Kinesin associated protein 3
DEVELOPMENTAL BIOLOGY

The Drosophila homolog of the non-motor accessory subunit of kinesin-II motor complex has been identified. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future (Sarpal, 2002).

Zygotic DmKAP expression begins at stage 6 when a high level of staining is observed in the cells around the morphogenetic furrows and in the neuro-ectoderm cells at this stage. The staining gradually condenses in a subset of cells in the CNS and these cells appear in every segment in the presumptive ventral ganglion (VNG) region of the developing brain. This staining persists until the end of embryogenesis and here a strong staining is observed in the brain lobes and the ventral ganglion. The DmKAP staining also appears in a subset of sensory neurons in the peripheral nervous system (PNS) at stage 13, and continues till late stage 17 when the embryo is completely developed. At this stage expression was found in (1) the lateral chordotonal organs, (2) Bolwig's organ, (3) and other anterior sense organs. In addition, there is staining in certain sensory neurons of the ventral and dorsal clusters of the chordotonal organ. The pattern of sensory neurons stained by DmKAP probe is identical to that of KLP68D and KLP64D (Sarpal, 2002).

The DmKAP expression in the CNS is found to continue to the first instar larva stage when a strong staining is observed in most of the ventral ganglion and brain lobe cells. In the third instar larva stage the staining is restricted to a few cells in the presumptive optic lobe region while a weak staining is visible in the ventral cortical region. DmKAP positive cells in the optic lobe are present at the insertion site of the optic stalk. This region is known to have cell bodies of cholinergic interneurons. A similar pattern of staining has been seen with a KLP64D specific probe. These data suggests that DmKAP could act in association with KLP64D and KLP68D in the neurons of CNS and PNS (Sarpal, 2002).

This hypothesis is based on co-localization of the respective gene expression in different neurons. Some direct experimental evidence would be necessary to establish this hypothesis. In addition to the nervous system cells, the epithelial cells of the imaginal disc peripodium are also stained at the third instar larva stage. This indicates that DmKAP may play a role in imaginal disc development as well. DmKAP in the disc epithelial cells may function in association with other kinesin-II components or it may function independent of KLP64D and KLP68D. This can be resolved by studying the corresponding expression pattern of these two genes in the imaginal disc and other non-neural tissue (Sarpal, 2002).

Since KLP64D and KLP68D have dissimilar levels of expression in the ovary, the in situ hybridization pattern in this tissue was analyzed using both the DmKAP cRNA and KLP64D cDNA probes, respectively. In addition, KLP68D localization was examined in this tissue using a purified antibody. RNA in situ hybridization data has revealed that DmKAP is expressed in follicle cells and in nurse cells at all stages of oogenesis. There is, however, no staining in other somatic tissue like the stalk cells that links individual vitellarium, and cells of the terminal filament of an ovariole. DmKAP mRNA is also present in stem cells and in all developing cyst cells inside germarium. The staining in nurse cells and in follicle cells persists throughout development. However, the level of expression in nurse cells is elevated from stage 10b. This is likely to contribute to the maternal component of the oocyte. DmKAP gene expression is also observed in all follicle cells that cover the egg chambers at this stage. This data suggests that DmKAP may play an important role during oogenesis. In contrast, the KLP64D cDNA probe revealed no staining during the early developing stages of oogenesis. Only the nurse cells of stage 10b egg chamber stained for KLP64D mRNA and this is likely to contribute as maternal component to oocyte. The KLP64D cDNA probe stains in an exactly identical pattern in the embryos. Furthermore, both the DmKAP cRNA and KLP64D cDNA probes stained in an identical pattern in the embryonic and larval nervous system. This suggests the DmKAP and KLP64D staining patterns in the ovary are a true reflection of tissue specific expressions of the respective genes. It is therefore concluded that the KLP64D does not express itself during early stages of oogenesis (Sarpal, 2002).

KLP68D antigen is present in all the germ line cells in the ovary in a pattern very similar to that of DmKAP mRNA. This suggests that KLP68D and DmKAP genes are simultaneously expressed in a subset of neurons and in all germline cells. Further, genetic analysis using mutants in the KLP68D and DmKAP gene would reveal their functions in the respective tissues (Sarpal, 2002).

