RESEARCH ARTICLE 2055
Development 137, 2055-2063 (2010) doi:10.1242/dev.047175
© 2010. Published by The Company of Biologists Ltd
Dynamic shape changes of ECM-producing cells drive
morphogenesis of ball-and-socket joints in the fly leg
Reiko Tajiri1, Kazuyo Misaki2, Shigenobu Yonemura2 and Shigeo Hayashi1,3,*
SUMMARY
Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan
manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology
remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to
ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton
must have precisely reciprocal shapes to fit each other. Here we found that, in the development of ball-and-socket joints in the
Drosophila leg, the two sides of ECM form sequentially. We show that distinct cell populations produce the ‘ball’ and the ‘socket’,
and that these cells undergo extensive shape changes while depositing ECM. We propose that shape changes of ECM-producing
cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. Our results highlight
the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture.
INTRODUCTION
Regulation of the body shape has been a central issue in
developmental and evolutionary biology. The skeleton provides the
framework of animal body shape. In vertebrates, bones act as the
inner core of the body, although there are some skeletal elements
that appear and function on the exterior, such as teeth, beaks, scales
and antlers. Skeletons of invertebrates are predominantly external:
cuticles on the body surface. Intensive studies have revealed that
genetically programmed body plans ultimately dictate skeletal
patterns. However, cellular mechanisms that directly control
skeletal morphology downstream of those genetic programs remain
unknown.
The skeleton is composed of extracellular matrix (ECM),
consisting of molecules secreted from cells. Bones comprise
collagen and proteins secreted by osteoblasts (Hall, 2005), whereas
invertebrate cuticles consist of proteins and chitin, a long-chain
sugar polymer, released by the underlying epidermal cells
(Chapman, 1998). The final morphology of skeletal ECM should
reflect not only the terminal arrangement of ECM-secreting cells
but also the history of how their number, position, shape and
activity have changed during the entire ECM formation period. To
understand the cellular mechanisms for skeletal morphogenesis, we
therefore need to look at the dynamic behaviors of those cells and
to determine how they relate to ECM morphology.
Stringent regulation of ECM morphology is particularly
important and prominent in the joints, which are the flexible parts
of the skeleton that permit movement. The human skeleton
1
Laboratory for Morphogenetic Signaling, and 2Electron Microscope Laboratory,
RIKEN Center for Developmental Biology, Kobe 650-0047, Japan. 3Department of
Biology, Kobe University Graduate School of Science, Kobe, Hyogo 657-8501, Japan.
*Author for correspondence ([email protected])
Accepted 21 April 2010
possesses joints of various configurations, such as ball-and-socket,
hinge, saddle and pivot (Standring et al., 2005). In each
configuration, opposing sides of the ECM must be shaped in a
precisely reciprocal manner to form a tightly interlocking structure
for efficient and controlled motion. Although the vertebrate
synovial joints have long received attention for their medical
importance, the mechanisms by which they actually form during
embryogenesis have been poorly understood (Khan et al., 2007;
Pacifici et al., 2005).
The exoskeletal system offers some technical advantages to
address this issue. The epidermis consists of a single, continuous
sheet of epithelial cells that are polarized along the apicobasal axis:
the apical surfaces of the cells face the body surface and secrete
cuticle. The epidermis maintains its integrity and apicobasal
polarity and continues to adhere to the cuticle without mixing with
it (Chapman, 1998). Within the epidermal sheet, cell identities are
specified by two-dimensional positional cues (Cohen, 1993; Payre,
2004). This specification process is separated in time from the later
differentiation, when cells actually make specific parts of the
cuticle (Fristrom and Fristrom, 1993). Moreover, morphogenesis of
the adult cuticle appears to be completed before the muscles start
to contract just prior to eclosion. All of these features make the
exoskeleton a promising system for studying the dynamic
behaviors of cells during ECM formation and how they are linked
to the final ECM morphology.
We focus on the ball-and-socket joints in the tarsus of the
Drosophila leg. How their positions are determined in the leg
primordium before pupariation has been studied extensively
(Kojima, 2004), and the central role of the Notch signaling
pathway therein has been demonstrated (de Celis et al., 1998;
Rauskolb and Irvine, 1999). The subsequent folding (Manjon et al.,
2007), unfolding and re-constriction (Fristrom and Fristrom, 1993;
Mirth and Akam, 2002) of the epidermis in the joint regions have
also been described. By contrast, the way in which the joint cuticles
develop afterwards and how the cells behave during that period are
largely unknown. As it takes more than three days from
DEVELOPMENT
KEY WORDS: Joint, Ball-and-socket, Cuticle, Chitin, Epidermis, Cell shape change, Leg, Drosophila
2056 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly strains and confocal microscopy
Fly strains used were: sqh-GFP-Moesin (Kiehart et al., 2000), Myosin
Regulatory Light Chain (MRLC)-GFP (Royou et al., 2004), UASSerp(CBD)GFP (Luschnig et al., 2006), UAS-GFP-TTras (Kato et al.,
2004), UAS-krotzkopf verkehrt (kkv) RNAi (Dietzl et al., 2007), Distallessmd23 (Dll-GAL4) (Calleja et al., 1996), big brainNP5149 (bib-GAL4) and
fringeNP6583 (fng-GAL4) (Hayashi et al., 2002), neuralizedP72 (neur-GAL4)
(Bellaiche et al., 2001), UAS-KO, ubi-KOnls (Kaido et al., 2009), ubiGFP.S65Tnls, UAS-GFP.S65T and dauterless (da)-GAL4 (Bloomington
Stock Center). sqh-GFP-Moesin or MRLC-GFP was used as wild type
unless otherwise noted.
