branchless
See the embryonic expression pattern of bnl at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
Early branchless expression (Stage 5) occurs around the dorsal aspect of the cephalic furrow [Images] and the posterior transverse furrow. By stage 8 ventral expression occurs around the cephalic furrow, while dorsal furrow expression diminishes.
branchless is expressed in epidermal clusters outside the developing tracheal system at essentially every position where a major tracheal branch will bud and grow, suggesting that BNL is an attractive factor that induces and guides the outgrowth of the major branches. The expression pattern is complex and dynamic. At stage 11, just before tracheal branching begins, bnl expression appears in five small clusters of epidermal cells arrayed around the tracheal sac, at positions where the five primary tracheal branches will soon bud. As the primary branches grow by cell migration over the next 2 hours (stages 12 and 13), expression in the clusters decreases. This appears to occur in a specific spatial pattern: the bnl-expressing cells closest to or contacting the growing tracheal branches lose expression first, with the tracheal cells continuing to migrate toward the remaining bnl-expressing cells. Two more cell clusters begin expressing bnl as expression in the other clusters turns off, presaging the subseuent outgrowth of specific branches. For example, the ganglionic branch, which initially grows toward one cluster, continues toward a cluster that expresses bnl later, finally reaching the central nervous system. A branch of bnl-expressing cells in the head marks the path of the pharyngeal branch.
Localized ectopic expression of bnl induces branching toward the ectopic source, while general overexpression disrupts the normal pattern of branching, with fine branches growing out in random directions. Expression is observed in a metameric pattern in the central nervous system (Sutherland, 1996).
branchless activates late programs of tracheal branching. Secondary branches sprout from the ends of primary branches and from a few internal positions. Ectodermal bnl is expressed near positions where secondary branch markers begin to be expressed, suggesting that bnl might also play a role in selecting where secondary branches sprout (Sutherland, 1996).
In the developing tracheal system of Drosophila, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic signaling is strictly
required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, it has been found that the two signaling systems affect different cellular functions required for branching
morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth (Ribeiro, 2002).
To investigate possible dynamic cell shape changes accompanying tracheal cell movement in vivo and link them to the different signaling systems, three-dimensional reconstructions were used of confocal images of living embryos expressing different GFP-tagged proteins in the developing tracheal system. Expression of GFP-actin, driven in tracheal cells by the btl-Gal4 driver line, revealed fine cellular protrusions from cells at the tip of growing branches after initiation of germ band retraction when migration starts. Such cell extensions are most prominently observed in the developing dorsal and ganglionic branches as well as in the dorsal trunk anterior and posterior. During the early stages of branch outgrowth, these cellular extensions were generally short and relatively few in number (Ribeiro, 2002).
In order to visualize possible cell shape changes during later migratory phases, a GFP protein fused to the myristilation site of the Src protein was expressed under the indirect control of the btl enhancer. This GFP fusion protein labels cellular membranes and thus traces the outline of tracheal cells. Three-dimensional reconstruction of dorsal branches using a stack of optical sections through a living embryo expressing this construct revealed that the two leading cells form numerous membranous extensions in all directions; extensions from more proximal cells of the dorsal branch or from cells of the dorsal trunk were only seen very rarely. To ascertain that these membranous extensions contain actin, embryos of the same developmental stage expressing the GFP-actin construct were also examined. Clearly, a similar network of cell extensions was also discernable with actin-coupled GFP. The diameter of these extensions was in the range of 0.3 to 0.4 µm (Ribeiro, 2002).
To investigate the dynamics of the formation of these cellular extensions, a time-lapse confocal analysis was performed of actin cytoskeletal activity in tracheal cells during the migration process, with special emphasis on dorsal and ganglionic branches. In both cases, actin-containing extensions were seen most prominently in the cells at the tip of the branches. Each of the two leading cells in the dorsal branches formed numerous dynamic cellular outgrowths. In the ganglionic branches, cell extensions were most prominently seen in the single leading cell. The formation of cell extensions was extremely dynamic and their topology changed dramatically with time. Some extensions were found to be short lived; others were more stable and rather long (up to 20 µm). It is concluded from these data that tracheal cell migration is accompanied by the formation of thin, dynamic actin-containing cell extensions, referred to here as filopodia (Ribeiro, 2002).
Until now, the only known chemoattractant for tracheal cells is the FGF-like protein encoded by the branchless (bnl) gene. Since bnl is expressed in nontracheal cells adjacent to the tip of migrating branches, it is likely that tracheal cells form extensions as a result of the activation of the Bnl/FGF signaling cascade. To test this prediction, the cytoskeletal activity of tracheal cells was examined in mutant embryos in which Bnl/FGF signaling was disrupted. The failure of tracheal cells to migrate in btl mutants is accompanied by a failure to form filopodial extensions. Lack of filopodia is also observed in embryos mutant for bnl and dof. These experiments demonstrate that Bnl/FGF signaling is required for filopodia formation. Ectopic expression of bnl in all tracheal cells leads to the formation of ectopic filopodia. These experiments provide clear evidence that tracheal cells react to the Bnl chemoattractant with the formation of dynamic actin-containing filopodial extensions. Further experiments show that Bnl/FGF signaling induces cytoskeletal dynamics in the absence of transcriptional induction of any known gene, and it is likely that the signaling input directly influences cytoplasmic events in the absence of changes in nuclear transcription (Ribeiro, 2002).
The formation of tracheal branches via directed cell migration requires input
from other signaling systems in addition to Bnl/FGF. Activation of the Dpp signal transduction cascade is essential in dorsal and ventral tracheal cells prior to migration for the subsequent formation of dorsal and ventral (ganglionic and lateral trunk anterior and posterior) branches. In the absence of the Dpp receptors Thick veins (Tkv) or Punt (Put), dorsal branches completely fail to develop and ventral branches are strongly affected. Dpp induces the expression of the genes kni and knrl in the ventral and dorsal cells of the placode; in the absence of these two nuclear proteins, dorsal branches are absent and ventral branches are strongly abnormal (Ribeiro, 2002).
Knowing that Bnl/FGF acts as a chemoattractant for tracheal cells, and having shown above that Bnl/FGF signaling induces filopodial activity, one must wonder why cells need input from the Dpp signaling cascade for a directed movement to the Bnl/FGF source. Is the Dpp response a prerequisite for the subsequent induction of filopodia by Bnl/FGF? Or do dorsal branch cells respond to Bnl/FGF with the formation of filopodia even in the absence of Dpp signaling input, yet fail to migrate properly? In order to find out how these different signaling systems interact in vivo, the cytoskeletal activity of tracheal cells was examined in the absence of Dpp signaling, with particular emphasis on dorsal branches. However, both tkv and put mutants lack dorsal expression of bnl; therefore, they not only lack the Dpp signaling input but also the Bnl/FGF signaling input. In line with the absence of dorsal bnl expression, cellular extensions were not observed in dorsal tracheal cells in put mutants when analyzed in vivo using the GFP-actin fusion protein (Ribeiro, 2002).
In the absence of Dpp signaling, tracheal cells close to the dorsal bnl-expressing ectodermal cells are able to form actin-containing filopodial extensions and initiate dorsal migration. However, the lack of Dpp signaling, which results in the lack of expression of the kni/knrl target genes, leads to failure to form a dorsal branch, and the short, bud-like dorsal outgrowths eventually reintegrate into the main dorsal trunk. Consistent with this interpretation, cells forming the initial dorsal outgrowth in Dad-expressing embryos in rare cases generated a dorsal trunk-sized lumen. These dorsally directed stumps of dorsal trunk were also visible in third instar larvae. Such dorsal trunk-like buds are also seen in mutants that lack Dpp-induced kni/knrl in the tracheal system, indicating that dorsal migration also takes place in these mutants. These buds are never observed in put mutants, presumably due to the lack of dorsal expression of the chemoattractant Bnl/FGF (Ribeiro, 2002).
These results demonstrate that while Bnl/FGF signaling is necessary and sufficient for the induction of filopodial activity in tracheal cells and for cell migration in the strictest sense (cells do start to migrate dorsally when Dpp signaling is inhibited by Dad), Bnl/FGF is apparently not sufficient to allow productive dorsal branch outgrowth. For dorsal branches to grow out and form, Dpp signaling input is strictly required, in addition to filopodial activity induced by Bnl/FGF. Thus, Dpp signaling does not appear to collaborate with Bnl/FGF in filopodia production and motility, but instead to target cellular functions distinct from those targeted by Bnl/FGF signaling. Thus, despite the essential and crucial role of Bnl/FGF, chemoattraction is not sufficient for successful tracheal branching and, despite the requirement of Dpp for dorsal branch formation, migration per se is not affected (Ribeiro, 2002).
Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target
tissues to facilitate gas exchange. The tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved
axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct
orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by
Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. It is proposed that the
attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface (Englund, 2002).
The tracheal system develops from 20 clusters of ectodermal cells, each containing about 80 cells. After
invagination and without further cell division, each epithelial cluster sequentially extends primary, secondary, fusion and terminal branches to generate
the tubular network that facilitates larval respiration. The regular outgrowth pattern of the primary branches is determined by the localized expression of signaling factors in the surrounding tissues. Among these signals, Branchless (Bnl), a member of the Fibroblast Growth Factor family, first directs the outgrowth of multicellular branches to its site of expression, and it then induces the activation of a set of terminal branching genes in the leading cells of the primary branches. Single terminal cells then form a
unicellular branch, migrate over substantial distances and finally stretch and bind to distinct parts of the target tissue to facilitate respiration. A single
terminal cell of each ganglionic branch (GB), for example, targets each hemisegment of the embryonic ventral nerve cord (VNC). A cluster of
bnl-expressing cells just outside the CNS attracts the GB toward the CNS. The GB cells migrate ventrally along the intersegmental nerve (ISN), but
just before reaching the entry point into the CNS, they break their contact with ISN and turn posteriorly to associate with the segmental nerve (SN). This substrate switch is promoted by the expression of adrift (aft), a bnl-induced gene required in the trachea for efficient
entry into the CNS. Inside the CNS, the GB1 cell extends over a distance of about 50 µm, from the entry point into the CNS
via four different neural and glial substrata to its target on the dorsal side of the neuropil. During the first 20 µm of its journey
inside the CNS, the GB1 cell moves its cell body and nucleus along the exit glia, the SN and ventral longitudinal glia towards the midline. The rest of the path is covered by a long cytoplasmic projection that turns dorsally at the midline and reaches the dorsal part of
the neuropil by the end of embryogenesis. The signals that guide GB1 migration inside the CNS are not known but the substrata that the GB contacts along its path could potentially provide important guidance cues (Englund, 2002).
