Download Muscle length and myonuclear position are

RESEARCH ARTICLE 3827
Development 139, 3827-3837 (2012) doi:10.1242/dev.079178
© 2012. Published by The Company of Biologists Ltd
Muscle length and myonuclear position are independently
regulated by distinct Dynein pathways
Eric S. Folker1, Victoria K. Schulman1,2 and Mary K. Baylies1,2,*
SUMMARY
Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei.
The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in
disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development
and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the
regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length
determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically,
microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate
myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting
that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the
cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear
positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function.
INTRODUCTION
The size, shape and organization of tissues and individual cells
directly impact their physiology. A pressing question in cell and
developmental biology is how different morphogenetic processes
that are necessary for the functional development of a common
tissue are coordinated. Skeletal muscle provides an ideal system
with which to study structure-function relationships as it is highly
organized with a physiologically testable output.
The cellular unit of all metazoan muscle is the myofiber, a
syncytial cell that is formed from the iterative fusion of myoblasts.
The correlation between structure and function is highlighted by
the organization of myosin and actin within the sarcomere that
provides the contractile apparatus (Squire, 1997). However, other
aspects of skeletal muscle structure, including myofiber size and
organelle positioning, are also crucial for its function.
Myofiber size encompasses a variety of measurements,
including length, width, depth and volume, and size is important
for muscle output, as smaller myofibers provide less contractile
force than larger muscles (Fitts et al., 1991). During muscle
formation in Drosophila, muscle cell growth occurs in three
distinct phases (Volk, 1999; Schnorrer and Dickson, 2004). As
fusion begins, the muscle cell breaks symmetry, adds mass, and
elongates to form polarized structures with a long axis and a short
axis. Once the long axis is established, the muscle cell extends
filopodia at each end in search of an attachment with a tendon cell.
Finally, the filopodial protrusions stop, the muscle cell poles
become smooth, and stable muscle-tendon attachments are made.
In Drosophila embryos, muscle size has often been correlated with
1
Program in Developmental Biology, Sloan Kettering Institute, New York, NY 10065,
USA. 2Cell and Developmental Biology, Weill Cornell Graduate School of Medical
Sciences, Cornell University, New York, NY 10065, USA.
*Author for correspondence ([email protected])
Accepted 16 July 2012
the number of fusion events (Bate, 1990), but it is unlikely that
fusion alone determines myofiber size. In Drosophila larvae and in
mammals, a number of signaling pathways have been demonstrated
to control muscle size through growth regulation (Demontis and
Perrimon, 2009; Rommel et al., 2001). Although several guidance
cues and signaling mechanisms are known to regulate the direction
of muscle growth (Schejter and Baylies, 2010; Schweitzer et al.,
2010), the factors and mechanisms that directly provide the
necessary forces that promote oriented and regulated muscle
growth during early myogenesis are not known.
The importance of organelle positioning is highlighted by the
localization of the myonuclei. In skeletal muscle, nuclei reside
above the sarcomere at the periphery of the muscle fiber and are
positioned to maximize their internuclear distance. Moreover,
mispositioned myonuclei, in conjunction with smaller myofibers,
are a hallmark of many muscle disorders and have been used
diagnostically for decades (Pierson et al., 2007; Romero, 2010).
The mechanisms by which nuclei are moved throughout the muscle
(Metzger et al., 2012) and anchored at the neuromuscular junction
(NMJ) (Grady et al., 2005; Lei et al., 2009) are beginning to
emerge; however, only a small number of factors necessary for
these processes have been identified. Additionally, potential links
between muscle size and myonuclear position have not been
explored. In other systems, the microtubule cytoskeleton regulates
both polarized growth and nuclear position. Cytoplasmic Dynein
(Dynein) is a crucial factor in both processes. During nuclear
movement in the first cell division of C. elegans and in migrating
neurons, cortically anchored Dynein pulls microtubule minus ends
and the attached nuclei towards itself (Gönczy, 2002; Tsai et al.,
2007). During polarized cell growth, Dynein contributes to the
establishment of the polarity axis (Etienne-Manneville and Hall,
2001; Palazzo et al., 2001) that is necessary to localize the factors
for cell protrusion (Prigozhina and Waterman-Storer, 2004;
Schmoranzer et al., 2003). Given these functions, Dynein may
contribute to both myofiber size and myonuclear positioning and
might integrate the two processes.
DEVELOPMENT
KEY WORDS: Drosophila, Dynein, Nuclear positioning, Muscle size
3828 RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila genetics
Stocks were grown under standard conditions. Stocks used were apMENLS::dsRed (Richardson et al., 2007), Dhc64C4-19 and Dhc64C6-10 (Gepner
et al., 1996), UAS-Dhc64C-RNAi (Vienna Drosophila RNAi Center,
v28053), dlc90F05089 (Caggese et al., 2001), lis1K11702 (Lei and Warrior,
2000), lis1G10.14 (Liu et al., 1999), UAS-Lis1-RNAi (Vienna Drosophila
RNAi Center, v6216), clip190KG06490 (Bloomington Drosophila Stock
Center, 14493), UAS-Clip190-RNAi (Vienna Drosophila RNAi Center,
v107176), raps193 (Parmentier et al., 2000), inscP49 (Kraut and CamposOrtega, 1996) and UAS-Glued-RNAi (Vienna Drosophila RNAi Center,
v3785). Mutants were balanced and identified using CTG (CyO, twi-Gal4,
UAS-2xeGFP), TTG (TM3, twi-Gal4, UAS-2xeGFP), TM6B, CyO
P[w+wgen11lacZ] and TM3 Sb1 Dfd-lacZ.
Immunohistochemistry
Embryos were collected at 25°C and fixed with 4% EM-grade
paraformaldehyde (Polysciences, 00380) diluted in PBS (Roche,
11666789001) and an equal volume of heptane to allow permeabilization.