Confocal microscopy and leg staining
Our method is based on that described by Mirth and Akam (Mirth and
Akam, 2002). Briefly, pupal thoraces were dissected in PBS and transferred
to 4% paraformaldehyde in PBS. After removal of pupal cuticles, the legs
were fixed for 20-30 minutes at room temperature and washed with PBST.
For GFP and KO, the legs were directly stored overnight in Vectashield with
or without DAPI (Vectashield) and then mounted on glass slides. For
quantification of GFP (see Fig. S3D in the supplementary material), confocal
images of the legs were captured [Olympus FV-1000, ⫻60 water immersion
lens, NA1.2; LD473 nm laser (15 mW) at 0.1% intensity, PMT 600 V,
aperture 120 m] and fluorescence intensities were measured by manually
outlining cells of interest using ImageJ (National Institutes of Health, USA).
For tissue staining, fixed legs were blocked in PBST containing 3%
normal goat serum for 30 minutes, incubated overnight at 4°C with
antibodies or fluorescent probes and washed with PBST before mounting.
The following probes and dilutions were used: mouse anti--galactosidase
antibody (Promega, 1:100), Cy3-conjugated anti-mouse IgG (Molecular
Probes, 1:400), rhodamine-conjugated chitin-binding probe (New England
Biolabs, 1:100), and Fluostain (Calcofluor White Stain, Fluka, 2 g/ml).
Antibody staining was effective for stages before cuticle deposition [~48
hours after puparium formation (APF)]. Chitin binding probe labels the ball
and the socket cuticles. The staining within the ball was non-uniform,
probably reflecting incomplete penetration of the probe due to its size (48
kDa; see Figs 1 and 3). Fluostain gave more uniform and reproducible
patterns in the ball (Moussian et al., 2005).
Verification of kkv RNAi
In order to verify the effect of RNAi against kkv, progeny from the crossing
of males with two copies of UAS-kkv RNAi and da-GAL4 females were
stained with Fluostain (Moussian et al., 2005) or processed for cuticle
preparation with or without the vitelline membrane in Hoyer’s medium
(Sullivan et al., 2000).
The effect of kkv RNAi on chitin accumulation in the ball cuticle was
quantified as follows (see Fig. S3A-C in the supplementary material). Legs
from RNAi (bib-GAL4/UAS-kkv RNAi; UAS-GFP-TTras/+) and sibling
control (CyO/UAS-kkv RNAi; UAS-GFP-TTras/+) pupae (at least four for
each genotype) were pooled and divided into two batches: one for
Fluostain staining and the other for negative control for background
measurement. Confocal images were taken [Olympus FV-1000, ⫻60 water
immersion lens, NA1.2; LD405 nm laser (50 mW) at 0.1% intensity,
PMT 550 V, aperture 120 m] and fluorescence intensity was measured
using ImageJ (see Fig. S3A⬙ in the supplementary material).
Time-lapse imaging
Pupae were removed from pupal cases and placed on glass-bottomed
dishes. Time-lapse images were taken with an Olympus FV1000 confocal
scanning microscope, and processed using iSEMS (Kato and Hayashi,
2008).
Electron microscopy
Thoraces were dissected from pupae in PBS, and legs were immediately
transferred to fixation buffer (2.5% glutaraldehyde, 2% formaldehyde,
0.1 M cacodylate buffer). Pupal cuticles were removed in the buffer, and
legs were incubated for >2 hours at room temperature. Fixed legs were
embedded in 1% agarose and subjected to post-fixation with 1% OsO4 in
0.1 M cacodylate buffer for 8 hours on ice, and then to en bloc staining in
0.5% uranyl acetate aqueous solution overnight at room temperature.
Samples were subsequently dehydrated in ethanol and embedded in
Poly/Bed812 (Epon 812, Polysciences). Sections stained with uranyl
acetate and lead citrate were viewed in a JEOL JEM-1010 transmission
electron microscope at 100 kV.
Fig. 1. Mature joint morphology. (A)Cuticle preparation of the adult leg (tarsus). ta1-ta5, tarsomeres 1-5. Arrows indicate the joints focused on
in this study; arrowhead indicates the joint between ta4 and 5. (B-D)Mature structure of the three proximal inter-tarsomere joints (arrows in A).
(B-B⬙) Electron micrograph of the mature joint. Boxed areas in B are enlarged in B⬘ and B⬙. (C,C⬘) Confocal microscope image of the mature joint.
(C)Differential interference contrast (DIC) image. (C⬘)The ball and socket cuticles are labeled by a chitin-binding probe (magenta). GFP-Moesin
labels actin enrichment at the cell cortices (green). (D)Serp(CBD)GFP (green) is overexpressed using Dll-GAL4 in the mature joint. Cuticles are
labeled by a chitin-binding probe (magenta). (E,E⬘) The joint between ta4 and ta5, labeled as in C and C⬘. Dorsal is up and distal is to the right in
this and all subsequent figures unless otherwise noted. so, socket cuticle; lu, lubricant matrix. Scale bars: 2m in B; 1m in B⬘, B⬙; 5m in C-E⬘.
