© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 665-671 doi:10.1242/dev.115964
RESEARCH REPORT
Communication between distant epithelial cells by filopodia-like
protrusions during embryonic development
Sagar1,2,3, Felicitas Prö ls1, Christoph Wiegreffe4 and Martin Scaal1,2,*
Long-range intercellular communication is essential for the regulation
of embryonic development. Apart from simple diffusion, various
modes of signal transfer have been described in the literature. Here,
we describe a novel type of cellular extensions found in epithelial cells
of the somites in chicken embryos. These filopodia-like protrusions
span the subectodermal space overlying the dorsal surface of the
somites and contact the ectoderm. We show that these protrusions
are actin- and tubulin-positive and require Rac1 for their formation.
The presence of glycophosphatidylinositol-anchored proteins and net
retrograde trafficking of the transmembrane Wnt-receptor Frizzled-7
along the protrusions indicate their role in signal transport and
distribution. Taken together, our data suggest a role of filopodia-like
protrusions in mediating signaling events between distant epithelial
cells during embryonic development.
KEY WORDS: Somite, Dermomyotome, Filopodia, FiLiPs, Frizzled-7
INTRODUCTION
The formation of a complex multicellular organism is a highly
orchestrated process with a tight spatial and temporal control of gene
expression during development. The regulation of embryogenesis
depends on molecular signaling events between different cells.
Various mechanisms of signal transport from the signaling to the
receiving cell have been described, including simple diffusion,
transcytosis and migrating cells (Crick, 1970; Entchev et al., 2000;
Serralbo and Marcelle, 2014). Recent studies have also implicated a
role of actin-based cellular extensions in transducing signals from
one cell to another, namely cytonemes in the wing imaginal disks of
Drosophila and the recently discovered specialized filopodia of
mesenchymal cells in chicken limb buds (Ramirez-Weber and
Kornberg, 1999; Sanders et al., 2013).
Somites are metameric mesodermal structures that originate from
the paraxial mesoderm. Initially, a somite resembles an epithelial
sphere, but soon the ventral half undergoes epithelial-tomesenchymal transition (EMT), forming the sclerotome, which
eventually gives rise to the axial skeleton. The dorsal part remains
epithelial and forms a sheet-like structure harboring myogenic and
dermogenic progenitor cells; this structure is called dermomyotome
(Brent and Tabin, 2002; Christ and Ordahl, 1995). From the
dermomyotome, cells translocate into a third compartment, the
1
Institute of Anatomy II, Department of Vertebrate Embryology, University of
2
Cologne, Joseph-Stelzmann-Straße 9, Cologne 50931, Germany. Institute of
Anatomy and Cell Biology, Department of Molecular Embryology, University of
3
Freiburg, Albertstraße 17, Freiburg 79104, Germany. Faculty of Biology, University
4
of Freiburg, Schaenzlestraße 1, Freiburg 79104, Germany. Institute of Molecular
and Cellular Anatomy, University of Ulm, Albert-Einstein-Allee 11, Ulm 89081,
Germany.
*Author for correspondence ([email protected])
Received 29 July 2014; Accepted 11 December 2014
myotome, to form embryonic muscle fibers (Gros et al., 2004). All
these events in somite maturation are known to depend on molecular
signals from the neighboring embryonic structures.
In chick and mouse embryos, it has been shown that signals from
the dorsal surface ectoderm regulate the formation of epithelial
somites and the patterning of the dermomyotome (Correia and
Conlon, 2000; Fan and Tessier-Lavigne, 1994). Intriguingly,
dermomyotomal development requires contact-dependent molecular
signals from the overlying ectoderm, notably Wnt6, despite the fact
that somites and ectoderm are physically separated by the somitic
basal lamina and the subectodermal space containing extracellular
matrix proteins (Fan and Tessier-Lavigne, 1994; Geetha-Loganathan
et al., 2006; Rifes and Thorsteinsdottir, 2012; Schmidt et al., 2004).
The mechanism of transfer of paracrine signaling molecules such as
Wnts between ectoderm and somites remains unknown. Here, we
show that, in chicken embryos, the subectodermal gap is bridged by a
special kind of cytoplasmic extensions formed by the epithelial
cells of the dermomyotome. These filopodia-like protrusions are
actin- and tubulin-based, contain cytoskeletal regulatory proteins,
such as fascin and cofilin, and require Rac1 for their formation.