In all cases, embryos were devitellinized by vortexing in a 1:1
methanol:heptane solution. Larvae were dissected in ice-cold HL3.1 as
previously described (Brent et al., 2009) and fixed with 10% formalin
(Sigma, HT501128-4L). Embryos and larvae were mounted in ProLong
Gold (Invitrogen) for fluorescent stainings. Antibodies were preabsorbed
where noted (PA) and used at the following final dilutions: rabbit antidsRed (PA, 1:400, Clontech 632496), rat anti-Tropomyosin (PA, 1:500,
Abcam ab50567), mouse anti-GFP (PA, 1:200, Clontech 632381), mouse
anti-DHC (1:50, Developmental Studies Hybridoma Bank), mouse antiTubulin (1:500, Sigma T9026), chicken anti--galactosidase (PA, 1:1000,
Novus Biologicals NB100-2045), mouse anti--PS-Integrin (1:100,
Developmental Studies Hybridoma Bank) and mouse anti-Discs large
(1:200, Developmental Studies Hybridoma Bank). We used Alexa Fluor
488-, Alexa Fluor 555- and Alexa Fluor 647-conjugated fluorescent
secondary antibodies (1:200), Alexa Fluor 647-conjugated phalloidin
(1:100) and Hoechst 33342 (1 g/ml) for fluorescent stains (all Invitrogen).
Fluorescent images were acquired on a Leica SP5 laser-scanning confocal
microscope equipped with a 63⫻ 1.4 NA HCX PL Apochromat oil
objective and LAS AF 2.2 software. Images were processed using Adobe
Photoshop CS4. Maximum intensity projections of confocal z-stacks were
rendered using Volocity Visualization software (Improvision).
Nuclear position and muscle length measurements
Embryos were staged based on standard laboratory procedures: overall
embryo shape, the intensity of the apRed and Tropomyosin signals, gut
morphology, and the morphology of the trachea (Beckett and Baylies,
2007). Measurements were taken from confocal projections of embryos
and acquired using the Line function of ImageJ software (NIH). For each
genotype, all four lateral transverse (LT) muscles were measured in three
hemisegments from each of 20 embryos from four independent
experiments. Three measurements were taken within each LT muscle: (1)
myotube length and (2, 3) the shortest distance between the myotube poles
and the outermost edge of the nearest nucleus (supplementary material Fig.
S1). The edges of the nuclei and ends of the myotubes were defined by the
clear change from signal to background within the relevant viewing
channel. Similar measurements were performed using -PS-Integrin to
identify the ends of the LT muscles. The statistical significance of
differences in measurements was assessed using a Student’s t-test to
compare each experimental genotype with wild-type apRed controls.
Statistical analysis was performed with Prism 4.0 (GraphPad).
Because the live stage 17 embryos offer few markers for accurate
staging, staging was achieved by timed lays: after a pre-lay period,
embryos were collected for 15 minutes and then aged for 16.5 hours.
Embryos were then dechorionated and mounted on coverslips in
halocarbon oil and images were acquired on a Zeiss Axioplan 2 microscope
with a 20⫻ PlanNeoFluor 0.50 NA objective and an Axiocam MRm
camera. Nuclear spread measurements were performed using the Line
function in ImageJ to measure the distance between the dorsal edge of the
dorsal nucleus and the ventral edge of the ventral nucleus. Additionally,
these same images of stage 17 embryos were used to count the number of
nuclei per hemisegment, which was used as a measure of the number of
myoblast fusion events.
Microtubule organization measurements
Confocal z-stacks were sequentially acquired to maximize the signal and
resolution at the myofiber poles and to minimize interference from the
internal muscles and the epidermis. Images were acquired with a 63⫻ 1.4
NA HCX PL Apochromat oil objective and a 10⫻ optical zoom. z-stacks
through a depth of 2 m with a step size of 0.25 m were acquired for all
analyzed images. z-stacks were then assembled into confocal projections
using Volocity, which were then exported to ImageJ for analysis. Analysis
was performed on 20 embryos from four independent experiments. To
count the number of microtubules in contact with the muscle pole, a 2⫻2
m box was drawn from the tip of the muscle toward the center and the
number of microtubules in this region were counted.
To determine the average intensity, the Measure Average Intensity
Function in ImageJ was performed on the distal 3 m of the myofiber. The
intensity of Tubulin staining was standardized against the intensity of
Tropomyosin staining to control for sample variation. For each genotype,
all four LT muscles were measured in three hemisegments from each of 20
embryos from four independent experiments. Student’s t-test was used to
DEVELOPMENT
To investigate how myofiber size and myonuclear positioning
are regulated, and whether the two processes are co-dependent, we
have studied the role of Dynein during muscle morphogenesis in
Drosophila. We establish muscle length, which is an aspect of
muscle size, and myonuclear positioning as independent aspects of
muscle morphogenesis. We further show that the microtubule
cytoskeleton contributes to both processes. Specifically, Dynein
regulates muscle length and myonuclear positioning with
overlapping, but distinct, sets of proteins. The Dynein heavy chain
(Dhc64C), Dynein light chain (Dlc90F; Tctex1 in mammals) and
Partner of inscuteable [Pins; Rapsynoid (Raps) – FlyBase] are
necessary regulators of both muscle length and myonuclear
positioning. Subsequently, the two pathways become distinct:
muscle length is regulated by a Dynein-Lis1-dependent
mechanism, whereas myonuclear positioning is regulated by a
Dynein–CLIP-190–Glued-dependent mechanism.