DEVELOPMENT
specification of the joint regions to the completion of the joint
cuticle architecture, the essence of ECM morphogenesis must lie
in how cells act during this period.
In this study, we describe the development of the ball and the
socket cuticles and the concomitant cellular morphogenesis, which
occur long after cell specification. Through chronological
observation of cellular and cuticular architectures, we reveal that
the ball and the socket cuticles develop in a sequential manner, and
that the two parts are formed by distinct cell populations that
undergo extensive shape changes while secreting cuticle. We
propose that the shape changes of ECM-producing cells result in
the sequential formation of the ball and the socket, permitting their
morphological coupling in a ‘mold-and-cast’ manner.
Development 137 (12)
Ball-and-socket joint morphogenesis
RESEARCH ARTICLE 2057
Nikkomycin injection
Pupae at 40-45 hours APF (a few hours prior to the onset of cuticle
formation) were sterilized by soaking in 70% ethanol and placed on plastic
dishes with adhesive tape. Nikkomycin Z (Sigma-Aldrich) was dissolved
in water (2.5 mg/ml) and 0.5~1.0 l of the solution was injected into the
thoracic region of pupae by piercing a hole in the pupal case with a glass
needle. Water injection was used as a control. Approximately 24 hours after
injection at 25°C, the legs were prepared for viewing under a confocal
microscope. Cuticle preparations in Gary’s Magic Mountant (Roberts,
1986) were made from pupae incubated for more than 48 hours after
injection.
Sequential development of the joint
The leg primordium is composed of an epithelium with deep
segmental folds during the prepupal period (de Celis et al., 1998)
that then elongate to become a straight and smooth cylinder by the
prepupa-pupa transition (apolysis) (Fristrom and Fristrom, 1993;
Mirth and Akam, 2002). At this stage (~20 hours APF, Fig. 2A),
cells in the joint region were aligned along the circumference (Fig.
2A⬘). They underwent apical constriction (data not shown),
resulting in the constriction of the leg in the joint regions (Fig.
2B,B⬘). When the entire leg subsequently became thinner (Fig. 2C),
the cells in the joint region remained neatly aligned and constricted
(Fig. 2C⬘). Cells on the dorsal side of the joint constricted further
and infolded proximally (Fig. 2D-D⬙). By ~45 hours APF, the
furrow grew deeper and resulted in a deep cavity (Fig. 2E-E⬙).
Cuticle deposition in the joint region began at 45-48 hours APF
when the chitin-binding probe revealed signals covering both the
dorsal and the ventral sides of the cavity (Fig. 3A-A⬙). The cuticle
in the deepest part (bottom) of the cavity then thickened to form
the ball cuticle (Fig. 3B-D⬙). Electron micrographs indicated that
layers of consistent thickness were added successively to the ball
cuticles (Fig. 4B,C). At 63-66 hours APF, the ball cuticle reached
its full size.
At this same stage, the socket cuticle was formed only on the
dorsal side of the cavity (Fig. 3D,D⬘; Fig. 4C-C⬙). The socket
cuticle then expanded gradually in the dorsal-to-ventral direction
Fig. 2. Cellular morphogenesis before cuticle formation. The
tarsus morphology under DIC (A-E), and cell arrangements in the joint
regions (A⬘-E⬘,D⬙,E⬙) at several stages before the onset of cuticle
formation. Cells are outlined by MRLC-GFP (A⬘-D⬘,D⬙) or GFP-Moe
(E⬘,E⬙). Boxes in A-E indicate the joint regions shown in A⬘-E⬘,D⬙,E⬙.
(A,A⬘) At 18-22 hours APF, cells in the joint region are aligned along the
leg circumference (arrows). (B,B⬘) By 24-26 hours APF, the leg has
shrunk in the joint regions (brackets). (C,C⬘) Cells in the joint region at
32-34 hours APF continue ordered alignment (brackets). (D-D⬙) A
projected image showing the leg surface (D⬘) and a single section at the
midline of the leg (D⬙) at 38-40 hours APF shows that cells on the
dorsal side invaginate proximally (arrows). (E-E⬙) By 45-48 hours APF, a
deep cavity has formed on the dorsal side of the joint region (arrows).
E⬘ is a projected image as in D⬘; E⬙ is a single section as D⬙. Scale bars:
in A, 50m for A,B,C,D,E; in A⬘, 20m for A⬘-E⬘,D⬙,E⬙.
towards the bottom of the cavity (Fig. 3E-E⬙), until eventually
covering the entire cavity at ~75 hours APF (Fig. 3F-F⬙; Fig. 4D).
During this time, the ‘lip’ of the ball cuticle also developed (Fig.
3E-F⬙; Fig. 4D).
Thus, the ball and the socket are produced in a sequential
manner: the socket cuticle develops throughout the cavity only
after the ball has reached its full size. We propose that the serial
formation of the ball and the socket serves as ‘mold casting’, which
permits their morphological coupling (see Discussion).
It has been reported that most of the final cell divisions are
complete by about 30 hours APF (Fristrom and Fristrom, 1993),
suggesting that the sequential formation of the ball and the socket
cuticles shown in Fig. 3 progresses without cell proliferation. We
next addressed the cellular mechanism underlying such
sequentiality.
