© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4279-4284 doi:10.1242/dev.112045
RESEARCH REPORT
A non-cell-autonomous role for Ras signaling in C. elegans
neuroblast delamination
Jean M. Parry1,2 and Meera V. Sundaram1, *
Receptor tyrosine kinase (RTK) signaling through Ras influences
many aspects of normal cell behavior, including epithelial-tomesenchymal transition, and aberrant signaling promotes both
tumorigenesis and metastasis. Although many such effects are cellautonomous, here we show a non-cell-autonomous role for RTK-Ras
signaling in the delamination of a neuroblast from an epithelial organ.
The C. elegans renal-like excretory organ is initially composed of
three unicellular epithelial tubes, namely the canal, duct and G1 pore
cells; however, the G1 cell later delaminates from the excretory
system to become a neuroblast and is replaced by the G2 cell. G1
delamination and G2 intercalation involve cytoskeletal remodeling,
interconversion of autocellular and intercellular junctions and
migration over a luminal extracellular matrix, followed by G1
junction loss. LET-23/EGFR and SOS-1, an exchange factor for
Ras, are required for G1 junction loss but not for initial cytoskeletal or
junction remodeling. Surprisingly, expression of activated LET-60/
Ras in the neighboring duct cell, but not in the G1 or G2 cells, is
sufficient to rescue sos-1 delamination defects, revealing that Ras
acts non-cell-autonomously to permit G1 delamination. We suggest
that, similarly, oncogenic mutations in cells within a tumor might help
create a microenvironment that is permissive for other cells to detach
and ultimately metastasize.
KEY WORDS: Caenorhabditis elegans, EMT, Delamination, Epithelia,
Junction, Ras
INTRODUCTION
Whereas some epithelial cells maintain their identities throughout
their lifetime, others will undergo a transition to form another cell
type. This is a common occurrence during normal developmental
programs such as epithelial-to-mesenchymal transition (EMT) or
neuroblast delamination and in disease states such as cancer cell
metastasis (Acloque et al., 2009; Lim and Thiery, 2012). When
epithelial cells transition to form another cell type, they require
many cell-autonomous changes including altered gene expression,
gaining motility, remodeling junctions, and detaching from or
invading through the extracellular matrix (ECM). Furthermore,
neighboring cells that remain within the epithelium must loosen
their connection to the departing cell and reseal the tissue after its
departure. Most studies of epithelial transitions have focused on the
cell-autonomous changes, while less is known about the role of
neighboring epithelial cells in facilitating such transitions.
1
Department of Genetics, University of Pennsylvania Perelman School of Medicine,
2
415 Curie Boulevard, Philadelphia, PA 19104, USA. Department of Biology,
Georgian Court University, 900 Lakewood Avenue, Lakewood, NJ 08701, USA.
*Author for correspondence ([email protected])
Received 29 April 2014; Accepted 5 September 2014
Many signaling pathways, including receptor tyrosine kinase
(RTK) signaling through Ras, promote EMT, neuronal delamination
and/or cancer cell metastasis (Yang and Weinberg, 2008). One clear
role for RTK-Ras signaling during cell transitions in vivo is as a cellautonomous trigger for transcription factors such as Snail and Twist
that repress epithelial identity (Lim and Thiery, 2012). On the other
hand, work in cell culture has shown that RTK signaling can alter the
adhesive dynamics of cells in various other ways (Janda et al., 2006;
Lu et al., 2003; Orlichenko et al., 2009; Palacios et al., 2001, 2002).
Here we show that RTK-Ras signaling in a cell that remains in the
epithelium is crucial to allow delamination of a neighboring cell.