The Drosophila tracheal system is an interconnected tubular respiratory network, which is formed by directed
stereotypic migration and fusion of branches. Cell migration and specification are determined by combinatorial
signaling of several morphogens secreted from the ectoderm. A group of ectodermal cells,
marked by Stripe (Sr) expression coordinates tracheal cell migration in the dorsoventral axis. Sr, an EGR family
transcription factor, is known to regulate muscle migration. Sr ectodermal cells also provide signals that are utilized for tracheal migration. These cues are separated in the time course of embryonic
development. Initially, tendon-precursor cells are in close proximity to the tracheal cells, and later, when tracheal
migration is complete, the muscles displace the trachea and attach to the tendon cells. sr-mutant embryos exhibit
defects in migration of all tracheal branches. Although the FGF ligand Branchless (Bnl) is expressed in a subset of
tendon-precursor cells independently of Sr, Bnl functions cooperatively with proteins induced by Sr in attraction of tracheal branches (Dorfman, 2002).
Sr mutant and misexpression experiments imply that Sr
induces an essential, yet noninstructive, signal for tracheal
migration. To assess whether Sr is able to modulate or
enhance the chemoattractive ability of Bnl, Sr
or Bnl were expressed alone or simultaneously, in the salivary glands of
larvae, which normally lack any trachea. Tracheal branches are attracted
to the salivary glands upon ectopic expression of Bnl. Misexpression of Sr in the salivary glands attracts muscles. However, misexpression does not attract tracheal branches. In contrast, misexpression of both Sr and Bnl leads
to dramatic tracheal sprouting in the salivary glands, much
more pronounced than following Bnl misexpression alone. This experiment demonstrates that Sr can enhance the chemoattractive activity of Bnl.
One possible way by which Sr cells may facilitate Bnl
activity is through expression of an extracellular component
by the tendon-precursor that is able to trap secreted
Bnl, thus increasing the local concentration of this potent
chemoattractant. Similarly, the activity of Bnl has been shown
to depend on heparan sulfate proteoglycans. This mode of action is also employed by tendon cells via the activity of Kakapo to concentrate Vein in
tendon/muscle junctions. An
alternative way for Sr to provide synergizing cues to the
tracheal cells is through induction of cell-cell contact or
cell-matrix interaction between the tendon and the tracheal
cells. However, the putative Sr-target genes that may
mediate the tendon-tracheal interaction remain to be identified (Dorfman, 2002).
In conclusion, it has been shown that ectodermal cells expressing
Sr provide sequential signals for migration of tracheal and
muscle cells. While the signal for muscles is instructive,
the cue for tracheal migration synergizes with the restricted
and dynamic Bnl signal. Sr and Bnl functioned cooperatively
to attract trachea to the salivary glands upon ectopic
expression. Based on this result, it is tempting to speculate
that, in a similar manner, the tendon cells normally mark
the correct path to the tracheal cells. This may be achieved
by expression of cell surface molecules that restrict the
diffusion of Bnl and thus necessitate tight association
between the trachea and ectoderm for proper migration (Dorfman, 2002).
Development of the ectodermally derived Drosophila tracheal system is based on branch outgrowth and fusion that interconnect metamerically arranged tracheal subunits into a highly stereotyped three-dimensional tubular structure. Recent studies have revealed that this process involves a specialized cell type of mesodermal origin, termed the bridge-cell. Single bridge-cells are located between adjacent tracheal subunits and serve as guiding posts for the outgrowing dorsal trunk branches. Bridge-cell-approaching tracheal cells form filopodia-like cell extensions, which attach to the bridge-cell surface and are essential for the tracheal subunit interconnection. The results of both dominant-negative and gain-of-function experiments suggest that the formation of cell extensions require Cdc42-mediated Drosophila fibroblast growth factor activity (Wolf, 2002).
Drosophila FGF signalling is used reiteratively during the different developmental steps of tracheal organogenesis. It triggers primary branch outgrowth, controls secondary branch sprouting and mediates terminal branching in response to the signals produced by oxygen-starved cells. Evidence is provided that FGF also acts as a growth factor that stimulates the development of tracheal cell extensions necessary for tracheal branch fusion. This conclusion is based on the observations that tracheal cell extensions are missing in FGF-signalling mutants while the formation of ectopic extensions is induced by ectopic FGF/Bnl. Gain-of-function experiments suggest that the FGF/Bnl-dependent cell extension formation is mediated via the Rho-like GTPase Cdc42 (Wolf, 2002).
What is the function of the tracheal cellular extensions? During early tracheal development, FGF/Bnl is instructive for tracheal branch outgrowth. However, gain-of-function experiments indicate that the dorsal trunk forms independently of FGF/Bnl-guidance, suggesting that FGF/Bnl provides a permissive rather than an instructive signal for the dorsal trunk formation. The mesodermal bridge-cell guides the dorsal trunk branches. The results establish that the FGF/Bnl-induced tracheal cell extensions are necessary for bridge-cell-mediated dorsal trunk formation. Several observations support this conclusion. (1) Bridge-cells are in direct contact with the leading edges of the outgrowing dorsal trunk branches that form cell extensions. (2) While the extensions grow out in an anterior or a posterior direction, they are in direct association with the bridge-cells. (3) The cell extensions interconnect adjacent tracheal metameres ~2.5 h before the dorsal trunk branches fuse. (4) Ectopic expression of dominant-negative Cdc42, which represses the formation of cell extensions, frequently induces the lack of dorsal trunk branch interconnections. (5) Cdc42-activated ectopic extensions partially rescue fusion of dorsal trunk rudiments in embryos that lack FGF/Bnl (Wolf, 2002).
The ability of cell extensions to mediate branch fusion via bridge-cells is restricted to dorsal trunk formation. This specific function is likely to be essential since dorsal trunk branch fusion is a multicellular process while all other tracheal interconnections are single cell fusions. Furthermore, dorsal trunk fusion precedes the other fusion processes although all branches bud out at the same time. In addition, different surrounding cell matrices may require various mechanisms for branch outgrowth. In fact, previous work has shown that dorsal branch cells follow a path along a pattern of grooves left between the muscle precursor cells of adjacent metameres, whereas the dorsal trunk branches remain in association with a contiguous population of mesodermal cells (Wolf, 2002).
Interestingly, FGF signalling has also been implicated in the outgrowth of cytonemes, which are thought to function in the distribution of morphogens during Drosophila imaginal disc development. However, cytonemes are remarkably long and microtubule-free cell extensions with a diameter of 0.2 microm. Thus, they differ from the cell extensions described here in size and cytoskeletal composition, i.e., tracheal cell extensions are more than double in diameter and contain microtubules in addition to filamentous actin (Wolf, 2002).
It is speculated that the bridge-cell recognition, the sliding of the tracheal cell extensions along the bridge-cell surface and finally the fusion process are likely to involve extracellular matrix and/or cell adhesion molecules that are associated with the tubular cell extensions and the bridge-cell surface. The functional characterization of such proteins will provide further insights into the guidance mechanisms of cell extensions along specialized cells (Wolf, 2002).
FGF signaling is a central regulator of branching morphogenesis processes, such as angiogenesis or the development of branched organs including
lung, kidney, and mammary gland. The formation of
the air sac during the development of the Drosophila
tracheal system is a powerful genetic model to investigate
how FGF signaling patterns such emerging
structures. This article describes the characterization
of the Drosophila matrix metalloprotease Mmp2 as
an extracellular inhibitor of FGF morphogenetic function.
Mmp2 expression in the developing air sac is
controlled by the Drosophila FGF homolog Branchless
and then participates in a negative feedback and lateral inhibition mechanism that defines the precise pattern of FGF signaling. The signaling function
for MMPs described here may not be limited to
branching morphogenesis processes (Wang, 2010).
To explore their potential role in morphogenesis, the
expression patterns of Drosophila mmp1 and mmp2 were studied using GFP reporter strains. Consistent with previously published in situ
hybridization data (Page-McCaw, 2003), it was found that
both Drosophila mmp genes are active in the larval air sac primordium (ASP) as this structure forms and migrates across the wing imaginal disc,
invading larval tissues. In the course of air sac outgrowth, mmp1
is evenly expressed throughout the tubular structure, whereasmmp2 levels become progressively more prominent
in the distal end of the air sac and the tip cells (Wang, 2010).
To investigate whether the striking expression pattern of the
Drosophila mmp genes in the ASP might point to a role in air
sac formation, Mmp1 or Mmp2 function in the
tracheal system of Drosophila third instar larvae was disrupted. This was
achieved by the expression of specific RNAi constructs for either
gene (UAS-mmp1RNAi and UAS-mmp2RNAi, respectively) under
the spatial control of the trachea-specific btlGal4 driver and the
temporal control of a temperature-sensitive Gal80ts suppressor.
The efficacy of the RNAi-mediated knockdown was validated
by PCR and antibody staining. Whereas knockdown
of mmp1 had only a subtle effect, the phenotype
caused by loss of mmp2 was dramatic: air sac extension was
impaired, resulting in a severely deformed structure. The characteristic
elongated and pointed shape of the air sac was lost.
Instead of a single well-defined tip, mmp2-
deficient air sacs displayed multiple tips
and sometimes had a multilobed appearance. Proliferation
appeared unaffected upon knockdown of mmp2 in the ASP as
shown by anti-phospho H3 staining, indicating that the mmp2 loss-of-function (LOF) phenotype is not caused by an insufficient supply of tracheoblasts for air sac development. Expression of Drosophila Timp, a specific MMP inhibitor, under btlGal4/Gal80ts control resulted in a multitip phenotype indistinguishable from the one elicited by mmp2RNAi. Coexpression of Mmp2, but not Mmp1, largely reverted the ASP defect caused by TIMP. Moreover, the function of Mmp2 is continuously required to maintain the ordered outgrowth of the ASP (Wang, 2010).