Sites of ball/socket production revealed by
electron microscopy
In order to study the spatio-temporal pattern of cuticle secretion at
the cellular level, we used EM analysis to monitor the appearance
of the histological structure called plasma membrane plaques
DEVELOPMENT
RESULTS
Mature joint morphology
The tarsus comprises five segments (tarsomeres), separated by
joints (Fig. 1A). On the dorsal side of the inter-tarsomere joints, the
cuticle has the ball-and-socket architecture (Fig. 1). The joint
between tarsomeres 4 and 5 had a slightly different ball-and-socket
morphology (Fig. 1E), with a smaller ball and a thicker socket than
the other three (Fig. 1C,D). In this study, we focused on the three
proximal inter-tarsomere joints, which develop simultaneously.
Both the ball and the socket were labeled by a chitin-binding probe
(Fig. 1C⬘,D,E⬘). The ball-and-socket structure was tucked under the
ridge, on which the socket cuticle spreads (Fig. 1B-C⬘). On the
ventral aspect of the ball, a distally oriented process, which we
named the ‘lip’, grew into the epithelium (Fig. 1B-C⬘). Electron
microscopy (EM) revealed a strikingly regular stratification of the
ball cuticle, consisting of 70-80 layers of approximately 100150 nm thickness (the variation might depend on the angles of thin
sections; Fig. 1B⬙). It also uncovered the matrix that filled the space
between the ball and the socket, sealed by the membranous cuticle
(Fig. 1B). A secreted form of GFP [Serp(CBD)GFP (Luschnig et
al., 2006)] was enriched in this matrix when it was expressed by
underlying cells (Fig. 1D). This matrix was labeled poorly with the
chitin-binding probe and we speculate that it acts as a lubricant in
the moving adult legs.
Fig. 3. Sequential formation of the ball and the socket cuticles.
Sequence of ball-and-socket cuticle formation observed by confocal
microscopy. (A-F)DIC images. (A⬘-F⬘) Cuticles are stained with a chitinbinding probe (magenta), and myosin or actin enrichment at cell
cortices is labeled by MRLC-GFP in B⬘-E⬘, or by GFP-Moe in F⬘ (green).
(A⬙-F⬙) Schematic representations. (A-A⬙) At 45-48 hours APF, cuticles
are detected on the dorsal (arrow) and ventral (asterisk) sides of the
cavity. (B-B⬙) By 52-55 hours APF, the ball cuticle on the ventral side of
the cavity has thickened (B⬘, asterisk). An arrow marks the edge of the
socket cuticle. (C-C⬙) The ball cuticle has further enlarged (asterisk) by
55-63 hours APF. The socket cuticle has not yet extended significantly
(arrow). (D-D⬙) At 63-66 hours APF, the ball has reached its full size
(asterisk). The socket is detected only on the dorsal side of the cavity
(arrow). (E-E⬙) By 70-72 hours APF, the socket is extending along the
cavity into the lateral side of the ball (arrow) and the ‘lip’ is developing
(arrowhead). (F-F⬙) By 72-75 hours APF, the socket has extended to the
ventral side of the ball (arrow) and the lip has fully formed (arrowhead).
Scale bar: 5m.
(PMPs). PMPs are electron-dense regions of the plasma membrane
located at the tips of microvilli in contact with newly synthesized
cuticles of various insects (Chapman, 1998; Locke and Huie,
1979), and are regarded as the sites of chitin microfibril release
and/or cuticle-cell adhesion.
At the onset of ball formation, the apical surfaces of the cells
surrounding the ball cuticle contained many PMPs (Fig. 4A,A⬘).
The PMPs were in contact with fibrous materials, presumably
chitin microfibrils, arranged parallel to the ball cuticle (Fig. 4A⬘).
Thus, these cells contacted the ball cuticle, and were probably
adding chitin microfibrils to it. The thin and scattered socket cuticle
was evenly associated with PMPs of the cells on the dorsal side
(Fig. 4A).
Development 137 (12)
Fig. 4. Distribution of plasma membrane plaques (PMPs) at
different stages of cuticle formation. (A-D)Electron micrographs
of joints at different stages of cuticle formation. Yellow arrows
indicate PMPs. (A)At 55 hours APF, the ball cuticle is thickening
(asterisk) and the socket cuticle is composed of thin and scattered
pieces (bracket). White arrow indicates the edge of the socket.
(A’)Magnification of the boxed area in A. Brackets indicate
presumptive chitin microfibrils. (B)At 58 hours APF, the ball continues
to enlarge (asterisk). The edge of the socket is indicated by white
arrow. (B⬘,B⬙) Enlargement of the boxed regions in B. White arrows
indicate pieces of the socket cuticle. (C)By 65 hours APF, the ball has
reached its full size (asterisk). The lip has not yet formed in this
sample. (C⬙)Enlargement of the boxed region in C showing that PMPs
are scarce on the lateral side of the ball. (C⬘)Magnification of the
boxed region in C showing that the socket cuticle is associated with
PMPs. (D)By 72 hours APF, the socket has formed throughout the
cavity, reaching the ventral side of the ball cuticle (edge indicated by
arrow). (E)Changes in the distribution of PMPs contacting the ball
cuticle and those contacting the socket. Scale bars: 1m in A,B,C,D;
500 nm in A⬘,B⬘,B⬙,C⬘,C⬙.
DEVELOPMENT
2058 RESEARCH ARTICLE
Ball-and-socket joint morphogenesis
RESEARCH ARTICLE 2059
During its growth, the ball cuticle continued to be surrounded by
PMPs (Fig. 4B,B⬘). However, when it had fully grown, the PMPs
were relatively sparse on its lateral side (Fig. 4C,C⬙). The socket
cuticle, which was still thin and scattered at this point, continued
to be associated with PMPs (Fig. 4B,B⬙,C,C⬘). When the socket
cuticle formed in the bottom of the cavity, it was uniformly lined
with PMPs (Fig. 4D). PMPs were also found enriched along the
elongated lip.