RESULTS AND DISCUSSION
Background
The C. elegans excretory system is composed of three unicellular
epithelial tubes, namely the canal, duct and pore cells, which
connect in tandem to provide a conduit for fluid waste excretion
(Buechner, 2002; Nelson et al., 1983; Nelson and Riddle, 1984)
(Fig. 1A). In the first (L1) larval stage, the initial pore cell, named
G1, delaminates from the excretory system and divides to form a
pair of neurons (Stone et al., 2009; Sulston and Horvitz, 1977;
Sulston et al., 1983) (Fig. 1B,C). Simultaneous with its departure,
the G1 cell is replaced as excretory pore by the neighboring G2
epidermal cell. As G1 departs the excretory system, it must remodel
its junctions to the duct cell and to G2 and the epidermis, as well as
eliminate an autocellular junction (AJ) that maintains it in the shape
of a tube. Correspondingly, the duct cell must remodel its
intercellular junction (IJ) to detach from G1 and connect to the
entering G2 cell.
G1 and G2 migrate over a luminal matrix during delamination
and intercalation
To visualize G1 delamination, we marked the duct and G1 cell
bodies with dct-5pro::mCherry and apical junctions with AJM-1::
GFP or HMR-1::GFP (Fig. 1; supplementary material Fig. S1). We
imaged carefully staged L1 larvae hourly at 25°C (see Materials and
Methods) to construct a timeline of events (Fig. 1B,C). Beginning
4 h after hatch, the G1 cell began to extend cytoplasm dorsally and
then migrated in an anterior/dorsal direction as its AJ gradually
shortened. Concomitantly, the G2 cell invaded at the base of the G1
cell and began to wrap into the shape of a tube and form a
lengthening AJ. At 7 h post hatch, the G1 cell retained only a
remnant of junction at its base, where it contacted the duct and now
fully incorporated G2 pore. Around this time, G1 divided in the
right/left plane to produce daughter cells G1.l and G1.r (Sulston and
Horvitz, 1977) (supplementary material Fig. S2).
As G2 invaded the excretory system, we noted a thickening of the
AJ at the moving G1-G2 cell boundary (Fig. 1B,C). Archival
transmission electron microscope (TEM) images reveal that this
thickening corresponds to adjacent junctions at a short region of
overlap where the G1 and G2 cells transiently share the pore lumen
4279
DEVELOPMENT
ABSTRACT
RESEARCH REPORT
Development (2014) 141, 4279-4284 doi:10.1242/dev.112045
G1 pore
A
canal
duct
AJ
WT
cuticle
Hatch
4 hours
5 hours
6 hours
iii
ii
7 hours
v
iv
Cell Body, Junctions
B i
duct
G1
G2
G1
G2
C i
ii
duct
iii
duct
iv
duct
duct
G1
G2
G2
D
F”
IJ
G2
G2
G2
F’
AJ
AJ
C
duct
G1
G1
G1
G1
G1
v
IJ
IJ
C
C
IJ
AJ
AJ
Fig. 1. G1 delaminates and G2 intercalates over a luminal matrix. (A) Schematic of C. elegans wild-type excretory system at hatch. (B) Excretory system at the
indicated times post hatch at 25°C. Duct and G1 pore cell are marked by dct-5pro::mCherry (red, outlined), whereas junctions are marked by AJM-1::GFP
(green, shown alone as inverted grayscale in bottom row). Arrows indicate G1-G2 overlap. (C) Idealized schematic interpretations of images above in B. Duct cell,
yellow; G1, blue; G2, pink; heavy black lines indicate junctions. (D) Archival TEM from L1 larva at stage corresponding to ∼4-5 h post hatch. Three consecutive
slices show the G2 cell wrapping around the ventral base of the pore lumen and forming an AJ, while the G1 cell unwraps its AJ. C, cuticle; AJ, autocellular
junction; IJ, intercellular junction. Scale bars: 5 µm in B; 1 µm in D.