Immunohistochemistry reveals the presence of microtubule motors,
including kinesin and dynein. Characterizing their functions, we
found that they contain glycophosphatidylinositol-anchored proteins
(GPI-APs) and retrogradely transport the Wnt-receptor Frizzled-7
(FZ7). Therefore, we propose a role of dermomyotomal filopodia-like
protrusions in long-range molecular signaling during somite
development.
RESULTS AND DISCUSSION
Dermomyotomal cells possess filopodia-like protrusions
Electroporation of a construct encoding a membrane-bound form
of green fluorescent protein (mGFP) into the dorsal part of the
newly formed epithelial somites revealed cellular extensions in the
subectodermal space formed by the dermomyotomal cells, which
we named filopodia-like protrusions (FiLiPs) (Fig. 1A,B). In
contrast to specialized filopodia in chicken limb buds (Sanders
et al., 2013), FiLiPs can be preserved by paraformaldehyde (PFA)
and can be further subjected to regular histological procedures.
Three-dimensional (3D) reconstruction of z-stack confocal images
of cultured transverse slices of the trunk region of Hamburger–
Hamilton stage 16 (HH16) embryos stained with CellMask orange
plasma membrane stain revealed that an average of 82% (s.e.m.±
2.3, n=8, 3D reconstructions) of the extensions are in stable contact
with the overlying surface ectoderm (Fig. 1C,D). Time-lapse
experiments showed no change in their length for at least 10 min
(Fig. 1E; see supplementary material Movie 1). Therefore, we
categorized these protrusions as ‘stable FiLiPs’. A subset of
extensions is not in stable contact with the ectoderm. These
extensions have a highly dynamic nature, extending in all
directions. We categorized these as ‘dynamic FiLiPs’. We
calculated the average extending and retracting velocities of the
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ABSTRACT
Development (2015) 142, 665-671 doi:10.1242/dev.115964
Fig. 1. Detailed characterization of dermomyotomal filopodia-like protrusions (FiLiPs). (A) Electroporation scheme. Left, the first four newly formed somites
of HH16 embryos were electroporated. Top right, DNA was injected into the somitocoel. Bottom right, schematic showing electroporated dermomyotomal cells
(green) after re-incubation. (B) Top left, low magnification image showing a fixed transverse section of somite VIII of a HH18 embryo electroporated with a
membrane-bound form of GFP (mGFP) at HH16. Nuclei (blue) were stained with DAPI. Bottom left, higher magnification image of the same section. Right, higher
magnification image of the same section merged with DAPI (blue) and differential interference contrast (DIC). White dashed lines mark the surface ectoderm.
(C) Percentage of stable and dynamic protrusions at a given time point (n=8, HH17, interlimb-level somites). (D) Representative 3D reconstruction of z-stack
images of a live embryo slice culture (transverse section) stained with CellMask orange plasma membrane stain. White arrowhead represents a stable FiLiP, red
arrowhead represents a dynamic FiLiP. Asterisk marks the surface ectoderm. Colors represent depth coding (see H). (E) Time-lapse images of an mGFPelectroporated dermomyotomal cell exhibiting stable FiLiPs (HH18, somite VII). White dashed lines indicate overlying surface ectoderm. (F) Time-lapse images
from a slice culture experiment show the dynamic nature of a representative FiLiP (HH18, somite VII). (G) Top, representative kymograph characterizing the
dynamics of the dermomyotomal FiLiP shown in F. Bottom, The instantaneous extending velocities of the same FiLiP. Velocity was calculated using kymographic
analysis. (H) Left, movement of a representative FiLiP (HH17, somite VI) in z-direction. Right, scale showing depth coding in z-direction. (I) Extension and retraction
speed of dynamic FiLiPs (n=10 each, HH17-18, interlimb-level somites). (J) Thickness distribution of FiLiPs (n=50; HH17, somites VI-VIII). Thickness range: 200500 nm (red box). (K) Frequency distribution showing the number of protrusions formed by the dermomyotomal cells (n=50; HH17, interlimb-level somites).