In order for outgrowth to proceed normally, the ASP has to be
patterned into stalk and tip cells. Under wild-type conditions,
characteristic actin-rich filopodia emanate from the migrating
ASP tip and extend toward the source of Bnl/FGF signaling. The multitipped, migration-deficient ASP caused by mmp2 LOF, however, are characterized by the widespread appearance of such filopodia. It is concluded that the patterning into stalk and tip cells might be disturbed under mmp2 LOF conditions, resulting in an expansion of tip territory. To confirm this interpretation, the expression of the tip cell marker esg was monitored in mmp2 LOF air sacs using a GFP reporter under the control of an esgGal4 driver or a straight esg-LacZ reporter. Expression of mmp2RNAi either in the tip cell domain (using the esgGal4 driver) or throughout the tracheal system (using btlGal4) causes a significant expansion of esg expression (Wang, 2010).
It is concluded that Mmp2 function is required to spatially
constrain the tip cell region. It is plausible that the failure to
migrate and the multitip phenotype are direct consequences
of an expansion of the tip cell domain at the expense of the
stalk cells (Wang, 2010).
The esg transcription factor controls tip-cell-specific functions
in the developing tracheal system of the Drosophila embryo. It is
reasonable to assume that esg would similarly confer tip cell
specification in the larval ASP. If that were the case, one might
expect that the expanded domain of esg expression that was
have seen under conditions of reduced mmp2 expression might
be causal for the multitip phenotype. To test this possibility, esg was overexpressed throughout the third instar ASP under the
control of btlGal4/tubGal80ts, thereby expanding the expression
of esg beyond the prospective tip domain. Interestingly,
this manipulation caused a multitip phenotype resembling that
of mmp2 LOF conditions. It is concluded that
the expansion of the esg expression domain is sufficient to
mediate the mmp2 LOF phenotype. This finding supports the
idea that the mmp2 mutant phenotype is a consequence of
a patterning defect caused by an expansion of tip cell fate (Wang, 2010).
Tip cell fate is specified by FGF signaling. Therefore, the expansion
of the tip cell domain under mmp2 LOF conditions might be caused by
a spread of FGF signaling activity. Such FGF signaling activity in the tip region can be visualized by staining with a phospho-specific (dpERK)
antibody, which recognizes the doubly phosphorylated, active
form of Drosophila ERK. Consistent with previous reports, dpERK staining was found to be restricted to the tip area of the
wild-type ASP. However, upon suppression of Mmp2 activity,
either by expression of mmp2RNAi or of timp, dpERK staining
was expanded broadly throughout the air sac. This
result suggests that the mmp2 LOF phenotype is caused by an
expansion of FGF signaling. Consequently, tracheoblasts that
would otherwise become part of the stalk are misspecified and
adopt ectopic tip cell fates. Consistent with this interpretation,
deliberate activation of FGF signaling by overexpression of
FGF receptor throughout the air sac can phenocopy the Mmp2
LOF phenotype. These data suggest that Mmp2
can prevent FGF signaling in prospective stalk cells and thereby
restrict FGF activity to the tip cell domain (Wang, 2010).
To directly confirm that Mmp2 can suppress FGF signaling, experiments were conducted in Drosophila S2 cells. Transient coexpression of Bnl and Btl potently activates ERK phosphorylation, as monitored by immunoblotting with the dpERK antibody. This ERK response can be abrogated by coexpression
with Mmp2. A catalytically inactive mutant, Mmp2E258A,
however, has no effect. In agreement with the in vivo data, this
result suggests that Mmp2 can interfere with Bnl/Btl signaling.
To investigate whether this effect is specific for the FGF pathway
or whether other receptor tyrosine kinases might also be
affected, a similar experiment was conducted in which ERK was activated by expression of Drosophila EGF receptor and a soluble form of its ligand, Keren. Expression of EGF-R and sKrn causes ERK phosphorylation to a similar
degree as Bnl/Btl expression. Significantly, however, this activation
is insensitive to the presence of Mmp2. It is
concluded that the regulatory function of Mmp2 on air sac development
is selective for the FGF pathway. Mmp2 signaling might
therefore control the balance between EGF-regulated cell proliferation
and FGF-mediated patterning and cell migration (Wang, 2010).
Many examples of branching morphogenesis require a lateral
inhibition process that serves to spatially restrict a tip domain
in the outgrowing organ. Lateral inhibition is mediated by an
inhibitory signal that is released by distal cells once they have
adopted tip cell identity, to stop their neighbors from doing the
same. In this manner, spreading of tip fate into the adjacent stalk
cell area is prevented, assuring the correct patterning and structure
of the forming organ. The data presented so far are compatible with the idea that Mmp2 is part of a tip-cell-specific lateral inhibition mechanism. Consistent with this model, this study found that FGF signaling itself can induce Mmp2 expression in the ASP. This conclusion was further confirmed by real-time RT-PCR and western blotting (Wang, 2010).
The lateral inhibition model predicts that Mmp2 expression is
required in the tip cells themselves, to restrict expansion of tip
cell territory. To test this notion, a clonal analysis
strategy was adopted. Using MARCM technology, random clones of
GFP-marked cells were generated that were homozygous for themmp2 LOF allele mmp2G535R or the mmp1 LOF allele mmp1Q273*. In parallel, mmp2RNAi or Timp was clonally expressed
using the flp-out Gal4 driver system in developing larvae. All strategies
resulted in the generation of GFP-labeled clones that lacked the
capacity to express Mmp2 activity. The location of these clones
within the mRFP-labeled air sac was recorded. Both strategies
showed that the mmp2-deficient cells rarely contributed to tip territory, whereas control clones that lack Mmp1 function or express
GFP only were randomly distributed across the whole area of
the air sac, including the tip. This is interpretated to mean
that mmp2-deficient cells, even if they were the first to
receive an FGF signal, would not be able to maintain tip fate, as they could not inhibit FGF signaling in their wild-type neighbors.
Those neighbor cells expressing Mmp2 normally would then exert
a lateral inhibition effect preventing mmp2-deficient clonal cells
from receiving FGF signaling. In other words, cells of the ASP
compete with each other to contribute to the tip. Cells that lack Mmp2 activity are at a disadvantage and will likely lose the ability to respond to FGF signals, and assume a subsidiary stalk cell role (Wang, 2010).
Finally, an experiment wss designed to directly visualize the
paracrine effect of Mmp2 on FGF signaling in the ASP. To this
end, a small number of clones expressing Mmp2
were induced in the ASP. The strain used here also carried the hs-bnl transgene. Thereby, Bnl expression can be ubiquitously activated
by exposing wandering third instar larvae to a mild heat treatment.
The resulting elevated levels of FGF throughout the ASP
made it easier to observe inhibitory functions of Mmp2. Strikingly,
areas were found of diminished ERK phosphorylation adjacent to Mmp2-expressing clones, as monitored by dpERK staining. Control clones that expressed only GFP never caused such an effect (Wang, 2010).
Several observations in this experiment are noteworthy. First,
the inhibitory effect of Mmp2 expression on ERK signaling is
strictly nonautonomous. Only cells adjacent to the Mmp2-
expressing clones showed decreased ERK activity. The clonal
cells themselves are impervious to the inhibitory activity of
Mmp2. This finding explains the persistent FGF activity in the
ASP tip cells even after Mmp2 expression is induced, and
supports the concept that tip cells act as classical organizers
that secrete signals to which neighboring cells respond but to
which they themselves are insensitive (Wang, 2010).
Second, the paracrine inhibitory effect that Mmp2-expressing
cells exert on FGF signaling in their neighbors is not gradual. The
affected cells adjacent to the Mmp2-expressing clones have
either normal or dramatically decreased ERK signaling activity,
but none show intermediary levels. This suggests that Mmp2
activity influences a yes/no decision. Such a mechanism would
be consistent with the proposed function of the FGF-Mmp2
signaling circuit to distinguish between two distinct cell fate
choices: tip or stalk (Wang, 2010).
Third, not all cells touching Mmp2-expressing clones show
decreased ERK activity. The basis for this anisotropic effect of
Mmp2 is not clear, but it might be related to the previous point:
cells can adopt either an ERK on (tip cell) or an ERK off (stalk
cell) state, a decision that is influenced by the interplay between
FGF, FGF receptor, and Mmp2. Stochastic variations in signaling
might tip the balance one way or the other, especially in the
experimental setting employed here, in which high ubiquitous
levels of FGF are present (Wang, 2010).
Matrix metalloproteases have long been implicated in invasion
and branching morphogenesis. Whereas many
studies focus on MMP-dependent extracellular matrix (ECM)
remodeling in this context, a different role for
Mmp2 was documented in this study: controlling the spatial pattern of FGF signaling. It should be noted that the signaling function of Mmp2 documented
here by in vivo and cell-culture evidence does not rule out a
mechanical contribution of MMP to air sac outgrowth and
invasion. Interestingly, Guha (2009) has very recently
reported that Mmp2 clears ECM components around the outgrowing ASP, which may facilitate the movement of the structure (Wang, 2010).
The function of Mmp2 as a modulator of FGF signaling and as
part of a lateral inhibition mechanism can be explained by the
following model (Figure 4C): tracheal cells that receive the FGF
signal first will activate ERK to induce gene expression programs
that direct budding and air sac formation. Among the activated
transcription units is the mmp2 gene, which is required for the
release of an inhibitory signal that nonautonomously prevents
further FGF responses in adjacent cells. This Mmp2-mediated
lateral inhibition mechanism would thereby restrict the spreading
tip cell fate through the prospective air sac. The nature of the
inhibitory signal that is delivered by the Mmp2-expressing tip
cells is still unknown (Wang, 2010).
It is likely that the mechanisms described here for the
Drosophila air sac are also employed by other species and developmental
processes. For example, it has been shown that cells
with high levels of FGF activity have a competitive advantage
in populating the tips or 'terminal end buds' of invading ducts
during murine mammary development (Lu, 2008), a finding
that is indicative of a lateral inhibition process. Interestingly, it is
well established that both MMPs and FGF signaling make critical
contributions to mammary development. It is therefore tempting to speculate
that the regulatory interplay between MMPs and FGFs operates
broadly in invasive growth and branching morphogenesis (Wang, 2010).