Mechanistically, in mutant embryos that lack Dynein
(Dhc64C4-19) or fail to localize Dynein to the muscle pole (raps193),
both muscle length and myonuclear positioning are affected,
suggesting that Dynein activity at the muscle pole is required for
both processes. However, Dynein localizes to the muscle pole in
both Lis1 and CLIP-190 mutant embryos, indicating that Dyneindependent regulation of muscle length and Dynein-dependent
regulation of myonuclear positioning are specified downstream of
its arrival at the muscle pole. Lis1 mutant embryos exhibit
hyperaccumulation of Dynein at the muscle pole with a tighter
focus of peak intensity, suggesting that Lis1-dependent activation
of Dynein retrograde trafficking is essential for muscles to achieve
their proper length. CLIP-190 mutant embryos have fewer
microtubules near the myofiber poles despite normal Dynein
localization, suggesting that CLIP-190 promotes microtubule-cell
cortex interactions that are necessary for Dynein-dependent
myonuclear positioning. Together, these data suggest that both
CLIP-190 and Lis1 work downstream of Dynein localization to the
muscle pole and thus specify Dynein function within this single
location. Differential regulation of Dynein activity within a single
location is novel and likely to be important during morphogenetic
processes for which proper timing is essential. Finally, both muscle
length and myonuclear positioning are important for muscle
function, as defects in either process correlate with larvae that
crawl more slowly than controls.
Development 139 (20)
compare each experimental genotype with wild-type apRed controls.
Statistical analysis was performed with Prism 4.0.
Dynein localization measurements
Embryos were collected and fixed with 10% formalin diluted 1:1 in
heptane for 20 minutes, then rinsed three times in PBS containing 0.6%
Triton X-100, then fixed with 4% paraformaldehyde diluted 1:1 in heptane
for 20 minutes. Antibody incubations were performed in PBS
supplemented with 1% BSA and 0.6% Triton X-100. Image acquisition and
processing were as for microtubule quantification. Dynein
immunofluorescence intensity was assessed by linescan analysis using
ImageJ. Positions of linescans were chosen randomly on the Tropomyosin
image and the line then copied to the identical position on the Dynein
image. The intensity of Dynein immunofluorescence at each point was
measured relative to Tropomyosin intensity at that same point to control
for sample variation, and the ratios were multiplied by 100. Three
hemisegments from 20 embryos from four independent experiments were
analyzed. The greatest pixel intensity from each embryo was used to
determine the peak intensity. To determine the width of peak intensity, the
pixels that were ≥80% of peak intensity were determined and the distance
between the first pixel and the last pixel that fitted these criteria was
measured. For each genotype, all four LT muscles were measured in three
hemisegments from each of five embryos. Student’s t-test was used to
compare each experimental genotype with wild-type apRed controls.
Statistical analysis was performed with Prism 4.0. The heat map
representation of Dynein immunofluorescence was generated using the
Cool application of the standard Lookup tools in ImageJ.
Larval behavior
Larval speed was assessed as previously described (Louis et al., 2008;
Metzger et al., 2012) with minor modifications. Stage 16 and 17 embryos
were selected for the absence of the fluorescent balancer and placed on
yeast-coated apple juice agar plates at 22°C overnight. L1 larvae were
selected the following day and placed into vials of standard fly food
containing Bromophenol Blue (Bio-Rad, 161-0404). L3 larvae were picked
from the vial 6 days later and individually tracked as they crawled towards
an odor source of ethyl butyrate (3.3%; Sigma, E15701) diluted in paraffin
oil (Fluka, 76235). Their movements were recorded with a CCD camera
for either five total minutes or until they reached the odor source or the
walls of the apparatus. The tracks were processed using Ethovision
software (Nodlus). At least 20 larvae were tracked for each genotype.
Tracked larvae were dissected, stained and analyzed as follows.
Internuclear distance was measured in ImageJ by drawing lines between
the center of each nucleus and its nearest neighbor using projection images.
Muscle size was measured by determining the area of confocal projections
of each muscle. To calculate the internuclear distance as a function of
muscle size, the average internuclear distance for a given muscle was
divided by the area of that specific muscle. Nine muscles from three larvae
were used for quantification.
RESULTS
Dynein regulates muscle length and myonuclear
position
Each body wall muscle in the Drosophila embryo and larva
consists of a single syncytial myofiber similar to the individual
myofibers in mammalian muscle. To assess the role of Dynein
during Drosophila muscle morphogenesis, the distribution of nuclei
within the lateral transverse (LT) muscles was examined in wildtype embryos and in embryos in which Dynein was zygotically
removed with either the Dhc64C4-19 (null) or the Dhc64C6-10
(hypomorphic) allele (Gepner et al., 1996). The myonuclei in the
LT muscles were visualized by expression of the apterous
mesodermal enhancer fused to a nuclear localization signal and the
fluorescent protein DsRed (apRed) (Richardson et al., 2007), which
specifically labels the nuclei of the LT muscles. Using this reporter,
live stage 17 embryos [16.5-20 hours after egg lay (AEL)] were
RESEARCH ARTICLE 3829
examined. Dhc64C mutant embryos had the same number of
apRed nuclei per hemisegment as controls, indicating that myoblast
fusion was unaffected (Fig. 1A,B). Although the nuclei were
aligned in columns within each LT muscle in embryos of each
genotype, the distance between the most ventral and the most
dorsal nuclei was reduced in Dhc64C mutant embryos (Fig. 1A,C).
To determine whether the difference in the distribution of nuclei
was caused by defective nuclear positioning or effects on muscle
size, we examined late stage 16 embryos (16 hours AEL), which
are amenable to whole-mount immunostaining.
The general muscle pattern as revealed by Tropomyosin staining
(Fig. 1D) was normal in Dhc64C mutant embryos. Additionally, PS-Integrin staining was normal. Thus, all muscles were present,
properly oriented and attached to tendon cells, indicating that
specification, guidance and attachment mechanisms are functional.
In late stage 16 (16 hours AEL) control embryos, the myonuclei
were in two clusters within each LT muscle: one cluster at the
dorsal pole and the other at the ventral pole (Fig. 1D;
supplementary material Fig. S1) (Metzger et al., 2012). In embryos
homozygous for either Dhc64C allele, the nuclei were also in two
clusters; however, the clusters were mispositioned 50% further
from the muscle poles than in wild-type and heterozygous controls
(Fig. 1D,E; supplementary material Fig. S2). Additionally, the
length of the LT muscles was reduced by 15% in embryos
homozygous for either Dhc64C allele as compared with wild-type
and heterozygous controls (Fig. 1D,F; supplementary material Fig.