Re-organization of the actin cytoskeleton precedes G1
delamination
Epithelial cells in transition often gain motility by stimulating Factin protrusions (Ridley, 2011). We visualized actin dynamics in
the G1 and duct cells using a GFP-tagged VAB-10 (spectraplakin)
actin-binding domain (ABD) (Liegeois et al., 2007) under control of
the dct-5 promoter (Fig. 2). From hatching to just before 3 h post
hatching, actin was very strongly enriched along the G1 AJ and, to a
lesser degree, along the duct lumen and at the IJ between the duct
and canal cell. However, at 3 h post hatch, the actin relocalized away
from the AJ to become generally distributed throughout the
4280
cytoplasm; this occurred ∼1 h before G1 began to migrate. As
delamination completed, actin accumulated at a narrow, anteriorly
protruding tip of the G1 cell.
sos-1 is required for G1 delamination
RTK signaling promotes many epithelial transitions. In C. elegans,
the RTK LET-23/EGFR signals through a well-described pathway
that includes the guanine nucleotide exchange factor (GEF) SOS-1
and LET-60/Ras (Sundaram, 2013). During embryogenesis, LIN-3/
EGF is expressed in the excretory canal cell and stimulates the
EGFR-Ras pathway in the duct cell to specify the duct versus G1
cell fate (Abdus-Saboor et al., 2011; Yochem et al., 1997). A
temperature sensitive (ts) allele of sos-1 that is specifically defective
in the Ras GEF domain (Rocheleau et al., 2002) revealed a
continued requirement for signaling during the L1 stage in order to
maintain excretory system integrity (Abdus-Saboor et al., 2011).
These observations suggested a possible role for Ras signaling in
the process of G1 delamination.
We tested a requirement for SOS-1 by shifting sos-1(ts) animals
to non-permissive temperature (25°C) directly after hatching and
then examining the dynamics of G1 departure (Fig. 3). In upshifted
animals, cell and junction morphology and cytoskeletal
organization initially appeared normal. Actin relocalized away
from the G1 AJ, G2 began to intercalate and G1 began to extend
cytoplasm and migrate dorsally with approximately normal timing
DEVELOPMENT
as G2 intercalates dorsally (Fig. 1D). Thus, the departing G1 cell
appears to unzip, first converting its AJ to IJ before losing junctions
entirely, while the entering G2 cell zips up beneath it, with a short
region of two-celled tube at their intersection.
The excretory duct and pore cell lumen is lined with a thick
collagenous cuticle that is contiguous with the epidermal cuticle and
will be shed during molting, ∼6 h after G1 departs the excretory
system (Mancuso et al., 2012; Nelson et al., 1983). The cuticular
lining of the duct/pore channel remains clearly visible by TEM
during delamination and intercalation (Fig. 1D), suggesting that the
G1 and G2 cells migrate over this lumenal cuticle as they change
positions, although we cannot exclude the possibility of some new
matrix synthesis.
RESEARCH REPORT
Development (2014) 141, 4279-4284 doi:10.1242/dev.112045
Hatch
3 hours
ii
Bi
duct
5 hours
iii
ii
duct
7 hours
iv
iii
duct
cell body
F-Actin
Ai
iv
duct
G1
G1
G1
G2
G1
G
G1
G2
G2
G2
Fig. 2. Cytoskeletal reorganization precedes G1 delamination. (A) Actin dynamics in the excretory system at the indicated times post hatch. F-actin is marked
by dct-5pro::VAB-10(ABD)::GFP (green, inverted grayscale in bottom row); duct and G1 pore cell bodies are marked by dct-5pro::mRFP (red), and the duct cell
body is also marked with lin-48pro::mRFP. Pore cell body is outlined. Arrows indicate region with intense actin accumulation. (B) Idealized schematic
interpretations of images above in A, with actin in gray. Scale bar: 5 µm.
