rib expression during development was analyzed by in situ hybridization of whole-mount embryos. rib transcript is detected in the developing tracheal system, as the tracheal precursor cells invaginate to form tracheal sacs and primary branches bud and grow out from the sacs. rib is also expressed in a variety of other tissues, in a complex and dynamic pattern that coincides with various morphogenetic movements. For example, rib is first detected at stages 5-8 (~2.5-3.5 hours AEL) in the primordia of the anterior and posterior midgut as the primordia invaginate to form tubes; expression is turned on again in the tubes a couple hours later (stages 10-14, ~5-11 hours AEL) as the tubes extend and form the central portion of the gut. rib expression is also seen in the developing mesoderm at or just after its ectodermal invagination and epithelial-to-mesenchymal transition, as the mesodermal cells begin their dorsal migration. rib is also expressed in the stomodeum and proctodeum, as these tissues invaginate to form the foregut and hindgut, and as the Malpighian tubules bud. rib is expressed broadly in the epidermis from stage 10 to 15 (~5-13 hours AEL) as it migrates during dorsal closure. Some of these sites of rib expression are also associated with morphological defects in rib mutants (Shim, 2001).
Although rib is expressed during many morphogenetic movements, some morphogenetic events are not associated with rib expression. For example, rib is not expressed strongly during cephalic furrow formation or formation of the anterior and posterior transverse folds (Shim, 2001).
In embryos doubly mutant for Wg and Dpp signaling, only visceral branch (VB) cells migrate. Similarly, in embryos doubly mutant for rib and thick veins (tkv), which encodes one of the receptors essential for Dpp signaling, only VB cells migrate, a phenotype identical to that of embryos doubly mutant for tkv and armadillo (arm), which encodes an essential component of Wg signaling. Thus, like Wg signaling, rib plays an instructive role in DT formation (Bradley, 2001).
In spalt (sal) mutants, dorsal trunk (DT) cells migrate dorsally instead of forming the DT, whereas in Wg signaling mutants, DT cells are stalled at the transverse connective (TC). Thus, WG signaling must regulate other genes, in addition to sal, that control migration. Given that rib appears to phenocopy the loss of WG signaling in the DT, and that rib functions downstream of or in parallel to Wg signaling, rib itself might be a target of Wg signaling. rib RNA is expressed throughout the epidermis and is not obviously upregulated in the trachea. Thus it is unlikely that rib is transcriptionally controlled by Wg or other signaling pathways (Bradley, 2001).
rib mutants fail to complete dorsal closure, the process by which the cells of the lateral epidermis move dorsally to encompass the amnioserosa and seal the dorsal surface of the embryo. The Jun N-terminal kinase (Jnk) signaling pathway and the Wg signaling pathway are required for dorsal closure. Both pathways are necessary for the characteristic cell shape changes in the leading edge cells and the transcriptional activation of dpp. rib mutants also lack the characteristic elongation of the cells at the leading edge, and at late stages, these cells are large and misshapen (Blake, 1998; Bradley, 2001).
To determine whether the dorsal closure defects in rib mutants are related to defects in Jnk or Wg signaling, the dorsal cuticle of larvae carrying different allelic combinations of rib mutations was examined. In the allelic combinations that could be scored (i.e., those in which sufficient cuticle was produced), approximately two-thirds of the larvae had a large dorsal hole, and one-third had a small anterior dorsal hole with a puckering of the remaining dorsal cuticle. This range of phenotypes is similar to the defects in larvae with loss-of-function mutations in either Jnk or Wg pathway components. Whether dpp expression is maintained in the leading edge cells of rib mutants was investigated. Unlike mutations in either Jnk or Wg signaling components, in which dpp expression is absent in leading edge cells, dpp expression is observed at high levels in the leading edge cells in rib mutants. At late stages, dpp expression is often observed in lateral patches. This apparent increase of dpp expression in rib mutants could be due to increased numbers of cells expressing dpp, increased size of leading edge cells and/or loss of cell cohesion (which could cause cells to collapse or remain in more ventral positions). In any case, this experiment reveals that, as in the trachea, rib is not an upstream activator of Jnk or Wg signaling and that Jnk- and Wg-dependent activation of dpp is not mediated by rib. Mutations in rib cause defects at an earlier step in dorsal closure than dpp mutations: the leading edge cells fail to change shape in rib mutants, whereas Dpp signaling is required for cell shape changes and movement of the ectodermal cells just ventral to those at the leading edge. Thus, if rib functions downstream of the Jnk or Wg pathway to mediate dorsal closure, it must be acting in parallel to dpp activation (Bradley, 2001).
