short stop/kakapo
The expression patterns of KAK mRNA and protein during various stages of embryonic development have been characterized.
KAK mRNA expression is restricted to ectodermally derived cells, and is initially observed in extended germ band embryos during
late stage 11. During germ band retraction, the mRNA is expressed by cells along the future segmental grooves and in a small cluster
of ectodermal cells at the middle of each hemi-segment. Since at these stages the somatic myotubes have not yet
extended their leading edge into their final position, it is assumed that the initial activation of KAK transcription is independent of
muscle-dependent cues (Strumpf, 1998).
The expression of Kak protein was studied using a polyclonal antibody raised against a bacterially expressed cDNA fragment from
the COOH-terminal region. The protein expression pattern of Kak follows its mRNA expression and is apparent from stage 14 of
embryonic development. During the final stages of muscle development (e.g., stage 16), high levels of Kak protein are detected in
muscle-bound tendon cells, as demonstrated in embryos double-labeled for Kak and myosin. Kak protein is
concentrated at the circumference of the tendon cell, presumably along the tendon cell plasma membrane. Low levels of
Kak protein are observed in other tissues, including epidermal cells and chordotonal organs (Strumpf, 1998).
Strong Kakapo expression in epidermal tendon cells is observed at mid-late stage 16, which is ~4
h after the muscles first start to attach to the epidermis. During the last two stages of embryogenesis (stages 16 and 17) attachment of the epidermal cells to the muscles or the tendon matrix is elaborated by expansion of the
hemiadherens junctions, accumulation of tendon matrix, and increased expression of beta1 tubulin. No expression of Kakapo is detected in the muscles, even though
comparable hemiadherens junctions (characterized by membrane-proximal electron-dense plaques into which cytoskeletal
stage 14 elements insert) are formed there and the adhesion is also integrin-dependent. This difference could
indicate that Kakapo has a function that is incompatible with muscle contraction, such as forming stable anchoring structures.
Kakapo is strongly expressed in two internal structures as well: the pharynx and the proventriculus.
In the pharynx, Kakapo is strongly expressed in the endodermal cell layers that attach to the pharyngeal muscles. As
with the epidermal cells that attach to the somatic muscles, Kakapo is found both at the basal surface that contacts
the mesoderm and at the apical surface, while the PS integrins are localized just at the basal surface. In
the proventriculus, Kakapo is expressed in a ring of cells at the anterior margin of the outer layer of this three-layered
structure. Expression of the PS integrins is found primarily at the interface between the outer endodermal
layer of the proventriculus and the surrounding visceral mesoderm. Therefore, in this tissue, the integrins are
expressed in more cells than Kakapo. It is not clear why strong expression of Kakapo might be needed at this site, but one
phenotype of integrin mutations is that the inner layers of the proventriculus become pulled out, suggesting that some resistance to mechanical stress is normally necessary to maintain the integrity of the proventriculus
structure. Modest Kakapo expression is detected in the scolopale of the chordotonal organs of the peripheral nervous
system. A function for the PS integrins in these cells has not been observed to date, but, like muscle attachment
cells, it is a site of stabilized beta1 microtubule based rigidity (Gregory, 1998).
In the epidermal muscle-attachment cells, the PS integrins are localized to the basal surface, which
contains large hemiadherens junctions. Microtubules extend from these basal junctions to the apical
hemiadherens junctions, which connect to the exoskeleton (cuticle). The microtubules appear to be serving a similar structural
role to that of keratin filaments in the epidermal cells of vertebrates, since intermediate filaments have yet to be identified in
Drosophila. When the subcellular localization of Kakapo was examined in more detail, it was found to be present at both
apical and basal surfaces of the muscle attachment cells. Kakapo can be seen to be positioned at the termini of the
microtubule bundles extending from the apical to the basal surface, as well as faintly along their length. This
subcellular pattern is distinct from other proteins that share the alpha-actinin type actin-binding domain, such as betaH-spectrin, demonstrating that this domain does not by itself direct the intracellular localization of
proteins containing it. The apical domain of Kakapo expression overlaps with the apical localization of the protein
4.1 superfamily protein Coracle. Reduced staining of Coracle was found on the lateral surface at this stage, when
compared with earlier stages. The 4.1 superfamily of proteins is used to link
transmembrane proteins to the cortical cytoskeleton, so the colocalization indicates that Kakapo is found at the cell cortex (Gregory, 1998).
Kakapo expression is adjacent to PS integrin expression, which consists of expression on the basal surface of the
epidermis and at the termini of the attaching muscles. There is no significant expression of Kakapo in the muscles, but the
basal expression of Kakapo in the epidermal muscle attachment cells is detected immediately next to, while not overlapping,
the epidermal integrin localization. Thus, Kakapo is not only present at the basal surface where the PS integrins
are located, but also at the apical surface, where as yet no adhesion receptors have been described (Gregory, 1998).
Shot mRNAs are expressed in the CNS and PNS during embryonic stages 13 and 14 when most axon outgrowth begins,
and this expression persists to later stages of embryonic development when synapse formation occurs. Intriguingly, mRNAs encoding
the actin binding domain containing isoforms A and B are expressed in the CNS, but mRNAs encoding isoforms C and D are not
detectably expressed in the CNS. The mRNAs encoding isoforms A and B are also transiently expressed in the developing PNS at
stage 13. This difference in the expression of the actin binding and non-actin binding isoforms parallels the differences
observed in the expression of neuronal, actin binding and epidermal, non-actin binding isoforms of BPAG1/dystonin. Otherwise, the mRNAs
encoding the N-terminal isoforms reported here, and those identified previously (Strumpf, 1998), are expressed in identical
patterns in the embryo: in the epidermis at low levels and in muscle attachment cells at high levels (Lee, 1999).
An antiserum against Kak (Strumpf, 1998) was used to investigate whether the Shot/Kak proteins are expressed in developing
axons. This antibody is raised against epitopes present in the 22 triple helical repeats of the long isoforms of Shot/Kak. Therefore it does not
detect the shorter isoforms of Shot and instead recognizes a major band of at least 400 kDa on Western blots (Strumpf, 1998). In embryos, Shot/Kak protein can be detected in axons in the CNS and PNS. Although the expression level in
growth cones is difficult to determine because Shot is expressed in surrounding epidermal cells, Shot is present in CNS axons as early as stage
12 and can be detected in the intersegmental nerve (ISN). In addition, it accumulates in cortical regions of the neuronal cell
bodies of chordotonal and dorsal cluster sensory neurons, consistent with an association with the actin cytoskeleton (Lee, 1999).
How E-cadherin controls the elaboration of adherens junction-associated cytoskeletal structures crucial for assembling tubular networks was investigated. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Short stop (Shot), a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to ß-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly (Lee, 2003).
Junctional contacts between cells are important for organizing the
cytoskeleton and regulating cell polarity. The large size
of plakins and their modular abilities to bind different cytoskeletal elements
make them potentially well suited to play key organizational roles.
However, except in the case of desmosomes, where the plakin desmoplakin
appears to be a crucial for organizing junction-associated cytoskeleton,
functional association of plakins with other cell-cell junctions has not been
described (Lee, 2003).
In selected cell types, Shot localizes with proteins of
the adherens junction and may play a role in adherens junction-mediated
organization of the cytoskeleton. It is proposed that Shot and E-cadherin form a
feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new
contacts between the fusion cells and Shot stabilizes the contacts. The
cytoskeleton organizes around these contacts because adherens junction
associated Shot promotes the assembly of an F-actin/microtubule-rich track.
This track grows to span the fusion cells, extending the reach of the
junctions through the cells. The recruitment mechanism may be indirect in that
new adherens junctions in fusion cells are centers for cytoskeletal assembly,
and Short Stop binds F-actin and microtubules. Alternatively, Shot may
associate directly with E-cadherin or associated proteins. The assembly of
Shot with F-actin and microtubules may stabilize E-cadherin contacts simply by
bringing in cytoskeletal proteins that bind E-cadherin or associated proteins.
For example, EB1, which is present in the fusion track, co-immunoprecipitates
with a C-terminal fragment of Shot in cultured cells and associates with APC. APC interacts with ß-catenin to control tubulogenesis in vitro (Lee, 2003).
It is proposed that the assembly and maturation of a cytoskeletal intermediate
are two E-cadherin-dependent steps in tracheal cell fusion. Imaging of fixed and
live embryos suggests that fusion proceeds through the assembly and maturation
of a cytoskeletal track associated with adherens junctions. The track forms
after contact between the fusion cells, and persists for ~1 hour before
fusion occurs (Lee, 2003).
In this model, the ß-catenin-binding site and the juxtamembrane site
in the E-cadherin cytoplasmic domain operate sequentially and in the same
E-cadherin molecule to promote fusion. In mutant embryos in which either
ß-catenin or its binding site is defective, fusion cells make contact but
track assembly is not observed. These data suggest that E-cadherin may
initiate track assembly via ß-catenin. A mutation in the juxtamembrane
site dominantly inhibits track maturation. Microtubules are generally absent
from fusion tracks in these embryos, though some F-actin and Shot assembly
occurs. In E-cadherin/shotgun (shg) mutant embryos,
E-cadherin bearing this juxtamembrane mutation supports a low level of
F-actin/Shot track formation, but the tracks do not mature. In addition, this
juxtamembrane mutant E-cadherin causes progressive delocalization of the
apical tracheal cytoskeleton in shg mutant embryos (Lee, 2003).
