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First published online 19 September 2007
doi: 10.1242/dev.007328
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1 Institut de Biologia Molecular de Barcelona (IBMB-CSIC), Parc Cientific de
Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.
2 Institut de Recerca Biomedica de Barcelona (IRB), Parc Cientific de Barcelona,
Josep Samitier 1-5, 08028 Barcelona, Spain.
* Author for correspondence (e-mail: mlcbmc{at}cid.csic.es)
Accepted 2 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ttk, Tramtrack, Tracheal system, Drosophila, Morphogenesis, Organogenesis, Tubulogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hogan and Kolodziej,
2002; Lubarsky and Krasnow,
2003). Full identification of the genetic circuits and molecular
mechanisms used to control these cellular responses will represent a step
forward in our understanding of organogenesis.
The Drosophila tracheal system represents one of the best models
for organogenesis and tubulogenesis. We use this system to approach how
morphogenesis is controlled both genetically and at the cellular level.
Tracheal patterning occurs via different cellular processes, including cell
migration and intercalation, branch fusion and formation of luminal
structures. Throughout the tracheal tree, these processes involve the
acquisition of different cell fates and the ability of cells to respond to
both intracellular and extracellular cues
(Manning and Krasnow, 1993).
Over the last decade, several studies have identified genes required for the
orchestration and coordination of these aspects. Transcription factors have
been reported to play key roles (reviewed in
Ghabrial et al., 2003). For
instance, Trachealess (Trh) and Ventral veinless (Vvl) are involved in
orchestrating early events by inducing many early tracheal-specific genes. At
later stages, subpopulations of cells giving rise to different branches with
distinct properties express specific transcription regulators, such as Knirps
(Kni) and Spalt (Sal). Other transcription factors, such as Grainy head (Grh)
are broadly expressed later to fulfil other specific requirements, such as the
control of apical membrane growth.
The transcription factor Tramtrack (Ttk) was first identified as a
zinc-finger protein involved in the regulation of the pair-rule gene fushi
tarazu (ftz) (Harrison and
Travers, 1990). ttk encodes two isoforms, Ttk69 and
Ttk88, which share N-terminal sequences containing a BTB/POZ domain, but
differ in their C-terminal region, in which their DNA-binding zinc-fingers
reside. For this reason, it is assumed that they have different DNA-binding
specificities and functions (Read and
Manley, 1992).
Ttk has been most extensively characterised in the developing embryonic
nervous system, in which it acts as a repressor
(Badenhorst, 2001;
Giesen et al., 1997;
Guo et al., 1995), and during
photoreceptor differentiation, during which it additionally plays a positive
role (Lai and Li, 1999).
During early embryogenesis, Ttk regulates the pattern of several pair-rule
genes (Brown et al., 1991;
Brown and Wu, 1993;
Read and Manley, 1992). In
addition, a role for Ttk in cell cycle regulation has also been proposed
(Audibert et al., 2005;
Badenhorst, 2001;
Baonza et al., 2002). To date,
most of the described requirements for Ttk rely on its ability to regulate
cell fate specification. Conversely, very little is known about other roles of
Ttk in morphogenesis regulation downstream of cell fate determination
(French et al., 2003).
In this study, we have analysed tracheal developmental dynamics with an emphasis on the functional orchestration of diverse morphogenetic steps. In addition to previously defined roles, we report here that Ttk controls various cellular responses downstream of cell fate specification. We find that Ttk is autonomously involved in a pathway leading to cell rearrangements and intercalation, most probably via the regulation of cell shape and the remodelling of adherens junctions (AJs). Remarkably, Ttk also controls tube size autonomously, regulating septate junction (SJ) activity and cuticle formation. Moreover, we define Ttk as the first identified regulator of intracellular lumen formation, and as a factor required autonomously and non-autonomously to specify different tracheal cell identities. The non-autonomous requirement, mediated by branchless (bnl) modulation, is also involved in the establishment of primary branching. In summary, we propose that Ttk plays a key role in the regulation of multiple steps during tracheal development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The wild-type strain used was yw. Drosophila stocks and crosses were kept on standard conditions at 25°C. Overexpression experiments were conducted at 29°C.
Molecular analysis
The GS element in line 346 was mapped by inverse PCR techniques
following standard protocols (BDGP,
http://www.fruitfly.org/about/methods/index.html).
