polychaetoid
Drosophila muscles originate from the fusion of two types of
myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To
better understand muscle diversity and morphogenesis, a
large-scale gene expression analysis was performed to identify genes differentially
expressed in FCs and FCMs. Embryos derived from
Toll10b mutants were employed to obtain primarily muscle-forming
mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM
fate, respectively. The transcripts present in embryos of each genotype were
compared by hybridization to cDNA microarrays. Among the 83 genes
differentially expressed, genes known to be enriched in FCs or FCMs,
such as heartless or hibris, previously characterized genes
with unknown roles in muscle development, and predicted genes of unknown
function, were found. These studies of newly identified genes revealed new patterns of gene
expression restricted to one of the two types of myoblasts, and also striking
muscle phenotypes. Whereas genes such as phyllopod play a crucial
role during specification of particular muscles, others such as
tartan are necessary for normal muscle morphogenesis (Artero, 2003).
Notch and Ras signaling pathways interact during muscle progenitor
segregation. The results suggest that phyl and
polychaetoid (pyd) may be additional links between the two
signaling pathways in FCs. phyl and pyd both interact
genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during
specification of SOPs and their progeny. This study
shows a similar regulation in muscle cells, where Notch signaling represses
phyl expression and Ras signaling increases phyl expression. Likewise, in the
nervous system, the segregation of SOPs requires pyd, a Ras target
gene, to negatively regulate ac-sc complex expression. Similarly, Pyd
may restrict the muscle progenitor fate to a single cell, perhaps by
regulating lethal of scute transcription. Thus, Pyd would collaborate
with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).
FCMs appear to integrate Ras and Notch signaling differently. Two genes
whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).
Embryos that lack or ectopically express phyl have morphological
defects in specific muscles, for example, in LL1 and DO4 in response to
diminished phyl function, and in DT1 and LT4 in response to increased
phyl function. The morphological defects in the loss-of-function
embryos appear to be due to a failure to specify particular FCs, a conclusion
that is based upon missing or abnormal production of the FC marker Kr. In eye
development and SOP specification, Phyl directs degradation of the
transcriptional repressor Tramtrack. In a subset
of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack
for degradation. The presence of Tramtrack would contribute to the specific
identity program of the muscle. Since Tramtrack is expressed in the mesoderm,
this possibility is likely. Alternatively, Phyl may be required for targeted
degradation of some other protein in a subset of FCs. The molecular partner
for Phyl during muscle differentiation is unknown, although preliminary data
suggest that sina is also expressed in somatic mesoderm and thus may
be its partner. These studies have
identified a new role for Phyl in muscle progenitor specification and suggest
the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).
Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).
As with the transcription factors Trh and Vvl, which
are involved in orchestrating early events of tracheal development, Ttk plays
a role in orchestrating several late tracheal events. Ttk69 has been found to
act mostly as a repressor. This study identified Ttk targets that appear to be
negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others
appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In
this latter case, Ttk might be converted into a positive regulator, as already
described during photoreceptor development (Araújo, 2007).
This study identified multiple tracheal requirements for Ttk. Interestingly, most
of them depend on Ttk regulating events downstream of cell fate specification,
at the level of cellular responses. Additionally, a few other
requirements depend on cell fate specification, as has been described for most
other functions of Ttk in other developmental situations. For instance, Ttk
regulates fusion cell specification by acting as a target and mediator of Notch,
as occurs during sensory organ development and
oogenesis. Such regulation of Ttk by N might be post-transcriptional,
as occurs during sensory organ development.
Remarkably, it was found that, although Ttk is sufficient to repress esg
expression in fusion cells, it might not be the only esg- and fusion
fate-repressor, because absence of Ttk does not increase the number of
Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).
Cell rearrangements during development are common to most animals and
ensure proper morphogenesis. During tracheal development, many branches grow
and extend by cell intercalation. Several cellular and genetic aspects of tracheal
intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).
This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect
intercalation. In fact, modulation of AJs has been proposed to play a role
during intercalation. However, Pyd cannot be the only Ttk effector of
intercalation, because the pyd mutant phenotype is much weaker than
that of ttk mutants. Accordingly, it was found that, in ttk
mutants, cells in branches that usually intercalate remain paired and
cuboidal, and appear unable to change shape and elongate. Although other
explanations could account for the impaired intercalation detected in
ttk mutants, it is proposed that inefficient cell shape changes represent
the main cause, and might prevent the proper accomplishment of several events,
such as the sliding of cells, formation of a first autocellular contact and
zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape
changes, particularly cell elongation, are an obligate requisite for different
steps of intercalation. Other targets of Ttk might presumably be regulators or
components of the cytoskeleton involved in cell shape changes. It is relevant
to point out here that Ttk has also been proposed to regulate morphogenetic
changes required for dorsal appendage elongation (Araújo, 2007).