Both the ß-catenin and juxtamembrane binding sites are required for
E-cadherin localization to adherens junctions, although only the juxtamembrane
mutation seems to interfere with endogeneous E-cadherin localization. The
results suggest that like mammalian E-cadherin, an evolutionarily conserved
juxtamembrane site is required for some E-cadherin functions. Similar effects
of mutations in the juxtamembrane site were observed in mammalian tissue
culture cells. However, juxtamembrane site function in Drosophila
E-cadherin probably does not require p120 (Lee, 2003).
Dominant effects on localization appear sensitive to expression levels,
whereas effects on fusion are less so, suggesting that defects in localization
are not enough to explain the defects in track maturation. Possibly, effects
on localization also reflect defects in organizing the cytoskeleton, as has
been observed in studies in which dominant alleles of Rho family GTPases
affect cadherin localization in culture (Lee, 2003).
It is proposed that the ß-catenin-binding site and ß-catenin are
required for track assembly, and that the juxtamembrane site regulates other
proteins involved in a later maturation step. This later step
likely requires microtubules. The microtubules or associated proteins may
reinforce the initial F-actin assembly in the track, as F-actin in fusion
tracks appears to be abnormally or poorly assembled in embryos expressing
AAA-JXT mutant E-cadherin in tracheal cells. The microtubules appear to be also
required for remodeling the fusion cell apical surfaces and also for bringing
them together to fuse. In embryos expressing AAA-JXT mutant E-cadherin in
tracheal cells, fusion cell apical surfaces do not develop or seal gaps at
appropriate times, and fusion tracks persist substantially longer, if they
resolve at all (Lee, 2003).
The microtubule regulated steps during fusion therefore likely involve
effects on F-actin dynamics. Microtubule-associated factors that may regulate
the F-actin cytoskeleton include Rac GTPase and exchange factors for Rho GTPase. Rac1 affects E-cadherin dependent adhesion in tracheal cells and a mutation in the juxtamembrane site in mammalian E-cadherin analogous to the one described in this study affects Rac activation. RhoA activation inhibits fusion track assembly.
Downstream interactions between F-actin and microtubules, such as those
mediated by Shot, may vary with cell type to produce distinct morphogenetic
outcomes. Further studies of tracheal tube fusion, a genetic system in which
adherens junction associated structures can be visualized in living embryos,
promises to identify the regulatory molecules that allow E-cadherin to direct
F-actin and microtubule assembly from the ß-catenin binding and
juxtamembrane domains (Lee, 2003).
Notch signalling is an evolutionarily conserved cell interaction mechanism; its role in controlling cell fate choices has been studied extensively. Recent studies in both vertebrates and invertebrates have revealed additional functions of Notch in proliferation and apoptotic events. Evidence suggests an essential role of the Notch signalling pathway during morphogenetic cell movements required for the formation of the foregut-associated proventriculus organ in the Drosophila embryo. The activation of the Notch receptor occurs in two rows of boundary cells in the proventriculus primordium. The boundary cells delimit a population of foregut epithelial cells that invaginate into the endodermal midgut layer during proventriculus morphogenesis. Notch receptor activation requires the expression of its ligand Delta in the invaginating cells and apical Notch receptor localization in the boundary cells. The movement of the proventricular cells is dependent on the short stop gene that encodes the Drosophila plectin homolog of vertebrates and is a cytoskeletal linker protein of the spectraplakin superfamily. short stop is transcriptionally activated in response to the Notch signalling pathway in boundary cells, and it has been shown that the localization of the Notch receptor and Notch signalling activity depend on short stop activity. These results provide a novel link between the Notch signalling pathway and cytoskeletal reorganization controlling cell movement during the development of foregut-associated organs (Fuss, 2004).
The proventriculus is a multiply folded, cardia-shaped organ that functions as a valve to regulate food passage from the foregut into the midgut of Drosophila larvae. It is derived from the stomodeum, which gives rise to the foregut tube and to parts of the anterior midgut in the early embryo. Cell shape changes are initiated at stage 12 when cell proliferation has been completed within the proventriculus primordium. Anti-Forkhead (Fkh)/anti-Defective proventriculus (Dve) double immunostainings which specifically visualize ectodermal and endodermal cells, respectively, reveal that the first step of proventriculus morphogenesis involves the formation of a ball-like evagination at the ectoderm/endoderm boundary of the posterior foregut tube. The formation of this evagination is initiated by a local constriction of apical membranes at the ectoderm/endoderm boundary leading to an accumulation of membrane-associated markers such as Arm towards the luminal (apical) side. It is notable that the ectodermal part of the ball-like evagination localizes in a mesoderm-free region, whereas the surrounding cells of the developing foregut and the midgut are covered by visceral mesoderm. At stage 14, a constriction forms at the boundary of the ectodermal and the endodermal cells. This results in the formation of the 'keyhole' structure. From stage 14 onward, cells from the anterior portion of the ectodermal keyhole part (in the mesoderm-free area) begin to move inward into the endodermal part of the keyhole and a heart-like structure is formed. The ectodermal keyhole cells continue to move inward until late stage 17 and give rise to the recurrent layer of the proventriculus; it links the outer endodermal layer (derived from the endodermal keyhole cells) and the inner layer of the proventriculus which is a continuation of the esophagus. The cells at the tip of the invaginating ectodermal keyhole cells thatderive from the most anterior region of the keyhole, are not covered by visceral mesoderm. It has been observed before that these cells assume a stretched appearance with long cytoplasmic extensions (Fuss, 2004 and references therein).
Immunohistochemical analysis demonstrates that the ligands of the Notch
receptor, Delta and Serrate are expressed in the ectodermal keyhole cells that invaginate into the endodermal cell layer during proventriculus development. Their expression becomes downregulated in the anterior and posterior boundary cells in which the Notch receptor is elevated and in which the Notch signalling pathway is activated, as demonstrated by the Notch-dependent Gbe-Su(H)m8-lacZ reporter construct. Whereas there is no proventricular phenotype in Ser mutants, the invagination movement of the ectodermal keyhole cells is defective in mutants of other components of the Notch signalling pathway, such as Notch, Delta, fng or Su(H). This strongly suggests that the boundary cells play a crucial role for cell movement during proventriculus development. It is not known whether
the cell movements are driven by the anterior boundary cells, dragging the
esophageal cells behind or whether the major force for the inward movement is contributed by the ectodermal foregut cells changing their shapes from a cuboidal to a more stretched appearance. The latter is known to occur during mid and late stages of embryogenesis when the foregut and the hindgut elongate dramatically increasing their size by two- to threefold. It has been shown for dorsal closure that multiple forces contribute to cell sheet morphogenesis. A similar scenario may apply for proventriculus morphogenesis. Genetic mosaic studies have revealed that the activity of the Notch receptor occurs in cells that are adjacent to the ligand-expressing cells. Therefore, the downregulation of Delta in the boundary cells may be a prerequisite for Notch signalling and cell movement, which would be consistent with the observation that a Notch-like proventriculus phenotype is induced when Delta expression is maintained in the anterior and posterior boundary cells (Fuss, 2004).
Recent studies on neural crest cells in the mouse also have suggested a
role for the Notch signalling pathway during cell migration. The
neural crest cells in vertebrates give rise to a wide range of cell types,
including nerve cells, pigment cells, as well as skeletal and connective
tissue. These cells constitute a migratory cell population that leaves the dorsal neural tube to migrate along specific tracks to their final destinations in the periphery of the body. In Delta1 knockout mice, the local expression of Ephrin receptors and ligands, which are guiding molecules, is reduced in the caudal region of the sclerotome, as well
as in neural crest-derived peripheral ganglia. A connection of Notch signalling with the modulation of cytoskeletal architecture has not been shown in these mutants. From loss-of-function experiments in the case of the proventriculus cell migration, the alternative view that Notch signalling may determine the fate of the boundary cells rather than directly controlling cell movement cannot be excluded. However, when the Notch pathway was ectopically activated by misexpressing NICD in the proventricular endoderm, this did not result in a change of cell fates of the endodermal cells towards ectodermal boundary cell fates. Furthermore, the link between Notch signalling and the activation of shot, a known cytoskeletal regulator, provides good evidence of a more direct role for Notch in controlling cell movements rather than determining cell fates (Fuss, 2004).
The results further demonstrate that the shot gene is
directly or indirectly transcriptionally regulated by the Notch signalling
pathway. Members of the spectraplakin superfamily such as Shot in flies or dystonin/BPAG1 or MACF1 in mammals share features of both the spectrin and plakin superfamilies and produce a large variety of giant proteins of up to almost 9000 amino acids in length. These proteins contain motifs interacting with all three elements of the cytoskeleton (the actin, the microtubules and the intermediate filaments), and they contribute to the linkage between membrane receptors and
the cytoskeletal elements. shot is strongly expressed during
embryogenesis at the muscle attachment sites, which are the most prominent
sites of position-dependent integrin adhesion. An
essential role for Shot has been shown for muscle-dependent tendon cell
differentiation. In the shot mutant tendon cells, Vein, a
neuregulin-like factor that activates the EGF-Receptor signalling pathway,
fails to be localized properly at the muscle-tendon junctional site; Vein is dispersed and its level is reduced. In these cells, Shot is concentrated at the apical and basal sides. Similarly,
the results place shot both upstream and downstream of Notch
signalling during proventricular development. In the posterior boundary cells, shot transcription is activated in response to Notch signalling; Shot protein, in turn, is required in the posterior boundary cells for Notch receptor localisation and/or stability as receptor expression and Notch signalling activity in the posterior boundary cells are affected in shot mutants. This indicates a feedback loop, as has been suggested for Crumbs-dependent localization of the Notch receptor in the boundary cells of the hindgut. It is not clear how shot expression is regulated in the endodermal part of the keyhole, in which it may require additional inputs from other yet unknown signalling pathways. Further molecular and biochemical experiments will have to demonstrate whether there exists a direct interaction between the Notch receptor and the cytoskeletal Shot protein (Fuss, 2004).