Immunostaining, in situ hybridisation and permeabilisation assays
Embryos were staged according to Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1985) and stained following standard protocols. Immunostainings
were performed on embryos fixed in 4% formaldehyde for 20-30 minutes, except
for DCAD2 (encoded by shotgun - FlyBase) stainings, which were fixed
for 10 minutes. The following antibodies were used: anti-Ttk69 (C. Murawsky
and A. Travers, MRC-LMB, Cambridge, UK), anti-Dys (S. Crews, University of
North Carolina, USA), anti-Sal (R. Schuh, Max Plank Institut, Göttingen,
Germany), anti-Cora (R. G. Fehon, University of Alberta, Canada), anti-FasIII
(7G10, Developmental Studies Hybridoma Bank, DSHB), anti-Verm (S. Luschnig,
University of Bayreuth, Germany), anti-Lac (M. Strigini, IMBB, Crete, Greece),
mAb2A12 (DSHB), anti-DSRF (2-161, Cold Spring Harbor Laboratory, CSHL),
anti-Kni (developed by J. Reivitz and provided by M. Ruiz-Gomez, CBM, Madrid,
Spain), anti-DE-cad (DCAD2, DSHB), anti-Trh (made by N. Martín in J.
Casanova's laboratory, IRB, IBMB-CSIC, Barcelona, Spain), anti-GFP (Molecular
Probes and Roche), anti-ßGal (Cappel and Promega) and anti-Pio (from M.
Affolter). Biotinilated or Cy3-, Cy2- and Cy5-conjugated secondary antibodies
(Jackson ImmunoResearch) were used at 1/300. For HRP histochemistry the signal
was amplified with the Vectastain-ABC kit. For fluorescent staining, the
signal was amplified using TSA (NEN Life Sciences) when required. Chitin was
visualised with Fluostain (Sigma) at 1 µg/ml or CBP (NEB) at 1:500.
Permeabilisation assays were performed by injecting rhodamine-labelled dextran
(Mr 10,000; Molecular Probes) into the body cavity of
embryos (Lamb et al., 1998).
In situ hybridization was performed according to standard protocols, with
ribo-pyd probe (gift from M. Neumann, University of Basel,
Switzerland). ribo-bnl and ribo-mmy were generated using the
whole cDNA as template and using the Megascript kit (Ambion). Photographs were
taken using Nomarski optics or fluorescence in a Nikon Eclipse 80i microscope.
Confocal images were obtained with a Leica TCS-SPE or TCS-SP2 system.
Unless otherwise stated, in all panels labelled `GFP' the embryos carried btlGal4 driving GFP-fusion proteins (btl>xGFP in Figures). btlGal4 also drove the expression of other indicated UAS constructs. We used mAb2A12 or CBP to visualise the lumen.
Luminal vesicle quantification
We quantified the number of 2A12-positive vesicles in the same
two-dimensional areas of confocal projections in both fusion and terminal
cells in wild-type and ttk embryos. We counted 2A12 vesicles within
these areas and subtracted the background measured outside fusion and terminal
cells in each case. We used the AnalySIS software v.3.2 (Soft Imaging System
GmbH) to quantify the number of vesicles.
Electron microscopy
Wild-type and ttk mutants at stage 16-17 were selected under a
stereomicroscope, and cryo-fixed and analysed according to Araujo et al.
(Araujo et al., 2005).
Time-lapse experiments
Embryos carrying btlGal4UAS-srcGFP;ttkD2-50 or
btlGal4UAS-srcGFP were collected at 25°C and dechorionated for 2
minutes with sodium hypochlorite diluted 1/100. They were glued to a coverslip
and mounted in 10S Voltaleff oil with the hanging drop method to improve
optics and to avoid desiccation in an oxygen-permeable chamber. Images were
collected from stage 14 embryos at 21°C on a Leica TCS-SP2-AOBS or
TCS-SP5-AOBS system, Leica DM IRE2 microscope and LCS software. The 488 nm
emission line of an Argon laser was used for excitation and sections were
recorded every 4 or 5 minutes over a 3- to 6-hour period. Laser intensity was
kept at a minimum to minimise phototoxicity. TIFF projection images were
processed into 3D and 4D LCS software, and the movie was assembled using
ImageJ (NIH Image).