Recent data have demonstrated a crucial role for the transcription factor SRF (serum response factor) downstream of VEGF and FGF signalling during branching morphogenesis. This is the case for sprouting angiogenesis in vertebrates, axonal branching in mammals and terminal branching of the Drosophila tracheal system. However, the specific functions of SRF in these processes remain unclear. This study establish the relative contributions of the Drosophila homologues of FGF [Branchless (BNL)] and SRF [Blistered (BS)] in terminal tracheal branching. Conversely to an extended view, it was shown that BNL triggers terminal branching initiation in a DSRF-independent mechanism and that DSRF transcription induced by BNL signalling is required to maintain terminal branch elongation. Moreover, increased and continuous FGF signalling can trigger tracheal cells to develop full-length terminal branches in the absence of DSRF transcription. These results indicate that DSRF acts as an amplifying step to sustain the progression of terminal branch elongation even in the wild-type conditions of FGF signalling (Gervais, 2011).
The results contribute to clarification of the roles of BNL and DSRF in terminal branch formation. First, DSRF transcription is dispensable for terminal branch initiation but is a crucial requirement for the progression of this process in wild-type embryos. Second, a constitutively activated form of DSRF still requires BNL signalling to achieve terminal branch formation, thereby indicating that an additional outcome from BNL signalling is required for terminal branch development. Third, high levels of BNL signalling give rise to terminal branches independently of DSRF transcription. All these observations indicate that branches with an intracellular lumen can initiate their development in the absence of DSRF activity. As these are the specific features of terminal cells, it can be concluded that DSRF is not a general determinant of terminal cell fate (Gervais, 2011).
On the basis of these results, the following model is proposed for terminal branch development. On the one hand, BNL signalling triggers the initial phases of cell elongation and intracellular lumen formation. This step is independent of DSRF transcription, probably because BNL levels at this stage are high enough to promote terminal branch initiation. On the other hand, BNL signalling activates DSRF, which in turn allows the progression of cell elongation and intracellular lumen formation. Indeed, as mechanical tension has been proposed as a means to active DSRF expression, it could well be that the same elements involved in the triggering of cell elongation by BNL signalling might also mediate activation of DSRF expression in the terminal cell. Irrespective of the mechanism promoting its expression, DSRF activity can be considered to be a boosting mechanism that, together with other outputs from BNL signalling, ensures that the cellular modifications required for elongation and intracellular lumen formation are kept active in the wild-type conditions of BNL signalling. Consequently, this process appears to spatially restrict induction of terminal branching to places of high BNL signalling, which are often found at the tip of the branches. Finally, although high levels of BNL signalling do not reproduce physiological wild-type conditions, the observation that such high levels bypass the requirement of DSRF transcription for the growth of terminal branches can be of relevance in stress conditions, such as in hypoxia. Likewise, a similar mechanism could be of significance for the induction of angiogenesis in disease (Gervais, 2011).
The Drosophila adult has a complex tracheal system that forms during the pupal period. The derivation of part of this sytem, the air sacs of the dorsal thorax, has been studied. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila; they correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role (Sato, 2002).
Metamorphosis presents special challenges to the tracheal system. The process that transforms the Drosophila larva into an adult fly consumes larval tissues and creates new organs using imaginal cells that were prepared during larval development. A new tracheal system that will satisfy the aerobic requirements of the specialized tissues of the adult must be built. But, in addition, the organs of the pupa must be kept oxygenated while the adult develops. Transformation of the tracheal system begins during the third larval instar, when imaginal tracheoblasts start to divide. These proliferating tracheoblasts spread over the larval tracheal system, using it as a scaffold to form an extensive branching network before the larval cells histolyze. Some tracheoblasts elaborate coiled structures that are unique to the pupa. Others grow to form the air sacs of the adult. Air sacs are large reservoirs that are juxtaposed with major muscle systems and with the brain. These structures have been thought to form as dilations of the main tracheal trunks, which are the direct descendants of the main tracheal trunks of the embryo and larva. The work described in this study shows that in the dorsal thorax these air sacs originate independently from a distinct population of cells (Sato, 2002).
The majority of the adult thorax, including most of the dorsal thoracic epidermis, the wing, and flight muscle, is produced by the wing imaginal disc. This organ arises as a tubular invagination of the epidermis, and when it grows and flattens during the larval periods, it develops four distinct cell types. It has squamous peripodial cells on one surface, columnar epithelial cells on the other surface, a distinct group of adepithelial cells that nestle against the most proximal columnar epithelial cells, and stalk cells that connect the disc to the epidermis. A large tracheal branch attaches in some manner to the columnar epithelial surface. It orients along the dorsal/ventral axis of the disc, but it does not ramify to generate multiple contacts with the disc cells. Therefore, no framework exists to serve as a template for the tracheolar network that provides oxygen to the thoracic cells of the adult. The mechanisms responsible for the branch formation and path finding that produce the extensive and complex tracheolar network in the adult thorax remain to be identified (Sato, 2002).
The work described in this study represents an effort to understand the role of FGF in wing disc development. These investigations led to the identification of a new cell type in the wing disc that migrates and proliferates in response to FGF. These cells contact FGF-expressing cells across at least one cell layer by extending long cytoneme-like filopodia. These cells are destined to form the air sacs that associate with the flight muscles in the adult thorax, but they are distinct from the cells that form the larval trachea or from the group of imaginal precursors that are programmed to generate tracheae in the pupa and adult (Sato, 2002).
To investigate the role of FGF in Drosophila wing disc development, patterns of expression of bnl and the two FGF receptor genes, breathless (btl) and heartless (htl), were examined in third instar discs. bnl/FGF expression is restricted to a small group of cells in the columnar epithelium. An exact count of their number was problematic: since the apparent level of expression in many cells is very low, their number is roughly estimated to be between 15 and 60 in early third instar discs and between 80 and 150 in late third instar discs. These cells straddle the anterior/posterior compartment border and are dorsal to the region of the prospective wing blade. The cells that express bnl most strongly are ventral to the progenitor of the aPA machrochaete, as indicated by double staining for Achaete protein. They are in a region that contributes to the notal wing processes: the cuticle located between the adult scutum and wing hinge (Sato, 2002).
Expression of btl and Htl is restricted to cells in the adepithelial layer of the wing disc and is absent from the cells of the columnar epithelium. The adepithelial cells give rise to the adult musculature; they also express twist, which controls htl and is a signature of all mesodermal cells and muscle precursors. It was confirmed that the wing disc adepithelial cells express both Htl and Twist. In addition, a small group of adepithelial cells was identified that expresses a btl enhancer trap line but expressed neither Htl nor Twist. Stumps, a putative adapter protein required for both Btl and Htl signaling, is expressed in both btl- and Htl-expressing adepithelial cells (Sato, 2002).
The btl-expressing cells in the adepithelial layer had not been identified previously, and their presence was unexpected, since one of the principal domains of btl expression is the trachea. Tracheal cells are almost invariably associated with tubules with cuticle-lined lumen, and the adepithelial cells have no such distinct structures. Nevertheless, the adepithelial btl-positive cells appear to maintain continuity with the cells of the main tracheal branch. Evidence is presented that the btl-expressing adepithelial cells are the precursors of the adult tracheal air sacs (Sato, 2002).
To better understand the origin and fate of the btl-expressing adepithelial cells, they were tracked during larval and pupal development. In early third instar wing discs, no btl expression was detected in adepithelial cells; only tracheal branch cells are btl positive. However, as third instar discs mature, btl-positive cells are detected budding from the tracheal branch that adheres to the wing disc. This bud forms at a stereotypical position just dorsal to the wing hinge progenitors and adjacent to the group of 15–60 bnl-expressing cells in the columnar epithelium. During development of the third instar, the number of btl-expressing cells increase, and the bud expands posteriorly toward the region of greatest bnl expression. In late third instar discs, the btl-expressing cells form a coherent group surrounded by Htl-expressing cells. These btl-expressing cells do not express Htl. Possible explanations for the complementarity of the patterns of btl and Htl expression are that btl cells displace Htl-expressing cells or that the expression of these genes is mutually exclusive. However, in the early third instar discs, Htl expression is already absent from the region that will be occupied by btl-expressing cells in older discs; therefore, these are not likely to be sufficient explanations (Sato, 2002).
To characterize these cells further, their relationship to the larval tracheal system was examined. The larval tracheal branch that adheres to the wing disc is called the first transverse connective. It has a small offshoot called the spiracular branch, where imaginal tracheoblasts, precursors of the adult tracheae, are located. These imaginal tracheoblasts do not express btl but do express escargot (esg) and trachealess (trh). Since esg inhibits endoreplication of imaginal cells and the imaginal tracheoblasts are assumed to be diploid, expression of esg in these cells was not unexpected. The presence of Trh, a transcription factor that directly activates btl transcription, may presage btl expression at a subsequent stage. The btl-expressing adepthelial cells also express Trh. They do not express esg early in the third instar; therefore, it is unlikely that they derive from the imaginal tracheoblasts. However, in mid third instar and thereafter, esg expression is evident in the most posterior cells of the group (Sato, 2002).
The capacity of tracheal branch cells to proliferate was unexpected because the cells lining the main tracheal branches had been considered to be both terminally differentiated and polyploid. Neither state is expected to be compatible with a mitotic cell cycle program. The morphology and fluorescence of DAPI-stained discs was examined to better understand the nature of the disc-associated tracheal cells. Most of the cells that line the lumen of the main tracheal branch, the first transverse connective, as well as the imaginal tracheoblasts, have nuclei that are similar in diameter (5–6 µm), are relatively small, and have a similar level of fluorescence. Some cells with large nuclei and >3× the fluorescence intensity of the smaller nuclei were observed populating other branches that connect to the first transverse connective. Assuming that the imaginal tracheoblasts are diploid, these observations suggest that the cells that respond to Bnl-FGF have a similar ploidy and that the tracheal branches that associate with the wing disc include both diploid and polyploid cells (Sato, 2002).