How does Ttk relate to the known genetic circuit (Sal-dependent) involved
in intercalation? Being a transcription factor, Ttk initially appeared as an
excellent candidate to participate in this genetic network by regulating
sal and/or kni expression. However, both these
genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus
sal upregulation. For instance, although both situations block
intercalation, cells expressing sal, unlike those lacking
ttk, are still able to undergo a certain change in shape, from
cuboidal to elongated. Therefore, the results
fit a model in which Ttk acts in a different and parallel pathway to Sal
during intercalation. Consistent with this model, it was found that Ttk is not
sufficient to promote intercalation on its own, because its overexpression
cannot overcome the inhibition of intercalation imposed by Sal in the DT.
Finally, genetic interactions also favour this
model, because it was found that: (1) ttk overexpression did not rescue
lack of intercalation produced by sal overexpression (even though it
rescued the intercalation defects of ttk mutants), and (2) absence of
sal (by means of the constitutive activation of the Dpp pathway) does
not overcome the intercalation defects of ttk mutants. Therefore, it is
proposed that Ttk promotes intercalation by endorsing changes in cell shape,
but absence of Sal is still required to allow other aspects of intercalation
to occur (Araújo, 2007).
Tube size regulation is essential for functionality. It was found that Ttk is
involved in such regulation. Tube expansion and extension relies on a luminal
chitin filament that assembles transiently in the tracheal tubes. The
metabolic pathway that leads to chitin synthesis involves several enzymes,
among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the
proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm
and Serp. SJs are also required to regulate tube size and it was
proposed that they exert this activity, at least partly, via the control of
the apical secretion of chitin modifiers. The current
results revealed that ttk acts as a key gene in tube size control,
playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).
SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss
of the transepithelial diffusion barrier, whereas accumulation of other
markers and SJ localisation remained apparently unaffected. It is speculated that
Ttk transcriptionally controls one or several SJ components that contribute to
maintain the paracellular barrier and to control a specialised apical
secretory pathway. As a result, chitin binding proteins such as Verm or Serp
are not properly secreted (Araújo, 2007).
It was also found that mmy is transcriptionally regulated by Ttk.
mmy tracheal expression positively depends on a mid-embryonic peak of
the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).
ttk mutants are defective in the formation of terminal and fusion
branches. These defects are due, in part, to non-autonomous, secondary and/or
pleiotropic effects of ttk. For instance, ttk mutants
exhibit a dorsal closure defect, which prevents the approach and fusion of
contralateral dorsal branches. Additionally, terminal and fusion branches depend on
correct cell type specification, which did not reliably occur in ttk
mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation.
These tracheal cell identity specification defects might be related to
non-autonomous requirements of ttk. For instance, DSRF is not
properly expressed in ttk mutants because of an abnormal expression
of its regulator, Bnl (Araújo, 2007).
It is important to note that, in spite of these non-autonomous and cell
fate specification defects, two pieces of evidence indicate that ttk
also plays a specific and autonomous role in the formation of terminal and
fusion tubes. First, markers for fusion and terminal cell specification were
expressed in many tracheal cells of ttk mutants, but yet most of
these cells did not form terminal or fusion branches. Second, only the
tracheal expression of ttk in ttk mutants (but not the
constitutive activation of the btl pathway, which regulates the
terminal and fusion identity) was able to restore the formation of terminal
branches (Araújo, 2007).
A common feature of terminal and fusion branches is that they both display
intracellular lumina that lack detectable junctions. The cellular events that
precede the formation of fusion and terminal branches differ, but the
mechanisms by which their intracellular lumina form has been proposed to be
comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However,
neither of these two cell types finalised the cellular events leading to tube
formation. It has been proposed that the lumen of terminal and fusion branches
forms by the coalescence of intracellular vesicles that use a 'finger' tip
provided by the neighbouring stalk cell as a nucleation point.
Interestingly, it was found that vesicles containing luminal material are less
abundant in ttk mutants. These observations suggest a new role for
Ttk in the formation of intracellular lumina in distinct cell types.
Intracellular lumen formation also occurs in other branched tubular
structures, such as in vertebrate endothelial cells and in
the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).
ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).
The Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. Multiple roles of Ttk have been discovered in the development of the tracheal system on the morphogenetic level. This study sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. Gene expression changes were profiled after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes (Rotstein, 2011).
The microarray results confirm previous observations and provide new data for the different Ttk tracheal requirements. For instance, the transcription factor Esg, which plays a pivotal role in fusion cell identity specification is lost when Ttk is over-expressed, but still present in Ttk loss-of-function conditions. The microarray data confirm this regulation, and in addition identifies other genes already shown to directly or indirectly modulate fusion fate as Ttk targets, like hdc, CG15252, or pnt. Similarly, polychaetoid (pyd), which has been identified as a Ttk target in in situ hybridisation analysis, is differentially expressed in the microarray conditions (it should be noted however that pyd is not formally a candidate due to inconsistencies between microarray replicates; in fact only splice variant pyd-RE shows a response), explaining in part the requirement of Ttk in tracheal cell intercalation. In addition, it is tempting to speculate about other candidate targets to mediate this function of Ttk in intercalation, like canoe for instance, which has been recently shown to act with pyd during embryogenesis (Rotstein, 2011).
