RESEARCH ARTICLE 4571
Development 139, 4571-4581 (2012) doi:10.1242/dev.083253
© 2012. Published by The Company of Biologists Ltd
Shroom3 is required downstream of FGF signalling to
mediate proneuromast assembly in zebrafish
Sandra Ernst1,2, Kun Liu1,3, Sobhika Agarwala1,2, Nicola Moratscheck1,2, Mehmet Ender Avci1,2,
Damian Dalle Nogare4, Ajay B. Chitnis4, Olaf Ronneberger1,3 and Virginie Lecaudey1,2,*
SUMMARY
During development, morphogenetic processes require a precise coordination of cell differentiation, cell shape changes and, often,
cell migration. Yet, how pattern information is used to orchestrate these different processes is still unclear. During lateral line (LL)
morphogenesis, a group of cells simultaneously migrate and assemble radially organized cell clusters, termed rosettes, that prefigure
LL sensory organs. This process is controlled by Fibroblast growth factor (FGF) signalling, which induces cell fate changes, cell
migration and cell shape changes. However, the exact molecular mechanisms induced by FGF activation that mediate these changes
on a cellular level are not known. Here, we focus on the mechanisms by which FGFs control apical constriction and rosette assembly.
We show that apical constriction in the LL primordium requires the activity of non-muscle myosin. We demonstrate further that
shroom3, a well-known regulator of non-muscle myosin activity, is expressed in the LL primordium and that its expression requires
FGF signalling. Using gain- and loss-of-function experiments, we demonstrate that Shroom3 is the main organizer of cell shape
changes during rosette assembly, probably by coordinating Rho kinase recruitment and non-muscle myosin activation. In order to
quantify morphogenesis in the LL primordium in an unbiased manner, we developed a unique trainable ‘rosette detector’. We thus
propose a model in which Shroom3 drives rosette assembly in the LL downstream of FGF in a Rho kinase- and non-muscle myosindependent manner. In conclusion, we uncovered the first mechanistic link between patterning and morphogenesis during LL sensory
organ formation.
INTRODUCTION
During morphogenetic processes in developing organisms, cells
must simultaneously proliferate, change their shape, differentiate
and, often, migrate. These biological processes must be tightly
orchestrated in time and space in order for the cells to form an
organ. Developmental patterning information, often in the form of
morphogens, plays a major role in coordinating these different
processes both within single cells and between neighbouring cells.
Important efforts have been made in recent decades to understand
the link between positional information and cell fate decisions.
However, how signals and genes that control cell fate also affect
the cytoskeletal and adhesion machinery that eventually drive
morphogenesis is still poorly understood, in particular in
vertebrates.
The zebrafish lateral line (LL) is a very good model system in
which to study how cell migration, cell shape changes and
differentiation are coordinated during organ formation. The LL
is a mechanosensory system that allows the detection of water
movements. It comprises mechanosensory organs, the
neuromasts, which are distributed over the body surface both on
the head (anterior LL) and along the trunk and tail (posterior LL
or pLL). Neuromasts are clusters of ~25 cells, including hair
1
Centre for Biological Signalling Studies (BIOSS), University of Freiburg,
Schänzlestrasse 18, D-79104 Freiburg, Germany. 2Department of Developmental
Biology, Faculty of Biology, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg,
Germany. 3Image Analysis Lab, Institute for Computer Science, University of
Freiburg, D-79110 Freiburg, Germany. 4Program in Genomics of Differentiation, The
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland, USA.
*Author for correspondence ([email protected])
Accepted 11 September 2012
cells in the centre surrounded by support cells (DamblyChaudière et al., 2003; Ghysen and Dambly-Chaudière, 2004;
Metcalfe et al., 1985; Winklbauer, 1989). During pLL
development, a group of ~100 cells, the primordium of the pLL
(pLLP), delaminates from a cranial placode posterior to the otic
placode and migrates posteriorly along the myoseptum to the tip
of the tail. As cells of the pLLP migrate collectively, they form
radially organized subgroups, termed rosettes, which are then
deposited repeatedly at the trailing end of the pLLP, where they
fully differentiate into mechanosensory organs (Chitnis et al.,
2012; Ghysen and Dambly-Chaudière, 2004; Ghysen and
Dambly-Chaudière, 2007). The assembly of these two to three
radially organized rosettes within the primordium is based on
coordinated apical constriction (AC) of the cells in the cluster
(Lecaudey et al., 2008), a process that requires Fibroblast growth
factor (FGF) signalling activity (Lecaudey et al., 2008;
Nechiporuk and Raible, 2008). FGF signalling is active in
approximately the back two-thirds of the pLLP where the FGF
receptor 1 (Fgfr1) is present. Fgfr1 is activated by two FGF
ligands, Fgf3 and Fgf10. Blocking FGF signalling
pharmacologically or genetically leads to a loss of apically
constricted rosettes and aberrant migration (Lecaudey et al.,
2008; Nechiporuk and Raible, 2008). The migration defect is the
consequence, at least in part, of downregulation of the G proteincoupled receptor-encoding gene cxcr7b in the absence of FGF
signalling (Aman and Piotrowski, 2008; Nechiporuk and Raible,
2008). Conversely, the morphogenetic defect has been proposed
to result from the failure of cells in the trailing part to become
epithelial in the absence of FGF activity (Lecaudey et al., 2008).