To determine the fate of the btl-positive adepithelial cells, their behavior and movements were examined throughout pupal development. They are located next to the posterior part of the prospective wing hinge in the late third instar disc. Analysis of fixed wing discs prepared at various times during the first 12 hr after puparium formation (APF) revealed that they remain in this location as a tight, rounded cluster of cells next to the prospective wing hinge (Sato, 2002).
To continue following the fate of these cells, five individual pupae that expressed btl-Gal4 UAS-GFP were observed during the pupal period, and photographs were taken at regular intervals. The pupal case is transparent to the GFP fluorescence; therefore, no surgical manipulations were necessary. The btl-positive adepithelial cells were identified by their proximity to the wing hinge at 12 hr APF, consistent with the observations of dissected, fixed discs. These btl-positive cells migrate dorsally between 12 and 23 hr APF then anteriorly and posteriorly to form three branches. At 32 hr APF, they cease their migrations and began to elaborate into air sacs. It is concluded that the btl-positive adepithelial cells are the precursors of the adult air sacs and that the air sacs of the adult thorax are derived from cells that are distinct from the imaginal tracheoblasts (Sato, 2002).
Although the air sac tracheoblasts do not form a tubular structure at 12 hr APF, these pupae do have numerous tracheae that project to the developing muscles. These tracheae are clearly distinct from the air sac tracheoblasts. They derive from the second dorsal branch and are present only during the pupal period. Air sacs are prominent and extensive in older pupae and in adults and are associated with numerous bundles of tracheae that extend from the air sacs and extensively interdigitate with flight muscle cells. At present, the structure of the air sacs is not understood enough to know how these tracheae either connect with or contribute to the function of the air sacs (Sato, 2002).
Thus, bnl/FGF expression is detected in a small group of columnar epithelial cells during the third instar and pupal periods. Although their fate in the adult has not been established, these cells are small in number and therefore cannot produce more than a small part of the adult cuticle. Nevertheless, their effect on the adult is profound. Through the action of Bnl-FGF, they induce a group of tracheal cells to initiate a program of proliferation and migration and to join with the cells in the disc adepithelial layer. Despite this intimate association with these mesodermal progenitors, the FGF-responsive cells retain their tracheal identity and go on to form the prominent adult tracheal air sacs that extend throughout much of the dorsal thorax (Sato, 2002).
The tracheal system of the Drosophila embryo has ten interconnected metameric units on either side of the animal; one unit derives from the second, or mesothoracic, segment. This mesothoracic component consists of portions of the dorsal and lateral trunks, a transverse connective that links these trunks to each other, a dorsal branch that connects the left and right sides, and numerous branches that radiate out to various tissues. During larval development, this general structure is retained, and, although the tracheal cells do not divide, many new branches form, and the diameter of the more proximal tracheae increase. The wing imaginal disc attaches to the transverse connective. Imaginal tracheoblast precursors of the adult tracheae populate a small spiracular branch at a location just dorsal to the disc attachment (Sato, 2002).
In constructing the adult tracheal system, the imaginal tracheoblasts use parts of the larval framework as templates and, in effect, remodel the dorsal and lateral trunks, the transverse connective, the dorsal branch, and the main pupal branches to the wing and leg. In contrast, the large and extensive air sacs do not correspond to earlier branches in any obvious way and have no apparent antecedent. The airs sacs of the dorsal thorax form de novo from a small group of wing imaginal disc cells. This study chronicles the transformation of these air sac tracheoblasts from a tight cluster of adepithelial disc cells to sculpted air sacs. These observations were made by expressing GFP under btl control and by following the GFP-containing cells through the pupal period. It was possible to directly account for three branches of the dorsal thoracic air sacs (e.g. the medioscutal, lateroscutal, and scutellar sacs) as products of the wing disc air sac tracheoblasts. All of the air sacs in the notum appeared to contain GFP in these animals, but the resolution of the analysis was not sufficient to make this a definitive conclusion nor did it allow for a conclusion that all of the air sacs derive from disc tracheoblasts. Nevertheless, this study did establish that the disc tracheoblasts generate air sacs and, by some process that is as yet unknown, form a tracheal lumen and tracheal network. It will be interesting to identify the intrinsic and extrinsic systems that direct the genesis of the air sacs, since they apparently develop in the absence of a preexisting framework (Sato, 2002).
Evidence is presented for air sac tracheoblasts in the wing imaginal disc. Data suggest that there may be similar strategies to make air sacs in other regions of the fly. This statement is based on the presence of nontracheal btl-positive cells and bnl-expressing cells in other imaginal discs. In leg discs, bnl expression is found in the stalk region, and the pattern of expression becomes more extensive and complex in the disc epithelium of pupal discs. In third instar discs, btl-positive cells are localized to an offshoot of a tracheal branch that attaches near the stalk and adjacent to the bnl-expressing cells. After puparium formation, the btl-positive cells migrate along the basal surface of the disc columnar epithelium to a position that roughly correlates with the region where air sacs will later form. In eye-antenna discs, bnl is expressed in cells surrounding the ocelli progenitors. Although btl- expressing cells were not found in larval eye-antenna discs, it was observed that in early pupae btl-positive cells assume a position underlying the presumptive ocelli cells. Air sacs in the adult head underlie most of the medial head cuticle and encircle the region where the ocelli form. These observations led to the suggestion that the process that induces the air sac tracheoblasts in the wing disc may be common to other discs as well (Sato, 2002).
Bnl-FGF is the key determinant of tracheal branching as the preadult tracheal system matures. In the embryo, it guides the migration of tracheal cells to form primary branches, induces secondary branches as the primary branches approach the cells expressing bnl, and regulates the process that generates terminal branches. To produce these different outcomes, the FGF signaling pathway acts through different, but related, mechanisms, but its role is, in effect, a single one -- to mold the tracheal cells and to influence where and how they extend. The roles that it plays during the early stages of air sac morphogenesis are different. As a signal in the wing imaginal disc, Bnl-FGF functions as a chemoattractant, inducing these cells to migrate from outside the imaginal disc to a location within the adepithelial layer. And it acts as a mitogen, inducing the tracheoblasts to proliferate. These properties are not manifested by Bnl-FGF during earlier stages of tracheal development. Thus, the morphogenic process that generates the air sacs is distinguished both by its independence from the tracheal framework of the embryo and larva and by the roles that FGF plays (Sato, 2002).
Perhaps the most surprising behavior this work identified is the response of the larval tracheal cells to Bnl-FGF. These cells form a tripartite tube that consists of an external basal lamina, a squamous epithelium, and a complex, multilayered luminal cuticle. They had been considered to be terminally differentiated and incapable of proliferation, having ceased cell division in the early embryo. It was found, instead, that many of the wing disc-associated tracheal cells can respond to Bnl-FGF by migrating out of the tracheal branch to embark on a program of proliferation and morphogenesis. Remarkably, this latent capacity for conversion to proliferative tracheoblast is shared by many (and perhaps all) of the tracheal cells that populate the disc-associated branch. These cells require only the action of Bnl-FGF to initiate the process. Although the possibility of a separate and distributed population of tracheal stem cells cannot be excluded, it is thought that the widespread capacity of tracheal cells to adopt a program of proliferation and migration makes this possibility unlikely. Instead, it is proposed that Bnl-FGF acts as an instructional determinant, reprogramming the cells in the tracheal branch to become air sac tracheoblasts. It is thought that the activity of Drosophila FGF to induce cells to dedifferentiate has no precedent (Sato, 2002).
Actin-based filopodia have been observed in many cells that send or receive signals. For purposes of illustration, four are mentioned here. (1) Neuronal growth cones are populated with many active filopodia that appear to probe their environment for guidance cues. (2) Long and highly dynamic filopodia extend from primary mesenchyme cells in the interior of early sea urchin embryos, apparently to contact and explore the overlying ectoderm. They are thought to relay information from the peripheral ectoderm that patterns internal skeletal elements. (3) Dendritic cells, professional antigen-presenting cells of the vertebrate immune system, are defined in part by their Medussa-like morphology. Their unusually large number of finger-like projections may maximize the likelihood that antigen presentation finds a suitable target cell. (4) Drosophila wing imaginal disc cells have thin filopodial extensions, 'cytonemes', that appear to connect cells with the organizer region at the anterior/posterior compartment border. Although the filopodia produced by these various cell types are similar in gross morphology, it is not known whether they all have a role in trafficking signals, have a common mechanism that receives and transduces signals, or are regulated in a similar manner (Sato, 2002).
The filopodia that the wing disc tracheoblasts produce have a number of properties in common with cytonemes. Both are actin-based, have a comparable size and appearance, and are similarly sensitive to standard conditions of fixation. Functional analysis indicates that the tracheoblast filopodia are dependent on Bnl-FGF and upon the ability of the tracheoblasts to carry out FGF signal transduction. There is no direct evidence that signaling cannot occur if they are absent, but the correlation between these structures and active signaling is strong. Moreover, their presence offers a possible mechanism to move the FGF signal from its source in the columnar epithelial cell layer across the adepithelial cell layer to the tracheal target cells. It is not known what alerts the target cells to the presence of a source of FGF. Two possibilities are that nonspecific cytoneme-like filopodia explore the extracellular environment for potential sources or that less efficient signaling can occur in the absence of direct contacts. It is assumed that, if this second possible mechanism is operative, once filopodia are induced and make appropriate contacts, signaling will be accelerated and more productive. The question of how the filopodia are induced and oriented is certainly important, but the very presence of these cellular extensions that make contact across these distances offers a mechanism to facilitate the ordered movement of signals and the concerted and directed migration of cells (Sato, 2002).
The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).
Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).
Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).
To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).
In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).
Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).
Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).
The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).
Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).
Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).
Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).
Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).
Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).
These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).
The Drosophila central nervous system is produced by two rounds of neurogenesis: one during embryogenesis to form the larval brain and one during larval stages to form the adult central nervous system. Neurogenesis caused by the activation of neural stem division in the larval brain is essential for the proper patterning and functionality of the adult central nervous system. Initiation of neuroblast proliferation requires signaling by the Fibroblast Growth Factor homolog Branchless and by the Hedgehog growth factor. The Branchless and Hedgehog pathways form a positive feedback loop to regulate the onset of neuroblast division. This feedback loop is initiated during embryogenesis. Genetic and molecular studies demonstrate that the absolute level of Branchless and Hedgehog signaling is critical to fully activate stem cell division. Furthermore, over-expression and mutant studies establish that signaling by Branchless is the crucial output of the feedback loop that stimulates neuroblast division and that Branchless signaling is necessary for initiating the division of all mitotically regulated neuroblasts in the brain lobes. These studies establish the molecular mechanism through which Branchless and Hedgehog signaling interface to regulate the activation of neural stem cell division (Barretta, 2008).