In the tracheal system, Shot is required for the formation of the
RhoA-dependent F-actin cytoskeleton in the fusion cells and to form the
lumenal connections between tracheal branches. It
has been suggested that Shot may function downstream of RhoA to form
E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization. The analysis of the actin cytoskeleton using phalloidin staining reveals a strong apical localization of F-actin filaments in the posterior boundary cells in which Shot also accumulates apically to a high level. By contrast, the density of the actin cytoskeleton is reduced in the anterior boundary cells that move inward and in which the contribution of Shot for Notch signalling activity seems minor. A stabilized cytoskeletal architecture in the posterior boundary cells may be required to provide stiffness/tension that may enable the inward movement of the anterior boundary cells. Lack- and gain-of-function results suggest that the small GTPase Cdc42, is one of several known cytoskeletal regulators, may play a major role to control cytoskeletal architecture during the inward movement of the proventricular cells. These results are consistent with the idea that Notch signalling controls cytoskeletal organization via the cytoskeletal linker protein Shot and they suggest a role for Cdc42 in this process, the specific involvement of which, however, has to be studied in more detail (Fuss, 2004).
Shot/Kak protein expression in neurons, epidermis, and epidermal muscle attachment cells cannot be detected in shot3, shotP1, shotP2, and
kakP2 mutant embryos. shot1 and shot2 mutants still express normal levels of Shot protein, consistent with their relatively weaker mutant
phenotypes. The phenotypes of shotP1 and shotP2 insertion mutants are also somewhat weaker than the phenotypes of shot3 and kakP2
insertion mutants, suggesting that Shot proteins may perhaps be present in the nervous system in these mutants at levels not detected by this
antibody. The Kak antiserum also reacts strongly with an antigen present in the wall of the tracheal lumen; this cross-reactivity remains
in all of the shot mutant alleles tested (Lee, 1999).
shot was identified in a screen for mutations affecting sensory axon morphology (Kolodziej, 1995). shot3 mutant embryos stained
with mAb 22C10, a reagent that labels all sensory neurons and their axons, were revealed to contain only rudimentary
sensory axons. Sensory axon bundles are more variably affected in shot2 mutants, and defects cannot be
detected with the mAb 22C10 antibody in shot1 mutants (Kolodziej, 1995). shot mutants appear normal with respect to other
aspects of neuronal differentiation and viability. To gauge the relative severity of the shot mutations more precisely, comparisons were made of the length of a bundle of four sensory axons in wild-type and
mutant embryos. mAb 49C4 specifically recognizes four of the five lateral chordotonal (LCH) neurons and their axons. In wild-type embryos, the LCH sensory axons grow anteriorly, turn ventrally when they join the ISN, and extend into the CNS by
stage 16. In shot1 embryos, the LCH axons arrest at variable points within the ISN. As quantitated here, the
LCH axons in shot2 and shot3 are more severely affected, and the LCH axons in shot3 mutants almost always stall before advancing
far along the ISN. In these severe alleles, the LCH axons never extend to the CNS border. The P-element insertions kakP1/kakP2,
shotP1, and shotP2 affect LCH axon growth on average less severely than the shot2 and shot3 alleles but show a similar range of stall
phenotypes. Although the long form of Shot protein cannot be detected in these P-element mutant embryos, these slight differences in phenotype may reflect low levels of the long Shot proteins or other isoforms still present in some of the
P-element alleles (Lee, 1999).
The shorter axons observed in shot mutants could reflect a delay in axonogenesis or a defect in the ability of growth cones to advance. Dye
filling of the chordotonal neurons allowed for the resolution of details of the timing of axonogenesis and the morphology of individual axons and growth
cones in wild-type and mutant embryos. In wild-type embryos, axonogenesis in the LCH neurons occurs before stage 15. In
shot2/shot3 mutant embryos, the LCH neurons also extend a growth cone toward the ISN by early stage 15. By early stage
16, the LCH axons in wild-type embryos reach the CNS. By early stage 16, no LCH axons in shotP2, shot3, or shot2/shot3
mutant embryos reach the CNS. Therefore, the stalled axon phenotype detected in stage 16 shot embryos does not
reflect a delay in axonogenesis. The inability of growth cones to advance in shot mutant embryos could reflect a defect in the formation of actin-based structures known to be
important for growth cone motility, such as filopodia or lamellopodia. The LCH axon morphologies observed in shot mutant
embryos are indistinguishable from those of wild-type embryos at the same point in the axon trajectory. LCH growth cones in wild-type
embryos extend numerous fine filopodia and have a wide lamellopodium before they join the ISN. As they advance along the ISN,
they become narrower and less complex. These morphological transitions are also observed in shot mutant embryos, with the growth
cones adopting the general morphology of wild-type growth cones at the place where they stall. Growth cones that fail to contact
the ISN are more complex than those that stall within the ISN. The direction of axon growth is also unaffected in shot mutant embryos. Thus,
growth cones form normally and orient correctly in shot mutant embryos but fail to continue advancing. Although growth cone morphology
appears to be unaffected, defects in LCH dendrite morphology are often observed in shotP2, shot3, and shot2/shot3 mutant embryos. This is consistent with the description of dendrite detachment reported (Prokop, 1998) in kak mutants (Lee, 1999).
In the abdominal hemisegments of wild-type embryos, 31 motor axons innervate 30 muscle fibers in a stereotyped pattern. These motor axons are organized into nerve bundles that innervate different muscle
fields. The main ISN motor axons innervate the dorsal muscles; the intersegmental nerve b (ISNb) motor axons innervate ventral muscles, and
the segmental nerve a (SNa) motor axons innervate ventrolateral muscles. shot was first identified as a gene specifically required for the outgrowth of the ISN and ISNb motor axons; SNa development is normal in shot1 mutants (Van Vactor, 1993). ISN motor axons in shot1 mutant embryos generally arrest near the dorsal trunk
of the trachea, just before they would normally make their two most dorsal arborizations. The ISNb motor axons arrest in the middle of the ventral
muscle field. Thus, shot was suggested to be required for the extension of only a subset of motor axons beyond a well defined point in their
development (Van Vactor, 1993). This previous analysis of shot's role in motor axon development is based on shot1, which still expresses Shot protein and has
a weaker effect on sensory axon development than the other shot alleles (Kolodziej, 1995). Other mutations in kak also affect
motor axon development but affect synapse formation without affecting axon outgrowth (Prokop, 1998). Thus, kak has been proposed
to be specifically involved in synapse formation and not to be required earlier in motor axon development. To investigate motor axon
development in a stronger allele, motor axons in shot3 and shot3/Df(2R)CX1 mutant embryos were analyzed, because shot3 appears to
affect the LCH axons most severely among the alleles that lack detectable Shot proteins (Lee, 1999).
In late stage 16/stage 17 wild-type embryos stained with mAb 1D4, the motor axons in the ISN pathway have reached the dorsal muscles and
formed three terminal arborizations. The motor axons in the SNa pathway have reached the ventrolateral muscles and bifurcated into two
branches: a lateral branch that extends posteriorly along the border of the ventral muscle field and a dorsal branch that extends more dorsally
into the ventrolateral muscle field. The ISNb motor axons have defasciculated from the ISN and have formed contacts with muscles 12, 13, and
6/7. In late stage 16/stage 17 shot3 mutant embryos, 90%-100% of the motor axons in all three major pathways (ISN, SNa, and ISNb)
stall prematurely. The ISN motor axons not only lack terminal arborizations (Prokop, 1998), but do not reach the
muscles that they normally innervate. Although SNa development appears normal in shot1 embryos, the SNa motor axons in stronger
shot mutants generally fail to form both branches, and they often stall at the entry to the ventrolateral muscles. The ISNb motor
axons defasciculate normally from the ISNb and correctly target the ventral muscle field, but they stall in the ventral muscle field or before they
enter it. The place where the motor axons in a given pathway stall is variable and does not suggest as well defined an arrest point as
proposed earlier from studies of the shot1 allele (Van Vactor, 1993). Some SNa and ISNb motor axon bundles fail to exit the
CNS successfully or stall earlier than the muscle field entry points, and some ISN motor axon bundles (17%) stall well before the dorsal muscles
in shot3/Df(2R)CX1 embryos. Thus, shot may also be required for motor axon extension before entry into the muscle fields. The rare
axons that reach their muscle targets generally fail to form the arborizations indicative of neuromuscular junction formation, or they form
arborizations of reduced size, as seen in weaker kak mutants (Prokop, 1998). shot does not appear to be required for motor
axons to choose the appropriate pathways or for selective fasciculation (Lee, 1999).
Motor axons in shot mutant embryos complete a substantially greater portion of their trajectory than do sensory axons. In wild-type embryos,
sensory neurons in the dorsal and lateral PNS clusters extend axons along the ISN motor axon pathway and fasciculate with the ISN motor
axons. Because the ISN motor axons extend beyond the lateral chordotonal cluster in shot mutant embryos, it is unlikely that defects in the
extension of the motor axons explain defects in the growth of sensory axons in shot mutant embryos. The ability of sensory and motor axons to
fasciculate appears normal in shot mutant embryos. The same range of defects is seen in shot3/Df(2R)CX1 mutant embryos as compared with shot3 mutants alone, and the frequency of the more
severe phenotypes is only modestly enhanced. These data, taken together with the absence of detectable long isoforms of Shot in
homozygous shot3 mutant embryos, suggest that shot3 is a null allele. Thus, Shot is only essential in motor axon development for the later steps
of outgrowth. If shot were required for the initial stages of motor axon outgrowth, one would expect to see some examples of segments with no
mAb1D4-stained axon bundles in the most severe alleles (Lee, 1999).