Quantification of the intercalation defects
ttkD2-50 mutants and ttkD2-50
mutants expressing ttk69 in the tracheal tissue (obtained from the
cross btlGal4/btlGal4; ttkD2-50/TM3lacZ x
ttkD2-50UASttk69/TM3lacZ and selected by the absence of
blue balancers) were immunostained with DCAD2. To determine the intercalation
state, we carefully analysed the presence of intercellular versus autocellular
AJs in each dorsal branch (DB) and lateral trunk (LT) of stage 15 or 16
embryos under the microscope. Each branch was classified into one of the four
categories we describe in Fig. S1 in the supplementary material.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to generate an original collection of lines whose tracheal
phenotypes were analysed by crossing with breathless-Gal4
(btlGal4) (C.C. and M.L., unpublished). GS line 346 was
selected because of its impairment of tracheal branch fusion
(Fig. 1A). This insertion was
mapped to 100D, close to the ttk gene. When an independent
UASttk69 line was crossed to btlGal4, the GS346
tracheal phenotype was reproduced (data not shown), indicating that the
tracheal phenotype was indeed due to ttk over- or mis-expression.
ttk is maternally supplied, subsequently declines and zygotic
expression reappears during germ band extension
(Harrison and Travers, 1990;
Read and Manley, 1992). From
stage 11 until the end of embryogenesis, clear expression of ttk is
observed in all tracheal cells (Fig.
1B,C). We found that ttk tracheal expression does not
depend on genes known to induce tracheal fate, such as vvl, trh or
kni, on their own (data not shown), suggesting that it might depend
on a combination of these inducers or directly on the same anteroposterior
(A-P) and dorsoventral (D-V) embryonic cues regulating tracheal inducers
(de Celis et al., 1995;
Wilk et al., 1996).
Strong tracheal pattern defects were detected in amorphic mutants
(ttkD2-50) whereas milder defects were observed in
hypomorphic mutants (ttkrM730)
(Fig. 1E and data not shown).
The ttk locus encodes ttk88 and ttk69
(Read and Manley, 1992).
Mutants for ttk88 (ttk1)
(Xiong and Montell, 1993) are
viable and do not show a tracheal phenotype. In addition, overexpression of
ttk88 did not result in tracheal defects (data not shown).
Conversely, mutants for ttk69 (ttk1e11)
(Lai and Li, 1999) displayed a
clear tracheal phenotype (Fig.
1F) and, as already indicated, overexpression of ttk69
affected tracheal development (Fig.
1A). These results suggest a specific role for ttk69
during tracheal development. In this work, we used either
ttkD2-50 or ttk1e11 to analyse
ttk tracheal requirements.
Ttk is non-autonomously required to establish proper tracheal identities
Early steps of tracheal development, such as tracheal induction and
invagination, proceeded normally in ttk mutants. The first tracheal
defects were visible from stage 13, when visceral branches (Vs.) were often
missing or reduced (Fig. 1G)
and, if present, contained fewer cells. Cell counts indicated that the rest of
the primary branches contained grossly the normal number of cells, except for
the transverse connective (TC), which contained more cells (19.2 cells,
n=10, in the TC of the fifth tracheal Metamora of ttk
mutants as compared with 8-10 cells in wild type)
(Samakovlis et al., 1996a),
suggesting that the TCs incorporate the presumptive VB cells.
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). The receptor Btl, expressed in all tracheal cells, is
activated by its ligand, Bnl, which is dynamically expressed outside the
tracheal tissue in positions towards which the primary branches will grow
(Sutherland et al., 1996). We
found that, from stage 12-13, bnl expression was slightly reduced and
disappeared earlier from the dorsal and from most ventral spots, and was
almost lost from the visceral mesoderm
(Fig. 1J), which is presumably
responsible for VB formation (Sutherland
et al., 1996). Consistent with a positive regulation of
bnl expression by ttk, we found that ttk is
co-expressed in bnl-expressing cells
(Fig. 1K).
Besides its role in primary branching, the Bnl/Btl pathway is also required
for terminal cell specification via the regulation of DSRF (also
known as blistered - FlyBase)
(Sutherland et al., 1996). We
investigated whether terminal cells were specified in ttk mutants and
found that cells expressing DSRF protein were generally present in some
branches [i.e. lateral trunk anterior (LTa)], but only occasionally present in
others, such as in dorsal branches (DBs), VBs and lateral trunk
posterior-ganglionic branches (LTp-GBs)
(Fig. 1M). This pattern of DSRF
is consistent with the defective pattern of bnl expression observed
in ttk mutants: in those spots in which bnl is lost or
reduced, DSRF is rarely expressed.
Altogether these results indicate that ttk regulates the allocation of cells to particular primary branches and the specification of the terminal cells by modulating bnl expression outside the tracheal tissue.
Specification of fusion fate requires Notch-mediated regulation of Ttk levels
In wild-type embryos, tracheal cells that mediate branch fusion express
specific markers, such as escargot (esg) and
dysfusion (dys) (Jiang
and Crews, 2003; Samakovlis et
al., 1996b; Tanaka-Matakatsu
et al., 1996) (Fig.