Number of protrusions formed by dermomyotomal cells: 2-11 (red box). Scale bars: 50 µm in B (top left); 10 µm in B (bottom left and right); 5 µm in D,H; 2 µm in E,F.
dynamic FiLiPs using kymographic images and found them to
be 53.76 nm/s (s.e.m.±1.73, n=10 protrusions) and 68.80 nm/s
(s.e.m.±1.54, n=10 protrusions), respectively (Fig. 1F-I; see
supplementary material Movie 2). Both stable and dynamic
FiLiPs showed considerable variability in their thickness,
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varying between 200 and 500 nm (n=50 protrusions; Fig. 1J).
Each dermomyotomal cell was found to form 2-11 protrusions
(mean=3.8 protrusions per cell, s.e.m.±1.0, n=50 cells; Fig. 1K)
and the overall length of dermomyotomal FiLiPs was limited by
the overlying surface ectoderm (≤20 µm).
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Molecular characterization of dermomyotomal FiLiPs
Filopodial structures have been shown to contain cross-linked actin
filaments (Small and Celis, 1978). Accordingly, phalloidin staining
revealed the presence of actin in the dense network of FiLiPs
within the subectodermal space (Fig. 2A,A′ and supplementary
material Movie 3). In order to further characterize the molecular
composition of dermomyotomal FiLiPs, we performed somite
electroporation experiments with constructs containing fascin,
cofilin and α-tubulin fused with GFP. In accordance with other
filopodia-like structures described for various cell types (Bernstein
and Bamburg, 2010; Kureishy et al., 2002), FiLiPs contain
fascin and cofilin (Fig. 2B-C′). FiLiPs can be visualized by
overexpression of the cytoplasmic form of GFP in dermomyotomal
cells (Fig. 2D,D′), which is in line with the observation of filopodia
in somite cells by Linker et al. (2005). Surprisingly, dermomyotomal
FiLiPs were also positive for tubulin, distinguishing them from
previously described protrusions, such as cytonemes and specialized
filopodia of limb bud mesenchymal cells (Fig. 2E,E′) (RamirezWeber and Kornberg, 1999; Sanders et al., 2013). Time-lapse
imaging of dermomyotomal cells co-electroporated with tubulinGFP and the red fluorescent tag pmKate2, as well as
immunohistochemistry against α-tubulin, revealed that the entire
population of FiLiPs is microtubule-based (Fig. 2F,G and
supplementary material Movie 4). Moreover, dermomyotomal
FiLiPs were also found to contain kinesins and dyneins, which are
motor proteins moving along the microtubule tracks to carry cellular
cargo. This indicates the potential role of dermomyotomal FiLiPs in
vesicular transport (Fig. 2H,I).
Rac1 inactivation leads to complete inhibition of FiLiPs
Rho family GTPases are known to have profound effects on
cytoskeleton architecture and cell migration (Burridge and
Wennerberg, 2004). It has been shown that Rac1, a member of
the Rho family of GTPases, regulates the formation of long cellular
protrusions via actin organization and microtubule stability (Bosco
et al., 2009). We co-electroporated a dominant-negative form of
Rac1 (dnRac1-IRES-GFP) and pCAG-DsRed into somites I-IV of
HH16 embryos to analyze the effect of Rac1 inactivation on the
formation of FiLiPs. Intriguingly, dermomyotomal cells expressing
dnRac1 exhibited complete loss of FiLiPs (Fig. 2J,J′). A complete
loss of cellular protrusions was also observed in cultured
dermomyotomal cells electroporated with dnRac1 (Fig. 2K,K′).
Together, this demonstrates that Rac1 is required for FiLiPs
formation.
Dermomyotomal FiLiPs exist at different somite levels and
embryonic stages, and surrounding tissues are not required
for their formation
As the dermomyotome is a structure of mature somites, we tested
whether FiLiPs are also exhibited by earlier stages, notably by presomitic mesodermal cells and by newly formed epithelial somites.
Actin staining of sagittal sections of HH16 embryos confirmed the
presence of FiLiPs in the subectodermal space overlying the cranial
presomitic mesoderm (PSM) and the epithelial somites (Fig. 3A-C).