These studies have demonstrated that Hh and Bnl act in a positive feedback loop in the larval brain to control the onset of neuroblast proliferation. The feedback loop acts at the transcriptional level, such that Hh signaling activity is essential to control the level of bnl expression and vice versa. Double mutant analyses showed that an absolute level of signaling by both Bnl and Hh are required to maintain normal neuroblast activation, rather than other possible models that would suggest a certain balance of signaling activity (for example more Bnl than Hh) is sufficient regardless of the exact magnitude of signaling activity. The discovery that Bnl signaling is the critical output of the feedback loop suggests that the main function of Hh signaling is to maintain the proper level of Bnl production and signaling. Furthermore, the observation that only the mushroom body and ventral lateral neuroblasts continue to divide in bnl null mutants regardless of the level of Hh signaling indicates that all the regulated neuroblasts, both optic lobe and central brain sets, require the input of the Bnl pathway to enter S phase. Thus the Hh-Bnl feedback loop appears to control cell cycle progression in all the mitotically arrested neuroblasts that begin cell division in first instar (Barretta, 2008).
Other developmental events that require Hedgehog and FGF signaling have used those pathways in different manners to achieve their goals. For example, in the mouse ventral telencephalon, Hedgehog and FGF/MAPK signaling operate as two independent pathways. FGF signaling is independent of Sonic Hedgehog (SHH) and does not affect expression of either SHH itself or its target gene and effector GLI1. Other systems have shown a linear dependence of FGF expression on SHH signaling and vice versa. During budding morphogenesis in the mouse lung Hedgehog signaling inhibits expression of FGF10 but up-regulates FGF7. In the Xenopus eye, expression of Banded Hedgehog increases expression of FGF8. In the zebrafish forebrain inhibition of Hh signaling decreases expression of FGF3, FGF8 and FGF19. Hedgehog also regulates FGF expression in the zebrafish mid/hindbrain. However, in the zebrafish forebrain HH expression requires FGF signaling. Inhibition of both FGF3 and FGF8 expression resulted in a downregulation of SHH. Alternatively, the HH and FGF pathways can integrate at the level of intracellular components. FGF has been shown to induce expression of GLI2, a transcription factor and HH signaling effector in ventroposterior development in zebrafish (Barretta, 2008).
Of course the classic example of FGF and SHH interplay is the development of the chick limb bud. In this system, several FGFs set up a signaling center at the tip of the bud that turns on expression of SHH in the posterior limb mesenchyme. In turn, SHH signaling is required for maintenance of FGF4, FGF9 and FGF17 expression in the bud tip. This function of SHH occurs through the expression of Gremlin, an inhibitor of Bone Morphogenetic Protein signaling. Gremlin inhibition of Bone Morphogenetic Protein signaling prevents down-regulation of the FGFs. Thus a positive feedback loop exists between SHH and FGFs, mediated by Gremlin (Barretta, 2008).
The model of the Hh-Bnl feedback loop proposed in this study is most similar to the classic SHH-FGF feedback loop described in the vertebrate limb bud. In is not yet known whether the regulation of bnl expression by Hh signaling is direct or if it is mediated by another signaling pathway such as the Gremlin/Bone Morphogenetic Protein connection that operates in the limb bud. However, like the distinct domains of FGF and SHH in the limb bud, bnl and hh expression also occur in distinct regions of the brain lobe. The fact that the Hh-Bnl feedback loop is activated during embryogenesis, but that the first regulated neuroblasts do not enter S phase until 8-10 h after larval hatching also suggests that additional events must take place downstream of Bnl signaling to permit mitotically arrested stem cells to transit through G1 to S phase. One such possibility is exposure to the steroid hormone ecdysone, which is necessary during first larval instar for the initiation of neuroblast division a few hours later. Both SHH and FGF2 have been shown to be necessary for the division of different subsets of neural stem cells in many different vertebrate and mammalian models and in multiple contexts. This is the first time that the interactions between these two pathways necessary to stimulate the reactivation of stem cell division in quiescent neural stem cells have been elucidated. The next challenge will be to determine whether different molecular mechanisms tying these two signaling pathways are used for different developmental decisions such as progeny cell fate, initiation of cell division and maintenance of stem cell identity (Barretta, 2008).
Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).
Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).
In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).
In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).
The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).
Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFRDN were recovered that co-expressed EGFRDN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).
Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).
Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).
To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).
Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).
The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).
To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).
Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv16713), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).
These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).
The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).
branchless was identified as a P transposon-induced mutation that reduces or eliminates tracheal branching, just like breathless mutation. At stage 16 in wild-type embryos, primary branches have budded and grown out from the tracheal sacs; secondary and some terminal branches have formed, and branch fusion has taken place to form the dorsal and lateral tracheal trunks. In branchless mutants, none of these events occur normally: almost every tracheal metamere appears as an unbranched, elongated sac of tracheal cells. Tracheal cells invaginate and form tracheal sacs normally in branchless mutants, but branches fail to grow out. As with breathless mutants, specific defects appear in the central nervous system (Sutherland, 1996).
The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream
of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic
mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding
stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore,
and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the
initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back
from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).
The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants,
invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a
lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).
Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).
p21-activated kinase (Pak) is known as a mediator of the activity of Rac
GTPase. Tracheal defects similar to those of Rac1, 2 mutants
are found in pak mutants. Furthermore,
Rac1, 2 and Pak mutations synergistically enhance tracheal
defects. Such results suggest that Rac and Pak are required for directed movement of tracheal branches (Chihara, 2003).
The loss of Rac activity also causes a defect in cell differentiation. Tips
of dorsal branch 1-9 are normally capped with terminal cells that extend terminal branch
in the ventral direction. In Rac 1, 2 mutant embryos, the loss of terminal
branches was observed with high penetrance. Consistently, serum response
factor (SRF), a marker protein for the terminal cell, also disappears, suggesting that terminal cell differentiation does not occur (Chihara, 2003).
Since directed cell migration and terminal cell differentiation are processes
requiring FGF signaling, it was asked whether Rac is involved in FGF signaling (a strong genetic interaction). Although tracheal patterning is only
mildly affected by half dose reductions of bnl (ligand), btl
(receptor) and dof (intracellular effector), the
phenotype is strongly enhanced by introducing one copy of Rac1, 2
mutant chromosome from mothers. A
similar genetic interaction was found between pak and bnl. These genetic
interactions suggest that Rac and Pak are required for the migration of
tracheal branches in response to FGF signaling (Chihara, 2003).
To determine the epistatic relationship between Rac and FGF signaling, the effect of constitutive activation of Rac was tested in btl mutants.
In the btl mutant, tracheal branching does not proceed beyond the
invagination at stage 11, and MAP kinase activation is absent (Chihara, 2003).
Expression of Rac1V12 partially restores the movement of tracheal cells, and
activates MAP kinase, as revealed by staining with the antibody against the
diphosphorylated form of MAP kinase (dp-MAPK). These results suggest
that Rac activation is an essential downstream event of tracheal cell motility
induced by FGF signaling (Chihara, 2003).
Extracellular signals that promote tracheal branching are good candidates
for regulators of Rac in tracheal cells. In this regard, the strong genetic
interaction between Rac and FGF signaling components observed suggests
an intriguing possibility that FGF signaling activates Rac within tracheal
cells to promote both cell motility and cell rearrangement. In support of this
idea, it was found that activated Rac 1 partially rescues tracheal cell motility
and MAP kinase activation in btl mutants. Involvement of Rac
in FGF-dependent events may not be limited to cell motility. Expression of SRF, the product of one of the target genes activated by FGF
signaling in the tracheal system, is lost in the mutant trachea with reduced
Rac activity because of Rac 1, 2 mutation or Rac 1N17. This result suggests that Rac also regulates transcription (Chihara, 2003).
Several lines of evidence suggest that FGF signaling is activated locally
at the tip of branches, and activation of FGF signaling in all tracheal cells prevents branching, suggesting that localized activation of FGF
signaling is essential for branching. Therefore the proposed function of Rac
in transducing FGF signaling must be localized at the tip of branches. How
does the proposed function of Rac in transducing FGF signaling relate to the
Rac function in regulating cell rearrangement? Since the effect of Rac 1N17 is
most clearly observed in cells destined to become tracheal stalk cells, the
location of tracheal cells requiring two of the Rac functions appears to be
different. One idea is that FGF signaling activated at the tracheal tip is
transmitted to tracheal stalk cells by a secondary signal that activates Rac
to promote cell rearrangement. It will be important to identify the upstream
signal regulating Rac in stalk cells (Chihara, 2003).
Epithelial organogenesis involves concerted movements and growth of
distinct subcellular compartments. Apical membrane enlargement is
critical for lumenal elongation of the Drosophila airways, and is
independently controlled by the transcription factor Grainy head. Apical
membrane overgrowth in grainy head mutants generates branches that
are too long and tortuous without affecting epithelial integrity, whereas
Grainy head overexpression limits lumenal growth. The chemoattractant
Branchless/FGF induces tube outgrowth -- it upregulates Grainy
head activity post-translationally, thereby controlling apical membrane
expansion to attain its key role in branching. A two-step model for FGF in branching is favored: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths (Hemphälä, 2003).
Bnl is the key morphogen co-ordinating branching that acts via the receptor
tyrosine kinase Breathless (Btl) and the adaptor protein Dof/Stumps. This
pathway leads to phosphorylation and activation of MAPK, which in
turn may alter the activity of regulatory proteins to control cell behavior.
During primary branching, actin-rich basal extensions are sent by the tracheal
cells towards the sources of Bnl, a process that is likely to involve
cytoskeletal modulation by the Rho family GTPases. Bnl
signalling is also required for the expression of cell-fate determining genes
in specific subsets of tracheal cells in each primary branch. Analysis of
these genes has identified key components of the patterning and guidance of
the unicellular secondary and terminal branches.