2A and data not shown). Protein expression of these markers was
not detected in btlGal4-UASttk embryos
(Fig. 2B and data not shown),
revealing that tracheal cells failed to acquire the fusion identity. This
results in an absence of branch fusions
(Fig. 1A).
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The fusion phenotype of ttk overexpression resembles that of
constitutive activation of the Notch (N) pathway, which also blocks fusion.
Indeed, N is active in fusion-adjacent cells, restricting the fusion
fate specification in that area (Ikeya and
Hayashi, 1999; Llimargas,
1999; Steneberg et al.,
1999). This pattern of N activity correlates with the
protein pattern of Ttk69. Furthermore, Ttk acts as an effector of N signalling
in several developmental contexts (Guo et
al., 1996; Jordan et al.,
2006; Okabe et al.,
2001). Interestingly, we found low Ttk69 protein levels in the
extra fusion cells present in N loss-of-function conditions [achieved
by overexpressing Hairless (H) (Llimargas,
1999), Fig. 2G].
Conversely, we did not find cells expressing low levels of Ttk69 protein in
constitutively activated N conditions (Fig.
2H). These results suggest that ttk acts as a downstream
effector of N during fusion cell type specification, although N might
have other targets to fulfil this function (see below and Discussion).
Ttk is involved in the pathway leading to tracheal cell rearrangements
By late embryogenesis, the tracheal pattern of ttk mutants
resembles that of stage 13 or 14 embryos, as if branches did not extend
properly (Fig. 1E,F). During
wild-type development, branches extend by directed cell migration and cell
rearrangements. Cell intercalation, a particular type of cell rearrangement,
occurs in most primary multicellular branches except the dorsal trunk (DT)
(Fig. 3A-B').
Intercalation has been divided into four steps
(Ribeiro et al., 2004): (1)
pairs of cells connected by intercellular AJs arrange side-by-side; (2) one of
the two cells reaches around the lumen with its distal end while the other
does it with its proximal end, thereby forming autocellular AJs at the points
at which the AJs of each single cell meet and seal; (3) the nascent
autocellular AJs elongate and zip up as the two cells arrange in an end-to-end
position; (4) the zipping-up process is stopped, leaving the two cells
connected by a small ring-like intercellular AJ.
We analysed cell intercalation in ttk mutants by monitoring DE-cadherin (DE-cad) protein accumulation, an marker of AJs (Fig. 3C-D'). Tracheal cells in branches in which intercalation usually occurs did not rearrange and remained positioned in side-by-side pairs by late embryogenesis (Fig. 3D). Autocellular AJs (visualised as lines after using AJ markers, Fig. 3B') only occasionally formed or zipped up. Indeed, we found several DBs with no signs of autocellular AJs (Fig. 3D', arrowhead; see Fig. S1 in the supplementary material) and others with short stretches of autocellular AJs followed by long stretches of intercellular ones (visualised as a mesh-like structure, Fig. 3D', arrows; see Fig. S1 in the supplementary material). Similar results were observed in the lateral trunk (LT) (Fig. 3E-F'; see Fig. S1 in the supplementary material). These results indicate that, in ttk mutants, the step involving reaching around the lumen is generally prevented and the zipping up, when it occurs, is incomplete.
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), in which it prevents intercalation
(Ribeiro et al., 2004).
Conversely, the transcription factors Kni and Knrl are expressed in all other
primary branches except the DT (Chen et
al., 1998), and they repress Salm, thereby promoting intercalation
(Ribeiro et al., 2004). Salm
and Kni proteins were correctly accumulated in ttk mutants
(Fig. 3H,I), indicating that
the allocation of cells to different primary branches was correct.
Additionally, we found that piopio (pio), required for the
intercalation process (Jazwinska et al.,
2003), was normally expressed and that Pio protein accumulated
normally (Fig. 3K) in
ttk mutants. We next asked how ttk was required for intercalation. We found that adding ttk to tracheal cells in ttk mutants rescued intercalation (Fig. 3G, see Fig. S1 in the supplementary material). This indicated an autonomous requirement for ttk during intercalation and ruled out the possibility that impaired intercalation was due to defects in other tissues (for instance, due to impaired dorsal closure).