These results from electron microscopy experiments are
summarized in Fig. 4E. Notably, the positions of the ball-contacting
area (blue) and the socket-contacting area (red), which presumably
comprise ball-producing activity and socket-producing activity,
respectively, shift extensively along the cavity during cuticle
formation. If cells remain stationary, the ball-producing and socketproducing activities must sequentially progress across the cells, in
a wave-like fashion. If, conversely, the ball-producing cells and the
socket-producing cells retain their respective identities as separate
populations, they must shift their apical surfaces along the cavity
by cell movement or shape changes. With this in mind, we next
examined the cell dynamics during cuticle formation.
Extensive cell shape changes in the joint region
during cuticle formation
We sought to label subpopulations of cells within the joint region
using the GAL4-UAS method (Brand and Perrimon, 1993) and to
monitor their locations and shapes at various stages. Among GAL4
drivers inserted near the genes reported to show segmental
expression in the tarsus, we found three drivers which were
expressed in different but overlapping populations of cells in the
joint region: big brain (bib)-GAL4, neuralized (neur)-GAL4 and
fringe (fng)-GAL4 (de Celis et al., 1998; Mirth and Akam, 2002;
Rauskolb and Irvine, 1999; Shirai et al., 2007). bib and neur were
expressed in the deep region of the cavity, flanked by fng
expression in cells projecting into the ridge (see Fig. 1C⬘) and cells
on the ventral side of the cavity (Fig. 5D,G,J). Simultaneous
detection of bib (bib-GAL4 and UAS-GFP) and neur (neurlacZnls) revealed that bib was expressed distal to neur, with an
overlap of about two cells (Fig. 5A-A⬙).
GAL4-expressing cells were outlined by a membrane-bound
GFP (Fig. 5B-J). Shortly after the onset of ball formation, the bibexpressing cells were in contact with the ball cuticle (Fig. 5B). The
DEVELOPMENT
Fig. 5. Cell shape changes during cuticle
formation. (A-A⬙) Comparison of bib and neur
expression patterns. (A)GFP (green) and anti--gal
immunostaining (magenta, shown in grey-scale in
A⬘) in the joint region of a bib-GAL4/+; neurlacZ/UAS-membrane-targeted-GFP pupa. The
sample was fixed at a stage comparable to that
shown in Fig. 3C,C⬘. neur expression is hardly
detected in the distal portion of bib-expressing
cells (bracket). (B-J)Membrane-targeted GFP is
expressed under the control of bib-GAL4 (B,E,H),
neur-GAL4 (C,F,I) or fng-GAL4 (D,G,J). Samples
fixed at different stages of cuticle formation are
shown. (B-D)The ball cuticle has started to grow
(asterisk). (B)bib-expressing cells contact the
dorsal part of the ball cuticle (arrow). (C)neur is
expressed in both the dorsal subset of bibexpressing cells (arrow) and the cells located dorsal
to them (arrowhead). (D)fng is expressed in cells
projecting into the ridge. Arrowhead indicates the
ventral edge of the fng expression domain.
(E-G)The ball has reached its full size (asterisk).
The lip and the socket are extending. (E)bibexpressing cells contact the ventral side of the ball
(arrow). (F)The ventral subset of neur-expressing
cells contact the ventral side of the ball (arrow).
The dorsal subset contact the extending socket
(arrowhead). (G)fng-expressing cells have
expanded their apical surfaces and broadly contact
the dorsal part of the socket cuticle (arrowhead).
(H-J)The socket cuticle has formed along the
entire cavity. Its edge is indicated by the ‘v’.
(H)bib-expressing cells extend past the ventral side
of the cavity and contact the lip (arrow). Their
basal sides remain in the proximal position
(asterisk). (I)The ventral neur-expressing cells
extend along the ventral side of the cavity (arrow).
The dorsal portion contacts the ventral part of the
socket (arrowhead). (J)fng-expressing cells have
further expanded their apical surfaces along the
cavity (arrowhead). (K)Summary of cell shape
changes, overlaid with the distribution of ball- and
socket-contacting PMPs (see Fig. 4).
expression of neur appeared to overlap largely with bib-expressing
cells and to extend dorsally to them (Fig. 5C; consistent with Fig.
5A-A⬙). bib and neur expression domains were four to six cells
wide along the anteroposterior axis (data not shown).
When the ball had fully grown, the apical surfaces of bibexpressing cells came into contact with the ventral side of the ball
(Fig. 5E), and then the apical surfaces further shifted distally
eventually contacting the newly formed lip (Fig. 5H).
Consistently, the apices of the ventral portion of neur-expressing
cells (co-expressing bib) relocated to the ventral side of the ball
(Fig. 5F,I), and the apical surfaces of their dorsal subsets moved
with the edge of the extending socket, until covering the ventral
part of the socket (Fig. 5F,I). Meanwhile, the dorsal fngexpressing cells expanded their apical surfaces (Fig. 5G,J), and
when the socket cuticle formed at the bottom of the cavity, they
covered a large dorsal portion of the socket cuticle (Fig. 5J). A
similar observation was made on legs in which all of the cells in
the joint region were labeled by Dll-GAL4 (data not shown).