S2). To account for the difference in muscle length, the distance
from each cluster of nuclei to the nearest muscle pole was
calculated as a percentage of muscle length (Fig. 1G). This
measurement was used with respect to all subsequent genotypes.
To validate the use of Tropomyosin as a marker for muscle ends,
we performed identical measurements using -PS-Integrin to mark
the muscle ends. These analyses produced identical results (Fig.
1D; supplementary material Fig. S2C,D).
To confirm that the effects of Dynein were autonomous to
muscle, we used the GAL4/UAS system to deplete Dynein
specifically in the mesoderm using twist-GAL4 to express UASDhc64C-RNAi (Dietzl et al., 2007). Tissue-specific depletion of
Dhc64C recapitulated the effects of the Dhc64C alleles: the LT
muscles were 12% shorter than controls and the nuclei were
positioned 40% further from the muscle poles than in controls (Fig.
1D-G). These data indicate that Dynein is specifically required in
the muscle to generate muscles of proper length and to properly
position myonuclei.
Given that myonuclear position seems to be affected similarly in
stage 16 and stage 17 embryos, we examined embryos at earlier
stages in their development to determine when the defects in
muscle length and myonuclear positioning become evident. Using
Tropomyosin to identify the muscles and a combination of trachea
morphology and gut morphology to assess embryo age, we
determined the length of muscles and the positioning of myonuclei
in late stage 14 (11 hours AEL), stage 15 (13 hours AEL) and stage
16 (16 hours AEL) embryos (Fig. 2). At stage 14, the length of the
muscles in control and Dhc64C4-19 embryos was similar. However,
by stage 15 and through stage 16 the muscles in the Dhc64C4-19
embryos were significantly shorter (17% and 14%, respectively)
than those in the control embryos.
The effects on myonuclear positioning at the different stages
were more complex. The nuclei were 30% further from the muscle
poles in late stage 14 Dhc64C4-19 embryos than in controls (Fig.
2C); however, only the positioning relative to the dorsal muscle
pole was statistically significant. At stage 15, myonuclei were
DEVELOPMENT
Dynein-dependent muscle formation
3830 RESEARCH ARTICLE
Development 139 (20)
similarly positioned in control and Dhc64C4-19 embryos (Fig. 2D).
In stage 16 embryos, the myonuclei were significantly
mispositioned (50% further from the poles) at both the dorsal and
ventral poles of the muscle in Dhc64C4-19 mutant embryos relative
to controls (Fig. 2E).
These data indicate that the defects in muscle length arise prior
to muscle-tendon attachment and, together with the muscle-specific
depletion of Dhc64C, suggest that the effects are muscle
autonomous and not due to defects in tendon specification.
Dynein-interacting proteins contribute to Dyneindependent regulation of muscle length and
myonuclear positioning
To determine whether the roles of Dynein in specifying muscle
length and myonuclear positioning are functionally coupled, we
tested known Dynein-interacting proteins for their contributions to
each process. We focused on factors that are general regulators of
Dynein activity or have been found to specifically contribute to
Dynein-mediated nuclear movements in other systems. We
examined Dynein light chains, which can regulate Dynein activity
both positively (Sönnichsen et al., 2005) and negatively (O’Rourke
et al., 2007); Glued, which increases Dynein motor processivity
and mediates Dynein-cargo interactions (Gill et al., 1991; Vaughan
et al., 2002; Waterman-Storer et al., 1997); Pins, which anchors
Dynein at the cortex to move nuclei in other systems (Gotta et al.,
2003; Hughes et al., 2004); Lis1, which maintains Dynein in an
active state and has been implicated in nuclear movements in yeast
and neurons (McKenney et al., 2010; Mesngon et al., 2006;
Sheeman et al., 2003); and CLIP-190, which works with Lis1 to
regulate Dynein activity (Tanenbaum et al., 2008).
We tested two Dynein light chains, Cdlc2 and Dlc90F, which are
the Drosophila orthologs of Dynein light chain 1/2 and Tctex1,
respectively (Caggese et al., 2001; Goldstein and Gunawardena,
2000), by RNAi-mediated depletion (Dietzl et al., 2007). Depletion
of Cdlc2 specifically in mesoderm and muscle did not affect
muscle length or myonuclear positioning, whereas depletion of
Dlc90F resulted in shorter muscles with mispositioned nuclei.
Additionally, embryos that were homozygous for dlc90F05089
(Caggese et al., 2001) or the pins allele raps193 (Parmentier et al.,
2000), produced muscles that were 12% shorter with nuclei that
were 40-50% further from the muscle poles than in wild-type and
heterozygous control embryos (Fig. 3A-C; supplementary material
Fig. S2). These data indicate that Dlc90F and Pins contribute to
both muscle length and myonuclear positioning.
Embryos homozygous for either of two Lis1 alleles, lis1K11702
(Lei and Warrior, 2000) and lis1G10.14 (Liu et al., 1999), produced
muscles that were 15% shorter than those of wild-type and
heterozygous controls. However, myonuclear positioning was
normal in Lis1 homozygous embryos. Conversely, embryos that
were depleted of Glued (Dietzl et al., 2007) in muscle and embryos
that were homozygous for clip190KG06490 (Bellen et al., 2004)
produced muscles of similar length to controls. However, in these
embryos the nuclei were positioned 50% further from the muscle
poles than in wild-type and heterozygous controls (Fig. 3A-C;
DEVELOPMENT
Fig. 1. Cytoplasmic Dynein regulates muscle length and
myonuclear position. (A)Fluorescence images of apRed nuclei
in the lateral transverse (LT) muscles of live stage 17 Drosophila
embryos (16.5-20 hours AEL) of the indicated genotypes. (B)The
number of nuclei per hemisegment in stage 17 embryos of the
indicated genotypes. (C)The distance between the dorsalmost
and ventralmost nuclei in stage 17 embryos of the indicated
genotypes. (D)Immunofluorescence images of stage 16 (16
hours AEL) embryos of the genotypes noted to the left and
antigen listed at the top of the first image. Green, Tropomyosin
(muscle); red, DsRed (nuclei); blue, -PS-Integrin in merge.