LET-23 and SOS-1 act upstream of Ras in the duct cell to nonautonomously permit G1 delamination
lin-3/EGF and let-23/EGFR reporters continue to be expressed in
the canal cell and duct/pore, respectively, during remodeling,
consistent with SOS-1 acting in the canonical EGF-Ras-ERK
pathway to control G1 delamination (Abdus-Saboor et al., 2011)
(supplementary material Fig. S4). To confirm the involvement of
LET-23, we examined hypomorphic let-23(sy97) mutants in which
initial duct versus pore cell fate specification was normal. let-23
(sy97) mutants had a G1 delamination defect similar to that of sos-1
(ts) mutants (Fig. 4A,B). To confirm that SOS-1 acts through Ras
and to determine where it carries out its function, we used a
transgenic approach to express a constitutively active version of
LET-60/Ras (G13E) in a tissue-specific manner. The lin-48
promoter drives expression in the duct, rectum and neuronal
support cells (Abdus-Saboor et al., 2011; Johnson et al., 2001),
whereas the dpy-7 promoter drives expression in G1, G2 and the
epidermis (Johnstone and Barry, 1996; Stone et al., 2009)
(supplementary material Fig. S4). Using these tissue-specific
promoters, we found that expression of LET-60 (G13E) in the
duct and G1 cells, or in the duct cell only, but not in the G1 cell only,
significantly rescued the junction-retention defects in the G1 cell
(Fig. 4C,D). Consistent with these data, removal of let-60 from G1
or G2, but not the duct, is compatible with normal excretory
function and animal viability (Yochem et al., 1997). Thus, SOS-1
acts through Ras, and Ras signaling in the duct cell acts non-cellautonomously to permit G1 delamination.
Notably, LET-60 (G13E) did not significantly affect the timing
or execution of G1 delamination in a wild-type background
(Fig. 4C), suggesting that Ras acts permissively, rather than
instructively, to allow G1 delamination. Furthermore, G1
delamination still initiated in earlier upshifted sos-1(ts) animals
that had a two G1, no duct phenotype (Fig. 4E); thus, the duct cell is
not required to stimulate G1 delamination. Previous work showed
that G1 delamination also occurs in the absence of G2 (AbdusSaboor et al., 2011). Together, these data suggest that a G1-intrinsic
clock or a signal from somewhere other than the duct or G2
stimulates G1 delamination at the appropriate time, and that Ras
signaling in the duct permits successful execution of the
delamination program.
How does Ras signaling in the duct facilitate G1 remodeling?
Several (non-mutually exclusive) models could explain the non-cellautonomous effects of Ras signaling. First, Ras signaling in the duct
could directly alter junction components on the duct side of the ductG1 connection, thereby allowing freer G1 mobility and remodeling.
Extensive work in cell culture has shown that Ras signaling can
stimulate cadherin endocytosis (Janda et al., 2006; Lu et al., 2003;
Orlichenko et al., 2009; Palacios et al., 2001, 2002), although the
in vivo significance of this role is unclear. Second, Ras signaling in
the duct could act indirectly by stimulating the production of another
signaling ligand. Much earlier in development, Ras signaling in the
duct appears to trigger an unknown, non-Notch-mediated lateral
inhibitory signal that prevents G1 from also adopting a duct fate
(Abdus-Saboor et al., 2011; Sulston et al., 1983; Yochem et al.,
1997); potentially, continued signaling could facilitate later G1
behaviors such as junction remodeling. Third, Ras signaling might
alter components of the luminal matrix that the duct shares with G1,
facilitating detachment of G1 from that matrix. Ras signaling in
mammalian cells can upregulate the expression of various matrix
metalloproteinases (Sanchez-Laorden et al., 2014), and we showed
previously that mutations that disrupt apical matrix organization
lead to premature separation of the duct and pore cells (Mancuso
et al., 2012). Further studies of mutants with G1 delamination
4281
DEVELOPMENT
(Fig. 3A-C). However, G1 retained most of its junction (Fig. 3D;
supplementary material Fig. S3) and remained inserted in the
excretory system dorsal and adjacent to the entering G2 cell
(Fig. 3A,B). The duct lumen began to dilate, suggesting that the
pore channel might have closed. G1 rarely formed a protruding tip,
and actin remained partly associated with the remaining G1 junction
and accumulated at the sites of duct lumen dilation (Fig. 3C).
Despite its failure to delaminate, G1 still divided with
approximately normal timing, with the G1.r cell maintaining a
junction (supplementary material Fig. S2). Thus, sos-1 is required
for G1 junction loss and detachment from the excretory system, but
is not required for many other G1 behaviors, such as initial actin and
junction reorganization, gain of motility and cell cycle re-entry.