Furthermore, tracing the cell shapes in electron micrographs
confirmed extensive changes in the morphology of the cell
contacting the ball and socket cuticles (see Fig. S1 in the
supplementary material).
We also took a live-imaging approach in order to directly
monitor the dynamic behaviors of bib- and neur-expressing cells.
Pupal legs were doubly labeled with fluorescent proteins expressed
by bib-GAL4 or neur-GAL4, and ubiquitously expressed nuclear
localized markers (see Movies 1-3 in the supplementary material)
and imaged over 5 hours. The movies allowed us to continuously
trace the bib-GAL4- and neur-GAL4-expressing cells, which
moved their apical surfaces extensively along the cavity.
Fig. 5K shows a summary of the cell shape changes, overlaid on
the distribution of PMPs at respective stages. Notably, the
movement of the apical surfaces of the cells continuously
paralleled the distribution changes of PMPs. This parallelism is
consistent with the model that the ball-producing cells and the
socket-producing cells retain their respective identities as separate
populations (see Discussion).
Identification of ball-producing and socketproducing cell populations
To identify which cutcicular elements are produced by each of the
bib-, neur- and fng-expressing cells, we attempted to interfere with
cuticle production in a cell-specific manner using transgenic RNAi
constructs of krotzkopf verkehrt (kkv), encoding Chitin Synthase 1.
The UAS-kkv RNAi strains used here caused defects phenocopying
those of kkv mutants: attenuation of chitin accumulation in the
tracheal lumen and its failure to expand uniformly (Devine et al.,
2005; Tonning et al., 2005), deformation of the head skeleton, and
reduction of cuticle strength (Moussian et al., 2005; Ostrowski et
al., 2002) (see Fig. S2 in the supplementary material).
Measurement of the chitin-staining dye Fluostain demonstrated that
kkv RNAi robustly reduced the amount of chitin in the ball cuticle
(see Fig. S3A-C in the supplementary material).
As a positive control for chitin synthesis attenuation, we
injected Nikkomycin Z, a GlcNAc analog that competitively
inhibits chitin synthase activity (Tellam et al., 2000), into the pupal
thoraces before the onset of cuticle formation. Fig. 6B shows the
typical morphology of the joint cuticle that developed in wild-type
pupae injected with Nikkomycin. The cuticle consisted of multiple
amorphous bulges: one relatively large bulge on the ventral side
of the cavity that presumably derived from the ball, and a few
smaller ones on the lateral and dorsal sides that probably
Development 137 (12)
Fig. 6. Attenuation of chitin synthesis in different cell
populations exerts differential defects on the ball and the
socket. (A-E)Joint cuticle morphology in situations where chitin
synthase is inhibited. (A)Control (wild-type, injected with water)
showing normal mature cuticle morphology. (B)Nikkomycin Z-injected
wild type. The large bulb (asterisk) and the smaller bulbs (arrows)
presumably represent the ball and the socket, respectively. (C-E)RNAi
against kkv is induced by bib-GAL4 (C), neur-GAL4 (D) or fng-GAL4 (E).
(C)The presumptive ball cuticle (asterisk) is severely misshaped, similar
to in B. The socket cuticle is partly formed (arrow). (D,E)The ball cuticle
has normal morphology. (D)The ventral portion of the socket cuticle is
missing (arrow). (E)The socket cuticle has some bulbs (arrows).
(F)Correlation between spatial relationship of bib-, neur- and fngexpressing cells and the types of cuticle that they produce.
correspond to the socket. Thus, the morphology of the entire balland-socket cuticle was severely disrupted by the global attenuation
of chitin synthesis.
We next used bib- and neur-GAL4 drivers for knock-down
experiments. Activities of those drivers were approximately the
same, as assessed by the measurement of GFP signals expressed by
those drivers (see Fig. S3D in the supplementary material). When
kkv RNAi was induced by bib-GAL4, the morphology of the ball
cuticle was disrupted as severely as seen in Nikkomycin-injected
samples (Fig. 6C; see also Fig. S3E in the supplementary material).
The socket formation was also inhibited, although we could often
discern the dorsal portion of the socket. Conversely, when neurGAL4 was used, the ball cuticle was largely intact, whereas the
ventral part of the socket was missing (Fig. 6D; see also Fig. S3E
in supplementary material). Thus, neur>kkv RNAi preferentially
affected the socket rather than the ball, whereas bib>kkvRNAi
strongly affected both (Fig. 6C,D; see also Fig. S3E in
supplementary material). The overlap in their phenotypes in the
socket cuticle is likely to reflect the overlap of bib and neur
expression (Fig. 5A-A⬙; see Discussion). Use of fng-GAL4 caused
yet another phenotype: the ball morphology was largely normal,
whereas the socket cuticle had a bloated appearance and a few
bulges (Fig. 6E).
Altogether, attenuation of chitin synthesis in specific cell
populations differentially affected both the ball and the socket, and
the ball-producing activity and the socket-producing activity were
mapped to different cell populations along the proximodistal axis,
DEVELOPMENT
2060 RESEARCH ARTICLE
Ball-and-socket joint morphogenesis
RESEARCH ARTICLE 2061
as shown in Fig. 6F. Importantly, this map is consistent with the
part of the cuticle that each cell population continues to associate
with during cuticle formation (Fig. 5K; see Discussion).