However, the number of FiLiPs formed by the PSM cells was
considerably lower compared with that visible in epithelial somites
and in the dermomyotome (Fig. 3D). The earliest developmental stage
studied was HH10, with FiLiPs already present in the subectodermal
space of somite I, whereas the latest stage studied was HH22, with
FiLiPs in the dermomyotome of somite stage VIII just prior to EMT
(Fig. 3E,F). To investigate whether ectoderm is required for the
formation of dermomyotomal FiLiPs, we surgically removed the
Development (2015) 142, 665-671 doi:10.1242/dev.115964
surface ectoderm overlying the electroporated somites and incubated
the operated embryos for 3 h before time-lapse imaging. Despite the
absence of overlying ectoderm, dermomyotomal cells were found to
form FiLiPs (Fig. 3G,H and supplementary material Movie 5).
However, such protrusions were longer and lost their orientation
towards the ectoderm. We also dissociated and cultured the
electroporated dermomyotomal cells in vitro, and observed
protrusions emanating from the cultured dermomyotomal cells
without any evident contact with other cell types (Fig. 3G, inset).
These results suggest that the ability to form FiLiPs is an intrinsic
property of dermomyotomal cells.
Dermomyotomal FiLiPs show retrograde membrane
transport and contain GPI-APs
We further hypothesized a role of FiLiPs in signal transduction
through membrane trafficking. Our hypothesis was supported by the
observation of ‘membrane puncta’ along the FiLiPs of the mGFPelectroporated dermomyotomal cells (Fig. 1E). Live-imaging of
transverse slices of mGFP electroporated somites revealed a retrograde
transport of these membrane dots along the FiLiPs (Fig. 4A,B and
supplementary material Movie 6). Using kymographic analysis, the
average retrograde transport velocity of such puncta was found to be
56.25 nm/s (s.e.m.±2.78, n=5 puncta) (Fig. 4C). We also tested
whether FiLiPs contain glycophosphatidylinositol-anchored proteins
(GPI-APs). GPI anchors facilitate the attachment of proteins to the
outer leaflet of the plasma membrane, and GPI-APs have been
shown to be organized in special microdomains rich in cholesterol and
sphingolipids, called lipid rafts (Mayor and Riezman, 2004; Varma
and Mayor, 1998). Various studies have implicated the role of
lipid rafts in signal transduction (Simons and Toomre, 2000). To
determine the presence of GPI-APs in dermomyotomal FiLiPs, we
electroporated GFP-GPI into the somites and analyzed their
localization in the protrusions. Our results showed the presence of
GPI-APs puncta along the actin filaments in the dermomyotomal
FiLiPs (Fig. 4D). To exclude the possibility of artificial results
concerning the subcellular localization of overexpressed proteins, the
mitochondria-specific aconitase fused with GFP (mAcn-GFP) was
electroporated into the somites of HH17-18 embryos at a high
concentration (7 µg/µl). According to its mitochondrial destination,
mAcn-GFP should be solely present in the mitochondrial
compartment and should be distinct from the pattern obtained with
any of the other overexpressed fusion proteins, thus serving as control.
Our results show specifically labeled mitochondria in the
dermomyotomal cells and no labeling in the FiLiPs, thereby
confirming the reliability of the experimental approach used to
characterize the dermomyotomal FiLiPs (Fig. 4F,G).
Wnt receptor Frizzled-7 is retrogradely transported along
dermomyotomal FiLiPs
As FiLiPs connect two different cell types, ectodermal and somitic
epithelia, we investigated their potential functional role in mediating
signaling events during dermomyotome differentiation. The role of
Wnt signaling during the various stages of somite patterning and
differentiation has been well studied. It has been shown that three
G protein-coupled transmembrane receptors for Wnt signaling,
namely Frizzled-1, Frizzled-2 and Frizzled-7, are expressed in
epithelial somites and in the dermomyotome, whereas Wnt6, which
regulates dermomyotomal development, is expressed by the
overlying surface ectoderm (Geetha-Loganathan et al., 2006;
Linker et al., 2003; Schmidt et al., 2004). As Wnts are lipidmodified proteins, which makes them hydrophobic and barely
diffusible (Nusse, 2003), we tested whether dermomyotomal FiLiPs
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Development (2015) 142, 665-671 doi:10.1242/dev.115964
Fig. 2. Molecular machinery required for the formation and dynamics of dermomyotomal FiLiPs. (A) Phalloidin-labeled F-actin (red) in FiLiPs
(transverse section, HH16, somite IV). (B-E) Dermomyotomal cells electroporated with (B) fascin-GFP (HH18, somite VI), (C) cofilin-EGFP (HH17, somite
VII), (D) cytoplasmic GFP (HH18, somite VIII) and (E) tubulin-GFP (HH18, somite VII) reveal dermomyotomal FiLiPs spanning the subectodermal space.