RESEARCH REPORT
Development (2014) 141, 4279-4284 doi:10.1242/dev.112045
sos-1(cs41)
4 hours
5 hours
6 hours
ii
7 hours
iii
iv
Cell Body, Junctions
^
^
A i
^
B i
ii
duct
iii
duct
G2
Hatch
G1
G2
3 hours
G2
5 hours
ii
7 hours
iii
iv
Cell Body, F-actin
C i
^
G1
G1
G2
duct
^
^
G1
iv
duct
^
D
G1 Junction Length
n.s.
**
**
**
**
µm
µm
n.s.
**
G2 Junction Length
Wildtype
sos-1(cs41)
Wildtype
urs
7 ho
6 ho
urs
urs
5 ho
4 ho
urs
urs
7 ho
urs
6 ho
urs
5 ho
4 ho
urs
*
sos-1(cs41)
defects should provide insights into the mechanisms by which Ras
signaling non-autonomously promotes this epithelial transition.
Implications for the role of EGFR and Ras in cancer cell
metastasis
Activating mutations in EGFR and Ras are among the most
common changes in epithelial-derived cancers (Pylayeva-Gupta
et al., 2011). The lethal effects of such cancers are predominantly
due to tumor cell metastasis (Nguyen and Massagué, 2007).
Although cell-autonomous effects of EGFR-Ras signaling clearly
4282
contribute to tumorigenesis and metastasis, the results presented
here emphasize the possibility that mutant cells that remain in the
original tumor could also help create a microenvironment that is
permissive for other cells to detach and ultimately metastasize.
MATERIALS AND METHODS
Strains and transgenes
See supplementary material Tables S1-S3 for strains, transgenes and
plasmids used. All strains were grown at 25°C under standard conditions
(Brenner, 1974) unless specifically noted otherwise.
DEVELOPMENT
Fig. 3. sos-1 is required for G1 delamination but not for cytoskeletal reorganization or gain of motility. (A) Delamination fails in sos-1(cs41ts) mutants.
Mutants were upshifted to non-permissive temperature at hatch. Cells are marked as in Fig. 1. Carets indicate duct lumen dilations. (B) Idealized schematics
as in Fig. 1. (C) Actin dynamics in sos-1(cs41ts) mutants. F-actin and cells marked as in Fig. 2. (D) Quantification of AJ length in G1 and G2. n≥15 for each
timepoint except N2 at 4 h (n=12). n.s., not significant; *P<0.01, **P<0.001 by Wilcoxon test. Scale bars: 5 µm.
RESEARCH REPORT
Development (2014) 141, 4279-4284 doi:10.1242/dev.112045
G1 Junction Area
*
*
A
D
B
E i
i
sos-1(cs41)
sos-1(cs41)
^
)
let-23(sy97)
G1’
sy
97
G1
le
s-
t-2
1(
ii
pore::Ras(GF)
3(
cs
41
)
Ty
pe
so
W
ild
ii
G2
Junctions
duct::Ras(GF)
cell body, Junctions
cell body, Junctions
µm²
G1’
G1
G1 Junction Area
C
**
*
n.s.
n.s.
**
n.s.
n.s.
*
µm²
**
sos-1:
Ras (GF):
WT
-
cs41
-
cs41 cs41 cs41
- duct
duct
+ pore
cs41 cs41
- duct
cs41 cs41
- duct
cs41 cs41 cs41 cs41
- duct
- pore
cs41 cs41
- pore
WT WT
- duct
Embryos were hand-picked at 1.5-fold stage, incubated at 15°C until hatch,
and then incubated at 25°C to reach the designated stage. Staging was
confirmed based on the morphology of seam hypodermal cells, which are
actively dividing and migrating during L1 (Austin and Kenyon, 1994;
Podbilewicz and White, 1994; Sulston and Horvitz, 1977).
and dct-5p::mCherry as two separate populations; then, the intersection of
these populations was calculated.
All box and whisker plots were generated using Graphpad Prism software
(Hyndman and Fan, 1996). The boxes represent the interquartile range
(25-75%) with a line at median, and the whiskers extend from 10%-25% and
from 75%-90%, with outliers as individual points.
