct role for dac in branch generation or patterning at metamorphosis. Additionally these results demonstrate that a regulator of distal leg development, Krn, which encodes an EGF ligand, is required for normal mandible elongation. This finding underscores the complex relationship between homology at the levels of anatomy and developmental patterning (Angelini, 2012).

Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle, Dermestes maculatus

A set of pair-rule segmentation genes (PRGs) promote the formation of alternate body segments in Drosophila melanogaster While Drosophila embryos are long-germ, with segments specified more-or-less simultaneously, most insects add segments sequentially as the germband elongates. The hide beetle, Dermestes maculatus, represents an intermediate between short- and long-germ development, ideal for comparative study of PRGs. This study shows that eight of nine Drosophila PRG-orthologs are expressed in stripes in Dermestes. Functional results parse these genes into three groups: Dmac-eve, -odd, and -run play roles in both germband elongation and PR-patterning. Dmac-slp and -prd function exclusively as complementary, classic PRGs, supporting functional decoupling of elongation and segment formation. Orthologs of ftz, ftz-f1, h, and opa show more variable function in Dermestes and other species. While extensive cell death generally prefigured Dermestes PRG RNAi cuticle defects, an organized region with high mitotic activity near the margin of the segment addition zone likely contributes to truncation of eve(RNAi) embryos. These results suggest general conservation of clock-like regulation of PR-stripe addition in sequentially-segmenting species while highlighting regulatory re-wiring involving a subset of PRG-orthologs (Xiang, 2017).

A regulatory program for excretory system regeneration in planarians

Planarians can regenerate any missing body part, requiring mechanisms for the production of organ systems in the adult, including their prominent tubule-based filtration excretory system called protonephridia. This study identified a set of genes, Six1/2-2, POU2/3, hunchback, Eya and Sall, that encode transcription regulatory proteins that are required for planarian protonephridia regeneration. During regeneration, planarian stem cells are induced to form a cell population in regeneration blastemas expressing Six1/2-2, POU2/3, Eya, Sall and Osr that is required for excretory system formation. POU2/3 and Six1/2-2 are essential for these precursor cells to form. Eya, Six1/2-2, Sall, Osr and POU2/3-related genes are required for vertebrate kidney development. Planarian and vertebrate excretory cells express homologous proteins involved in reabsorption and waste modification. Furthermore, novel nephridia genes were identified. These results identify a transcriptional program and cellular mechanisms for the regeneration of an excretory organ and suggest that metazoan excretory systems are regulated by genetic programs that share a common evolutionary origin (Scimone, 2011).

C. elegans odd-skipped homologs and gut development

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans has been initiated. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family (Buckley, 2004).

Mammalian odd-skipped homologs

The Drosophila pair-rule gene odd-skipped (odd) and two related genes, sister of odd (sob) and bowel (bowl), encode zinc finger containing proteins, two of which play important roles in embryonic development. The cloning and expression analysis of a mouse gene related to odd, odd-skipped related 1 (Osr1), is reported in this study. During early embryogenesis Osr1 is expressed in the intermediate mesoderm and in a dynamic pattern during limb and branchial arch development (So, 1999).

A new mouse gene, odd-skipped related 2 (Osr2), has been identified that encodes a zinc finger containing protein related to Drosophila Odd-skipped. The putative OSR2 protein shares 65% amino acid sequence identity overall and 98% sequence identity in the zinc finger region, respectively, with the previously reported Osr1 gene product. During mouse embryonic development, Osr2 expression is first detected at E9.25, specifically in the mesonephric vesicles. By E10.0, Osr2 expression is also observed in the rostro-lateral mandibular mesenchyme immediately adjacent to the maxillary processes. In the developing limb buds, Osr2 is expressed in a unique mesenchymal domain and the onset of Osr2 expression follows a distinct dorsal to ventral developmental time sequence beginning in the forelimb and then in the hindlimb. Osr2 exhibits a dynamic expression pattern during craniofacial development, in the mandibular and maxillary processes as well as the developing palate. Osr2 is also expressed at sites of epithelial-mesenchymal interactions during tooth and kidney development (Lan, 2001).

Development of the mammalian secondary palate involves multiple steps of highly regulated morphogenetic processes that are frequently disturbed during human development, resulting in the common birth defect of cleft palate. Neither the molecular processes governing normal palatogenesis nor the causes of cleft palate is well understood. In an expression screen to identify new transcription factors regulating palate development, the odd-skipped related 2 (Osr2) gene, encoding a zinc-finger protein homologous to the Drosophila odd-skipped gene product was isolated; Osr2 mRNA expression is specifically activated in the nascent palatal mesenchyme at the onset of palatal outgrowth. A targeted null mutation in Osr2 impairs palatal shelf growth and causes delay in palatal shelf elevation, resulting in cleft palate. Whereas palatal outgrowth initiates normally in the Osr2 mutant embryos, a significant reduction in palatal mesenchyme proliferation occurs specifically in the medial halves of the downward growing palatal shelves at E13.5, which results in retarded, mediolaterally symmetric palatal shelves before palatal shelf elevation. The developmental timing of palatal growth retardation correlates exactly with the spatiotemporal pattern of Osr1 gene expression during palate development. Furthermore, the Osr2 mutants exhibit altered gene expression patterns, including those of Osr1, Pax9 and Tgfb3, during palate development. These data identify Osr2 as a key intrinsic regulator of palatal growth and patterning (Lan, 2004).

