costa/costal2

DEVELOPMENTAL BIOLOGY

Embryonic

Prior to cellularization, Costa is distributed uniformly within the cortical cytoplasm, at apical and basal focal planes. Cos2 is cytoplasmic and is not associated with microtubule spindles. In late syncytial blastoderm embryos just prior to cellularization, Cos2 accumulates between, and apical to, nuclei. Cos2 accumulation forms rays perpendicular to the surface of the embryo and staining is punctate rather than uniform within a honeycomb lattice. Cos2 is associated with furrow canals throughout cellularization. Furrow canals are located at the leading edge of newly forming membrane between adjacent somatic nuclei, and these canals later expand and then fuse with one another in a process that seals off new cells from the embryo's interior. Cos2 is found in the cytoplasm of cellular blastoderm embryos and after the onset to gastrulation (Sisson, 1997).

Transcripts are uniformly distributed in the early embryo, while the protein is present in a striped pattern. Faint stripes along the germband are first observed in late stage 9 [Images] embryos and become prominent by stage 10. Each stripe is continuous along the dorsal-ventral axis in both the ectoderm and the underlying mesoderm. the stripes persist throughout stage 11 and decay before germband retraction. The stripes appear to form just anterior of parasegmental grooves in anterior compartment cells (Sisson, 1997).

Larval

In imaginal discs COS2 mRNA is uniform within wing discs but protein levels are elevated in the anterior compartment. Cos2 protein, detected in a posterior disc extract, has a slower mobility than anterior protein (Sisson, 1997).

Effects of mutation or deletion

costa mutation results in an absence of thoracic denticle belts and an abnormal shape and reduced width in abdominal belts. The denticles are all small and the segment boundaries are duplicated. Pattern duplication is observed in all segments of the body: the proboscis shows additional pseudotracheae, the head shows duplicate aristae, extra sex combs are observed on the forleg, halteres and turgites are duplicated, extra clasper teeth are observed in male genitalia, and extra vaginal teeth are seen in female genitalia. Mutations in Costal-1 and costal2 produce wing duplications in flies simultaneously heterozygous for the mutations Cos-1 and cos2. Costal-1 mutations are dominant; when heterozygous, they cause local pattern duplications of wings and halteres. Penetrance and expressivity of the dominant phenotype is variable but flies can be selected to give high or low penetrance. In order to recover revertants of Cos-1 mutations it is necessary to find conditions in which the penetrance is 100%. This is done by placing Cos-1 mutations in trans with deletions of the cos2 locus. Reversion of Cos-1 leads to viable, fertile, and morphologically normal flies. Presumably a number of revertants are null alleles; thus Cos-1 is a gene that is not required for viability, fertility or normal morphology. The pattern duplications seen in flies heterozygous for dominant Cos-1 alleles are indistinguishable from those seen in flies homozygous for leaky mutant alleles of cos2. It is thought that the dominant phenotype seen in flies heterozygous for Cos-1 is a result of a reduced level of functioning of the cos2 locus. Lowering the number of wild-type copies of cos2+ leads to a more extreme dominant phenotype of Cos-1 heterozygotes, whereas the addition of extra copies decreases the dominant phenotype. cos2 also acts maternally and the stringest mutant phenotype is seen when both the female parent and the zygote are mutant (Grau, 1987).

Mutations in the extracatalytic domain of Fused abolish both the biological function of the protein and its association with Cos2 (Robbins, 1997).

Su(fu) enhances a cos2 phenotype and cos2 mutations interact with fu in a way similar to Su(fu). A close relationship might exist between fu, Su(fu) and cos2 throughout development. The Fu+ kinase might be a posterior inhibitor of Costal2+ while Su(fu)+acts as an activator of Costal2+. The expression pattern of wingless and engrailed in fu and fused-Su(fu) mutant embryos supports this interpretation (Preat, 1993).