Effects of Mutation or Deletion

To obtain mutations in the DmKap gene, the genomic transgenes P(213w+) and P(219w+) were used in a chromosome walking strategy. This yielded two PlacW insertion alleles, DmKapKP1 and DmKapKP2, and two EMS-induced alleles, DmKapV5 and DmKapV6. Although the DmKap homozygous flies die at or before the pupal stage, careful culture conditions allowed several homozygous/hemizygous escapers to be obtained as pharate adults. Some of these mutant pupae, except for DmKapV5, even emerged as uncoordinated adults. DmKapV5 adults never emerge by themselves, but if rescued from the pupal case, they survive for 2-4 days on moist filter paper. The uncoordinated behavior of these mutants is similar to that described for the Klp64D alleles (Ray, 1999) and is reminiscent of nomp mutations (Kernan, 1994). The mutant flies cannot stand or right themselves when turned on their back, and their legs get entangled in an attempt to walk. Such behavioral defects were completely rescued by both genomic transgenes, indicating that the observed phenotype arose from mutations in the DmKap gene (Sarpal, 2003).

To determine the cellular basis for this lethality and uncoordinated behavior, a UAS-Kap cDNA transgene was constructed. This was expressed in specific tissues with different Gal4 drivers in DmKapV6 males and DmKapV6/DmKapV5 females to test for the rescue of the recessive lethality. The Gal4C155 DmKapV6/Y; UAS-Kap/+ males and Gal4C155 DmKapV6/DmKapV5; UAS-Kap/+ females as well as the DmKapV6/Y; Gal4SG18.1/+; UAS-Kap/+ males were perfectly motile and fertile, whereas the DmKapV6/Y; UAS-Kap males (with no driver) were uncoordinated. Gal4C155 (elav-Gal4) expresses in all neurons during development and in the adult, and Gal4SG18.1 expresses in a majority of sensory neurons plus a subset of neurons in the central nervous system in larvae and adults. Therefore, these rescue data clearly indicate that DmKAP activity is mainly required in a subset of neurons, including the ciliated sensory neurons, and they are consistent with earlier in situ hybridization results (Sarpal, 2002; Sarpal, 2004).

RNA in situ analysis has suggested that the DmKap gene expresses in the embryonic chordotonal organ neurons at a higher level in comparison to other sensory neurons (Sarpal, 2002). These neurons contain long sensory cilia and are involved in proprioception in the larva. In adult flies, similar ciliated neurons innervate the JO in the second antennal segment (Eberl, 2000). Since Kinesin II motor activity is implicated in ciliogenesis in other organisms (Marszalek, 2000a), the cilia structures of these neurons in DmKap alleles was investigated. The sensory cilia are absent in the mutant animals and specific expression of the UAS-Kap transgene in these neurons with Gal4JO15 rescues the phenotype. This suggested that DmKAP activity in the JO neurons is required for ciliogenesis in a cell autonomous manner (Sarpal, 2003).

The JO neurons can detect acoustically induced antennal vibrations and respond to the male courtship song (Eberl, 2000). It was therefore reasoned that the measurement of sound-evoked potentials from the JO neurons in wild-type and mutant animals could provide a quantitative physiological assay for the analysis of DmKap function in these sensory cilia. The sound-evoked potential was completely eliminated in DmKapV6 and in DmKapV6/Df(1)RA37 (breakpoints: 10A7-10B17) hemizygous adults, and it was rescued by the presence of genomic transgenes and the chromosomal duplication Dp(1;Y)v+y+. In addition, neuron-specific expression of UAS-Kap with Gal4C155 rescues the auditory defects in DmKapV6 males. It was further observed that DmKapV5 and DmKapKP2 hemizygous or homozygous adults are also deaf and all failed to complement DmKapV6. These experiments mapped the auditory defect to the DmKap gene. However, it is still formally possible that the auditory defects observed in homozygous DmKap alleles result from a general physiological defect in all neurons. To rule out this possibility, the electroretinogram responses from DmKapV6, DmKapV5, and DmKapKP2 hemizygous adults was measured; all these mutants responded like the wild-type control. Unlike vertebrate photoreceptor cells, the photoreceptor neurons of Drosophila have no connecting cilium, and therefore this result further indicates that the recessive auditory response defects of DmKap mutants are caused by loss of sensory cilia in the JO neurons (Sarpal, 2003).