Patterning in the ventral cuticle is also impaired in rib larvae, which exhibit both a narrowing of the lateral extent of denticle belts and a fusion of belts at the midline. At a gross level, these phenotypes are similar to those described for the Egfr signaling mutants, rho and spitz. The cuticle phenotypes of different allelic combinations of rib mutations were examined, scoring both the lateral extent of denticle belts and denticle diversity. The lateral extent of rib denticle belts is narrowed to 37%, 46% and 70%-100% of the wild-type width, consistent with an allelic series (rib2/rib2<rib2/rib1=rib2/Df<rib1/rib1=rib1/Df). rib1/rib1 and rib1/Df cuticles were often very hard to detect, suggesting that very little cuticle is secreted (Bradley, 2001).
The loss of denticle diversity in rib mutants also corresponds to the above allelic series. The least affected cuticles had the most diversity of denticle types (rib2/rib2), whereas more severely affected cuticles had only one or two denticle types (rib2/rib1 and rib2/Df), and the most severely affected cuticles had very few faint denticles that appeared to be of a single type (rib1/rib1 and rib1/Df). The denticle belts of rib larvae with a single denticle type looked notably similar to larvae simultaneously lacking the late activities of Wg and Egfr signaling, in which all denticles are type 5 (wgts, UAS-DN-DER, arm.Gal4). Unlike Wg/Egfr-deficient larvae, however, not all of the denticles in rib mutants are oriented posteriorly. Overall, the dorsal and ventral cuticle phenotypes, together with the tracheal defects, suggest that rib may function with a combination of signaling pathways. It is clear that rib does not function upstream of these pathways, nor does rib interfere with transcriptional activation of early target genes. Thus, rib functions downstream of or parallel to these pathways to promote cellular changes (Bradley, 2001).
Signaling pathways controlling cell migration in the embryonic salivary gland have not yet been identified. Nonetheless, the salivary gland, like the tracheal system, invaginates through a stereotypical process involving directed cell migration. The salivary glands form from two paired primordia that arise from the ventral ectoderm of parasegment two. Through changes in cell shape and migration, the primordia are internalized and ultimately give rise to two cell types: secretory and duct. The secretory cells are the first to invaginate and proceed in an ordered, sequential manner beginning with the cells in the dorsal posterior region of the primordium. The secretory cells move dorsally into the embryo, then turn and migrate posteriorly until the distal half of the gland reaches the level of the third thoracic segment. After the movements of head involution, the salivary glands lie closer to the anterior end of the embryo and are oriented along the anteroposterior axis. Concomitant with later secretory cell migrations, the duct cells undergo a complex set of morphogenetic movements to create a tubular structure. This tube starts at the larval mouth and then branches to connect to the two secretory glands (Bradley, 2001).
Although salivary glands have been reported to be abnormal in late stage rib mutant embryos, earlier stages were not analyzed. As with the tracheal primordia, the secretory gland primordia in rib mutants are indistinguishable from those in wild-type embryos, and the initial invagination proceeds normally; however, rib secretory cells do not migrate past the point at which wild-type cells turn and migrate posteriorly. Thus, the secretory cells in rib mutants never reach their final destination. At late stages, the lumina of the salivary glands are greatly enlarged, compared to wild-type glands, suggesting that rib may also play a role in maintaining organ shape once the salivary gland has formed. Expressing a rib transgene specifically in the secretory cells of rib mutants restores both directed migration and lumen size. Thus, rib function is required in secretory cells to control migration and organ shape (Bradley, 2001).