Odd-skipped related 2 (Osr2) gene is mouse homolog of Drosophila Odd-skipped gene. This study examined Osr2 expression regulation. The mouse Osr2 promoter region was cloned and characterized, and found to have two enhancer elements in the -1463/-1031 (distal) and -581/+3 (proximal) regions, and a repressor region (-4845/-1463, far distal). CCAAT/enhancer binding protein (C/EBP) binding sites were found in both the distal and proximal enhancer elements. Osr2 promoter activity was enhanced by C/EBPδ, a member of the C/EBP family, in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified GST-C/EBPδ bound to distal (-1295/-1261) and proximal (-89/-55) C/EBP binding motifs. Chromatin immunoprecipitation demonstrated that acetylated histones H3, H4, and C/EBPδ in the proximal region (-280/-43), but not the distal region (-1438/-1196), indicating that the Osr2 promoter proximal region was transcriptionally activated in C3H10T1/2 cells. These results suggest that Osr2 expression is regulated by C/EBP regulatory elements (Kawai, 2006).

Formation of kidney tissue requires the generation of kidney precursor cells and their subsequent differentiation into nephrons, the functional filtration unit of the kidney. The gene odd-skipped related 1 (Odd1) plays an important role in both these processes. Odd1 is the earliest known marker of the intermediate mesoderm, the precursor to all kidney tissue. It is localized to mesenchymal precursors within the mesonephric and metanephric kidney and is subsequently downregulated upon tubule differentiation. Mice lacking Odd1 do not form metanephric mesenchyme, and do not express several other factors required for metanephric kidney formation, including Eya1, Six2, Pax2, Sall1 and Gdnf. In transient ectopic expression experiments in the chick embryo, Odd1 can promote expression of the mesonephric precursor markers Pax2 and Lim1. Finally, persistent expression of Odd1 in chick mesonephric precursor cells inhibits differentiation of these precursors into kidney tubules. These data indicate that Odd1 plays an important role in establishing kidney precursor cells, and in regulating their differentiation into kidney tubular tissue (James, 2006).

Odd-skipped family of proteins (Odd in Drosophila and Osr in vertebrates) are evolutionarily conserved zinc finger transcription factors. Two Osr genes are present in mammalian genomes, and it was recently reported that Osr1, but not Osr2, is required for murine kidney development. In Xenopus and zebrafish both Osr1 and Osr2 are necessary and sufficient for the development of the pronephros. Osr genes are expressed in early prospective pronephric territories, and morphants for either of the two genes show severely impaired kidney development. Conversely, overexpression of Osr genes promotes formation of ectopic kidney tissue. Molecularly, Osr proteins function as transcriptional repressors during kidney formation. Drosophila Odd induces kidney tissue in Xenopus. This might be accomplished through recruitment of Groucho-like co-repressors. Odd genes may also be required for proper development of the Malpighian tubules, the Drosophila renal organs. These results highlight the evolutionary conserved involvement of Odd-skipped transcription factors in the development of kidneys (Tena, 2007).

odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate

The kidney and vasculature are intimately linked both functionally and during development, when nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms that underlie the differentiation of kidney versus blood/vascular lineages remain unknown. The odd skipped related1 (osr1) gene encodes a zinc-finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a complete, segment-specific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Histology revealed a subsequent absence of kidney tubules, an enlarged cardinal vein and expansion of the posterior venous plexus. Altered kidney versus vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function and blockade of endoderm development by knockdown of sox32/casanova rescued anterior kidney development. The results indicate that osr1 activity is required to limit endoderm differentiation from mesendoderm; in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney towards vascular development (Mudumana, 2008).

Osr1 acts downstream of and interacts synergistically with Six2 to maintain nephron progenitor cells during kidney organogenesis

Mammalian kidney organogenesis involves reciprocal epithelial-mesenchymal interactions that drive iterative cycles of nephron formation. Recent studies have demonstrated that the Six2 transcription factor acts cell autonomously to maintain nephron progenitor cells, whereas canonical Wnt signaling induces nephron differentiation. How Six2 maintains the nephron progenitor cells against Wnt-directed commitment is not well understood, however. This study reports that Six2 is required to maintain expression of Osr1, a homolog of the Drosophila odd-skipped zinc-finger transcription factor, in the undifferentiated cap mesenchyme. Tissue-specific inactivation of Osr1 in the cap mesenchyme causes premature depletion of nephron progenitor cells and severe renal hypoplasia. Osr1 and Six2 act synergistically to prevent premature differentiation of the cap mesenchyme. Furthermore, although both Six2 and Osr1 could form protein interaction complexes with TCF proteins, Osr1, but not Six2, enhances TCF interaction with the Groucho family transcriptional co-repressors. Loss of Osr1 was shown to result in β-catenin/TCF-mediated ectopic activation of Wnt4 enhancer-driven reporter gene expression in the undifferentiated nephron progenitor cells in vivo. Together, these data indicate that Osr1 plays crucial roles in Six2-dependent maintenance of nephron progenitors during mammalian nephrogenesis by stabilizing TCF-Groucho transcriptional repressor complexes to antagonize Wnt-directed nephrogenic differentiation (Xu, 2014).

date revised: 30 August 99

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