Microscopy
Immunostaining
Confocal microscopy utilized a Leica TCS CP. Images were processed using
ImageJ (Schneider et al., 2012). TEM data correspond to animal L1Q
(available at wormimage.org).
Embryos in supplementary materials Fig. S4A were freeze-cracked and
fixed in methanol as described previously (Duerr et al., 1999) and
incubated with primary antibodies [goat polyclonal anti-GFP (Rockland,
600-101-215; 1:50); rabbit polyclonal anti-DLG-1 (1:50 to 1:100;
Segbert et al., 2004)] overnight at 4°C and with secondary antibodies
(Jackson ImmunoResearch Laboratories, 705-095-003 and 111-295-003;
1:50 to 1:200) for 2 hours at room temperature.
Staging of larvae
Measurements of junction length
At least 15 animals per stage were analyzed for each genotype (except N2
4 h, n=12).
For manual measurements (Fig. 3), confocal z-stacks were processed as
average projections. ImageJ was used to draw a line from the base of the AJ
to the base of the G1 cell body (G2 AJ length), or from the base of the G1
cell body to the top of the AJ (G1 AJ length). G1 junction length did not
change from hatch to 4 h.
Volocity software (PerkinElmer) was used to quantify the area of overlap
between the G1 cell body and AJ (supplementary material Fig. S3; Fig. 4). A
single raw section from a confocal z-stack corresponding to approximately
the center of the excretory system was imported to Volocity as an image
sequence and a region of interest (ROI) was drawn around the G1 cell body.
The Find Objects protocol was used to automatically quantify AJM-1::GFP
Acknowledgements
We thank David Hall and the Center for C. elegans Anatomy (funded by NIH
OD010943) for providing TEM images of L1Q, which was prepared in the Brenner
lab at the MRC/LMB (Cambridge, UK), and forwarded by John White and
Jonathan Hodgkin for long-term curation; Michel Labouesse (Illkirch, France) for
VAB-10::ABD; Ishmail Abdus-Saboor for LET-23::GFP expression data; Hasreet Gill
and Michelle Kanther for technical assistance; and David Raizen, John Murray,
Amanda Zacharias and members of the M.V.S. lab for discussion and comments on
the manuscript. Some strains were obtained from the Caenorhabditis Genetics
Center (University of Minnesota), which is funded by NIH Office of Research
Infrastructure Programs (P40 OD010440).
4283
DEVELOPMENT
Fig. 4. SOS-1 acts downstream of LET-23/EGFR and upstream of LET-60/Ras in the duct cell to permit G1 delamination. (A) Volocity quantification of
overlap between G1 cell body and AJ in wild type, sos-1(cs41ts) and let-23(sy97) mutants. (B) Excretory system at 7 h post hatch in let-23(sy97). Cells are
marked as in Fig. 1; bracket indicates G1 cell body/AJ overlap. (C) Volocity quantification as in A. Multiple independent lines were scored for each transgene, and
non-transgenic siblings were scored as controls. n=15 each. n.s., not significant; *P<0.01, **P<0.001 by Wilcoxon test. (D) Excretory system at 7 h post hatch in
sos-1(cs41) mutants with indicated transgenes. Cells are marked as in Fig. 1. duct::Ras(GF) refers to lin-48pro::LET-60(G13E). pore::Ras(GF) refers to
dpy-7pro::LET-60(G13E). (E) Two examples of sos-1(cs41ts) animals with no duct and two G1-like cells at 3 h post hatch. Both cells are beginning to unzip the AJ
(arrows), and G2 is beginning to intercalate ventrally. Caret indicates lumen dilation. Scale bars: 5 µm.
RESEARCH REPORT
Competing interests
The authors declare no competing financial interests.
Author contributions
J.M.P. and M.V.S. jointly conceived the approach and prepared the manuscript.
J.M.P. performed the experiments and data analysis.
Funding
This work was supported by National Institutes of Health grants [GM58540 to
M.V.S.; T32 HD007516 and F32 DK093204 to J.M.P.]. Deposited in PMC for release
after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.112045/-/DC1
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