Ttk regulates cell shape changes and modulates AJs during intercalation
How does Ttk affect intercalation? To approach this question, we performed
time-lapse experiments in embryos carrying btlGal4 UAS-srcGFP, in
which the outline of tracheal cells is highlighted
(Fig. 3E-F',
Fig. 4 and see Movies 1-4 in
the supplementary material). In an otherwise wild-type background, paired
cells of intercalating branches showed a short period of rapid relative
movement followed by a directional sliding. These movements were accompanied
by a conspicuous change in shape, which transformed originally paired-cuboidal
cells into a single row of elongated ones. Strikingly, ttk mutant
cells remained cuboidal throughout development, and only occasionally could
weak signs of cell elongation be detected. These cuboidal-paired cells still
showed the relative movement (for longer periods than in wild type), but this
was not usually followed by a shift to a directional displacement.
ttk mutant cells appeared unable to undertake cell shape changes,
which we suggest (see Discussion) prevents the paired cells to slide one over
the other and intercalate.
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), was
working in ttk mutants. In wild type, numerous thin filopodia
extended at the tips of primary branches in response to Bnl
(Fig. 4, see Movies 1 and 2 in
the supplementary material) (Ribeiro et
al., 2002). Although in ttk mutants bnl
expression was affected, we detected filopodia at the tips of the branches,
presumably in response to Bnl (Fig.
4, see Movies 3 and 4 in the supplementary material). This result
suggests that the bnl mRNA remaining in ttk mutants should
be sufficient to drive intercalation. Supporting this hypothesis, we found
that ttk tracheal expression rescues the intercalation defects of
ttk mutants (Fig. 3G),
ruling out the possibility that impaired intercalation was due to the
non-autonomous requirement of ttk for bnl expression.
A correlation between the modulation of AJs and intercalation during
tracheal development has been recently established in a report on the role of
Polychaetoid (Pyd). pyd encodes a MAGUK protein that localises to
AJs, and loss of pyd prevents intercalation
(Jung et al., 2006). We found
that pyd is a target of Ttk; pyd expression was lost in
ttk embryos (Fig. 3M)
and enhanced by ttk overexpression
(Fig. 3N). Our results indicate
that ttk autonomously regulates intercalation not by regulating cell
fate but by allowing cell shape changes and by modulating AJs via Pyd.
Ttk is required for the proper fusion of branches
Besides its role in fusion fate specification (see above), we also found
that ttk mutants show impaired (in DBs and the LT) or delayed (in the
DT) fusion events. In particular, we observed that 30% of ttk embryos
showed a complete absence of LT fusion, 40% showed one single anastomosis out
of the total nine per hemisegment, and the remaining mutants never showed more
than three anastomosis. It is unlikely that this is caused by defects in
fusion fate specification, because fusion markers, such as esg and
dys (Fig. 5A and data
not shown), are expressed in a normal pattern but at slightly lower levels
than in wild type, although we cannot rule out this possibility. At the
cellular level, the fusion process has been well-characterised
(Lee et al., 2003;
Samakovlis et al., 1996b;
Tanaka-Matakatsu et al.,
1996). It begins when two fusion cells extend cytoplasmic
processes and make contact. Then they form a new DE-cad contact at the
interface (Fig. 5C), promoting
the formation of an actin-containing track that guides the invaginating apical
surfaces of the fusion cells. Finally, the two apical surfaces meet and fuse,
giving rise to two doughnut-shaped fusion cells containing an intracellular
junctionless lumen. It has been proposed that this intracellular lumen forms
by assembly and coalescence of luminal vesicles that appear at the tip of the
growing lumen (Uv et al.,
2003). We observed that the first steps of branch fusion appear
normal in ttk mutants: the presumptive fusion cells extended
filopodia and established contact, and they formed a new DE-cad contact at the
interface (Fig. 5D). However,
ttk mutant cells seemed defective in generating an intracellular
lumen that penetrates the fusion cell (Fig.
5B). In agreement with this, we detected fewer luminal vesicles in
ttk fusion cells as compared with wild type (0-5 vesicles in
ttk mutants, n=10, versus 10-20 in wild type, n=10;
see Materials and methods) (Fig.
5E',F', arrowheads). Altogether, our observations
suggest that ttk regulates a step downstream of fusion fate
specification: contact of fusion cells and formation of a new DE-cad
contact.
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At the cellular level, terminal branches are intracellular junctionless
tubes formed inside the terminal cells. Terminal cells form an F-actin-rich
structure and extend cytoplasmic protrusions that are invaded by an
intracellular lumen, which presumably grows by the fusion of intracellular
vesicles (Oshima et al., 2006;
Uv et al., 2003). Time-lapse
experiments of btlGal4 UAS-srcGFP embryos revealed the formation of
cytoplasmic protrusions in ttk mutants, although these filopodia were
never stabilised and no internal lumen was detected
(Fig. 4, and see Movies 3 and 4
in the supplementary material). In addition, we detected a lower density of
luminal vesicles in presumptive terminal cells in ttk mutants as
compared with wild type (0-3 vesicles in ttk mutants, n=10,
versus 20-30 in wild type, n=10; see Materials and methods)
(Fig. 5E',F',
arrowheads), suggesting a defect in intracellular lumen formation.