Arrows in the control panels denote points from which
measurements were made for all genotypes. The distance
between the points indicated by the yellow arrows in the
Tropomyosin and -PS-Integrin panels was used to determine
muscle length. The distance between the pair of gray arrows at
the top of the merged image was used to determine the
distance between the nuclei and the dorsal pole. The distance
between the pair of white arrows at the bottom of the merged
image was used to determine the distance between the nuclei
and the ventral pole. (E)The shortest distance between the
indicated pole of the LT muscles (gray, dorsal; white, ventral) and
the nearest cluster of nuclei in stage 16 embryos. (F)LT muscle
length in stage 16 embryos. (G)The shortest distance between
the indicated pole of the LT muscles (gray, dorsal; white, ventral)
and the nearest cluster of nuclei normalized for muscle length in
stage 16 embryos of the indicated genotypes. Error bars indicate
s.d.; **P<0.01, *P<0.05. Scale bars: 10m.
Fig. 2. Stage-dependent effects of Dynein on muscle length and
myonuclear positioning. (A)Immunofluorescence images of the
muscles (green, Tropomyosin) and nuclei (red, DsRed) at stage 14, 15
and 16 in control and Dhc64C4-19 Drosophila embryos. Scale bar:
10m. (B)The length of the muscles in control (gray) and Dhc64C4-19
(black) embryos at stage 14, 15 and 16. (C-E)The distance between the
nearest nucleus and either the dorsal (gray) or ventral (white) pole of
the muscle in stage 14 (C), 15 (D) and 16 (E) embryos. Error bars
indicate s.d.; *P<0.05, **P<0.01.
supplementary material Fig. S2). The effects seen on myonuclear
positioning in the clip190KG06490 homozygote were phenocopied in
embryos that were transheterozygous for clip190KG06490 and each
of three different deficiencies that removed CLIP-190:
RESEARCH ARTICLE 3831
Df(2L)BSC294,
Df(2L)Exel7068
and
Df(2L)Exel8036
(supplementary material Fig. S2). Additionally, we measured the
ventral longitudinal muscle VL3 and found that its length in the
embryo was similar in each genetic background (supplementary
material Fig. S2). These data suggest that a unique feature of the
LT muscles makes them more susceptible to Dynein levels and/or
activity with respect to muscle growth in the embryo.
The number and position of nuclei in stage 17 embryos were also
examined. Embryos from each genotype had the same number of
apRed nuclei per hemisegment, indicating that the defects in muscle
length did not result from impaired fusion (Fig. 1B). The clusters of
nuclei in each genotype did resolve into columns during stage 17, but
the distance between the ventral and dorsal nuclei was reduced by
15% compared with controls. Moreover, the distance between the
most distal nuclei was similar in stage 16 and stage 17 embryos for
each genotype (supplementary material Fig. S3).
To determine whether the Dynein-interacting proteins work with,
or independently of, Dynein, we examined embryos that were doubly
heterozygous for Dhc64C mutations and mutations in each of the
associated genes. In these, and all subsequent genetic interactions,
maternal effects were controlled for by providing each allele from
the mother or the father in reciprocal crosses. Both Dhc64C4-19 and
Dhc64C6-10 were used in combination with alleles for each putative
Dynein-interacting gene. Double heterozygotes of dlc90F05089 and
Dhc64C4-19 or of dlc90F05089 and Dhc64C6-10 produced muscles that
were 15% shorter than controls and with their nuclei 50% further
from the muscle poles (Fig. 4A-C). These data indicate that Dlc90F
(Tctex1) regulates Dynein activity for both muscle length and
myonuclear positioning. All Lis1–/+; Dhc64C–/+ doubly
heterozygous embryos produced muscles that were 15% shorter than
those in control embryos, but with properly positioned myonuclei at
the muscle poles (Fig. 4A-C). Conversely, CLIP-190–/+; Dhc64C–/+
doubly heterozygous embryos produced muscles that were the same
length as those of control embryos but with nuclei 40% further from
the muscle poles (Fig. 4A-C; supplementary material Fig. S3).
Dhc64C–, +/+, raps193 doubly heterozygous embryos and embryos
Fig. 3. Dynein-interacting proteins
regulate myotube length and/or
myonuclear position.
(A)Immunofluorescence images of
stage 16 Drosophila embryos of the
indicated genotypes. Green,
Tropomyosin; red, DsRed; blue, -PSIntegrin in merge. The antigen for
grayscale images is listed at the top of
the first image. Scale bar: 10m. (B)The
length of the LT muscles in stage 16
embryos. (C)The shortest distance
between the indicated pole of the LT
muscles (gray, dorsal; white, ventral) and
the nearest cluster of nuclei normalized
for muscle length. Error bars indicate
s.d.; *P<0.05, **P<0.01.
DEVELOPMENT
Dynein-dependent muscle formation
3832 RESEARCH ARTICLE
Development 139 (20)
Fig. 4. Interactions between Dynein and associated genes during muscle development. (A)Immunofluorescence images of stage 16
Drosophila embryos that are doubly heterozygous for Dhc64C4-19 and the indicated allele. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT
muscle length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele shown beneath the histogram and the listed alleles. (C)The
shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle
length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele indicated beneath the histogram and the listed alleles. Error bars
indicate s.d.; **P<0.01.