JO is a large chordotonal organ containing nearly 200 scolopidial units. Each scolopidium is innervated by two or three bipolar sensory neurons. Each sensory neuron grows a single slender dendritic cilium. The cilia are encapsulated in the extracellular scolopale space formed by the scolopale cell. The apical ends of the cilia are attached to the extracellular dendritic cap formed by the scolopale cell. At about three-quarters the length of the cilium from basal bodies, the cilium contains an electron-dense matrix called the ciliary dilation. The cilium is supported by an axoneme of nine microtubule doublets that assemble from electron-dense basal bodies, which contains the protein rootletin (Yang, 2002). A cross-section from the middle of the scolopale reveals two cilia in each JO scolopidium. Electron microscopic observations of ultrathin sections of JO from DmKap homozygous mutant animals revealed deformities in the ciliary substructure. Almost all sections through the dendritic cap were devoid of cilia, and sections at the midscolopale level showed lack of axonemal profiles; however, in more proximal sections, the basal body structures and roots were visible in many scolopales. Very rarely scolopales were found with thin, deformed membranous cilia extending from a distal basal body. Together, all these observations establish that DmKAP is required for axoneme formation in the cilia (Sarpal, 2003).

KLP64D and KLP68D have been identified as the two motor subunits of Kinesin II in Drosophila (Ray, 1999), and DmKap coexpresses with Klp64D in the ciliated sensory neurons (Sarpal, 2002). If DmKAP functions as part of the Kinesin II motor in the sensory cilia of JO neurons, then loss of KLP64D function should also affect the auditory response from JO. To test this hypothesis, the sound-evoked potentials were recorded from several viable combinations of Klp64D mutants. The Klp64Dk5 homozygous mutant animals, and those with heteroallelic combinations Klp64Dk1/k5 and Klp64Dl4/k5, survive as uncoordinated adults (Ray, 1999). Recordings from all three genotypes produced drastically reduced responses to the standard pulse stimulus, while the heterozygous control flies responded normally. To confirm that the Klp64D mutations are responsible for the reduced response, mutant flies rescued by the UAS-Klp64D and UAS-Kif3A (mouse homologue of KLP64D) transgenes were simultaneously tested. Even in the absence of a Gal4 driver, these two transgenes can partially rescue the response. The two transgenes rescue the lethality and the behavioral defects caused by mutations in the Klp64D gene (Ray, 1999). The amplitudes of sound-evoked potentials from the UAS-Klp64D; Klp64Dk5/k5 and UAS-Kif3A; Klp64Dk5/k5 flies were enhanced by introducing any of three different Gal4 drivers in the background. The Gal4MJ94 line expresses in chordotonal organs and a subset of other Type I sense organs, as well as in some CNS neurons. The Gal4C817 expresses in chordotonal organs and in a subset of CNS neurons, while the Gal4JO15 line mainly expresses in a subset of chordotonal organ neurons in JO. The homozygous Klp64Dk5/k5 produced an average maximum potential of 370 μV, which is increased to 1513 μV and 1198 μV, respectively, by introducing UAS-Klp64D and UAS-Kif3A transgenes. The response index was further significantly enhanced to 2277 μV and 1992 μV, respectively, in the presence of Gal4JO15. This suggests that Klp64D gene activity is required in the JO neurons to maintain the sound reception ability (Sarpal, 2003).

The Klp64D mutant alleles significantly reduce the sound-evoked potentials but still have some residual response to sound stimulus, probably because only alleles that allow some animals to survive to adulthood can be tested. To study whether the auditory defect has a correlation to ciliary structure, the JO scolopidia were analyzed from Klp64D mutant combinations. Often the scolopidia from Klp64Dk5/k5 adults contain complete sets of cilia. The basal body structures, ciliary roots, and desmosomal junctions between inner-dendritic segments appeared normal. However, the ciliary dilations were deformed and disorganized and were often located in the same plane as the dendritic caps. This might happen if the distal-most axoneme extension is compromised. The sensory cilia were absent in most of the JO scolopidia from Klp64Dk1/A8.n123 hemizygous adults, but the inner-dendritic segment and desmosomal junctions appeared normal. Together, these observations show that severe mutations in both DmKap and Klp64D would cause identical defects in JO neurons, while weaker hypomorphic mutations in the Klp64D locus, e.g., Klp64Dk5, would cause moderate levels of ciliary and axonemal damage. Interestingly, the levels of ciliary and axonemal defects in different Klp64D and DmKap alleles were directly correlated to the reduction of the auditory response. Since both DmKap and Klp64D functions are cell autonomous and map to the JO neurons, these gene products are involved in ciliogenesis in the JO neurons (Sarpal, 2003).