Cubitus interruptus (Ci), a Drosophila transcription factor, mediates Hedgehog (Hh) signaling during the patterning of embryonic epidermis and larval imaginal discs. In the absence of Hh signal, Ci is cleaved to generate a truncated nuclear form capable of transcriptional repression. Hh signaling stabilizes and activates the full-length Ci protein leading to strong activation of downstream target genes, including patched and decapentaplegic. A number of molecules have been implicated in the regulation of Ci. Mutations in these molecules effect changes in Ci protein level, and also influence the extent of Ci proteolysis and the expression of Ci target genes. This paper examines the regulation of Ci subcellular localization and activity. A bipartite nuclear localization signal (NLS) within Ci has been characterized. It is proposed that the subcellular distribution of Ci is affected by two opposing forces, the action of the NLS and that of at least two regions targeting Ci to the cytoplasm. The data also show that loss of PKA or Costal-2 activity does not fully mimic Hh signaling, demonstrating that Ci proteolysis and Ci activation are two distinct events which are regulated through different paths. It is proposed that there are three levels of apparent Ci activity, corresponding to three zones along the AP axis with different sets of gene expression and different levels of Hh signaling (Wang, 1999).

Previous studies have demonstrated that protein kinase A plays an important role in Ci proteolysis. In PKA loss-of-function clones, Ci protein level is greatly elevated. An extract from discs carrying large numbers of such clones exhibits reduces proteolysis. Furthermore, Ci mutated at putative PKA sites is resistant to cleavage. The resolution of Ci into phosphorylation isoforms enables a direct test of the action of PKA upon Ci. When cells of the cultured line cl-8 are treated for an hour with H-89, a potent PKA inhibitor, a slight increase in the amount of full-length Ci is observed, accompanied by a slight decrease of Ci-75. This suggests that the basal activity of PKA is low in cl-8 cells. When cl-8 cells were treated with the PKA stimulator forskolin, within an hour a decrease of Ci-155 is observed, but there is no marked change in the IEF pattern of Ci. However, there is a dramatic shift in the western pattern when cells are simultaneously treated with forskolin and MG132. The amount of the unphosphorylated isoform remained constant, while the highly phosphorylated isoforms accumulate (Wang, 1999).

Cells in the anterior compartment of wing pouch express different sets of Ci target genes depending on their distance from the AP boundary. Based on the expression profile of Ci and its target genes, cells along the AP axis can be divided into three zones. The Anterior Zone consists of cells more than 8-9 cell diameters away from the boundary. Cells in this zone express low level Ci, low level Ptc and no Dpp. The Intermediate Zone, marked by expression of high level Ci, low level Ptc and high level Dpp, corresponds to cells between 8-9 and 2-3 cell diameters away from the boundary. Cells immediately adjacent to the boundary (within 2-3 cell diameters) fall into the Boundary Zone, marked by medium level Ci, high level Ptc, and medium level Dpp. The Ci regulated enhancer element/reporter 4bslacZ is also expressed in this zone. Expression of ci target genes is a consequence of the overall activity of many individual Ci peptides, which is determined by both the potency of each peptide and the number of peptides present. In the following discussion, the overall Ci activity observed for a cell, judged by the expression of target genes, is termed the 'apparent activity' of Ci. The specific activity, or potency, of each peptide is defined as its 'activation state'. In a wild-type wing disc, there are three levels of apparent Ci activity corresponding to the three zones. In the Boundary Zone, Ci has the highest apparent activity, activating both ptc and 4bslacZ to high levels. dpp expression in this region is likely subjected to a partial repression by anterior En. In the Intermediate Zone, Ci exhibits an intermediate level of apparent activity, inducing strong expression of dpp but not high level ptc nor 4bslacZ. In the Anterior Zone, Ci has the lowest apparent activity (Wang, 1999).

Although there are three levels of apparent Ci activity, evidence has been found for only two activation states. 4bslacZ, whose sensitivity allows the monitoring Hh-induced Ci activation, is expressed only in the Boundary Zone, suggesting that Ci is activated only in this zone. Ci in the activated state, therefore, is given the name Ci^boundary. The high level of Ci in the Intermediate Zone indicates that cells in this zone must receive some level of Hh signaling, which inhibits Ci proteolysis. Despite the high protein level, Ci in the Intermediate Zone is not sufficiently activated to induce 4bslacZ expression, and is given the name Ci^default. The lack of proteolysis in the Intermediate Zone allows Ci^default to accumulate, resulting in high level expression of dpp. In the Anterior Zone, the majority of Ci is proteolytically cleaved into Ci-75. Although the Anterior Zone and the Intermediate Zone differ in Ci protein levels and the expression patterns of target genes, at present there is no evidence that they differ in the state of Ci activation. In fact, when Ci stabilization is mimicked in the Anterior Zone by making PKA loss-of-function clones, the expression of high level dpp is also mimicked. This observation is consistent with the idea that the two zones share the same activation state of Ci (Ci^default) but differ in the levels of full-length Ci. In summary, the three levels of apparent Ci activity correspond to Ci boundary, high level Ci^default, and low level Ci^default, respectively (Wang, 1999).