The two Dynein-dependent pathways are
separable
To confirm that Dynein-dependent myonuclear positioning and
Dynein-dependent determination of muscle length are regulated by
separate mechanisms, we examined the interactions between
Dynein regulatory proteins. Both Lis1 and CLIP-190 functionally
interacted with dlc90F05089, and these interactions recapitulated
their interactions with Dhc64C: lis1K11702/+; dlc90F05089/+ and
lis1G10.14/+; dlc90F05089/+ double heterozygotes had muscles that
were 20% shorter than those of control embryos, whereas
clip190KG06490/+; dlc90F05089/+ double heterozygotes had muscles
in which the nuclei were mispositioned, 30% further from the
muscle poles compared with controls. Similarly, pins functionally
interacted with both Lis1 and CLIP-190: lis1K11702/+; raps193/+
double heterozygotes had muscles that were 20% shorter than those
of control embryos and clip190KG06490/+; raps193/+ double
heterozygotes had normal sized muscles with mispositioned
myonuclei that were 60% further from the muscle poles compared
Fig. 5. The pathways controlling muscle length and myonuclear position do not genetically interact. (A)Immunofluorescence images of
stage 16 Drosophila embryos that are doubly heterozygous for the indicated alleles. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT
muscle length in stage 16 embryos that are doubly heterozygous for the indicated alleles. (C)The shortest distance between the indicated pole of
the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei in stage 16 embryos normalized for muscle length. Error bars indicate
s.d.; *P<0.05, **P<0.01.
DEVELOPMENT
depleted of Glued in the Dhc64C–/+ background died prior to stage
16 and could not be evaluated. These data indicate that Lis1 and
CLIP-190 regulate muscle length and myonuclear position,
respectively, via interactions with Dynein, and not by Dyneinindependent mechanisms.
Dynein-dependent muscle formation
RESEARCH ARTICLE 3833
with controls (Fig. 5A-C; supplementary material Fig. S3). The
interaction of dlc90F05089 with pins was also examined but resulted
in embryonic death prior to stage 16 and could not be evaluated.
Lis1 contributes only to muscle length, whereas CLIP-190
contributes solely to myonuclear positioning. We tested whether
Lis1 functionally interacts with CLIP-190. Lis1–, +/+,
clip190KG06490 double heterozygotes were similar to control
embryos in both muscle length and myonuclear positioning. This
indicates that Dynein-dependent muscle length and Dyneindependent myonuclear positioning are indeed mechanistically
distinct processes (Fig. 5A-C; supplementary material Fig. S3).
Lis1 affects Dynein localization and CLIP-190
affects microtubule-cortex interactions
We next examined the effects of Dhc64C, Lis1, CLIP-190 and raps
mutants on Dynein localization (Fig. 6A) and microtubule
organization (Fig. 7A) to understand how Dynein differentially
regulates muscle length and myonuclear positioning. Using
confocal projections, linescans of Dynein immunofluorescence
intensity (Fig. 6B) were employed to measure Dynein localization.
To control for sample variation, the intensity of Dynein
immunofluorescence was measured relative to the intensity of
Tropomyosin immunofluorescence at the same point. Both the peak
of Dynein immunofluorescence (Fig. 6C) and width of peak
immunofluorescence intensity (Fig. 6D) were quantified.
clip190KG06490 did not significantly affect Dynein localization. Lis1
and raps mutants did affect Dynein localization, although
differently. In raps193 mutant embryos, the peak intensity of Dynein
immunofluorescence was decreased compared with controls,
whereas the width of peak intensity was increased (Fig. 6A-D),
suggesting that Pins is required for Dynein localization to the
muscle poles. In Lis1 mutant embryos, the peak intensity of Dynein
immunofluorescence was increased compared with controls and the
width of peak intensity was slightly decreased (Fig. 6A-D). The
higher peak intensity combined with the sharper localization
suggested that Lis1 does not contribute to the initial localization of
Dynein to the muscle pole, but rather to the retrograde trafficking
of Dynein away from the muscle pole. Together, these data indicate
that proper muscle length requires Dynein localization to the
muscle pole, and that raps193 results in shorter muscles due to poor
accumulation of Dynein at the myofiber pole. Additionally, tightly
focused hyperaccumulation of Dynein, as seen in Lis1 mutant
embryos, also affects muscle length, suggesting that trafficking of
Dynein away from the myofiber pole is mediated by Lis1 and is
necessary to generate muscles of the proper length.
Similarly, confocal projections of embryos immunostained for
Tubulin and Tropomyosin were used to assess the presence of
microtubules near myofiber poles (Fig. 7A). Two different
measures – the number of microtubules in contact with the
myofiber pole (Fig. 7B) and the average intensity of tubulin
immunofluorescence in the distal 3 m of the myofiber (Fig. 7C)
– indicated that only clip190KG06490 homozygotes had significantly
fewer microtubules near the muscle pole than all other genotypes.
These data suggest that CLIP-190 is necessary to mediate
interactions between microtubules and the myofiber cortex that are
then utilized by Dynein to move nuclei towards the muscle pole.
Defects in muscle size and myonuclear position
impair muscle function
Larval crawling towards an odorant stimulus was used to assess the
importance of Dynein-dependent muscle size regulation and
Dynein-dependent myonuclear positioning to muscle physiology.
DEVELOPMENT
Fig. 6. Dynein hyperaccumulates in
Lis1 mutant embryos. (A)Confocal
projections of a single hemisegment
from stage 16 Drosophila embryos
immunostained for Tropomyosin
(green) and Dynein heavy chain (red).
The boxed regions are shown at higher
magnification to the right, with
Tropomyosin shown in grayscale and
Dynein shown as a heat map (the scale
indicates relative intensity). The regions
outlined by the green dotted lines
were used for linescan analysis. Scale
bars: left-hand panel, 5m; right-hand
panel, 10m. (B)Representative
linescan analysis indicating the
intensity of Dynein heavy chain
immunofluorescence as a function of
position across the muscle pole.
(C)The peak intensity signal for Dynein
heavy chain immunofluorescence in
the indicated genotypes. (D)The width
of the peak intensity of Dynein heavy
chain immunofluorescence. Error bars
indicate s.d.; *P<0.05, **P<0.01,
compared with control.