(A′-E′) Corresponding images merged with DAPI (blue) and DIC. (F) Representative time-lapse images of a dermomyotomal cell (HH18, somite VIII)
electroporated with tubulin-GFP and pmKate2 showing that all FiLiPs are microtubule-based. (G-I) Antibody staining against α-tubulin, dynein and kinesin
on frozen sections (HH17, interlimb-level somites). Dermomyotomal FiLiPs contain microtubules and the motor proteins dynein and kinesin. Nuclei are
stained with DAPI (blue). Phalloidin staining labels F-actin (green) in G. (J) A representative transverse section of somite VII of a HH18 embryo
co-electroporated with a dominant-negative form of Rac1 (dnRac1-IRES-GFP) and pCAG-DsRed. Merged image (dnRac1, green; and DsRed, red)
showing the inhibition of FiLiPs. (J′) Image merged with DAPI (blue) to visualize the ectoderm. (K) A representative image of a cell taken from the primary
culture of dermomyotomal cells electroporated with dnRac1-IRES-GFP. Cells were stained with phalloidin (red) and DAPI (blue). No protrusions are
visible. (K′) pCAG-DsRed-electroporated cells served as control. Green represents F-actin and blue represents nuclei in the composite images. Scale
bars: 10 µm; 5 µm in F.
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Development (2015) 142, 665-671 doi:10.1242/dev.115964
Fig. 3. Presence of dermomyotomal FiLiPs at different somite levels and embryonic stages and their dependence on surrounding tissues.
(A) High-magnification image of a sagittal section representing cranial presomitic mesoderm (PSM) of a HH14-stage embryo stained with phalloidin (green) and
DAPI (blue). (B,C) Representative images of the dorsal part of somite I (S I) and the dermomyotome of somite V (S V), respectively, from the same sagittal section
stained for F-actin (green) and nuclei (blue). (D) Quantification of mean fluorescence intensity (MFI) in the subectodermal space overlying PSM, S I and S V
(n=6). The average MFIs were found to be 6.8 for cranial PSM, 30.6 for S I and 37.4 for S V. (E) FiLiP contacting the ectoderm in somite I of a HH10 embryo (white
arrowhead) (F) A representative image of the dermomyotome of somite VIII of a HH22 embryo shows that dermomyotomal cells at this stage also exhibit
protrusions. F-actin is labeled by phalloidin (green). (G) Image of a pmKate2-electroporated dermomyotomal cell without overlying ectoderm (HH17, somite VII).
Inset, primary culture of PBX-pmKate2-electroporated, dissociated dermomyotomal cells (HH17, somites VI-IX) reveals the presence of protrusions in vitro
without any evident contact with other cell types. (H) Control dermomyotomal cell with intact overlying ectoderm indicated by white dashed line (HH17, somite VII).
Scale bars: 10 µm; 5 µm in G,H.
CONCLUSIONS
Here, we describe a novel type of filopodia-like cellular
protrusions in epithelial cells, which we called FiLiPs, and
provide evidence that these protrusions play a role in long-range
signaling events during somite development. Intriguingly, they are
formed from the basal part of the epithelial somites, thus
penetrating the basement membrane of the somitic epithelium,
and are not only actin-based structures but also contain
microtubules. Partly dynamic and partly stable, they bridge the
subectodermal gap and connect the somites to the ectoderm. We
provide evidence that these filopodia-like protrusions are involved
in ectodermal-dermomyotomal signaling, as the Wnt receptor
Frizzled-7 is retrogradely transported in punctate form along the
FiLiPs. In addition, the presence of GFP-anchored proteins and
retrograde transport of mGFP-labeled membrane puncta along
FiLiPs suggest their general role in signal transportation. Taken
together, our results add new support to the recent concept that
intercellular signaling through cytoplasmic projections is an
important principle during embryonic development.