The boundary between the Anterior Zone and the Intermediate Zone corresponds to a division between cells with low Ci levels and those with high Ci levels, and is likely to coincide with the anterior border of Hh signaling. Between this line and the AP boundary, cells in both the Intermediate Zone and the Boundary Zone receive some level of Hh signaling and show stabilization of Ci. (The relatively lower Ci level in the Boundary Zone probably reflects partial transcriptional repression by anterior En. However, the activation from Ci^default to Ci^bounday happens only in the Boundary Zone, suggesting that it takes place when the level of Hh signaling is above a certain threshold. The level of Hh signaling changes across the anterior compartment. In the Boundary Zone, cells express high level Ptc, which both transduces and sequesters Hh signaling. The presence of high level Ptc creates a steep decline of the Hh signal. Consequently, cells receiving Hh are divided into those receiving high level Hh signal (the Boundary Zone) and those receiving lower level Hh signal (the Intermediate Zone). Its role in regulating Hh distribution makes Ptc essential for proper regulation of the apparent activity of Ci (Wang, 1999).

Study of Hh-induced Ci activation has been complicated by the fact that in almost all the assays, a high level of Ci protein seems to suffice for the activation of Ci target genes. This is well illustrated in the case of loss-of-function PKA clones, in which elevated Ci protein levels are associated with strong activation of Hh target genes such as ptc and dpp. It has been difficult to tell whether the activation of downstream genes is due to elevated Ci alone, or if Ci is activated in addition to being stabilized in these clones. Progress was made in a recently published study through a combination of manipulating the expression of an uncleavable deletion construct of Ci and examining smo loss-of-function clones in the posterior compartment (Methot, 1999). Importantly, it demonstrates that inhibition of Ci proteolysis is not sufficient to activate Ci. However, the assays as described do not address the question of whether in vivo stabilization and activation of Ci are regulated simultaneously as two consequences of the same process, or regulated separately through different mechanisms. The sensitivity of 4bslacZ allows this question to be addressed. While a modest level of Ci in cells receiving high level Hh signal can activate 4bslacZ and clones lacking ptc activate 4bslacZ, high level Ci in PKA or cos2 loss-of-function clones cannot. It is concluded that Ci is stabilized but not activated in PKA or cos2 mutant clones. In other words, these mutations mimic one aspect of Hh signaling but not the other, and Ci in the clones exists as Ci default instead of Ci boundary (Wang, 1999).

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. This study analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. These results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh (Ho, 2005).

How Hh differentially regulates target genes is central to understanding how one signal generates multiple downstream effects and activates different target genes at different concentrations. Using mutant forms of Cos2, this study has investigated how components of the Hh signal transduction pathway form a sensitive switch that governs the difference between dpp-expressing cells and cells expressing both ptc and dpp. To assess the importance of the putative motor, neck and cargo domains to Cos2 in Hh signaling, deletion constructs of Cos2 were made lacking each domain. In addition, the Ser182 of Cos2 was changed to Asn (S182N) in the P-loop, which in other kinesins gives rise to a dominant-negative form that lacks ATPase activity. Using these mutant forms of Cos2, the roles of Cos2 and Su(fu) were investigated in the regulation of the Hh target genes (Ho, 2005).

Cos2 is required for the activation of the target gene en, and S182N expression or cos2-overexpression can block this activation, despite the presence of high levels of Hh. Cos2 has been proposed to act not only as a scaffold for Hh signaling components, but as a sensor of the Hh signal, playing a dual role as both an activator and a repressor of the pathway. Mutation of the P-loop of Cos2, which is designed to disrupt the ATPase activity of the protein, profoundly affects the activity of the protein, and through that the outcome of the pathway, in agreement with a role for Cos2 as a sensor for Hh signal (Ho, 2005).