It has been reported previously that shot mutants are defective in muscle attachment because of defects in the formation of epidermal
attachment cells. Moreover, Shot appears to be important for stabilizing the cytoskeleton of these attachment cells against contractile forces
exerted by muscle. On becoming contractile (stage 17 embryos or later), muscles rip loose from their attachment sites. Because defects in
muscle organization could affect motor axon growth and targeting, muscle morphology was examined in shot3 null mutant embryos using an
antibody against myosin. Muscle number and organization are normal in late stage 16 shot3 mutant embryos, a stage at
which motor axons have reached their target muscles in wild-type embryos. Muscles are also normal in embryos homozygous for the weaker
shot1 allele (Van Vactor, 1993). Moreover, the expression of Connectin, which marks a subset of lateral muscles, is also normal in shot mutant embryos. It is concluded that defects in muscle
development probably do not contribute to the motor axon growth defects observed (Lee, 1999).
The localization of Kakapo to both ends of the microtubule bundles suggests that Kakapo could have a role in connecting the
microtubules to the cortical actin network at the membrane, and may also directly or indirectly link to transmembrane
receptors such as the integrins. To test its function in the epidermal muscle attachment cells, kakapo mutant
embryos were studied. In stage 16 embryos no penetrant defects could be found in muscle attachment in embryos homozygous for
most kak alleles, although some stronger alleles produce severe disruptions in embryonic morphogenesis. However, in stage 17 embryos mutant for the weaker alleles, the muscles are found to detach from the
epidermis, but they stay attached end to end. In the living embryo one can see that although muscle
contraction occurs, it is no longer coupled to movement of the exoskeleton. In kak mutants the microtubule
bundles are no longer attached to the basal membrane, and as a consequence, the epidermal cell rips in half. This phenotype is
highly reminiscent of the BPAG and plectin phenotypes (Guo, 1995; McLean, 1997), and is distinct from the
PS integrin's mutant phenotype where each muscle detaches both from the epidermis and from the other muscles. Thus, Kakapo is required for epidermal attachment to muscles, but not for muscle-muscle attachment, consistent
with its expression pattern (Gregory, 1998).
Embryos mutant for
Kakapo were examined to see if more general defects that might be a consequence of loss of low-level Kakapo
expression could be detected. Embryos mutant for kakapo display a wide range of phenotypes, from almost normal development to severe
morphological abnormalities. The variability of the phenotype may be due to a partial redundancy of
function between Kakapo and another protein, or variable contribution of maternal protein. The former possibility is favored,
since generating germ line clones of one of the kakapo alleles does not enhance the phenotype (Walsh, 1998), and no detectable Kakapo protein is found before gastrulation (Gregory, 1998).
Consistent with the variability found in embryos deficient for kakapo, kakapo alleles display diverse
phenotypes. Most of the alleles isolated are embryonic lethal as homozygotes, although the two alleles develop normally through stage 16. At the end of embryogenesis
(stage 17), the mutant embryos have a phenotype where the epidermis detaches from the muscles. By examining the
phenotype of the different alleles, it was found that some have much stronger phenotypes, indicative of a more general function. An examination was made of the epidermally secreted cuticle, which reflects the pattern of the underlying epidermis -- of embryos homozygous for all kakapo alleles. In the majority of the alleles, the
homozygous mutant embryos have a normal epidermal pattern, although ~30% of these embryos showed modest cuticular
defects. Epidermal development was examined in more detail in the embryos homozygous for strong kakapo alleles using two markers
for epidermal shape. This examination revealed defects in the integrity of the epidermal cell layer: breaks
in the ventral epidermis in the middle abdominal segments of the fully germband-extended embryos. These breaks
appear to arise at the site of maximum strain during germ band retraction. Consistent with this observation, germband retraction
is arrested in some embryos. Other embryos successfully undergo germband retraction, but retain a hole in
the epidermis at this position of maximum strain, which could account for the disruptions observed in the
cuticular denticle belts. The homophilic adhesion molecule Fasiclin III is not present on the surface of the cells bordering the hole. Such breaks in the epidermis are not observed in embryos mutant for the PS integrins, suggesting that this Kakapo
function involves other cell adhesion molecules. Morphogenetic defects are observed in the internal tissues, but because they occur in embryos with severe epidermal defects, it is not yet certain whether this indicates a function
for Kakapo in these tissues, or whether the internal defects are a result of the epidermal disruption. The phenotypes in the
embryonic epidermis indicate that the low-level general expression of Kakapo is significant, and that Kakapo may play a general
role in mediating adhesion, possibly by mediating interactions between transmembrane proteins and the cytoskeleton (Gregory, 1998).
Neuronal growth requires a dynamic cytoskeleton.
Some kak mutant phenotypes suggest that kak function might be required for cytoskeletal organization. This is most obvious
for muscle attachments to the epidermis. At stage 17 kak mutation causes severe detachment
of muscles from the cuticle, but many muscles remain attached to one another. Muscle attachments are formed by
hemiadherens junctions, the adhesion of which depends on PS integrins. In late stage 17 kak mutant embryos the extracellular adhesion of
hemiadherens junctions is intact and, accordingly, PS integrin is expressed at
the muscle tips. However, a striking phenotype is observed on the intracellular face of hemiadherens junctions,
only on the epidermal side. Normally the intracellular face of epidermal hemiadherens junctions contains a thick layer of
electron-dense material, which connects to the stress-resisting microtubules. In kak mutant
epidermal cells the layer of dense material is thinner and microtubules, although present within
the cell, seem not to be attached to the remaining layer of dense material. As a result, epidermal cells
rupture, and the retracting muscles take with them the epidermal cell fraction around the
hemiadherens junction (Prokop, 1998). This phenotype is similar to BPAG1 mutant mice where intermediate filaments, the major stress-resisting
cytoskeletal elements in epidermal cells of vertebrates, fail to adhere to hemidesmosomes (Guo, 1995).
Epidermal cells at sites of muscle attachments contain beta 1-Tubulin, which is also strongly expressed throughout the
central nervous system and in the scolopidia, which are part of the peripheral nervous system (also called chordotonal organs). Because growth defects in the nervous system are restricted to small dendrites and neuromuscular
side branches, potential defects in the neural cytoskeleton are expected to be subtle, and so far no
specific defects have been pinpointed at the ultrastructural level. However, analysis of the more prominent scolopidia yields interesting results. In the
wild type, scolopidial sensory neurons form a long process stretched between cap and sheath cells and the soma bulges out
asymmetrically on one side. The neuronal processes
contain a typical ciliary apparatus with a cilium, basal body, and rootlet. The rootlets are surrounded by a circle of microtubules,
especially in the distal parts of the processes. This circle of microtubules is mostly absent or very poorly developed in kak
mutant embryos, and the prominent dendrites appear collapsed in 22C10-labeled specimens. The cilia contain a ring of nine microtubule doublets and are each located in a
lymph-filled capsule formed by scolopale and cap cells. The cilia are anchored with their apical ends in extracellular
matrix at the tip of the capsules. In kak mutant embryos the cilia look normal, however, they fail
to anchor at the capsule tips and are retracted. In some mutant embryos grey inclusions are found within the
extracellular matrix at the capsule tips, which might be remnants of the cilia. This could suggest an intracellular
detachment of the cilia similar to that seen in the epidermis, leaving behind pieces of fractured membrane (Prokop, 1998).
Directed intercellular interactions between distinct cell types underlie the basis for organogenesis during embryonic development.
This paper focuses on the establishment of the final somatic muscle pattern in Drosophila, and on the possible cross-talk between
the myotubes and the epidermal muscle attachment cells, occurring while both cell types undergo distinct developmental programs.
The stripe gene is necessary and sufficient to initiate the developmental program of epidermal muscle
attachment cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various
epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific
markers. Moreover, these ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited
distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis
of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell was performed in mutant
embryos in which loss of muscles, or ectopic muscles were induced. This analysis indicates that, although the initial expression of
epidermal muscle-attachment cell-specific genes including stripe and kakapo is muscle independent, their continuous expression is
maintained only in epidermal muscle attachment cells that are connected to muscles. These results suggest that the binding of a
somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Taken
together, these results suggest the presence of a reciprocal signaling mechanism between the approaching muscles and the epidermal
muscle attachment cells. First, the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion
to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells,
inducing the terminal differentiation of these latter cells into tendon-like cells (Becker, 1997).