MATERIALS AND METHODS
Expression constructs and in ovo electroporation
Constructs used for electroporation (both previously published and designed
by us) are listed in the supplementary material methods. Embryos at HH16
were electroporated in modified form as described previously (Scaal et al.,
2004). Fertilized chicken eggs (Gallus gallus domesticus, White Leghorn)
were incubated at 38°C and staged. Embryos at HH16 were electroporated as
described previously (Scaal et al., 2004). Eggs were fenestrated and the
vitelline membrane over somites I-IV was carefully removed. DNA
constructs (final concentration 5-7 µg/µl) were mixed with Fast Green
(Sigma-Aldrich, 1% final concentration) and injected into the somitocoel of
somites I-IV. To achieve mosaic and distinct single-cell pattern of the
electroporated cells wherever required, we reduced the concentration of
expression constructs to 0.5-1 µg/µl. For further details, see supplementary
material methods.
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could act as ‘antennae’ reaching out to the ectodermal cells to fetch
the Wnt ligand directly from the Wnt-secreting ectoderm. In order to
observe the localization of Frizzled receptors along these
protrusions, we electroporated an expression construct encoding
Frizzled-7 receptor fused to GFP (FZ7-GFP) into the
dermomyotomal cells. Confocal imaging of the electroporated
sections revealed the presence of Frizzled-7 along the FiLiPs in
punctate form, suggesting their potential role in mediating Wnt
signaling events (Fig. 4E). To study the dynamics of Frizzled-7
puncta along FiLiPs, we performed time-lapse experiments on the
transverse slices of FZ7-GFP-electroporated dermomyotomes of
HH17 embryos. We observed a net retrograde transport of Frizzled7 puncta along the dermomyotomal FiLiPs (Fig. 4H,I and
supplementary material Movie 7). Kymographic analysis revealed
that the instantaneous velocities of the Frizzled-7 particles are
relatively constant, varying between 55 and 75 nm/s (Fig. 4J).
Average retrograde transport velocity was found to be 63 nm/s
(s.e.m.±1.8, n=5 puncta) (Fig. 4K). Further analysis revealed that an
average of 96.7% (s.e.m.±3.3, n=20 protrusions) of these puncta
was transported along the stable FiLiPs (Fig. 4L), whereas the
dynamic FiLiPs did not seem to be involved in receptor trafficking.
An average of 28.5% (s.e.m.±4.11, n=20 protrusions) of the stable
FiLiPs were found to transport Frizzled-7 along them (Fig. 4M).
These results suggest a role for dermomyotomal FiLiPs in
transducing Wnt signaling events during somite differentiation.
Development (2015) 142, 665-671 doi:10.1242/dev.115964
Fig. 4. Dermomyotomal FiLiPs contain GPI-anchored proteins and transport Wnt receptor Frizzled-7. (A) Time-lapse images of a representative FiLiP
visible through the mGFP electroporation of a HH17 embryo showing the retrograde transport of a membrane punctum (white arrowheads). (B) Top,
representative kymograph showing the mGFP punctum transport along the dermomyotomal FiLiP shown in A. Bottom, instantaneous retrograde transport
velocities of the same punctum calculated using kymographic analysis. (C) The average retrograde transport velocity of mGFP puncta (n=5). (D) Left, a GFP-GPIelectroporated dermomyotome (somite VII, HH17) reveals that GPI-APs are present in punctate form along the FiLiPs (arrowheads), actin filaments labeled using
phalloidin (red). Right, higher magnification image showing GPI-AP (green, white arrowheads) distribution along the FiLiPs containing actin filaments (red,
phalloidin staining) in a representative protrusion. (E) A deconvoluted image from a transverse section of somite VI of HH18 embryo electroporated with FZ7-GFP
shows the presence of Frizzled-7 (white arrowheads) along a representative FiLiP stained for actin (red). (F) Electroporation of a construct expressing
mitochondria-specific aconitase (mAcn) fused with GFP specifically labels mitochondria (HH18, somite VII). (G) Composite image of mAcn (green) and DAPI
(blue) with DIC. (H) Time-lapse images of a FZ7-GFP-electroporated dermomyotome (HH17, somite VIII) showing retrograde transport of two Frizzled-7 puncta
(white and red arrowheads) along a representative FiLiP. White line marks a stable FiLiP with no Frizzled-7 punctum. (I) Top, a representative kymograph
showing the transport of Frizzled-7-GFP punctum along the dermomyotomal FiLiP shown in H (white arrowhead). Bottom, graph quantifying the instantaneous
retrograde transport velocities of the same Frizzled7-GFP punctum calculated using kymographic analysis. (J) Instantaneous retrograde velocities of Frizzled-7
puncta from five different embryos (HH17-18, interlimb-level somites). Velocities were calculated using kymographs. (K) Average retrograde transport velocity of
Frizzled-7-GFP puncta (n=5). (L) Graph quantifying the distribution of Frizzled-7 puncta on stable and dynamic FiLiPs. 96.67% of the Frizzled-7 puncta were
seen on stable FiLiPs (n=20). (M) The percentage of stable FiLiPs positive for Frizzled-7 puncta. 28.5% of the stable FiLiPs transport Frizzled-7 along them
(n=20). Scale bars: 10 µm in D (left), E-G; 2 µm in A,H; 1 µm in D (right).