Conventional kinesins require ATPase activity in order to move along microtubules. Studies have shown that mutation of the conserved Ser or Thr at a precise position in the P-loop causes the protein to become immobile, locking itself and its cargo along microtubules prematurely, before the final intracellular destination for the kinesin has been reached. Expression of such kinesin mutants specifically inhibits the movement of its endogenous partner, but not the movements of other kinesins or dyneins along the microtubule This knowledge about kinesins and the importance of their P-loops to design the equivalent mutation in Cos2. The mutation of amino acid 182 of Cos2 to a conserved Thr does not detectably alter the function of Cos2 in vivo, while mutation of the same residue to Asn clearly interferes with normal Cos2 activity. This clearly suggests that Cos2 is likely to use ATPase activity for either locomotion or conformational changes in response to Hh signaling. The movement of Cos2 along microtubules in vitro has yet to be demonstrated, but the importance of intracellular localization of various Hh signaling components has been clearly demonstrated. Among the examples: in response to Hh, Smo accumulates at the plasma membrane, and associates with Cos2 and Fu; Ci accumulates in the nucleus in response to Hh signaling; and in the absence of Hh signal, Smo is located in internal membranes in the cytoplasm of responding cells, and Ci is continually exported from the nucleus, phosphorylated by kinases, and processed into CiR by the proteasome. How do the components arrive at the appropriate places to affect the appropriate response? As a binding partner for all of these components and as a kinesin-related protein, Cos2 is in a unique position to orchestrate some of these events. Ideas for how it may accomplish are given below (Ho, 2005).

The data suggest that the activities of Cos2 and Su(fu) are independently regulated by different concentrations of Hh along the gradient that forms from posterior to anterior. In the anterior cells distant from the AP boundary, little or no Hh is received and target genes are silent. In these cells, Cos2 is required for proteolytic processing of Ci into its repressor form and possibly for the delivery of Ci^FL for lysosomal degradation. The data suggest that Cos2 requires an intact P-loop for its role in these events. Cos2 ATPase activity may be inhibited in cells receiving very low levels of Hh, preventing Ci proteolysis and stabilizing Ci^FL. The stabilization of Ci^FL results in the activation of dpp. Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes phosphorylated, inactivating its negative regulatory hold on Ci, while inhibition of the ATPase activity of Cos2 continues to allow stabilization of Ci. In this situation, ptc and dpp are transcribed. Finally, at the highest levels of Hh signaling adjacent to the AP border, Cos2 is required for activation of the pathway and the expression of en. S182N expression, or cos2 over-expression, inhibits the induction of en by endogenous Hh in these cells. The elements of this model are addressed below (Ho, 2005).

Ci plays a central role in determining which genes are repressed or activated in response to different concentrations of Hh. In order to activate target genes such as dpp or ptc, Ci must be stabilized in its full-length form. In wild-type discs, Hh stabilizes Ci by antagonizing molecular events that reduce the concentration of nuclear Ci^FL. In addition to the constitutive nuclear export of Ci, there are two ways Ci^FL concentration is reduced: full-length Ci is proteolytically processed into a repressor form; and Ci^FL is degraded by a lysosome-mediated process involving a novel protein called Debra. In these experiments, the stabilization of Ci^FL was accomplished by expressing S182N in responsive cells, which antagonizes Cos2 repressor activity and results in the accumulation of high levels of Ci^FL, with minimal effects on the levels of Ci^R. This same type of differential effect on Ci^R and Ci^FL is accomplished by Debra, which causes the lysosomal degradation of Ci^FL without affecting the production of Ci^R. Cos2 and Debra may act in concert to destabilize Ci^FL, while Cos2 may also aid in the production of Ci^R via a Debra-independent mechanism. This would involve presenting Ci to the kinases, PKA, CKI and GSKß (Shaggy) for phosphorylation and processing by the proteasome. Since Debra regulates Ci stability in limited areas of the wing disc but S182N can stabilize Ci throughout the anterior compartment, it is likely that S182N interferes with both Debra-dependent and Debra-independent mechanisms of Ci stability to achieve the observed effect: cell-autonomous stabilization of Ci^FL leading to derepression of dpp (Ho, 2005).

These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of Ci^FL in the nucleus and the activation of dpp (Ho, 2005).