FLP-FRT induced mitotic
recombination to generate clones of randomly induced mutations in the F1 generation
and these mutations were screened for those that cause wing blisters. All 11 nonintegrin loci are required on both
sides of the wing, in contrast to integrin subunit genes. Mutations in 8 loci only disrupt adhesion in the wing,
similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the
strongest integrin mutations, cause a 'tail-up' embryonic lethal phenotype, and mutant alleles of one of these loci strongly enhance
an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin
function Both bladderwrack (bad) and kakapo mutant embryos have more variable developmental defects. Mutations at both loci
cause much stronger phenotypes over a deficiency than when transheterozygous, suggesting that
amorphic alleles of these loci have not been recovered. However, embryos homozyogus for deficiencies of these genes also have variable phenotypes, suggesting that even amorphic alleles would give variable defects. For both loci, the transheterozygous
combinations of mutant alleles cause embryonic lethality but with relatively mild defects. Thus, the kak mutant embryos in
general look normal, but a few have a tail-up phenotype. Hemizygous mutant embryos, kak/Df(2R)CX, exhibit a range of
phenotypes, from apparently normal to the strongest phenotype, in which the embryo is much shorter, fails to involute the head,
and has very abnormal germ-band retraction and dorsal closure and a disrupted pattern of muscles. In addition,
in some kak mutant embryos, parts of the abdominal segments appear to be missing, leading to missing or fused denticle bands
in the cuticle preparations. The embryos transheterozygous for two bad mutant alleles have a low penetrance
tail-up phenotype and a higher penetrance of an anterior open phenotype. When these mutant alleles are
hemizygous over Df(2L)319, the frequency of the tail up phenotype in the mutant embryos is much higher (60%, a frequency
similar to that caused by the homozygous deficiency) and the abnormal head involution and dorsal closure is much clearer. The muscle pattern looks relatively normal although the muscles appear thinner and the pattern is
mildly disrupted. The localization of the PS2 integrin to the ends of the muscles appears normal (Walsh, 1998).
In each abdominal hemisegment of the Drosophila embryo, an array of 30 muscle fibers is innervated by about 34
motoneurons in a highly stereotyped and cell-specific fashion. To begin to elucidate the molecular basis of neural specificity in
this system, a genetic screen was conducted for mutations affecting neuromuscular connectivity. Focus was placed on 5 genes required
for specific aspects of pathway (beaten path, stranded, and short stop) and target (walkabout and clueless/abrupt) recognition. The
different classes of mutant phenotypes suggest that neural specificity is controlled by a hierarchy of molecular mechanisms:
motoneurons are guided toward the correct region of mesoderm, in many cases navigating a series of choice points along the
way; they then display an affinity for a particular domain of neighboring muscles; and finally, they recognize their specific muscle
target from within this domain (van Vactor, 1993).
Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and
cell-to-cell adhesions. In Drosophila the position-specific (PS) integrins mediate the formation and maintenance of junctions
between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in
integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing
surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. Seventy-six independent
mutations were isolated in 25 complementation groups, 15 of which contain more than one allele. Chromosomal sites were determined by
deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated.
Mutations in four known genes (blistered, Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new
genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three
other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide
valuable material for the study of integrin-dependent cell-to-cell adhesion (Prout, 1997).
Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse
morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. Methods have been developed to visualize
the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in
transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, several
genes have been identified that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include
kakapo, a large cytoskeletal protein related to plectin and dystrophin; starry night/flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a
substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis (Gao, 1999).
The peripheral neurons in each hemisegment of the Drosophila embryo are grouped into dorsal, lateral, and ventral clusters. The neurons within each cluster can be further
classified on the basis of their dendritic morphology; these categories are external sensory (es) neurons and chordotonal (ch) neurons, each containing a single dendrite; bipolar dendrite
(bd) neurons, each with two simple unbranched dendritic projections; and multiple dendrite (md) neurons with extensive dendritic arborizations. The md neurons are
thought to function as touch receptors or proprioceptors to sense body surface tension or deformation. The dendritic branching of md
neurons does not begin until 16 hr after egg laying (AEL) and continues until and beyond hatching. Because impermeable cuticle already forms at 16 hr AEL, md
neuron dendrites can not be visualized by standard antibody staining of whole mount embryos. It is possible to manually dissect individual embryos to allow antibody
access; however, this technique is too laborious to be useful for a large-scale mutant screen. To circumvent these technical problems,
an assay system was developed on the basis of expression of GFP in living embryos. First, a panel of Gal4 enhancer trap lines was screened to identify those that allow high levels of UAS-driven GFP expression in a subset of PNS neurons at the appropriate developmental stages. Of these, the
Gal4 line 109(2)80 was chosen. Recombination was performed to create a second chromosome harboring both the Gal4 109(2) 80 transgene and a
UAS-GFP transgene, but no background lethal mutations. A fly line homozygous for the Gal4 109(2) 80/GFP chromosome (denoted as Gal4 80/GFP) was then introduced. In the dorsal clusters of abdominal segments A1-A7, GFP expression labels both axons and dendrites of all six md neurons, one bd neuron, and
one tracheal innervating neuron, but not the es neurons. In addition, high levels of GFP expression are detected in the lateral and ventral clusters, and in the
antennomaxillary complex. Low levels of GFP fluorescence are also observed in a small subset of neurons in the central nervous system (CNS). The dendrites of dorsal cluster md neurons elaborate just underneath the epidermal layer. In larvae, these dendrites as revealed by Gal4 80/GFP are in tight
association with a layer of epidermal cells labeled by Kruppel-Gal4/GFP. It is thus possible to visualize the md neuron dendrites in the dorsal cluster in living animals.
A focus was placed on the development of these md neuron dendrites in wild-type as well as mutant embryos. To simplify this description, two types of
easily detectable dendrites were defined: dorsal branches grow toward the dorsal midline and lateral branches grow along the approximate anterior-posterior axis toward
segment boundaries (Gao, 1999).
The projection pattern of md neuron dendrites in a specific
hemisegment is largely invariant from embryo to embryo, on the basis of
observations on over thousands of embryos. The major characteristics of dendritic morphogenesis are summarized here. By
12-13 hr AEL, ch and es neurons have already sent out their initial
dendritic projections. At this stage, bd neurons have also extended
their dendrites. The primary dendrites of md neurons emerge at 13-14
hr AEL, 2 hr after the axons of PNS neurons have reached the CNS. The location of initial dendritic outgrowth and the orientation of this outgrowth are fairly invariant for md neurons. At 13 hr AEL, a dorsal
dendrite first emerges from one md neuron in the anterior of the dorsal
cluster; shortly after, a second dorsal dendrite emerges from a
posterior md neuron of the same cluster. Both dorsal dendrites extend
perpendicular to the anterior-posterior axis towards the dorsal
midline. Each md neuron (in the dorsal cluster only) sends out one
dorsally oriented primary dendrite; however, some md neurons have
additional primary lateral dendrites. The dorsal extension essentially
stops between 15 and 16 hr AEL, before the lateral branches start to
develop. Between 15 and 17 hr AEL, numerous transient lateral branches
extend and retract. These
branches undergo constant remodeling. Only a subset is
eventually stabilized between 18 and 20 hr AEL to become the final
lateral branches. At this stage, dorsal and lateral
branches are clearly distinguishable. The number of lateral branches in
a particular segment is similar from embryo to embryo. In addition, the
anterior and posterior dorsal branches within a hemisegment are clearly
separated by an area devoid of dendrites. Before hatching (23-24 hr
AEL), most lateral branches further elaborate tertiary branches before
and after they reach the segment boundary, but only a small number of
branches cross over into neighboring segments. At hatching, the dorsal branches have not yet reached the
dorsal midline so there is a clear dendrite-free zone near the dorsal
midline. After hatching, the dorsal branches resume elongation and
reach the dorsal midline by the second instar stage. The length and the
thickness of dendritic processes continue to increase with increasing
larval body size (Gao, 1999).
Before hatching, the lateral branches are regularly spaced and project toward the segment boundaries. This pattern is relatively invariant from embryo to embryo for a specific hemisegment. To investigate how the dendritic patterning develops, dendrite formation was monitored in living embryos from 15 to 16 hr AEL and time-lapse analysis was carried
out. Numerous lateral growth buds emerge
anterior or posterior to the dorsal branches and then retract.
Only a subset of these lateral branches elongates toward the segment
boundaries and becomes stabilized. During this process, the length and
orientation of dorsal branches remains largely unchanged. Numerous thin
processes at the tips of the lateral branches undergo rapid extension
and retraction. These thin processes are not labeled
by a Tau-GFP fusion protein, indicating that they might
not contain microtubules. This analysis reveals that dendritic
development is a dynamic process (Gao, 1999).
Two approaches were used to identify genes involved in dendritic
morphogenesis: (1) an investigation of the effects of previously isolated
mutations, and (2) a systematic mutant screen. It was reasoned
that dendrite development might share some common molecular mechanisms
with axon and tracheal development, because all of these processes
exhibit subcellular outgrowth and branching. The formation of lateral branches proceed in three steps, budding
from the dorsal branches, extension of numerous lateral branches, and
stabilization of a subset of these branches. Eight mutations in four complementation groups have been defined that result
in reduced numbers of lateral branches in the dorsal cluster of sensory
neurons. In one mutant the number of anterior lateral branches is
reduced from ~10 per hemisegment to ~4, whereas more branches
extend dorsally and appear to contact each other more extensively. The dendrite phenotype is seen in greater than 90% of the mutant
embryos. The individual dorsal branches may appear to have fused. This
is an illusion due to the strong GFP signals. Overall, this mutation
appears to primarily affect the extension of lateral branches. The lethal mutation was mapped to the cytological region between 50A3 and
50C6. The lethality of this mutant line is not complemented by
l(2)k03010, l(2)k04204, l(2)k15606, l(2)k05821, l(2)k05434, l(2)k10821,
and two alleles of short stop, shot1, and
shot2. The shot gene has been implicated in
axonal outgrowth and has been shown to be allelic to kakapo. kakapo encodes a protein of 5385 amino acids
with an alpha-actinin-type actin-binding domain and dystrophin-like
repeats. In late embryos, kakapo is expressed predominantly at muscle
attachment sites, with low levels of expression in the epidermal cell
layer and in neurons. The line
l(2)k03010 carries a P-element insertion in an intron of
kakapo, disrupting the normal kakapo expression. The other five lethal P lines, which fail to
complement a kakapo mutant, map to the same region as kakapo. Thus, it appears that the lethal mutation isolated is an allele of
kakapo; it was named kak30. Indeed,
kak30/kakk03010 embryos
exhibit the same dendritic phenotype as homozygous kak30. In addition, md neurons in kak30/shot2 embryos also
exhibited fewer lateral branches. Like other kak mutants,
kak30 homozygotes exhibit defects in axonal
outgrowth. kakapo
mutations are known to alter the dendritic sprouting of motoneurons. Taken together, these studies indicate that the kakapo
gene is required for the normal development of both dendrites and axons
in the CNS and PNS of Drosophila (Gao, 1999).
Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short stop (shot). In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led to an investigation of whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. It is therefore proposed that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization (Lee, 2002).
The tracheal lumen is initially closed at branch tips. Concurrent with branching morphogenesis, specialized cells at branch tips, known as fusion cells, join branches into a continuous tubular network. This process of anastomosis requires each fusion cell to recognize its partner in the adjacent hemisegment and to form a lumen that connects the two branches. Shotgun, the Drosophila homolog of the cell adhesion molecule E-cadherin is integral to the initial fusion cell contact. Mutations in shotgun affect tracheal branch extension and lumen formation at anastomosis sites, as do mutations in armadillo, the Drosophila homolog of its effector ß-catenin. E-cadherin and ß-catenin control cell polarity and tube extension in culture, suggesting an evolutionarily conserved role for cadherin-mediated cell adhesion in apical surface regulation (Lee, 2002).
Several studies suggest that these apical surface determinants such a Crumbs and E-cadherin regulate the cytoskeleton and therefore control lumen formation and morphology. For example, Crumbs may attach ßH-spectrin, an F-actin cross-linker, to the apical membrane in Drosophila and mutations in a C. elegans ßH-spectrin moderately enlarge the lumen of the excretory canal. E-cadherin interacts with F-actin via multiple mechanisms. These include binding to p120 catenin (p120ctn), a negative regulator of the RhoA GTPase. RhoA controls the formation of F-actin-containing focal adhesions and stress fibers in cultured cells. These interactions with RhoA also potentially regulate apical membrane protein targeting, a process important for lumen development in culture (Lee, 2002).
Little is known about the cytoskeletal structures required for lumen formation and how apical surface determinants are localized. This study identifies an F-actin-rich track that is associated with E-cadherin-dependent contacts between fusion cells that appears to guide deposition of apical surface determinants and lumen formation. During anastomosis, Short stop accumulates at these contacts and transiently along the track. Mutations in shot and constitutively active alleles of the Drosophila RhoA (RhoA) GTPase specifically disrupt this contact and the associated track. Remarkably, the interactions of Shot with F-actin and its binding to microtubules are functionally redundant in organizing the track, suggesting that Shot acts with other pathways to organize F-actin and microtubules, rather than as an F-actin/microtubule cross-linker. It is proposed that in fusion cells, RhoA antagonizes Shot to regulate E-cadherin-associated cytoskeletal structures required for apical surface determinant localization and lumen formation (Lee, 2002).
The results presented here provide further insights into how the cytoskeleton and associated proteins support contact formation and subsequent apical surface remodeling. The F-actin- and microtubule-binding domains of Shot are required to maintain and remodel E-cadherin contacts and to assemble a track of F-actin and Shot in fusion cells. This track initiates at the E-cadherin contact and extends outwards from it to connect with the existing apical assemblies of F-actin and Shot. It is proposed that the track guides new apical surface formation. Apical surface determinants and membrane appear to accumulate along the track, possibly by spreading from existing apical concentrations. This track may also enable the fusion cells to contract and to draw the existing lumenal surfaces closer, as fusion cells appear notably less compact in shot mutant embryos (Lee, 2002).
Loss-of-function shot and gain-of-function RhoA alleles have similar phenotypes in fusion cells, and RhoA disrupts Shot localization. It is therefore proposed that RhoA negatively regulates track assembly and E-cadherin contact remodeling by Shot. Apically organized F-actin and adherens junctions in other tracheal cells appear to develop normally in shot mutant and RhoAV14 embryos, suggesting specific requirements for shot and RhoA during new apical surface formation in fusion cells. It is proposed that Shot and RhoA regulate E-cadherin-dependent cell adhesion in selected developmental contexts (Lee, 2002).
shot is required in neurons for growth cone motility. shot is also required to remodel E-cadherin-containing contacts between tracheal fusion cells. Surprisingly, Shot proteins perform these distinct morphogenetic roles using different combinations of the same cytoskeletal interaction domains. In fusion cells, the binding sites for F-actin and microtubules appear functionally redundant. The F-actin binding domain is essential when the GAS2 microtubule binding site is absent, and the GAS2 microtubule binding site is essential when the F-actin binding site is absent. By contrast, during axon extension, the Shot behaves as an F-actin/microtubule cross-linker because the cytoskeletal interaction domains are both individually essential and required in the same molecule (Lee, 2002).
These observations suggest that direct interactions between Shot and cytoskeletal proteins organize the cytoskeleton in fusion cells. The F-actin and microtubule domains may directly enable the accumulation of their cytoskeletal partners at the E-cadherin contact. In support of this hypothesis, the structurally similar F-actin binding domain of plectin alters F-actin organization and the GAS2 motif stabilizes associated microtubules against depolymerization in cultured cells. Since Shot’s interactions either with F-actin or with microtubules suffice to organize both cytoskeletal elements, binding to either F-actin or microtubules may then enhance other organizing interactions between F-actin and microtubules (Lee, 2002).
These other interactions may involve molecules required for E-cadherin signaling. E-cadherins are physically linked to F-actin via the ß-catenin/alpha-catenin complex and to dynein, a microtubule-based motor, via ß-catenin. They can further regulate actin dynamics via association with p120, a RhoA antagonist; E-cadherins also stabilize microtubule minus ends in cultured cells. E-cadherin signaling may therefore affect other proteins mediating interactions between F-actin and microtubules. Candidates include other F-actin/microtubule cross-linkers, regulators of Rho family GTPases that bind to microtubules and F-actin-based motors that form complexes with microtubule-based motors. Further analysis will be necessary to identify these other molecules in fusion cells: these other cytoskeletal regulators may permit residual anastomoses in shot mutant embryos (Lee, 2002).
The analysis also indicates that RhoA is required for lumen formation, most probably by regulating the apical cytoskeleton or by affecting the transport of lumenal antigens. Similarities between RhoAV14 and shot mutant phenotypes suggest that RhoA could work either to antagonize Shot activity, or through parallel pathways acting on F-actin and microtubules. RhoA has many effectors that control F-actin distribution. RhoAV14 has been reported to stabilize subsets of microtubules in fibroblasts in culture via an F-actin-independent pathway. Shot localizes apically via its interactions with the cytoskeleton, and either these interactions or the cytoskeletal structures themselves may be RhoA-regulated (Lee, 2002).
In cells throughout the trachea, reduced RhoA activity disrupts lumen formation. Tracheal expression of RhoAN19 does not appreciably affect E-cadherin localization. In cultured epithelial cells, E-cadherin localization is also resistant to RhoAN19. These findings are consistent with RhoA functioning downstream of or parallel to E-cadherin. E-cadherin-associated p120ctn negatively regulates RhoA, but whether a similar pathway operates in Drosophila is unknown (Lee, 2002).
In fusion cells, RhoA can also function upstream of E-cadherin, as constitutively active RhoAV14 affects E-cadherin localization selectively in these cells. E-cadherin distribution is more dynamic in fusion cells than in other tracheal cells, and may therefore be more sensitive to RhoAV14. RhoAV14 also affects new E-cadherin contacts in culture. Further experiments will reveal whether Shot, RhoA and E-cadherin function in a common, evolutionarily conserved pathway to regulate apical surface remodeling in fusion cells (Lee, 2002).
Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, a genetic screen using the MARCM system was performed in mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains ~20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. Mutations that affect these different aspects of morphogenesis are reported; a subset has been phenotypically characterized. roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. These results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process (Reuter, 2003).
Adult neuroblast shot3 clones also display
abnormal processes projecting out from the calyx. They appear to
follow a curved route towards the antennal lobe, mimicking the trajectory of
the inner antennal cerebral tract (iACT), which contains the axons of a large
subset of projection neurons, the major input to the MB dendrites. In addition to these overextension phenotypes, neuroblast clones homozygous
for shot3, as well as for the new alleles identified in this study exhibit significantly reduced cell
number. Most or all neurons are gamma neurons, since their axons project to the
gamma lobe, suggesting a defect in the continuous generation of new neurons
from the neuroblast (Reuter, 2003).
Originally identified as a mutation in which embryonic motoneurons fail to
reach their targets, shot has subsequently been found to affect CNS and
PNS axon growth and dendritic morphogenesis in
Drosophila embryos. It also is required for morphogenesis of other
embryonic and imaginal epithelial and mesodermal tissues.
shot is allelic to kakapo, which encodes a large
cytoskeletal linker protein similar to vertebrate plakin.