Immunohistochemistry
To label actin filaments in the protrusions, the free-floating sections were
incubated with Alexa Fluor 488/Rhodamine Phalloidin (Life Technologies)
diluted to a final concentration of 0.2 µM in PBS containing 1% BSA for 1 h.
Immunohistochemistry was performed on 20 µm-thick cryosections. The
sections were washed three times in PBS containing 0.1% Triton X-100
(PBT) and incubated overnight at 4°C with desired primary antibodies (final
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concentration 5 µg/ml), which were diluted in 0.1% PBT solution
containing 10% serum. The sections were rinsed, washed frequently for
1 h in 0.1% PBT and incubated with the respective biotinylated secondary
antibodies (1:500, 10% serum/0.1% PBT) overnight at 4°C. The sections
were again washed frequently for 1 h in 0.1% PBT, incubated with Cy3conjugated streptavidin (1:500, 0.1% PBT) overnight at 4°C and mounted
with DAPI Fluoromount-G (Southern Biotechnology) after frequent
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washing for 1 h in 0.1% PBT. Further details and a list of antibodies are
provided in the supplementary material methods.
Embryo slice preparation for live imaging
Successfully electroporated embryos were immediately embedded in 2%
low-melting agarose (Sigma-Aldrich) and excised in blocks, which were
mounted on vibratome wells for cutting. Vibratome wells were surrounded
by ice and filled with ice-cold PBS. 200 µm-thick trunk-level transverse
sections were collected in ice-cold PBS, placed on 35 mm-high glassbottom dishes (Ibidi, Germany) that were coated with 0.1% poly-L-lysine
(Sigma-Aldrich) and containing slice culture media (neurobasal medium,
B27 supplement and penicillin-streptomycin). The sections were processed
for time-lapse confocal imaging as described below. All media reagents
were purchased from Life Technologies.
Confocal microscopy, image processing and statistical analysis
Glass-bottom dishes containing the electroporated chick embryo slices were
imaged in real-time using a Carl Zeiss LSM 510 laser-scanning confocal
microscope. Images were processed using Imaris software (Bitplane), and
time-lapse movies were created using ImageJ (NIH). Fixed sections were
imaged using a Leica TCS SP8 confocal laser-scanning microscope. Images
were acquired, processed and analyzed using Leica application suite –
advance fluorescence (LAS-AF) software. Fig. 4E was deconvoluted using
the Huygens professional deconvolution software package (Scientific
Volume Imaging). Statistical analyses were performed using Prism
software (GraphPad). Error bars represent s.e.m. For further details, see
supplementary material methods.
Acknowledgements
We thank Wilfred Denetclaw (SFSU, CA, USA) and Charles Ordahl (UCSF, CA,
USA) for valuable advice concerning the experimental approaches, Sebastian
Schildge for critical reading of the manuscript, and Ute Baur, Ellen Gimbel, Lidia
Koschny and Valentina Safronjuk for technical assistance.
Competing interests
The authors declare no competing financial interests.
Author contributions
M.S. and S. designed the experiments; S., F.P. and C.W. performed the
experiments; S. analyzed the data, and S, F.P. and M.S. wrote the manuscript.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft [SFB592 and
GRK1104 to M.S.] and the Imhoff-Stiftung (to M.S.).
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.115964/-/DC1
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