The salivary duct also fails to undergo proper morphogenesis in rib mutants. Two duct markers, Trh protein and BTL mRNA, are detected in a normal pattern in the duct primordia of rib mutants. In contrast, duct cells stain poorly for the Dead ringer (Dri) protein, which is normally expressed robustly by stage 13; only diffuse low levels of Dri expression are detected in rib mutants prior to stage 15. In late stage rib mutant embryos, no tubes or rudimentary individual tubes connected to the secretory glands were seen; these semi-tubular structures do not elongate and never elaborate into a normal duct. In embryos expressing a rib transgene in secretory cells of rib mutants, duct formation is restored. This result indicates that rib duct defects are indirect and suggests that duct formation requires proper secretory cell morphogenesis. While both salivary gland structures are abnormally formed in rib mutants, there is a specific requirement for rib in the secretory cells for their posterior migration, similar to the requirement for rib in the tracheal DT cells for their anteroposterior migration (Bradley, 2001).
In the more severe allele rib1, a single nucleotide change in the rib ORF results in a nonsense codon after residue 282. This mutation deletes the entire C-terminal half of the protein, and is likely to be null, consistent with phenotypic analysis. rib2 has a single base change that replaces arginine 58 with a histidine (R58H) in the BTB/POZ domain. A mutation in rib was also discovered on the zipper1 (zip1) chromosome (Blake, 1998). zip1 mutants fail to complement both rib alleles; the rib ORF on the zip1 chromosome was sequenced and the identical nucleotide change was found that created the R58H mutation in rib2. This result is consistent with the phenotypic report that, when recombined off the zip1 chromosome, the ribz1 allele behaves like rib2 and is not as severe as rib1 (Blake, 1998). It is not clear whether the zip1 chromosome recombined with rib2 at some point, or whether finding the identical residue substitution indicates the importance of R58 in RIB function. All other detected base changes that resulted in residue substitutions were detected on all rib chromosomes and/or on the balancer chromosome, suggesting that these other changes are polymorphisms not responsible for rib phenotypes (Bradley, 2001).
Imprecise excision of the Terminal-3 P[w+,lacZ] tracheal marker at cytological position 56A-B generates a mutation (ex12) that severely impairs tracheal branch outgrowth. In wild-type embryos, all primary branches have grown out and the dorsal trunk branches have fused by stage 13, as seen by staining with tracheal antiserum TL1. In ex12 mutants, there is little branch outgrowth, and the trachea remain as a series of elongate, mostly unbranched sacs, similar to bnl null mutants at this stage. The block in branch outgrowth in ex12 mutants is not absolute because after stage 14, although 99% of tracheal branches remain stalled, 1% form and grow out excessively and in aberrant directions. By stage 14, several other morphogenesis defects become apparent, including failure of epidermal dorsal closure and midgut constriction formation (Shim, 2001).
The TL1 tracheal antiserum stains the apical surface and lumen of the developing tracheal epithelium. To further characterize the tracheal migration defects, rib1, rib2, and ribex12 mutants were examined using the tracheal cytoplasmic marker 1-eve-1, a viable P[lacZ] insert in trachealess. Despite the severe defect in movement of the apical tracheal surface, the basal surface continues to extend actively in the mutants (Shim, 2001).
Whether Bnl FGF signaling is affected in rib mutants was examined. In situ hybridization for bnl and btl transcripts showed that both are expressed grossly normally in ribP7 embryos, a null allele of rib. Furthermore, unlike bnlP1 mutants, in which only an occasional tracheal cytoplasmic extension was detected, in rib mutants cytoplasmic extensions form and grow towards their normal targets, implying that the Bnl pathway is active (Shim, 2001).