However, the role of Bnl in the movement of the cell bodies and the growth of
the branch lumen remains unknown (Hemphälä, 2003).
One possible mechanism for regulation of Grh activity is through Bnl
signalling, which is instrumental in the formation and extension of all
tracheal branches. Initially, it was established that apical cell surface growth
is an intrinsic component of Bnl-induced tube extension, by combining alleles
of grh and bnl. This revealed that a subset of the branch
outgrowth defects seen in embryos that carry only one copy of the bnl
gene are partially rescued by a reduction in grh function
(grhs2140/grhs2140; bnlP1/+). Thus,
in embryos heterozygous for bnl, 40% of the ganglionic branches fail to reach the CNS, whereas the simultaneous removal of
grh restores this phenotype so that 78% of the
branches now enter the CNS. These data therefore show that Grh-mediated
modulation of the apical cell surface has an active inhibitory role on
Bnl-induced branch extension (Hemphälä, 2003).
In order to analyse whether tracheal Grh activity could be targeted by
Bnl/Btl signal transduction, GBE-lacZ expression was analyzed in
embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically
expressed in all tracheal cells, GBE-lacZ expression becomes
significantly upregulated, although the levels of Grh protein are not altered. This suggests that
Bnl controls Grh activity post-translationally, and surprisingly, upregulates
the expression of this artificial Grh target. Nevertheless, the effects of Btl
appear specific since with more limited Bnl expression using the
Term-Gal4 driver, GBE-lacZ expression becomes enhanced
specifically in the cells that respond to Bnl by ectopically expressing the
terminal marker DSRF. Similar enhancement of GBE-lacZ expression is evident
upon tracheal expression of an activated form of the Btl receptor itself
(UASBtl-Tor). In all instances the augmented
GBE-lacZ expression is dependent on Grh, since embryos that express
ectopic Bnl or the activated form of Btl, but lack Grh activity, do not
express GBE-lacZ. Furthermore, ectopic activation of Dpp,
another signalling pathway that promotes the growth of dorsal and ganglionic
branches during tracheal development, has no
effect on GBE-lacZ, indicating that the effects on
GBE-lacZ are specific for Bnl/Btl (Hemphälä, 2003).
Whether Bnl signalling is a prerequisite for the
transcriptional activity of Grh was tested by analysing the levels of GBE-lacZ
expression in mutants for bnl, btl or pointed (pnt). Tracheal
GBE-lacZ expression is both reduced and uniform in bnl and
btl mutant embryos, but is unchanged in pnt embryos that lack the
activity of a downstream transcriptional effector of the ETS family.
Since Grh is a substrate for activated MAPK (ERK2) in vitro, its
activity could be modulated directly during branching by Bnl-induced
phosphorylation. This would account for the fact that GBE-lacZ
expression is affected by mutations in bnl and btl, but not
by mutations in the nuclear effector pnt (Hemphälä, 2003).
It is concluded that Bnl
signalling converts Grh to a more potent activator of its GBE-lacZ
target. Since Grh becomes phosphorylated by MAPK in vitro, and MAPK
is a downstream effector of Btl signal transduction, the alteration in Grh
activity may be brought about by MAPK-mediated phosphorylation of the Grh
protein (Hemphälä, 2003).
Currently, two ways of explaining the biological consequence of the
regulation of Grh have been suggested. In the first model, the regulation of Grh by Bnl increases
its activity, and thereby delimits lumen growth. This invokes a hierarchical
two step function for Bnl in which it first promotes branching and tube
elongation and it then activates Grh to halt excess apical surface growth and
establish a functional lumen. In this model active restriction of
morphogenetic processes is required to achieve stereotyped tube dimensions and
is an intrinsic part of the program that induces branching morphogenesis. In
the second model, regulation by Bnl has differential consequences on Grh,
activating some functions (like the one necessary for GBE-lacZ
expression) and inactivating others, necessary for inhibiting apical membrane
growth. In this model, high levels of Btl signalling would temporarily
inactivate Grh, in order to allow for apical membrane expansion during the
process of branch extension. Both models are consistent with the genetic
interactions, which indicate an antagonistic relationship between grh
and bnl, and add the control of apical membrane growth to the
repertoire of cellular activities regulated by FGF signalling during
morphogenesis (Hemphälä, 2003).
Of the two models, the former, where Btl coordinates
branching through a sequence of activities, is currently favored since this model is consistent
with the activation of the GBE-lacZ reporter. It can also be well
integrated with the apical overgrowth phenotype of grh mutants, which
becomes apparent first in the branches that have reached their final length
and only after the completion of branch elongation at stage 16. If Grh were
acting to restrict membrane growth continuously, the grh mutant
phenotype would be expected to appear at earlier stages. A two step model
could also explain the inhibiting effect on tube elongation that is seen upon expression of activated forms of Btl receptors in all tracheal cells of
wild-type embryos (Hemphälä, 2003).
Mutations in the Drosophila terribly reduced optic lobes (trol) gene cause cell cycle arrest of neuroblasts in the larval brain. trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF (Branchless) and Hedgehog (Hh) signaling. Addition of human FGF-2 to trol mutant brains
in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in
wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are
consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through
both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system (Park, 2003).
Trol appears to display functions similar to mammalian
Perlecans, which are known to bind FGF-2 and to be required
for FGF signaling. Dominant enhancement of the
neuroblast proliferation phenotype of two different trol alleles
has been observed with mutations in bnl and the
Bnl receptor breathless (btl), but not with mutations in the orphan heartless (htl) receptor. The neuroblast proliferation phenotype of trol8
mutant brains was rescued in culture to control levels by
addition of human FGF-2. Addition of the MAPK
inhibitor PD98059 at 10 hs post-hatching decreased the
number of S-phase neuroblasts. Biochemical analysis
has shown that FGF-2 can be coimmunoprecipitated with Trol
and that the binding of FGF-2 to Trol can be competed by
added heparin. This suggests that, like mPerlecan, Trol
binds FGF-2 through heparan sulfate residues. These results
demonstrate that Trol-mediated FGF signaling is required
for initiation of neuroblast proliferation sometime in first
larval instar. This similarity to the function of mPerlecans in
mammalian FGF signaling and the implications of up-regulation
of mPerlecan in tumors strongly imply that trol
encodes a functional Drosophila Perlecan homolog (Park, 2003).
Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).
The Drosophila tracheal system develops from epithelial sacs of about 80 cells from which primary, secondary and terminal branches sprout without cell division or cell death. Primary branch sprouting is induced by Branchless (Bnl) FGF, a chemoattractant secreted by clusters of cells surrounding each sac, which activates Breathless (Btl) FGF receptor (FGFR), a receptor tyrosine kinase expressed on tracheal cells. Primary branches contain 3-20 cells that organize into a tube as they migrate out from the sac. Bnl also induces the expression of secondary branching genes, such as the transcription factor pointed (pnt), and specifies terminal cells at the ends of outgrowing branches. Terminal cells ramify in the larva in response to Bnl to form fine terminal branches. Other cells at the ends of primary branches become fusion cells that connect with neighbouring branches to form a continuous tracheal network. Terminal and fusion cell fate decisions are also influenced by the Notch, Dpp and Wingless signalling pathways. Dorsal branches, the primary branches that were focused on here in this study, typically consist of five or six cells: two cells near the branch tip, one (DB1) that becomes a terminal cell and another (DB2) that becomes a fusion cell, and three or four cells (DB3-DB6) that form the branch stalk (Ghabrial, 2006).
In a genetic mosaic screen, six mutants (724, 788, 1118, 1187, 1476 and 1684) were identified with a subtle phenotype: mosaic branches (+ / + , +/ - , -/ - cells) were grossly normal, yet homozygous mutant clones (- / - cells) rarely if ever included terminal cells. These were neither general nor terminal-cell-specific lethal mutations because homozygous mutant cells were readily recovered in all other tracheal positions, and there was no decrease in the overall number of cells in mosaic dorsal branches or the number of terminal cells. It was difficult to imagine how mutations could block clone generation in specific cells. It seemed more likely that the mutations caused cells otherwise destined to become terminal cells to switch fates with other tracheal cells (Ghabrial, 2006).
The six mutations compose a single lethal complementation group that mapped to the left arm of chromosome 3 and failed to complement breathlessLG18. DNA sequencing identified a single nucleotide change in each mutant resulting in a nonsense or missense mutation in btl. Five mutations (724, 788, 1118, 1476 and 1684) appear to be null btl mutations, whereas the sixth mutation (1187) causes partial loss of function. Thus, the 'no mutant terminal cells' gene is btl (Ghabrial, 2006).
The distribution of cells homozygous was quantified for btl null mutations (724 and 788), or homozygous for a wild-type btl allele as a control, in mosaic dorsal branches. Control clones were evenly distributed throughout the branch at the expected frequencies; for example, the ratio of stalk-cell to terminal-cell clones was about 3:1. By contrast, btl-/- cells showed a nearly complete bias against the DB1 position: the ratio of stalk-cell to terminal-cell clones was 51:1. The three exceptional mutant terminal cells may be cases in which the clone was induced after btl began to be expressed, allowing wild-type btl gene products to perdure in mutant cells. Hundreds of mosaic branches with one or more btl-/- cells present in positions DB2-DB6 were recovered without affecting cell or branch morphology. Indeed, branches composed largely or exclusively of btl-/- cells, except for a wild-type terminal cell, were morphologically indistinguishable from wild-type branches. Thus, although all tracheal cells normally express btl, and the receptor is activated by Bnl in most or all cells of budding branches, the receptor appears to be required in just a single leading cell (DB1). All other cells can migrate normally and form tubes in the absence of btl. It is concluded that there are two functionally distinct classes of cells in budding primary branches: lead cells, which require Btl FGFR and directly respond to Bnl FGF, and trailing cells, which do not require Btl but follow the lead cell and form the stalk (Ghabrial, 2006).