The microarray analysis pointed to a regulation of the Notch signalling pathway or its activity by Ttk, likely acting as a negative regulator. In contrast, it has previously been observed that Ttk acts as a downstream effector of N activity in the specification of different tracheal identitites. Indeed, it was shown that Ttk levels depend on N activity in such a way that when N is active, Ttk levels are high, whereas when N is not active, Ttk levels are low. Thus, lower levels of Ttk were observed in tracheal fusion cells due to the inactivity of N there. Therefore, Ttk acts as a target of N in fusion cell determination. Now, the results of the microarray add an extra level of complexity to the Ttk-N interaction. The observation that in turn Ttk also transcriptionally regulates several N pathway components suggests that Ttk is involved in a feedback mechanism that could play a pivotal role in biasing or amplifying N signalling outcome (Rotstein, 2011).
Interactions between Ttk and N have been observed in different developmental contexts, emphasising the importance of such regulations. Several examples illustrate the regulation, either positive or negative, of Ttk expression by N activity. In addition, a recent report provides evidence of a regulation of N activity by Ttk and proposes a mutually repressive relationship between N and Ttk which would also involve Ecdysone signalling. The results are consistent with many of these observations, indicating that they could represent general molecular mechanisms of morphogenesis. Thus, tracheal cell specification could serve as an ideal scenario to investigate the intricate, and often contradictory, interactions between N and Ttk and the complexity of N signaling (Rotstein, 2011).
It is suggested that the membrane-associated Polychaetoid protein is involved in the signaling
pathway that activates extramacrochaetae (emc) expression. The tam1 mutation shows a genetic interaction with emc and reduces the amount of the emc transcript. Achaete protein is hyperexpressed in tam1 mutants (Takahisa, 1996).
The major output of the JNK pathway in dorsal closure is Dpp. Dpp is thus considered to be the most downstream component of the pathway, involved in the induction of cell shape changes in lateral ectodermal cells ventral to the leading edge. Because mutations in some of the members of the JNK pathway have been shown to eliminate dpp expression in the leading edge cells, dpp expression was examined in canoe mutants. dpp expression in leading edge cells is decreased by canoe mutation (Takahashi, 1998).
The observations that pyd and cno mutations genetically interact, and that Pyd and Cno colocalize in ectodermal epithelia, prompted an examination of the possibility for direct binding of Pyd and Cno using the yeast two hybrid system. The binding of Cno and Pyd is mediated by two different domains of the respective proteins. The N-terminal half of Cno selectively binds to the N-terminal end of Pyd where PDZ12 and PDZ2 domains are present. In contrast, the C-terminal half of Cno specifically binds to the Pyd C-terminal fragment, including the proline-rich region. Using deletion constructs for Pyd reveals that a 162-amino acid segment at the very end of the proline-rich region is indispensable for the interaction of Pyd with the Cno C-terminus. Direct interaction of Pyd with Cno was also demonstrated using an in vitro binding assay (Takahashi, 1998).
The gene coding for Drosophila Cortactin was cloned as a Polychaetoid interacting protein using the yeast two hybrid technique. Drosophila Cortactin is a 559-amino acid protein highly
expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. An SH3 domain of approximately 60 amino acids dominates the C-terminal region of Cortactin; no other significant structural motif is found. The SH3 domain is known to bind to a PXXP motif often found in
proline-rich regions. The 139-amino acid region extending from
amino acid 1115 to 1253 of Pyd is sufficient for the interaction. This region contains four isolated PXXP motifs and
three overlapping motifs (PFKPVPPPKP). The Cortactin
SH3 domain binds to the PXXP motif located at the center of the Pyd C-terminal proline-rich domain, where three PXXP
motifs are clustered (Katsube, 1998).
To examine the in vivo association of Pyd and Cortactin, Canton-S wild-type embryo lysates were precipitated with the rabbit anti-Drosophila Cortactin antiserum, the rabbit anti-Pyd
antiserum, or the respective preimmune sera. The precipitates were analyzed by Western blotting. Western blotting with rat anti-Cortactin antiserum reveals that the Cortactin
105-kDa form is specifically coprecipitated by the anti-Pyd antiserum. These results clearly prove
that Cortactin associates with Pyd in Drosophila embryo cells (Katsube, 1998).
Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, this study demonstrated that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, a core antagonistic interaction was identified bet en Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, it is proposed that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization (Djiane, 2011; full text of article).
PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. This study screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns4,5P2-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. The transmembrane protein Crumbs was identifed as a Pyd PDZ2 ligand, and the relation between peptide and PtdIns4,5P2) binding was probed. Contrary to the prevalent view on PDZ/peptide/lipid binding, no competition was found between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns4,5P2 6-fold. These results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns4,5P2 binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals (Ivarsson, 2011).
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