To further test the functional interaction between the two Kinesin II subunits DmKAP and KLP64D during ciliogenesis, a dominant genetic interaction paradigm was used in which sound-evoked responses were recorded from flies carrying one copy of a DmKap mutation and a viable combination of Klp64D mutations. Reduction to a single copy of DmKapV6 significantly enhances the recessive lethality of different Klp64D combinations. This made it difficult to obtain viable adults carrying one copy of a DmKap mutant allele and two Klp64D alleles. Finally, a combination of DmKapV6/+; Klp64Dl4/Klp64Dk5 females was obtained, that survived to the adult stage with a reduced viability compared to that of Klp64Dl4/Klp64Dk5 females. The Klp64Dl4/Klp64Dk5 adults respond to a sound stimulus with intermediate efficiency; the average amplitude of the sound-evoked potential was 426 μV. In contrast, the sound-evoked response was completely absent in DmKapV6/+; Klp64Dl4/Klp64Dk5 females. This suggests that mutation in the DmKap locus is haplo-insufficient to compromised Klp64D, and therefore these two gene products are likely to interact with each other in JO neurons (Sarpal, 2003).

The interaction between DmKAP and KLP64D waa further established at the ultrastructure level. Klp64Dl4/k5 has the highest viability among the Klp64D mutant alleles and the JO of these mutant adults have an almost indistinguishable set of defects from those seen in Klp64Dk5/k5 animals. These ciliary defects are significantly enhanced in DmKapV6/+; Klp64Dl4/Klp64Dk5 animals. The proximal basal body structures are normal and the desmosomes between inner-dendritic segments are present. However, the cilia appear greatly deformed and disappear apically. Transverse sections through central and distal levels of the scolopales show variable presence of the axonemes within the dendritic caps, and some ciliary membranes appear inflated. This further established that DmKAP interacts with KLP64D for axoneme growth from the distal basal body in JO neurons (Sarpal, 2003).

The Kinesin II subunits KRP85 and SpKAP115 localize to the mid-piece and flagellum of sea urchin and sand dollar sperm. Additionally, Polaris, the mouse homolog of IFT88, is present in mature spermatids. These observations indicate that Kinesin II and the IFT particles are involved in the maintenance and the growth of sperm flagella. Therefore, to determine the universality of this hypothesis, the testes were examined from DmKapV6 hemizygous and Klp64Dk1/A8n123 males. It was surprising to find that seminal vesicles of these mutants had vigorously motile sperm. In addition, the DmKapV6 males, rescued with neuronally expressed UAS-Kap (with the Gal4C155 driver), were as fertile as the wild-type. Gal4C155 does not express in the germline cells of the testis. Hence, this observation established that the sperm axoneme growth is not affected in DmKap mutants. The morphology of sperm axonemes was examined in Klp64D mutants. Unlike sensory cilia, Drosophila sperm tails contain the classical 9+2 microtubule arrangement. Ultrastructural analysis revealed normal axoneme and other sperm tail structures in these mutants. This suggests that in Drosophila, Kinesin II is not required to generate or maintain sperm flagella. This result is consistent with the finding that the Drosophila nompB gene product, a homolog of the Chlamydomonas IFT88 protein, is also not required to generate motile sperm. This indicates that sperm development in Drosophila occurs by an IFT-independent mechanism (Sarpal, 2003).

Analysis of no mechanoreceptor potential mutants reveals that intraflagellar transport is required for assembly of sensory cilia but not for the extension or function of the sperm flagellum

Intraflagellar transport (IFT) uses kinesin II to carry a multiprotein particle to the tips of eukaryotic cilia and flagella and a nonaxonemal dynein to return it to the cell body. IFT particle proteins and motors are conserved in ciliated eukaryotes, and IFT-deficient mutants in algae, nematodes, and mammals fail to extend or maintain cilia and flagella, including sensory cilia. In Drosophila, the only ciliated cells are sensory neurons and sperm. no mechanoreceptor potential (nomp) mutations have been isolated that affect the differentiation and function of ciliated sense organs. The nompB gene is here shown to encode an IFT protein. Its mutant phenotypes reveal the consequences of an IFT defect in an insect. Mechanosensory and olfactory neurons in nompB mutants have missing or defective cilia. nompB encodes the Drosophila homolog of the IFT complex B protein IFT88/Polaris/OSM-5. nompB is expressed in the ciliated sensory neurons, and a functional, tagged NOMPB protein is located in sensory cilia and around basal bodies. Surprisingly, nompB mutant males produce normally elongated, motile sperm. Neuronally restricted expression and male germline mosaic experiments show that nompB-deficient sperm are fully functional in transfer, competition, and fertilization. It is concluded that NOMPB, the Drosophila homolog of IFT88, is required for the assembly of sensory cilia but not for the extension or function of the sperm flagellum. Assembly of this extremely long axoneme is therefore independent of IFT (Han, 2003).