To examine more directly whether rib– tracheal cells can sense and respond to Bnl, downstream components in the Bnl signaling pathway were examined. Expression of the terminal branch gene blistered/DSRF, which is normally induced by Bnl signaling at the ends of growing primary branches, is not induced to high levels in rib mutants. However, this appears to be a secondary consequence of the migration defect, because when a bnl transgene (UASbnl) is expressed ubiquitously in rib mutants to expose the stalled tracheal cells to high concentrations of Bnl, MAPK is activated and blistered/DSRF expression is induced throughout the tracheal epithelium, as in rib+ animals. It is concluded that rib mutant tracheal cells can respond to Bnl. This suggests that the migration defect results from either an inability to transmit the Bnl signal from the basal surface to the cell bodies and apical surface, or an inability of the cell bodies and apical surface to respond once they receive the signal (Shim, 2001).
The products of two genes, raw and ribbon, are required for the proper morphogenesis of a variety of tissues. Malpighian tubules mutant for raw or rib are wider and shorter than normal tubules, which are only two cells in circumference when they are fully formed. The mutations alter the shape of the tubules beginning early in their formation and block cell rearrangement late in development, which normally lengthens and narrows the tubes. Mutations of both genes affect a number of other tissues as well. Both genes are required for dorsal closure and retraction of the CNS during embryonic development. In addition, rib mutations block head involution, and broaden and shorten other tubular epithelia (salivary glands, tracheae, and hindgut) in much the same manner as they alter the shape of the Malpighian tubules. In tissues in which the shape of cells can be observed readily, rib mutations alter cell shape, which probably causes the change in shape of the organs that are affected. In double mutants raw enhances the phenotypes of all the tissues that are affected by rib but unaffected by raw alone, indicating that raw is also active in these tissues (Jack, 1997).
Mutations in the genes rib and raw cause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by the observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced in rib and raw mutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by either rib or raw mutations. Furthermore, mutations of zipper, the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected by rib and raw, and many of the phenotypes are similar to those of rib and raw. Therefore, the products of rib and raw may be required for proper myosin-driven contraction of the actin cytoskeleton (Blake, 1998).
rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zipper, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton (Blake, 1999).
The fact that zip mutations suppress many of the mutant phenotypes of rib and raw is inconsistent with the hypothesis that either rib or raw is directly required for contraction of the actin cytoskeleton by myosin. Nevertheless, the suppression of the rib and raw phenotypes by zip mutations might be observed if the rib and raw products regulate myosin contraction either by repression or by controlling the direction of contraction. Alternatively, both the effect of the mutations on cell shape and the suppression by zip mutations could be observed if rib and raw contribute to remodeling of the actin cytoskeleton by involvement in the organization of the actin filaments. The counteraction of rib and raw mutant phenotypes by zip mutations could then occur if the normal activities of the rib and raw products on the cytoskeleton oppose to some extent the activity of myosin (Blake, 1999).
The effect of the rib and raw mutations on hairs and denticles of the embryonic cuticle offers support for the hypothesis that the gene products are active in organizing actin. In late embryogenesis, bundles of filamentous actin form epidermal extensions around which cuticular structures are secreted. Some of these cuticular structures are the external apparatus of sensory organs and others are nonsensory projections, the dorsal and lateral cuticular hairs and ventral denticles. The denticles and hairs, both sensory and nonsensory, have various shapes and orientations and are organized in stereotypical, segmentally repeated patterns. The actin cytoskeletal supports of the cell extensions can be observed in stage 16 and 17 embryos by staining with rhodamine labeled phalloidin. Both rib and raw mutations alter the morphology of the F-actin structures, but mutations of each gene have different effects on the structures (Blake, 1999).
In normal embryos actin bundles form a prehair in the cells that secrete sensory hairs, but no prehairs form in rib embryos. rib mutants lack hairs and denticles almost completely, leaving only a few isolated cuticular hairs and denticles. The remaining hairs are either much longer than normal or are abnormally curved. At junctions of three or more cells, rib embryos display intense actin spots, some of which could be sockets of sense organs. In addition, F-actin, which in wild-type epidermal cells accumulates in the cytoplasm and subsequently dissipates, remains in the cytoplasm of rib epidermal cells. The observation of cytoplasmic F-actin accumulation that disappears prior to formation of the actin prehair is consistent with the possibility that actin filaments begin to form in the cytoplasm and are recruited into the prehair structures. The rib product is apparently required for the formation of the larger actin structures from smaller actin filaments that form in the cytoplasm (Blake, 1999).