What does it take to become the leader? The lead cell (DB1) is specified to become a terminal cell by Bnl-Btl signalling. If terminal cell specification is required, then null mutations in the downstream gene pnt, which abolish this function, should have the same effect as btl mutations. Cell clones homozygous for pntDelta88 or two new pnt alleles isolated in the screen (198 and 1318) failed to develop as terminal cells, as expected. However, unlike btl mosaic branches, pnt mosaic branches often lacked a terminal cell. When a terminal cell was missing, there was usually a pnt-/- cell in the stalk position nearest the tip, presumably the DB1 cell that failed to differentiate into a terminal cell. This suggests that pnt-/- cells are able to assume the lead position but fail to differentiate as terminal cells, and that the bias against btl mutant terminal cells is due to the earlier, pnt-independent, function of Btl in primary branch budding and outgrowth. If cells lacking Btl cannot migrate in response to Bnl during budding, they should not be able to move to the lead position necessary to be selected as a terminal cell. Consistent with this, genetic mosaic analysis of stumps (dof/heartbroken), which encodes a Btl adaptor required for cell migration, showed a dearth of terminal cell clones similar to btl (Ghabrial, 2006).
Two results demonstrate that the ability to sense Bnl and migrate in response to it is not enough to become the leader: cells compete for the lead position. The first involves btlBN (E796K mutation in the kinase domain), a weak btl allele isolated in a separate screen. Unlike btl-/- animals, which die in first larval instar and lack virtually all branches, btlBN homozygotes survived until L3 larval stage or beyond and had a normally patterned tracheal system with a full complement of terminal cells. The only defects detected were a reduced number and altered morphology of terminal branches, presumably due to the dosage-sensitive function of btl in terminal branch outgrowth. The late and subtle phenotype demonstrates that BtlBN protein retains sufficient activity for early migration and terminal cell specification events. However, in genetic mosaic animals, in which btlBN/BN cells must compete with btlBN/+ and btl+/+ tracheal cells, btlBN/BN cells rarely acquired the lead position (DB1) and developed as terminal cells. Indeed, homozygosity for btlBN conferred nearly as complete a bias against becoming a terminal cell as total loss of btl. Thus, Btl activity above the threshold necessary for migration and terminal cell specification is not sufficient to acquire the lead position and become a terminal cell: a cell must have more Btl activity than other cells in the branch (Ghabrial, 2006).
Similar conclusions derive from a second experiment in which marked wild-type (btl+/+) cells were analysed in heterozygous (btl+/-) animals. Whereas btl+/+ clones in control (btl+/+) animals were distributed evenly throughout the branch, btl+/+ clones in btl788/+ heterozygotes preferentially localized to the tip. Cells that did not occupy the lead (DB1) position took positions close to the tip. Similar results were obtained for btl+/+ clones in animals heterozygous for btl1187, a partial-loss-of-function allele. Clones mutant for sprouty, an FGF feedback inhibitor, also preferentially populated the tip. Together, the data show that there is competition for the lead position: cells with highest btl activity assume positions at or near the tip of the branch, whereas those with less or no activity segregate towards its base (Ghabrial, 2006).
Because small differences in btl dosage or activity affect a cell's ability to compete for the lead, whether lateral inhibitory mechanisms that amplify small differences in signalling might be operative was investigated. Data suggest that the Notch pathway, a lateral signalling pathway implicated in cell specification events including cell fate determination at tracheal branch tips, also affects cell arrangement. Nts embryos shifted to the restrictive temperature during budding formed branches in which most DB cells behaved like lead cells, resulting in large clusters of cells congregated at the lead position, whereas expression of constitutively active NACT throughout the tracheal epithelium had the opposite effect, arresting outgrowth and stalling cells near the base of the branch. It is proposed that Notch-mediated lateral inhibition among tracheal cells prevents extra cells from assuming the lead position (Ghabrial, 2006).
These results provide evidence for social stratification and dynamic social interactions between epithelial cells during branching morphogenesis. First, the results show that budding cells are functionally specialized. A cell at the branch tip requires btl and leads outgrowth towards the Bnl signalling center. Trailing cells do not require btl but nevertheless follow the lead cell towards the Bnl source. Because tracheal cells do not migrate or form tubes in btl-/- animals, trailing cells must receive a secondary signal generated by the lead cell that induces them to migrate and also activates their tubulogenesis program. This could be a secreted molecule or physical stimulus such as pulling or stretching the trailing cells (Ghabrial, 2006).
Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity become lead cells whereas those with less or no btl FGFR activity become trailing cells and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibitory signalling between tracheal cells, and it may also be influenced by positive feedback mechanisms such as increased activation and expression of Btl as cells approach the Bnl source. Third, there may be cooperation between cells, because in a genetically mosaic epithelium, tracheal cells with less Btl activity allow those with more activity to move ahead of them (Ghabrial, 2006).
There may be similar social interactions between budding cells in other branching organs. Studies of other branching processes have identified genes selectively expressed in tip cells of budding branches, and in some cases these cells display morphological specializations indicating that they might actively lead outgrowth. However, because most budding branches contain hundreds or thousands of cells, it is difficult to track and manipulate individual cells to investigate social behaviours like those described here. Recent analyses of chimaeric Ret+/Ret- mouse renal ureteric buds in culture and btl mosaic air sacs reveal that cells lacking these receptor tyrosine kinases are excluded from branch tips, indicating that RTK-dependent interactions similar to those described here might be operative in more complex branching events (Ghabrial, 2006).
Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. This study analyzed the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. It was confirmed that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, genetic approaches were utilized that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. This study demonstrated that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. It is contended that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable (Kadam, 2009).
These experiments demonstrate that the Drosophila
FGFs Pyr, Ths and Bnl have different functions and that the activation of FGF
receptors by specific ligands affects particular biological processes.
Examination of an allelic series of pyr and ths mutants
suggests that pyr and ths are not redundant in function:
both influence mesoderm spreading, whereas pyr is the dominant player
controlling Eve+ cell specification within the dorsal mesoderm. It has been demonstrated that ectopic expression of ths by twist-Gal4 and
69B-Gal4 in the Df(2R)BSC25 mutant background can support
Htl FGFR activation. However, this study assayed whether the expression
supported in distinct domains would support Htl activation. By a series of
'rescue' experiments, through ectopic expression of one ligand in the
Df(2R)BSC25 mutant background, evidence was obtained that localized expression of the ligands is important for proper mesoderm spreading. It was found, surprisingly, that the ligands exhibit differences in their functional range of action. In addition, using this same approach, it was found that either Pyr or Ths can support Eve+ cell specification within the dorsal mesoderm, but that Bnl cannot. Collectively, these data suggest that the Pyr and Ths FGFs function as ligands for the Htl FGFR and that specificity of FGF-FGFR interactions exists in Drosophila (Kadam, 2009).
The results demonstrate that both Pyr and Ths FGF ligands can activate the
Htl FGFR, whereas only the Bnl FGF ligand can activate the Btl FGFR. Specificity of FGFR activation was observed:
pyr or ths, but not bnl, expression is able to
activate Htl to affect expression of Eve, and bnl, but neither
pyr nor ths, is able to support tracheal specification. No
evidence was obtained that other cross-interactions occur (i.e. Pyr-Btl,
Ths-Btl or Bnl-Htl), which demonstrates that Gal4-mediated ectopic expression
does not simply 'swamp the system'. This experimental approach also 'levels
the playing field', since expression of each ligand is driven at the same time
and place and presumably at similar levels. It is concluded that only three
FGF-FGFR combinations function in Drosophila (i.e. Pyr-Htl, Ths-Htl
and Bnl-Btl), which supports the idea that FGFRs exhibit ligand-binding
preferences. Previous studies have investigated FGF signaling specificity by
analyzing the ability of other receptor tyrosine kinases to support cell
migration or by activating particular intracellular signaling pathways to
examine which are required to effect FGFR-dependent cell migration versus cell
differentiation. This work analyzed the specificity of FGF ligand-receptor interactions and how they contribute to particular developmental processes (Kadam, 2009).
When ligand expression is supported by twist-Gal4, Htl FGFRs
presumably become saturated because dpERK is ectopically activated in all
cells and spreading is negatively affected. One explanation for why this might affect mesoderm cell spreading is that these FGF-saturated mesoderm cells may no longer be competent to respond to endogenous ligands that provide directional cues. Recently, it has been shown that movement of the mesoderm cells during gastrulation is in fact
directional (McMahon, 2008). Pyr and Ths ligands are differentially expressed during
gastrulation and this might provide the necessary positional information
required to direct migration of the mesoderm. It is proposed that Pyr and Ths have different activities that fulfil aspects of FGFR activation required to support cell migration. Ectopic expression of Pyr within the ectoderm negatively affects mesoderm spreading, which suggests
that the refined expression domain of pyr within cells of the dorsal
ectoderm is normally required to guide the mesoderm cells toward dorsal
regions. However, even though ectopic expression of ths in the
ectoderm has no effect on mesoderm spreading, ths mutants also
exhibit defects in mesoderm spreading, demonstrating that both genes are
required, perhaps to control different aspects of the migration. The 'rescue'
experiments using the zenVRE.Kr-Gal4 driver support the view that Pyr
has a longer functional range than Ths. These differences
in range of function might correlate with different diffusion capabilities,
but an alternative explanation is that the ligands activate the receptor with
different affinities. Additional experiments will be necessary to distinguish
their exact functions and to uncover the molecular basis for the differential
functions of Pyr and Ths; it is suggested that in vivo imaging and quantitative
analysis (McMahon, 2008) of single-mutant phenotypes will provide insights (Kadam, 2009).
With regard to the FGF-dependent cell differentiation, the 'rescue'
experiments suggest that ectopic expression of either Pyr or Ths is sufficient
to support Eve+ cell specification. The reason why
loss of ths has less of an effect on Eve+ cell
specification is most likely because pyr is prominently expressed in
the vicinity of the future Eve+ cells; normally, Pyr supports this function, but Ths can support this activity if presented at sufficient levels
within the correct domain. Furthermore, it is proposed that FGF signaling might
not play an instructive role in supporting eve expression. Other signaling
pathways already provide positional information required for the specification
of Eve+ cells; FGF signaling pathway activation might simply serve
a permissive role, and in this context either ligand would suffice (Kadam, 2009).
branchless:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
date revised: 28 February 98Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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