3834 RESEARCH ARTICLE
Development 139 (20)
Fig. 7. Microtubule organization is disrupted in
CLIP-190 mutant embryos. (A)Confocal projections
of a single hemisegment from stage 16 Drosophila
embryos immunostained for Tropomyosin (green) and
Tubulin (grayscale). The boxed regions are shown at
higher magnification to the right. Yellow arrows
indicate individual and/or bundles of microtubules that
reach the muscle pole. Scale bars: 10m. (B)The
number of microtubules within 3m of the myofiber
pole in the indicated genotypes. (C)The average
intensity of Tubulin immunofluorescence in the 3m
near the myotube pole in the indicated genotypes.
Error bars represent s.d.; *P<0.05, compared with
control.
size and myonuclear position, suggesting that the defects seen in
the embryo are bona fide effects and not merely developmental
delays.
DISCUSSION
We have used the Drosophila musculature to investigate the
mechanisms that control muscle size and intracellular organization.
We find that muscle length is regulated independently of the
number of fusion events and demonstrate that perturbations that
affect embryonic muscle length correlate with decreased larval
muscle size and poor muscle function. Additionally, we find that
intracellular organization, specifically myonuclear positioning, is
essential for muscle function, consistent with our recent
observations (Metzger et al., 2012). Moreover, we have shown that
the length and intracellular organization of the myofiber are
mechanistically independent. Although a number of factors link
both processes, we have identified factors that contribute solely to
muscle length or myonuclear position and demonstrate that we can
independently manipulate each feature.
Dynein regulates both muscle length and myonuclear
positioning. Some Dynein-interacting proteins, such as Dlc90F and
Pins, are necessary for both processes. That these factors regulate
both muscle length and myonuclear positioning suggests that
specific aspects of muscle growth and the positioning of myonuclei
can indeed be coordinated. However, Lis1 affects muscle length
specifically, whereas CLIP-190 and Glued specifically affect
myonuclear position. Within the contexts of muscle length and
myonuclear position CLIP-190 and Lis1 do not genetically interact,
illustrating that, although linked, the two processes are
mechanistically distinct.
Our identification of CLIP-190 and Lis1 as regulators of Dynein
is not novel. CLIP-190 and Lis1 are known to interact with Dynein
both physically and functionally, and they usually cooperate toward
a single goal (Coquelle et al., 2002). Here, we provide the first
DEVELOPMENT
Each genotype tested crawled towards the stimulus, indicating that
all genotypes perceived and responded to the stimulus. However,
L3 larvae that were homozygous for clip190KG06940 or lis1G10.14
crawled towards the stimulus 33% more slowly than controls (Fig.
8A). Dhc64C4-19, Dhc64C6-10 and lis1K11702 were early larval lethal,
thus their locomotive ability could not be tested. However, when
Dhc64C was depleted during embryonic and larval development
by driving the UAS-Dhc64C-RNAi construct with the musclespecific driver DMef2-Gal4, these larvae also crawled towards the
stimulus 40% more slowly than controls (Fig. 8A,B). Additionally,
muscle-specific depletion of CLIP-190 and Lis1 inhibited larval
locomotion by 20% (Fig. 8A,B). That the depletion was muscle
specific confirms that the effects on crawling are muscle
autonomous. Furthermore, the NMJ, as measured by bouton
number and distribution, was similar to that of the control in each
of the genotypes tested (supplementary material Fig. S4). Finally,
lis1G10.14, +/+, clip190KG06490 doubly heterozygous larvae crawled
towards the stimulus at the same speed as control larvae (Fig.
8A,B), further indicating the independence of these two genetic
pathways.
The larvae were then dissected and their musculature was
examined. The internuclear distance was reduced by 50-60% in
lis1G10.14 and clip190KG06490 larvae and in muscle depleted of
Dhc64C, Lis1 or CLIP-190 (Fig. 8C,D). However, the muscles in
lis1G10.14, Lis1-depleted and Dhc64C-depleted larvae were 35%
smaller than in control and clip190KG06490 larvae (Fig. 8E). Because
the muscle size was variable between genotypes, internuclear
distance was normalized as a function of muscle size. Using this
measure, both muscle size and internuclear distance were
diminished in Dhc64C-depleted larvae compared with controls.
However, internuclear distance was specifically reduced in
clip190KG06490 and CLIP-190-depleted larvae, whereas muscle size
was specifically reduced in lis1G10.14 (Fig. 8D-F). These data
demonstrate a correlation between muscle output and both muscle
Fig. 8. Larval muscle organization and physiology are affected by
Dynein and associated proteins. (A,B)The average (A) and
maximum (B) speed of Drosophila larvae as they crawl towards a
stimulus. (C)Fluorescence images of VL3 muscles from L3 larvae that
were used in locomotion assays just prior to dissection. White,
phalloidin; green, Hoechst. Scale bar: 20m. (D)The average distance
between nuclei in larval muscles from the indicated genotypes. (E)The
surface area of the muscles in larvae of the indicated genotypes. (F)The
distance between nuclei in the larval muscles normalized for muscle
size. Error bars indicate s.d. *P<0.05, **P<0.01.
example of CLIP-190 and Lis1 serving completely independent,
Dynein-dependent functions.