Follicle separation during Drosophila oogenesis requires the activity of the Kinesin II-associated polypeptide Kap in germline cells

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP-driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin-associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane-bound organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. Kap mutants were generated and it was shown that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells (Pflanz, 2004).

In Drosophila, Kinesin II forms a heterotrimeric complex composed of two motor subunits, KLP68D and KLP64D, and a singular nonmotor protein, the Kinesin-associated protein 3 (Kap3), which acts as adaptor and regulator of the complex (Ray, 1999). Recent studies on KLP64D mutants suggest that Kinesin II is specifically required for the axonal transport of choline acetyltransferase in a subgroup of neurons (Ray, 1999). A similar phenotype was reported for Kap mutants (Sarpal, 2003), suggesting that Kap/Kinesin II complexes are only required for neural function. However, in mice, humans and rats as well as in sea urchins and algae, Kinesin II was shown to be necessary for the formation and maintenance of cilia/axoneme structures. In some organisms, a third motor protein, Kif3C, has been identified. Kif3C was proposed to be part of distinct Kinesin II complexes, which act in a spatiotemporal and/or cell-specific fashion. A Kif3C homolog has been identified in the Drosophila genome (CG17461), suggesting that the previous studies may not have revealed all aspects of Kinesin II-dependent Kap function in the fly (Ray, 1999; Sarpal, 2003). To test this possibility, Kap lack-of-function mutants were generated. In addition to the recently described neural function, Kap activity is also required in germ cells for proper follicle separation during oogenesis. The results suggest that Kap participates in signalling necessary for the establishment of follicle-separating stalk cells (Pflanz, 2004).

To assess Kinesin II requirement, mutations affecting the single non-motor component Kap of Drosophila were generated. A semilethal mutation, l(1)G0396, was recovered in which a P{lacW} element was inserted in the first intron of the Kap gene. In all, 95% of hemizygous l(1)G0396 males develop into pupae and die as pharate adults; about 5% hatch and show a paralytic phenotype as described for KLP64D (Ray, 1999). Precise excisions of the P-element resulted in wild-type flies, indicating that the P insertion is the cause of the mutation. Imprecise excisions were also made that represent hypomorphic Kap alleles, which have small internal deletions in the protein coding sequence. In addition, alleles such as Kap89 were obtained that lack the promoter and the first exon of the gene. Homozygous Kap89 mutants fail to express Kap as revealed by in situ hybridization. In addition, their phenotype was as strong as the phenotype of transheterozygous Kap89/Df(1)v-N48 mutant individuals. These findings indicate that Kap89 is a null mutation. Kap89 and the hypomorphic mutants could be rescued by Kap activity that was derived from an Actin5C enhancer-driven Kap cDNA transgene, confirming that the mutations affect only the Kap gene (Pflanz, 2004).

To determine the sites of Kap expression in the organism, RNA in situ hybridization was performed on staged ovaries and embryos. During oogenesis, Kap expression is observed in nurse cells from where transcripts are transported into the growing oocyte. The transcripts remain ubiquitously distributed in eggs and embryos until the blastoderm stage. Zygotic Kap expression is initiated during gastrulation in both ectoderm and mesoderm and is subsequently enriched in neurons. Based on the strong maternal expression of the gene, it was asked whether Kap also has a role during oogenesis in addition to its recently reported function in the nervous system (Pflanz, 2004).

To assess the function of maternal Kap activity, homozygous Kap mutant germline clones were generated using the FLP/ovoD1 system. Females with homozygous Kap89 mutant germ lines are sterile; germline mutant follicles degenerate after they reached stage 6 of oogenesis. Up to this stage, mutant follicles lacking Kap contain more cells than wild-type follicles. The supernumerary cells are either only nurse cells (type I follicles) or both nurse cells and oocytes (type II follicles). Type II follicles have multiple oocytes and a corresponding number (ratio 1:15) of extra nurse cells. Of all follicles scored, 50% showed mixed type I/II follicles. Of the remaining follicles, few (~5%) showed either type I or type II follicles only (Pflanz, 2004).