Recent structure-function analysis suggests that the actin and
microtubule-binding domains must be present on the same molecule for Shot to
function in axon extension (Reuter, 2003).
shot phenotypes has been characterized in larval MB neuronal
morphogenesis based on defects in axon fasciculation and misguidance, since many
'axons' project out of the calyx, rather than following the peduncles. In light
of the finding that shot also affects dendritic development in the
embryonic PNS and CNS, the processes projecting from the calyx were re-examined. A
fusion protein made of a microtubule motor Nod and ß-galactosidase
(Nod-ßgal) is highly enriched in MB dendrites and their tips but largely
absent from axons. The processes projecting out of the calyx stain strongly for
Nod-ßgal.
Strikingly, however, in shot neuroblast clones, axons that follow the
normal route through the peduncle are also strongly labeled for Nod-ßgal. Sometimes the
Nod-ßgal fusion protein is present in both the dorsal and medial lobes
all the way to their terminals, despite the fact that total axon staining becomes
progressively weaker as they are further from the cell bodies (reflecting axon
growth defects). This is in contrast to wild type, where Nod-ßgal
staining rapidly diminishes along the axonal peduncle. These
observations suggest that neuronal polarity, as measured by the microtubule
polarity in axons and dendrites, is perturbed in shot mutants (Reuter, 2003).
During Drosophila oogenesis a membranous organelle called the fusome has a key function in the establishment of oocyte fate and polarity, ultimately leading to the establishment of the major body axes of the animal. The fusome is necessary for the microtubule-driven restriction of markers of oocyte fate to the oocyte, but the mechanism by which the fusome organizes the microtubules is not known. The spectraplakin Short stop (Shot) has been identified as a component of the fusome. Spectraplakins are giant cytoskeletal linker proteins, with multiple isoforms produced from each gene. Shot is the sole spectraplakin in Drosophila. The phenotype caused by the absence of Shot is not similar to that of other components of the fusome but instead is similar to the absence of the downstream components that interact with microtubules: the dynein/dynactin-complex-associated proteins Egalitarian and BicaudalD. Shot is required for the association of microtubules with the fusome and the subsequent specification of the oocyte in 16-cell cysts. Shot is also required for the concentration of centrosomes into the oocyte, a process thought to be independent of microtubules because it still occurs in the presence of microtubule depolymerizing drugs. This suggests that Shot may protect some microtubules from depolymerization and that these microtubules are sufficient for this process. It is concluded that Shot provides the missing link between the fusome and microtubules within meiotic cysts, that is essential for the establishment of the oocyte. Shot associates with the fusome and is required for microtubule organization. It is suggested that Shot does this directly, via its microtubule binding GAS2 domain (Röper, 2004).
The restriction of oocyte fate to a single cell within a cyst of 16 cells is a crucial process during oogenesis in Drosophila. This is the first event to result in asymmetry in each cyst, and this asymmetry will later be amplified and translated into the two embryonic axes. A number of players in this process have been identified, but the molecular mechanism that leads to the selection of a single cell as the oocyte has still to be fully elucidated. The spectraplakin Shot has been identified as a new player in this process and it is proposed that Shot provides a previously missing component required for this mechanism (Röper, 2004).
Shot is a component of the fusome, and the fusome itself has been implicated in most processes important for oocyte determination: (1) the fusome is asymmetrically inherited by the cystocytes, and it has been proposed that the cell that inherits more fusome in the first division of the cystoblast will be the future oocyte; (2) the fusome organizes the microtubule cytoskeleton within each cyst, apparently so that oocyte-specific components can be transported into and concentrated within the oocyte, and (3) the centrosomes appear to migrate along the fusome into the oocyte, where they later contribute to the establishment of the body axes within the oocyte. It is proposed that Shot is recruited to the fusome by binding to a component of the fusome, where it contributes to the organization of the microtubules, so that Dhc, Egl, and BicD can transport factors to enrich them in the oocyte (Röper, 2004).
The analysis of mutant phenotypes is consistent with the placement of Shot between the structural fusome components and the second level of components, Dhc, Egl, BicD, and Orb, in the genetic pathway of oocyte determination. Two particular phenotypes, the distribution of the synaptonemal complex and the migration of centrosomes, allow discrimination between these components. The differing requirement of these components for initiation of meiosis, as revealed by the synaptonemal complex, has led to the idea of the balance between activating (Dhc and BicD) and repressing (Orb and Egl) activities. The finding that shot has a variable effect on the appearance of the synaptonemal complex has therefore not aided efforts to place Shot within the pathway. In contrast, the finding that Shot and Dhc are the only components required for centrosomal migration places them at the top of the pathway because they are the only ones required for the transport of all known oocyte-specific components (Röper, 2004).
The requirement for Shot in the organization of microtubules is just apparent after the cystocytes complete their synchronous divisions. Thus, Shot does not bind the centrosomes that nucleate spindle microtubules to the fusome, nor does the absence of Shot affect the highly dynamic microtubules in dividing cysts. This differential effect of Shot on centrosomes and microtubules is likely due to the differing characteristics of mitotic and meiotic cysts: (1) early centrosomes actively nucleate microtubules, whereas the migrating centrosomes do not, indicating a molecular difference between them; (2) microtubules in mitotic cysts are highly dynamic and have to reassemble into spindles in synchronous cycles, whereas fusome-associated microtubules are stable (Röper, 2004).
It has not been possible to definitely position Dhc and Shot relative to each other within the pathway. Shot is the only one of these second-level proteins that is clearly part of the fusome at all stages in the germarium. In addition, the localization of Shot on the spectrosome and early fusome, prior to the accumulation of microtubules on the fusome, suggests that Shot is responsible for the organization of microtubules rather than that it binds to them once they have been recruited to the fusome. This was also supported by the fact that upon colchicine-mediated depolymerization of microtubules, Shot still localized to the fusome. The most likely scenario is that Shot is required for recruiting microtubules to the fusome and that Dhc is required for transporting the oocyte-specific factors along them into the oocyte. This is in agreement with the loss of Dynein accumulation in the oocyte in the absence of Shot, which is probably a secondary effect caused by the absence of polarized microtubules with their minus ends concentrated in the oocyte. The one inconsistency with this view is that Dhc is required for the integrity of the fusome, whereas Shot is not, suggesting that microtubule organization or transport plays an essential role in the stability of the fusome, independent of Shot function (Röper, 2004).
The only other protein known to localize to the fusome and affect microtubule stability is Par-1. However, the loss-of-function phenotype of Par-1 is less severe than loss of Shot: the association of microtubules with the fusome appears normal, and markers of oocyte fate become restricted to one cell but then fail to translocate from the anterior to the posterior cortex of the oocyte; consequently, oocyte fate is lost (Röper, 2004).
How does Shot fulfil the role of an essential mediator between the fusome and the fusome-associated polarized microtubule cytoskeleton in meiotic cysts? Shot contains a bona fide microtubule binding domain, the GAS2 domain, in its C terminus. This domain is likely to be present on the fusome because all described Spectraplakin isoforms, including those of Shot, contain the GAS2 domain, and cytoplasmic RNAs containing this domain were detected in the germarium. Thus, Shot could bind the fusome via one of its other domains, leaving the GAS2 domain free to bind, stabilize, or bundle microtubules in the vicinity of the fusome. In accordance with this, loss of Shot leaves the fusome unaffected but abolishes fusome-associated and polarized microtubules. There are at least three ways that Shot could contribute to the organization of the microtubules within the meiotic cysts. Shot could be involved in the nucleation of microtubules, particularly because neither the centrosomes nor gamma-tubulin plays a role in the nucleation of microtubules on the fusome after completion of mitoses. This would be a novel role for Shot; in most other cases it is functioning in combination with other microtubule-organizing centers. Alternatively, given the ability of the GAS2 domain of spectraplakins to bundle and stabilize microtubules, this could be Shot's function on the fusome. However, Shot appears on the fusome prior to accumulation of microtubules, so it is not recruited to the fusome solely by interaction with microtubules, as is also indicated by its absence from the mitotic spindles. The idea of a population of microtubules that are stabilized by Shot and resistant to depolymerization by colcemid helps to explain a previously anomalous result: that centrosome migration into the oocyte still occurs in the presence of colcemid. Because both Shot and Dhc are required for this, it suggests that this is still a microtubule-dependent process but can occur on colcemid-resistant, stable microtubules. Because Egl- and BicD-dependent concentration of each other and Orb into the oocyte is blocked by colcemid, the stable microtubules seemingly cannot provide a sufficient track to transport them. This may be because the number of molecules to be transported is greater relative to the number of centrosomes or because the rate of diffusion out of the oocyte is greater. Alternatively, two distinct mechanisms may exist for microtubule-dependent transport into the oocyte (Röper, 2004).
In addition to being required for the accumulation of stable microtubules, Shot could also assist in polarizing the microtubules so that components can be transported into the oocyte. Such a role would be consistent with recent observations on the effect of Shot in axon outgrowth. Mutations in shot were picked up in a screen that analyzed neuronal morphogenesis in Drosophila. shot mutants showed defects in axonal microtubule polarity, and these defects led to mixed polarity of neurons in the axons that in the wild-type situation have the plus ends pointing distally. This possible function is difficult to analyze because the loss of Shot abolishes all fusome-associated microtubules in the 16-cell cysts (Röper, 2004).