We also attempted to locate the lubricant-producing activity. As
shown in Fig. 1D, Serp(CBD)GFP was enriched in the lubricant
when it was expressed by Dll-GAL4. When Dll-GAL4 was
replaced with either bib-, neur- or fng-GAL4, Serp(CBD)GFP still
localized in the lubricant (see Fig. S4 in the supplementary
material), suggesting that the lubricant-secreting activity is
distributed rather broadly in the joint region. Alternatively, it could
be that proteins secreted by cells outside of this region are
transported into the lubricant.
Fig. 7. Cell shape changes occur independently of normal cuticle
formation. The final shapes of bib-expressing cells (A,C) or fngexpressing cells (B,D) in Nikkomycin Z-injected animals, visualized by
membrane-targeted GFP. (A,B)Typical cuticular phenotype. The ball and
the socket appear as the large, amorphous bulb on the ventral side
(asterisk) and the smaller fragments on the dorsal side (arrowheads),
respectively. No obvious lip development. (A)bib-expressing cells are
extended along the ventral side of the cavity. Their apical surfaces are
located distal to the cavity (arrow), where the lip would have formed.
(B)The apical surfaces of fng-expressing cells are expanded ventrally
along the cavity (arrow). (C,D)Severe defects in cuticle morphology
observed in some Nikkomycin Z-injected animals. The ball is
fragmented into a small bulb on the distal side (asterisks) and multiple
smaller bulges (dots). bib-expressing cells are extended distally (C,
arrow), and fng-expressing cells expand their apices (D, arrow).
treatment did not completely eliminate the cuticle, it is possible that
the cuticle might play a permissive role in cell movement; for
example, by acting as a substratum for migration.
DISCUSSION
In this study, we revealed that the ball and the socket cuticles
develop sequentially. We have shown that the ball-producing
activity and the socket-producing activity are allocated to distinct
cell populations, and have found that shape changes of these cells
that occur simultaneously with their cuticle-secreting activities
result in the interlocking ball-and-socket structure. As the ball
cuticle builds up, concurrent cell shape changes drive the apical
domains of ball-producing cells out of the cavity and bring in the
apical domains of the socket-producing cells, resulting in close
enwrapment of the ball by the latter cell population. Accordingly,
the shape of the resulting socket cuticle conforms to that of the ball.
Synchronization between ECM formation and dynamic relocation
of the cell surfaces that mediate it thus underlies the building of the
complex ECM structure.
Mapping of ball-producing cells and socketproducing cells
The map of ball-producing and socket-producing cells shown in
Fig. 6F best summarizes the results of kkv RNAi, and is consistent
with the result indicating their continuous association with
respective parts of the cuticle during their formation (Fig. 5K). The
ball morphology was severely disrupted by bib>kkv RNAi but not
by neur>kkv RNAi, indicating that the ball-producing activity is
restricted to the distal subset of bib-expressing cells that do not
significantly express neur. Consistently, these cells are in constant
contact with the ball cuticle throughout its formation. The cuticle
phenotype of neur>kkv RNAi shows that neur-expressing cells are
DEVELOPMENT
Shape change of cuticle-secreting cells proceeds
even when cuticle morphology is severely
disrupted
As shown above, the cells progressively relocated their apical
surfaces in the proximal-to-distal direction (Fig. 5; see also Movies
1-3 in the supplementary material), and this was accompanied by
polarized growth of the cuticle in the same direction: stratification
of the ball progressed from dorsal to ventral; the socket and the lip
both elongated from proximal to distal. Interestingly, the apical
surfaces of the ball-producing cells moved together with the lip,
whereas those of the socket-producing cells followed the edge of
the socket cuticle (Fig. 5). These observations prompted us to test
whether the cuticle acts as a structural guidance cue informing the
cells of the direction or the destination of movement.
We used Nikkomycin treatment, which causes severe disruption
of the morphology and continuity of the entire joint cuticles (Fig.
6B), to determine whether the cuticle has a guidance role.
Nikkomycin was injected at the onset of cuticle formation into
pupae in which bib- or fng-expressing cells were labeled by
membrane-bound GFP, and the cell and ECM morphologies were
examined after 24 hours, when cell shape changes would have been
completed under normal conditions (cf. Fig. 5G-I). Typically, we
saw that the ball developed as an amorphous bulb without the ‘lip’,
and the socket disintegrated into multiple small bulges (Fig. 6B;
Fig. 7A,B). In severe cases, the ball even appeared to be
fragmented into small pieces (Fig. 7C,D).
To our surprise, the overall shapes of bib- and fng-expressing
cells in Nikkomycin-injected pupae were comparable to those in
controls. The apical domains of bib-expressing cells had relocated
to the ventral side of the cavity, where they would have normally
contacted the lip (Fig. 7A,C). Thus, the movement of the apices of
ball-producing cells appears to be capable of orienting correctly in
the absence of the normal cuticular morphology, including the lip.
Likewise, the apical surfaces of the dorsal fng-expressing cells
extended ventrally out of the ridge (Fig. 7B,D), similarly to how
they move in standard conditions. The shape changes of neurexpressing cells in Nikkomycin-injected pupae were also
comparable to those in the normal situation (data not shown). The
fact that all of the cells relocated their apical surfaces in the correct
direction and to the correct destination, even when the cuticle
morphology was severely disrupted, suggests that the normal shape
and continuity of the cuticle is dispensable for guiding the
migration of the apical surface of cuticle-secreting cells.