Ttk plays a key role in the control of tube size
By late embryogenesis, the tracheal tubes of ttk mutants appeared
thicker and more convoluted than those in wild type. On average, the diameter
of the largest part of the DT (between abdominal segments 6 and 8) is 25%
wider in ttk mutants than in wild type (n=12)
(Fig. 6A,B). This phenotype is
reminiscent of other mutants affecting tube size. To date, two different
systems have been reported to regulate tube size: the septate junctions (SJs)
and a transient chitin filament [(Swanson
and Beitel, 2006; Wu and
Beitel, 2004) and references within].
We investigated whether the intraluminal chitin matrix was properly organised in ttk mutants. Using a fluorescent chitin binding protein (CBP) or Fluostain, which label chitin fibrils, we detected clear differences between ttk and wild-type embryos (Fig. 6C,D and data not shown). Instead of an organised cylindrical filament composed of parallel chitin polymers, ttk mutants showed an amorphous, decreased and disorganised labelling, with a scratched perpendicular pattern. These results point to a defect in the assembly of the chitin filament, consequently affecting tube size.
Several genes participate in a pathway devoted to chitin synthesis in the
trachea. mummy (mmy) encodes a UDP-N-acetylglucosamine
pyrophosphorylase enzyme required for the synthesis of the building blocks of
chitin (Araujo et al., 2005;
Devine et al., 2005;
Tonning et al., 2006). We
found that mmy is a target of Ttk, because its expression was
increased in ttk embryos at stages in which chitin is being
synthesised and lowers when ttk is overexpressed
(Fig. 6E-G). The chitin
filament has to be properly assembled and modified to become functional.
serpentine (serp) and vermiform (verm, also
known as LCBP1 - FlyBase) encode two ChLD (Chitin and LDL-receptor
binding motifs) proteins required to assemble the intraluminal chitin filament
and restrict tube elongation (Luschnig et
al., 2006; Wang et al.,
2006). We detected a decrease in levels of these proteins in the
lumen of ttk mutants (Fig.
6K). These results indicate a defect of filament synthesis and
maturation.
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). Thus, we next analysed SJ complexes in detail. At confocal
resolution, we could only detect very subtle differences between ttk
and wild-type embryos using different markers. In particular, with antibodies
recognising Fasciclin III (FasIII, Fas3) or Lachesin (Lac), a slight decrease
and/or diffusion was detected in the trachea, salivary glands and hindgut of
ttk mutants, whereas we found no detectable differences in these
tissues with respect to Coracle (Cora) accumulation
(Fig. 6L,M and data not shown).
In agreement with this, transmission electron microscopy (TEM) analysis of
ttk embryos revealed the presence of properly localised SJs
(Fig. 7D). To further analyse
SJ functionality, we assayed for the trans-epithelial diffusion barrier of the
tracheal tubes by injecting 10 kDa rhodamine-labelled dextran
(Baumgartner et al., 1996;
Lamb et al., 1998). We
detected diffusion of this dye into the trachea of ttk mutants
(Fig. 6O). These results show
that, although the assembly of SJs is not grossly affected, the complexes are
not fully functional. This inefficient SJ activity might be responsible, at
least in part, for the tube size defects observed in ttk mutants.
Taken together, our results indicate that ttk regulates tube size by
controlling chitin filament formation and its proper assembly via SJ
activity.
Ttk is required for proper luminal cuticle formation
In ttk mutants, we found clear ultrastructural defects that were
related to abnormal chitin deposition. At late stages of development,
wild-type embryos display three distinguishable layers of cuticle (envelope,
epicuticle and procuticle). In addition, the luminal cuticle is decorated by
regular ridges known as taenidia. These taenidial folds are filled by the
procuticle, loaded with lamellar chitin, which, at TEM resolution, can be
recognised as a continuous and electron-dense material with an organised
aspect (Fig. 7A)
(Araujo et al., 2005;
Locke, 2001). In ttk
embryos, taenidia showed an irregular shape, size and pattern. In addition,
the material filling the taenidia (procuticle) was disorganised, discontinuous
and frequently contained inclusions of more electron-dense material
(Fig. 7B). The cuticle of the
larval epidermis was also affected in ttk mutants. Instead of the
characteristic lamellar organisation of wild-type procuticle
(Fig. 7E), ttk mutants
showed an amorphous, unstructured layer
(Fig. 7F). These observations
indicate that ttk is required for both epidermal and tracheal cuticle
formation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a similar fashion to the transcription factors Trh and Vvl (reviewed in
Ghabrial et al., 2003), which
are involved in orchestrating early events of tracheal development, Ttk plays
a role in orchestrating several late tracheal events. Ttk69 has been found to
act mostly as a repressor. Here we identify Ttk targets that appear to be
negatively regulated (such as mmy and esg) whereas others
appear to be positively regulated (such as pyd and bnl). In
this latter case, Ttk might be converted into a positive regulator, as already
described during photoreceptor development
(Lai and Li, 1999).