How does Shot being the link between the fusome and the polarized microtubule cytoskeleton help to explain how oocyte fate is restricted to one cell only? Starting with the first division of the cystoblast, the fusome is inherited asymmetrically, potentially leaving the future oocyte with the largest portion of fusome once the 16 cell stage is reached. Shot would act in establishing the fusome-associated microtubule-cytoskeleton in meiotic cysts, and slight differences in the distribution of factors that depend on the microtubule cytoskeleton could then be amplified over the process of cyst maturation from region 2a to region 3, leading to the selective enrichment of proteins first in the two pro-oocytes and later the oocyte. At a time when most structural fusome markers such as beta-Spectrin have disappeared, in region 3 and stage 1-2 of oogenesis, Shot is still highly enriched on structures that could be fusome remnants. Shot was still more concentrated toward the oocyte and strongly colocalized with the highly polarized microtubules, thus again placing it physically between the fusome and the microtubules, with the fusome acting as a template to mold this highly polarized microtubule cytoskeleton (Röper, 2004).
It is concluded that the spectraplakin shot is the only member of a class of proteins with similar loss-of-function or partial-loss-of-function phenotypes during oogenesis (this class includes Dynein heavy chain, the dynein/dynactin-complex associated proteins Egl and BicD, and the RNA binding protein Orb) to associate with the fusome and directly bind to microtubules. Short stop is the missing link between the fusome and the polarized microtubule cytoskeleton in meiotic cysts, essential for oocyte specification and establishment of the embryonic axes (Röper, 2004).
Certain Drosophila embryonic epidermal cells construct actin-based protrusions, called denticles, which exhibit stereotyped, column-specific differences in size, density and hook orientation. This precise denticle pattern is conserved throughout all drosophilids yet studied, and screening for mutations that affect this pattern has been used to identify genes involved in development and signaling. However, how column-specific differences are specified and the mechanism(s) involved have remained elusive. This study shows that the transcription factor Stripe is required for multiple aspects of the column-specific denticle pattern, including denticle hook orientation. The induction of stripe expression in certain denticle field cells appears to be the primary mechanism by which developmental pathways assign denticle hook orientation. Furthermore, the cytoskeletal linker protein Short stop (Shot) functions both cell-autonomously and non-autonomously to specify denticle hook orientation via interaction with the microtubule cytoskeleton. It is proposed that stripe mediates its effect on hook orientation, in part, via
upregulation of shot (Dilks, 2010).
The denticle field within most abdominal parasegments consists of seven columns of epidermal cells that can each produce several denticles. Although the posterior-most two columns of cells produce small, non-descript denticles, cell columns 1-5 produce denticles that are uniquely patterned from column to column. These cell columns differ from one another in the size, shape, number and hook orientation of the denticles they produce, as well as in the expression of certain cell fate markers and signaling components. Of note, cell columns 1 and 4 are the only columns that produce denticles that hook towards the anterior. This anterior hooking is unique, in the wild-type denticle field as well as in situations in which the field is artificially expanded. This prompted an examination of what it is that is unique about columns 1 and 4 that allows them to produce denticles that have a reversed hook orientation. To date, no specific cell fate determinant has been found to be common to these two cell columns that might provide a clue to this process (Dilks, 2010).
Screening for mutations that affect the cuticle pattern has been a powerful approach to identify genes involved in developmental decisions. Some years ago, it became clear that the column-specific differences in denticle shape and hook orientation occurred as a result of differential activation of the signaling pathways that pattern the epidermis. However, since then, no downstream targets have been identified that selectively affect column-specific denticle diversity, and the mechanisms involved have remained elusive. This study shows that stripe integrates signaling information and positional cues to specify denticle density and anterior hook orientation. Furthermore, stripe is shown to govern hook orientation, in part via the spectraplakin shot. Thus, the stripe-shot circuit has the potential to link the unpatterned blastoderm to the fully patterned cuticle (Dilks, 2010).
stripe is a genetic target of both the Hh and Egfr pathways, and is regulated by Hh (and Wingless) directly via distinct cis-regulatory elements. Thus, these data suggest that the signals responsible for the elaborate denticle pattern act by determining stripe expression in the appropriate cell columns (Dilks, 2010).
This work significantly advances the current understanding of how pattern diversity is produced within the cuticle. The allocation of cells into the smooth and denticle fields occurs when competitive signaling interactions between the Wingless and Egfr pathways result in restricted expression of the transcription factor Shavenbaby (Svb; Ovo -- FlyBase). Svb then regulates target genes that participate in actin filament, extracellular matrix and membrane morphogenesis to construct denticles. However, whereas svb is sufficient for denticle production, stripe is required for denticle diversity by regulating target genes that modulate denticle morphogenesis. How the Stripe targets interact with, and modify, the output of Svb targets in specific denticle columns will be interesting to explore (Dilks, 2010).
Taking the data together with previously published work, stripe has at least three functions in this epidermis: the specification of the muscle attachment sites, the reversal of denticle hooking orientation, and the determination of denticle density. At least some of these functions are separable, as it was shown that muscle attachment is not required for hook orientation. However, the ventral denticle pattern is conserved throughout all the drosophilids studied so far, which suggests that these three biological characteristics are functionally coupled. This, in turn, suggests an explanation for why select denticle columns are anteriorly hooked. As muscle attachments are required for larval movement, perhaps a reversal in hook orientation at the attachment site provides for more efficient traction. Similarly, the denticles produced by tendon cells, although fewer, are often more robust and might be better able to transmit tension. It is suggested that stripe could integrate patterning information to correlate muscle attachment with denticle number and shape, the latter via cytoskeletal remodeling or modulation of cuticle secretion components. It should be possible to design biophysical approaches to test these inferences (Dilks, 2010).
One Stripe target that this study has implicated is the spectraplakin shot. Shot acts intrinsically in denticle hooking, but is also important for the non-autonomous influence that one cell column exerts on another. The phenotypic similarities between stripe and its target shot strongly suggest that shot acts as part of a non-autonomous, stripe-controlled circuit, rather than in some parallel pathway. In turn, this suggests that the smaller, stripe-dependent Shot isoform functions in the non-autonomous circuit. In addition, although it was found that muscle tension is not required, the involvement of Shot suggests that the non-autonomous signal from tendon cells to the responding cells might still be mechanical in nature. This is the first work to demonstrate a functional role for either spectraplakins or microtubules in the shaping of actin-based protrusions (Dilks, 2010).
Although Shot is required intrinsically for columns 1 and 4 to construct properly hooked denticles, there are two pools of Shot to consider in this context. Thus, it is not possible to differentiate whether Shot is required intrinsically to respond to stripe-dependent cues or for a separate structural purpose. Of course, these possibilities are not mutually exclusive, and it seems likely that Shot is intrinsically required at multiple steps (Dilks, 2010).
Shot, actin filaments and microtubules are all found within the protrusion. Since Shot has the ability to bind both actin and microtubules simultaneously, the intrinsic role of Shot might be to physically link these cytoskeletal networks within the protrusion itself. In addition, it was recently demonstrated that MACF, a vertebrate homolog of Shot, possesses actin-induced ATPase activity, which is hypothesized to move microtubules along actin filaments (Wu, 2008). Thus, Shot could function within the protrusion by maintaining physical tension between the cytoskeletal networks (Dilks, 2010).
Conversely, as Shot is also enriched at the posterior cortex of the responding cells, this pool of Shot sits in close proximity to the denticles it influences. Thus, it is tempting to speculate that this pool of Shot is required to respond to the hooking cues given by the tendon cell. One unresolved issue is how Shot in column 1 becomes enriched along the 1-2 boundary. Since Shot protein fails to localize to the posterior cortex of columns 1 and 4 in the absence of stripe, it is tempting to speculate that stripe-dependent Shot stabilizes intrinsic Shot at select column boundaries (Dilks, 2010).
Although this study demonstrated that Shot is required in tendon cells for denticles in the adjacent column to hook properly, the specific molecular requirement for Shot in the tendon cell remains to be determined (Dilks, 2010).
The column 1-2 and 4-5 interfaces are enriched for a number of cytoskeletal components (Walters, 2006; Simone, 2010), and late stage embryos show dramatic enrichment of Shot protein at these interfaces. stripe does not control the special nature of these interfaces, and raises the wider question of what initially differentiates these interfaces from other column boundaries. It is speculated that the same signaling cues that initiate stripe expression (Hh and Spi) might also be responsible for the specialization of the 1-2 and 4-5 interfaces, and serve to coordinate cytoskeletal enrichment, muscle attachment and reversal of denticle hook orientation. It is speculated that stripe-dependent Shot could be required to stabilize this enrichment along the 1-2 and 4-5 column boundaries (Dilks, 2010).
Notably, in response to Stripe, a specialized microtubule array is formed within the tendon cell that runs from the basal to the apical cell surface. Shot localizes along this array in two places - the basal cell surface, where the EMA junction will form, and the apical portion of the cell, where the microtubule array links to the cortical actin cytoskeleton. It is not known whether the apical-basal microtubule array is responsible for the localization of Shot, or whether this array is required for denticle hooking, although this would be consistent with the requirement observed for microtubules (Dilks, 2010).
It appears that the non-autonomous stripe-shot circuit culminates in the localization of Shot protein across the boundaries where denticle hooks reverse. As spectraplakins can stabilize, localize and bundle microtubule arrays, as well as create specialized membrane domains via membrane protein clustering , a likely hypothesis is that Shot organizes a specialized microtubule array or other cytoskeletal complex at these interfaces (Dilks, 2010).
Since denticle hooks manifest themselves at the level of the cuticle and not in bending of the actin filaments themselves, it is speculated that this complex could control polarized secretion, extracellular matrix deposition or membrane-matrix attachment. Alternatively, since a role was discovered for microtubules in denticle hooking, perhaps Shot remodels the cytoskeleton via membrane-microtubule attachment, as it does in tracheal cells (Dilks, 2010).
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