The cells of raw mutant embryos do form projections, albeit abnormally shaped ones. In raw mutants, hairs are generally disorganized in appearance, may be inappropriately clustered, and are often forked or branched. These are the same types of abnormalities described for embryos mutant for forked (f) and singed (sn), two genes that encode actin bundling proteins. Thus, the raw product could have a role in bundling or otherwise organizing actin filaments. The formation of the F-actin prehair structures might be independent of the activity of myosin. Zygotic expression of zip is not obviously required for formation of actin prehairs and predenticles since both form normally in zip mutants. If myosin does not affect the organization of actin into prehair structures, zip mutations would not be expected to alter the phenotype of rib mutants with respect to the failure of formation of prehairs. However, zip mutation suppresses the rib phenotype, causing a substantial increase in the number of denticles and hairs present on the embryonic cuticle of rib;zip mutants, as compared to rib mutants. Thus, zip counteracts the effect of rib mutations for each of the rib phenotypes. The suppression of the cuticular hair phenotype of raw mutations by zip is less obvious, but the severity of the branching and forking, characteristic of the raw prehair phenotype, is reduced in raw;zip double mutants. The fact that a zip mutation causes actin prehairs and predenticles to form more normally in rib and raw mutants indicates that myosin antagonizes the formation of the actin structures (Blake, 1999).
Although rib and raw have similar effects on the ability of cells to elongate, the differences in the effects of mutations of the two genes on the actin structures that underly cuticular hairs suggest that the two gene products have different functions with respect to the actin cytoskeleton. Distinct functions for rib and raw products are consistent with the observation that raw;rib double mutants are far more defective than embryos mutant for either of the genes individually. In the double mutants many of the affected tissues are greatly reduced in size and the embryos are generally very delicate. The extreme phenotype of the double mutant could be the result of separate defects in the actin cytoskeleton. The evidence presented provides further support for the hypothesis that rib and raw products have functions necessary for cytoskeletal activity, either in a structural or regulatory capacity. The data also indicate that the gene products are not required for myosin to apply force to the actin cytoskeleton. Because the products are essential for formation of the actin models of cuticular hairs and denticles, they could function directly in organizing actin filaments. Defects in reorganization of actin filaments of the cortical cytoskeleton could also explain the abnormal cell shapes associated with rib and raw mutants. However, as is the case with other rib and raw phenotypes, lack of zygotic zip activity suppresses the effect of mutations of the genes on hair and denticle formation in the embryonic cuticle. Therefore the rib and raw products could also act by repressing myosin or controlling its activity in some other way. Analysis of the rib and raw products will likely be necessary to resolve the issue (Blake, 1999).
Blake, K. J., Myette, G. and Jack, J. (1998). The products of ribbon and raw are necessary for proper cell shape and cellular localization of nonmuscle myosin in Drosophila. Dev. Biol. 203(1): 177-88. 9806782
Blake, K. J., Myette, G. and Jack, J. (1999). ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton. Dev. Genes Evol. 209: 555-559.
Bradley, P. L. and Andrew, D. J. (2001). ribbon encodes a novel BTB/POZ protein required for directed cell migration in Drosophila melanogaster. Development 128(15): 3001-15. 11532922
Jack, J. and Myette, G. (1997). The genes raw and ribbon are required for proper shape of tubular epithelial tissues in Drosophila. Genetics 147: 243-253. 9286684
Nusslein-Volhard, C., Wieschaus, E. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster: I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193: 267-282
Shim, K., Blake, K. J., Jack, J. and Krasnow, M. A. (2001). The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis. Development 128(23): 4923-33. 11731471
date revised: 30 January 2002
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