Mechanistically, our data suggest that Dynein function, with
respect to the regulation of muscle length and myonuclear
positioning, is specified by Lis1 and CLIP-190 downstream of its
localization. That Dynein localization to the myofiber pole is
important is illustrated by pins (raps193) mutants in which Dynein
does not accumulate at the myofiber pole, and we observe defects
in both muscle length and myonuclear positioning. This suggests
that Pins recruits and stabilizes Dynein at the cortex as it does
during mitosis (Gotta et al., 2003; Hughes et al., 2004). Lis1 also
affects Dynein localization, but in Lis1 mutants Dynein is localized
to the muscle pole and is tightly restricted to the pole compared
with controls. We interpret this to mean that Lis1 is necessary for
RESEARCH ARTICLE 3835
retrograde trafficking of Dynein away from the myofiber pole. We
hypothesize that, in the absence of Dynein retrograde trafficking,
cellular components accumulate at the extending muscle end, thus
inhibiting the trafficking of factors necessary for further directed
growth. Thus, when Dynein is incapable of moving cargo away
from the myofiber pole, muscles are shorter than in control
embryos. Indeed, a similar correlation between retrograde
trafficking and directed growth was recently reported during
mechanosensory bristle growth and axonal transport (Otani et al.,
2011; Yi et al., 2011). Interestingly, it was recently reported that
decreased retrograde transport of Dynein results in longer processes
in both fibroblasts and neurons in culture (Rishal et al., 2012).
This contradictory finding raises several interesting questions. Is
there an opposing pathway in muscle or is another aspect of muscle
size increased? For example, does the length of the muscle impact
its volume? Complications due to muscle contraction in the live
embryo make such analysis difficult, however. Likewise, working in
vivo on a dynamically changing tissue located 30-150 m below the
surface of the developing embryo presents challenges for imaging
the rapid cellular processes that underlie motor activity, microtubule
organization, organelle positioning and cell size. Nevertheless, the
link between different aspects of cell size and their relationships to
each other and to motor activity are being explored.
It is not clear why LT muscle growth/length is more sensitive to
Dynein activity than the VL muscles in the embryo. A simple
explanation is that the maternally loaded Dynein persists longer in
the VL muscles than in the LT muscles. Additionally there might
be a physical explanation. Although a cluster of potential tendon
cells for the VL muscles exist, they are clustered at the segment
border. Therefore, the size of the hemisegments, and thus the
distance from segment border to segment border, determines
muscle length. Conversely, the cluster of potential tendon cells for
the LT muscles might be more broadly dispersed and the location
of the muscle pole at the time of tendon specification determines
the length of the muscle. Under this hypothesis, inefficient
extension of the LT muscles would result in the muscles being
shorter during tendon specification/maturation and therefore shorter
throughout embryonic development. Alternatively, differences in
guidance/signaling systems could explain why the LT muscles, but
not the VL muscles, are shorter when Dynein activity is
compromised. Different signaling mechanisms are employed by
these different muscle types (Schnorrer and Dickson, 2004; Volk,
1999; Schweitzer et al., 2010; Schejter and Baylies, 2010). For
example, Derailed (Drl) plays a crucial role in the ability of LT
muscles to recognize their target (Callahan et al., 1996). Perhaps,
altered trafficking of Drl or another factor in the signaling pathway
causes slight, but significant, changes in LT muscle length.
It is interesting that the LT muscles are smaller in Dhc64C,
Dlc90F, pins and Lis1 mutant embryos, but that other muscles are
unaffected, whereas in larvae all muscles appear to be smaller.
During the larval stages, muscles remain stably attached to tendon
cells and grow through insulin signaling/Foxo- and dMycdependent pathways (Demontis and Perrimon, 2009). We interpret
our observations to mean that Dynein and its regulatory proteins
Dlc90F, Pins and Lis1 contribute to insulin receptor-mediated
muscle growth as previously described (Huang et al., 2001; Varadi
et al., 2003). Indeed, it would not be surprising to find that Dyneindependent cellular trafficking is essential to that signaling pathway.
CLIP-190 does not dramatically affect Dynein localization, but
it does affect microtubule organization, which, in turn, affects
nuclear positioning. CLIP-190 mutant embryos have fewer
microtubules at the myofiber pole, suggesting that, similar to its
DEVELOPMENT
Dynein-dependent muscle formation
functions in other systems, the role of CLIP-190 is to stabilize
microtubule-cortex interactions (Neukirchen and Bradke, 2011;
Watanabe et al., 2004), which Dynein then uses to move nuclei
towards the myofiber pole. The specification of Dynein function,
downstream of its localization, is novel. Although phosphoregulation has been shown to alter Dynein function during mitosis
(Whyte et al., 2008) and the possibility for competitive regulation
of Dynein has been suggested (McKenney et al., 2011), this is, to
our knowledge, the first example in which Dynein at a single
location has its activity modified through interactions with unique
binding partners. The ability to specify Dynein function without
dramatically altering its localization is likely to be an important
factor during development when temporal constraints are high.
With regards to physiology, the small, but significant, changes
in myonuclear positioning and muscle size seen in the embryo
continue throughout larval development and are associated with
impaired muscle function. Additionally, that the same defects and
impairments are found in larvae that were depleted of Dynein, Lis1
or CLIP-190 specifically in the muscle shows that the effects are,
at least in part, muscle specific.
Many muscle myopathies are characterized by smaller myofibers
and mispositioned nuclei. However, it is unclear whether these
pathologies are linked and which of these defects are paramount in
causing the muscle weakness associated with these myopathies. We
have shown that in Drosophila these two processes are linked via
the requirement for Dynein activity at the muscle pole. We have
further shown that these two processes are mechanistically distinct,
yet that both are necessary for muscle function. Together, these data
suggest that therapeutics aimed at improving the functional
capacity of diseased muscles must counteract effects on both
muscle size and myonuclear positioning. This highlights
Drosophila as an ideal model system with which to identify the
genes and mechanisms required for distinct aspects of muscle
morphogenesis and to shed light on key features of muscle disease.
Acknowledgements
We thank the Bloomington Stock Center and the Vienna Stock Center for fly
stocks and the Developmental Hybridoma Bank for antibodies.
Funding
Our work is supported by Muscular Dystrophy Association (MDA) and National
Institutes of Health (NIH) [GM078318 to M.K.B.]. V.K.S. is supported by an NIH
Training Grant in Developmental Biology [T32HD060600]. Deposited in PMC
for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.079178/-/DC1
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DEVELOPMENT
Dynein-dependent muscle formation