We identified different tracheal requirements for Ttk. Interestingly, most
of them depend on Ttk regulating events downstream of cell fate specification,
at the level of cellular responses (see below). Additionally, a few other
requirements depend on cell fate specification, as has been described for most
other functions of Ttk in other developmental situations. For instance, Ttk
regulates fusion cell specification by acting as a target and mediator of N,
as occurs during sensory organ development
(Guo et al., 1996;
Okabe et al., 2001) and
oogenesis (Jordan et al.,
2006). Such regulation of Ttk by N might be post-transcriptional,
as occurs during sensory organ development
(Okabe et al., 2001).
Remarkably, we found that, although Ttk is sufficient to repress esg
expression in fusion cells, it might not be the only esg- and fusion
fate-repressor, because absence of Ttk does not increase the number of
Esg-positive cells, as does downregulating N
(Ikeya and Hayashi, 1999;
Llimargas, 1999;
Steneberg et al., 1999). Other
N targets might be redundant with Ttk, and such redundancy could reinforce
N-mediated repression of fusion fate in positions in which inductive signals
(such as Bnl, Dpp and Wg) (Ikeya and
Hayashi, 1999; Llimargas,
2000; Steneberg et al.,
1999; Chihara and Hayashi,
2000; Llimargas and Lawrence,
2001) are very high, particularly near the branch tips.
The role of Ttk during cell rearrangements
Cell rearrangements during development are common to most animals and
ensure proper morphogenesis. During tracheal development, many branches grow
and extend by cell intercalation (Neumann
and Affolter, 2006; Pilot and
Lecuit, 2005). Several cellular and genetic aspects of tracheal
intercalation have been well described
(Ribeiro et al., 2004).
However, targets of Sal (which inhibits intercalation) are currently
unknown.
Here, we identify Ttk as a new and positive regulator of intercalation. We
found that Ttk is involved in cell junction modulation by transcriptionally
regulating pyd, the only junctional protein shown, so far, to affect
intercalation (Jung et al.,
2006). In fact, modulation of AJs has been proposed to play a role
during intercalation (Neumann and
Affolter, 2006). However, Pyd cannot be the only Ttk effector of
intercalation, because the pyd mutant phenotype is much weaker than
that of ttk mutants. Accordingly, we found that, in ttk
mutants, cells in branches that usually intercalate remain paired and
cuboidal, and appear unable to change shape and elongate. Although other
explanations could account for the impaired intercalation detected in
ttk mutants, we propose that inefficient cell shape changes represent
the main cause, and might prevent the proper accomplishment of several events,
such as the sliding of cells, formation of a first autocellular contact and
zipping up, thereby blocking intercalation. Hence, we propose that cell shape
changes, particularly cell elongation, are an obligate requisite for different
steps of intercalation. Other targets of Ttk might presumably be regulators or
components of the cytoskeleton involved in cell shape changes. It is relevant
to point out here that Ttk has also been proposed to regulate morphogenetic
changes required for dorsal appendage elongation
(French et al., 2003).
How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, we found both these genes to be normally expressed in ttk mutants, and we detected several differences in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated (our unpublished observations). Therefore, our results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, we found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions (our unpublished results) also favour this model, because we found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, we propose that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur.
Ttk in tube size
Tube size regulation is essential for functionality. We found that Ttk is
involved in such regulation. Tube expansion and extension relies on a luminal
chitin filament that assembles transiently in the tracheal tubes (reviewed in
Swanson and Beitel, 2006). The
metabolic pathway that leads to chitin synthesis involves several enzymes,
among which are Mmy and Kkv (Araujo et al.,
2005; Devine et al.,
2005; Tonning et al.,
2006; Tonning et al.,
2005). In addition, other proteins are known to participate in the
proper assembly and/or modification of the chitin filament, such as Knk, Rtv
(Moussian et al., 2006), Verm
and Serp (Luschnig et al.,
2006; Wang et al.,
2006). SJs are also required to regulate tube size
(Behr et al., 2003;
Hemphala et al., 2003;
Llimargas et al., 2004;
Paul et al., 2003;
Wu et al., 2004) and it was
proposed that they exert this activity, at least partly, via the control of
the apical secretion of chitin modifiers
(Wang et al., 2006). Our
results revealed that ttk acts as a key gene in tube size control,
playing at least two roles: it regulates chitin filament synthesis and SJ
activity.