The supernumerary cells in type I follicles suggest that the mutant germline cells have undergone more than the normal four rounds of mitotic divisions. To confirm this proposal, the number of ring channels and the fusome of the germline mutant ovaries were inspected. Both structures derive from an incomplete separation of daughter cells after the division, resulting in a maximum of four ring channels in the case of the wild-type oocyte and in a branched fusome structure that interconnects the germ cells of a follicle. Kap mutant germ cells were found that have more than four ring channels and fusomes connecting up to about 50 cells. This indicates that at least one extra round of mitotic divisions occurs in Kap mutant follicles. The use of oocyte markers, such as gurken (grk) and oskar (osk) mRNA, identified either one oocyte or multiple oocytes in ectopic locations within the follicles. Collectively, these findings indicate that the determination and the initial stages of oocyte differentiation do not depend on Kap activity, whereas the cellular processes underlying the formation of individual follicles and regulation of germ cell proliferation do (Pflanz, 2004).

Kinesin I has previously been shown to participate in the transport of distinct mRNA species and associated protein to the posterior pole of the Drosophila oocyte. To assess the integrity of this type of microtubule-dependent transport in Kap89 mutant follicles, expression and localization of the oo18 RNA-binding protein (Orb) were examined. Orb is necessary for the directed transport and localized translation of grk and osk mRNA, the axial polarity determinants of the growing oocyte. The expression of Orb is initiated normally, although it is not maintained subsequently. The oocyte determination factor Bic-D and ß-tubulin are expressed normally, and oskar RNA is normally expressed and transported in Kap89 mutant follicles. Thus, cytoskeletal structures and the transport of oskar mRNA into the growing oocyte appear not to be directly affected by the lack of Kap activity (Pflanz, 2004).

Separation of follicles during early oogenesis involves the formation of a characteristic intervening stalk structure. This structure derives from a small group of precursor cells (located in germarium region 2B), which develop into polar follicle cells and the stalk cells. Their proper development depends on cell-cell communication events between the germline and soma cells as shown by Notch/Delta, JAK/STAT and Hedgehog signalling mutants, which develop fused follicles and lack morphologically distinct stalks. Since such a phenotype was also observed with the Kap mutant ovaries, it was asked whether polar follicle cells (which can be visualized by A101-lacZ expression) and stalk cells are formed. In Kap mutant follicles, supernumerary A101-lacZ expressing polar follicle cells were observed, whereas follicle-separating stalk cells were absent. Identical results were obtained by anti-FasIII antibody staining, a different marker for polar follicle cells. These observations suggest that stalk precursor cells fail to differentiate, remain in the follicle epithelium and express the molecular characteristics of follicle cells as has been observed in Notch mutants. Clonal analysis shows that stalk cells are formed properly in the absence of Kap activity in the somatic epithelial cells. Thus, Kap activity has a cell autonomous effect on germline cell proliferation, and the absence of Kap in germ cells also affects somatic precursor cells in a non-cell autonomous manner (Pflanz, 2004).

The non-autonomous Kap effect on follicle cell determination could be explained by a requirement for Kap for the transport and/or localization of components that are needed to signal cell fate to the surrounding somatic precursor cells. Therefore Kap mutant ovaries were examined for the expression of Delta, which activates Notch signalling, hedgehog and the JAK/STAT-activating ligand unpaired. Delta and hedgehog expressions were not affected, whereas unpaired failed to be expressed in polar follicle cells of Kap germline mutant ovaries. This observation suggests that lack of Kap activity interferes with the signalling-mediated crosstalk between germline and somatic cells (Pflanz, 2004).

These results provide evidence that Kap activity in germline cells is required for the proper differentiation of a distinct group of epithelial cells, the stalk cells, which separate individual follicles during early oogenesis. The absence of Kap in germ cells prevents somatic target cells from differentiating into proper polar follicle cells, because they fail to express the ligand Unpaired. Unpaired activates JAK/STAT signalling and thereby induces stalk cell fate in the respective precursor cells. The details of the germline cell/polar follicle cell/stalk cell signalling cascade are currently being investigated to address the cellular mechanism of these interactions (Pflanz, 2004).


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Reference names in red indicate recommended papers.

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Kinesin associated protein 3: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 10 February 2014

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