We also noted that the apical surfaces of socket-producing cells
expanded so as to wrap around the misshapen or fragmented ball
cuticle (Fig. 7B,D), indicating that the final shapes of socketproducing cells respected the morphology of the remaining ball
cuticle. This local adjustment of cell shape might reflect the effect
of remaining cuticle components. Because the Nikkomycin
responsible for forming the ventral part of the socket cuticle, with
which they continue to associate. Likewise, fng-expressing cells
contribute to the formation of the remainder of the socket. Partial
disruption of the socket by bib>kkv RNAi (Fig. 6C; see Fig. S3E
in the supplementary material) should be, to some extent, due to
direct blocking of socket production in cells co-expressing bib and
neur. Additionally, the impairment of ball formation might
somehow interfere with socket formation. Occasional deformation
of the ball by neur>kkv RNAi (see Fig. S3E in the supplementary
material) might be caused by marginal expression of neur in the
presumptive ball-producing cells, which is barely detectable in Fig.
5A-A⬘.
Role of dynamic cell behaviors in regulating ECM
morphology
Patterns of ECM-producing tissues do play a major role in the
regulation of ECM morphology. Previous studies have unraveled
how global positional information affects skeletal patterns through
the regulation of specification, differentiation and proliferation of
ECM-producing cells (Hall, 2005; Payre, 2004). There, the
morphology of ECM was assumed to be synonymous with that of
the cells or tissues that secrete it. Our present study illustrates that
the skeletal morphology reflects not only the pattern of those cells
at one point in time, but also the history of their dynamic behaviors
during ECM formation. Secreted apically by the epidermis, the
cuticle is monolayered in most parts. In the joints, however,
relocation of the secretory surfaces enables formation of a cuticle
beneath a pre-formed layer. Cell motility thus allows a tissue of
simple configuration to build a complex and essential threedimensional ECM structure. We envision that movements of ECMsecreting cells probably play important roles in ECM
morphogenesis in other systems, especially in formation or
adjustment of intricate skeletal structures.
Mechanism for cell shape changes
The morphology of the cuticle, as well as how it develops,
correlated well with cell shape changes (Fig. 5K). This suggested
either that the cell-shape changes govern the morphology of the
cuticle, and/or vice versa. We found that the movement of the
apical surfaces of the cells was correctly oriented even when the
shape of the cuticle was disrupted (Fig. 7), indicating that the
morphogenesis of the ball-and-socket cuticle is primarily controlled
by the way the cells change their shapes as they deposit the cuticle.
How do the cells know which way to move? In other words, what
is the molecular mechanism that mediates global proximodistal
polarity of the leg to direct cell movement? In mutants of wellknown planar cell polarity genes, such as frizzled, dishevelled and
prickled, extra joints of reverse proximodistal polarity are formed
(Held et al., 1986). Nonetheless, the ball-and-socket structure of
individual joints remains largely intact (data not shown), indicating
that cell shape changes are correctly guided by a mechanism other
than this pathway. Analysis and local disruption of cytoskeletal
architecture in the joint region could help us to answer this
question.
As mentioned earlier, our results do not rule out the possibility
that the cuticle plays a permissive role in cell movements. The
ECM generally affects cell shape and motility (Alberts et al., 2002),
and chitin-based ECM has been shown to regulate epithelial
morphogenesis in some Drosophila tissues (Devine et al., 2005;
Moussian et al., 2005; Tonning et al., 2005). Whether the cuticle
provides a permissive environment for cell shape changes in the
joint is an important issue to address in future work.
Development 137 (12)
‘Mold casting’ – a general mechanism for joint
development?
The formation of reciprocally shaped interfaces is vital for the sake
of joint function. The serial progression of ball-and-socket
morphogenesis shown here can be compared to ‘mold casting’: (1)
the ball enlarges rapidly through stratification, and the cavity
expands to accommodate it; and (2) the enlarged cavity then serves
as the ‘mold’ along which the socket cuticle is formed. Hence, the
shape of the ball is transmitted to the socket (the ‘cast’). Whether
or not this model also applies to vertebrate synovial joints is an
intriguing question. Pacifici et al. speculated that, in the chick digit
joints, chondrogenic cell differentiation on the distal side might
promote its expansion to become convex; at the same time,
proliferation of peripheral cells on the proximal side might permit
them to grow and wrap themselves around the distal side, thereby
becoming concave (Pacifici et al., 2005). If this were the case, that
model can be regarded as a modified version of our ball-and-socket
morphogenesis, one side fitting to the other through cell
proliferation instead of cell shape changes. It will then become
important to study how cells and ECM collectively undergo
morphogenesis in other types of joints and in other species.
Unraveling similarities and differences in the modes of joint
development would be crucial in a medical sense as well, for
understanding various joint pathologies and designing therapies to
treat them.
Acknowledgements
We thank Drs S. Luschnig, D. Kiehart and F. Matsuzaki, the Bloomington Stock
Center, the Kyoto Stock Center and the Vienna Drosophila RNAi Center for fly
strains. We are grateful to Drs S. Kuratani, G. Sheng, N. Niwa, T. Noguchi and
T. Kondo for helpful comments on the manuscript, and to all members of
Hayashi lab for valuable discussions. This work was supported in part by a
Research Grant for Genome Research from MEXT Japan to S.H. R.T. is a RIKEN
Special Postdoctoral Fellow.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.047175/-/DC1
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Ball-and-socket joint morphogenesis