SJ regulation by Ttk appears functional rather than structural: we detected mild defects in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. We speculate that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted.
We also found that mmy is transcriptionally regulated by Ttk.
mmy tracheal expression positively depends on a mid-embryonic peak of
the insect hormone 20-hydroxyecdysone
(Tonning et al., 2006).
Therefore, we propose that Ttk and ecdysone exert opposing effects on chitin
synthesis. Excess of mmy mRNA results in the abnormal deposition of
the chitin filament (S.J.A., unpublished), as occurs in ttk mutants.
Defects in chitin deposition might lead to the irregular organisation of
taenidia and the faint larval cuticle observed in ttk mutants (our
unpublished observations). Strikingly, Ttk is also required for normal chorion
production (French et al.,
2003), which represents another specialised secreted layer.
Ttk is required for intracellular tube formation downstream of cell fate specification
ttk mutants are defective in the formation of terminal and fusion
branches. These defects are due, in part, to non-autonomous, secondary and/or
pleiotropic effects of ttk. For instance, ttk mutants
exhibited a dorsal closure defect, which prevented the approach and fusion of
contralateral DBs. Additionally, terminal and fusion branches depend on
correct cell type specification, which did not reliably occur in ttk
mutants. For instance, DSRF was missing in some presumptive terminal cells of
ttk mutants, impairing terminal branch formation
(Guillemin et al., 1996).
These tracheal cell identity specification defects might be related to
non-autonomous requirements of ttk. For instance, DSRF is not
properly expressed in ttk mutants because of an abnormal expression
of its regulator, Bnl (Sutherland et al.,
1996).
It is important to note that, in spite of these non-autonomous and cell
fate specification defects, two pieces of evidence indicate that ttk
also plays a specific and autonomous role in the formation of terminal and
fusion tubes. First, markers for fusion and terminal cell specification were
expressed in many tracheal cells of ttk mutants, but yet most of
these cells did not form terminal or fusion branches. Second, only the
tracheal expression of ttk in ttk mutants (but not the
constitutive activation of the btl pathway, which regulates the
terminal and fusion identity) (Samakovlis
et al., 1996a) was able to restore the formation of terminal
branches.
A common feature of terminal and fusion branches is that they both display
intracellular lumina that lack detectable junctions. The cellular events that
precede the formation of fusion and terminal branches differ, but the
mechanisms by which their intracellular lumina form has been proposed to be
comparable (Uv et al., 2003).
We found that, in ttk mutants, terminal and fusion cells engage in
the correct cellular changes before intracellular lumen formation. However,
neither of these two cell types finalised the cellular events leading to tube
formation. It has been proposed that the lumen of terminal and fusion branches
forms by the coalescence of intracellular vesicles that use a `finger' tip
provided by the neighbouring stalk cell as a nucleation point
(Uv et al., 2003).
Interestingly, we found that vesicles containing luminal material are less
abundant in ttk mutants. These observations suggest a new role for
Ttk in the formation of intracellular lumina in distinct cell types.
Intracellular lumen formation also occurs in other branched tubular
structures, such as in vertebrate endothelial cells
(Kamei et al., 2006) and in
the excretory cell of Caenorhabditis elegans, presumably by the
coalescence of vesicles (Buechner,
2002). Importantly, a crucial role for vesicle formation and their
fusion during intracellular tube formation has been demonstrated
(Kamei et al., 2006).
To our knowledge, ttk is the first gene described to be involved
in intracellular lumen formation during tracheal development. Possible targets
of Ttk might be genes related to the apical surface and the underlying
cytoskeleton, because several of these genes are involved in C.
elegans excretory canal formation
(Buechner, 2002;
Gobel et al., 2004).
Additionally, genes involved in intracellular vesicle trafficking might also
be good candidates, as has been recently reported for C. elegans
(Liegeois et al., 2007). In
this respect, we have detected several abnormalities in ttk mutants
that might reflect defects in vesicle trafficking.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/20/3665/DC1
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ACKNOWLEDGMENTS |
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