The human ortholog of Suppressor of fused is a tumor suppressor gene. Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli proteins, through specific protein-protein interactions. Through these interactions, Su(fu) controls the nuclear shuttling of Ci and Gli, as well as the protein stability of Ci^FL and Ci^R. Flies homozygous for Su(fu) loss-of-function mutations are normal, so the importance of Su(fu) becomes evident only when other gene functions are thrown out of balance, as in a fu mutant background, with extra or diminished Hh signaling caused by ptc, slimb and protein kinase A mutations or when altered Cos2 is produced (Ho, 2005).

To activate ptc transcription in the wing disc, two conditions have to be met simultaneously: Ci^FL must be stabilized, and the activity of Su(fu) must be reduced. Removal of Su(fu) changes S182N from a ptc repressor into a ptc activator. Removal of Su(fu) may result in the modification, activation or relocalization of Ci^FL, or in further sensitizing the system to stabilized Ci^FL. In Su(fu) homozygous animals, the quantity of Ci^FL and Ci^R proteins is greatly diminished, and Su(fu) mutant cells are more sensitized to the Hh signal. The lower levels of both Ci^FL and Ci^R in mutant Su(fu) cells may contribute to the sensitivity of these cells to Hh, since a small Hh-driven change in the absolute concentration of either form of Ci would result in a significant change in the ratio between the two proteins. Both Ci^FL and Ci^R bind the same enhancer sites, so their relative ratio is likely to be important in determining target gene expression. S182N expression tips the ratio of Ci^FL to Ci^R toward Ci^FL, and reducing the absolute quantities of both Ci isoforms by removing Su(fu) will enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the cytoplasm in a stoichiometric manner Reducing the amount of Su(fu) should release more Ci^FL to the nucleus to activate ptc (Ho, 2005).

The activity of Su(fu) must be regulated or overcome so that target genes can be activated at the right times and places in response to Hh. The regulation of Su(fu) activity may occur by Hh-dependent phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs where GAL4 was used to drive extra Hh expression. At high concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by overexpression of cos2 or either of the cos2 mutants, suggesting that phosphorylation of Su(fu) occurs independently of Cos2 function. One kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused, a well-established component of Hh signal transduction. It is not known whether the phosphorylation of Su(fu) by Fu is direct or indirect (Ho, 2005).

The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of Ci^FL in the nucleus and the activation of Hh target genes such as ptc (Ho, 2005).

Anterior en expression was used as an in vivo reporter of high levels of Hh signaling. cos2 mutant cells at the AP boundary fail to activate en, suggesting that Cos2 plays a positive regulatory role in en regulation. S182N, S182T and Cos2 overexpression mimics the cos2 loss-of-function condition with respect to en: en remains off in these cells. One interpretation of these data is that all the Cos2 proteins are able to associate with another pathway component, such as Smo, and overproduction of any of them inactivates some of the Smo in non-productive complexes not capable of activating en (Ho, 2005).

In contrast to the activity of all the other mutations generated, deletion of the C terminal domain creates a protein (Cos2DeltaC) that represses normal dpp, ptc and en expression in the wing disc. In this in vivo assay, Cos2DeltaC acts just like wild-type Cos2. A similar deletion has been shown to retain function in cell culture assays. This mutant, expressed under the control of its endogenous promoter, rescues the lethality and wing duplication phenotypes of a cos2 loss-of-function allele over a cos2 deficiency. The results of the rescue experiment bring up a new possibility: that the C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of Cos2, is not necessary for repressor activities of Cos2. Alternatively, Cos2DeltaC could complement or boost the activity of the hypomorphic allele cos2¹¹, which was used for the rescue experiment (Ho, 2005).

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. Precocious non-cell autonomous differentiation was observed in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth (Christiansen, 2012).

Hh signaling is known to regulate proliferation. Consistently, this study shows that deregulated, ligand-independent Hh signaling due to loss of the negative regulators cos2 and ptc causes overgrowth phenotypes of mosaic eyes and heads. Paradoxically, however, cos2 and ptc mutant cells have a growth-disadvantage and are eventually eliminated by apoptosis. In mosaic discs, proliferation is increased at the border to adjacent cos2⁺ tissue suggesting that the overgrowth is mediated through induction of non-cell autonomous proliferation. This effect is position-dependent and restricted to cos2 clones in or anterior to the MF. Finally, it was demonstrated that cos2 clones not only cause non-cell autonomous precocious proliferation, but also non-cell autonomous differentiation (Christiansen, 2012).

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date revised: 5 August 2016 

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