However, the precise molecular mechanisms induced
downstream of activated Fgfr1 that mediate apical constriction
and proneuromast formation are not known.
DEVELOPMENT
KEY WORDS: Lateral line, Apical constriction, Rosette, Shroom3, Rho kinase, Collective cell migration, Morphogenesis, Zebrafish
Development 139 (24)
4572 RESEARCH ARTICLE
MATERIALS AND METHODS
Zebrafish lines and maintenance
Zebrafish (Danio rerio) were raised and staged according to standard
protocols (Kimmel et al., 1995). The Tg(−8.0cldnb:lynEGFP)zf106
(cldnb:gfp), Tg(hsp70l:dnfgfr1-EGFP)pd1 and Tg(hsp70l:fgf3-Myc)zf115
transgenic lines have been described previously (Haas and Gilmour, 2006;
Lecaudey et al., 2008; Lee et al., 2005).
Cloning of cDNAs, transgenic constructs and transgenic lines
Full-length Shroom3 was amplified from cDNA of 36 hours postfertilization (hpf) zebrafish embryos and two splice variants, v1 and v2,
were identified by 5⬘RACE (Roche). The sequences for shroom3v1 and
shroom3v2 have been deposited in GenBank with accession numbers
JX455752 and JX455753, respectively. All constructs were generated using
the Tol2kit (Kwan et al., 2007) and Multisite Gateway (Invitrogen). gfpmyh9 (Blaser et al., 2006) was PCR-amplified and cloned into pCS2.
Capped mRNAs were transcribed with the SP6 mMessage mMachine Kit
(Ambion). See supplementary material Table S1 for primer sequences.
Whole-mount in situ hybridization and immunohistochemistry
In situ hybridization (ISH) and immunofluorescence (IF) staining were
performed according to standard procedures (Lecaudey et al., 2004). The
following antibodies were used: mouse anti--tubulin (1/100; Sigma),
mouse anti-ZO1 (1/500; Invitrogen), rabbit anti-PMLC2 (1/50; Cell
Signaling), rabbit anti-GFP (1/500; Torrey Pines Biolabs) and mouse antiGFP (1/500; JL8, Clontech) and secondary Alexa dye-conjugated
antibodies (1/500; Molecular Probes). Phalloidin stainings were performed
with Rhodamine-Phalloidin (1/500; Molecular Probes).
Morpholino and rescue experiment
Mo_Shr3e6i6 sequence was 5⬘-CCTAATAAATTGTTACCTGACTAAC3⬘ (Gene Tools). 4.2 pmol of morpholino were injected in the cell at the
one-cell stage. Because morpholinos with a GC content <40% might work
better at lower temperatures (Schuster et al., 2010), we kept Mo_shr3e6i6injected embryos at a constant temperature of 25°C. For the rescue
experiment, Mo_Shr3e6i6 was injected into hsp70l:shr3v1-tagRFP and
hsp70l:shr3v2-tagRFP embryos. At 26 hpf, embryos were heat-shocked
for 30 minutes at 39°C and fixed after 4 hours.
Heat-shock and inhibitor treatment
Tg(hsp70l:dnfgfr1-EGFP)pd1, Tg(hsp70l:fgf3-Myc)zf115, hsp70l:shr3v1tagRFP and hsp70l:shr3v2-tagRFP embryos (28 hpf) were heat-shocked
for between 15 minutes and 1 hour at 39°C. For inhibitor treatment, 28-hpf
embryos were treated with 5 or 10 M of SU5402 for 4-6 hours
(Calbiochem), 50 M of blebbistatin for 2-5 hours (Sigma), or 50 to 200
M of Rockout for 30 minutes (Calbiochem).
For the experiment shown in Fig. 8, hsp70l:shr3v1-tagRFP and
hsp70l:shr3v2-tagRFP embryos were stimulated 3 to 4 hours after the start
of the heat-shock (HS) with 5 M SU5402 for 4 hours before overnight
fixation.
Imaging, image processing and statistical analysis
Live embryos were anesthetized in 0.01% MESAB (Sigma) and mounted
in 1% low melting point agarose. Time-lapse movies were performed on
an inverted Zeiss Spinning Disc or LSM510 confocal microscopes. z-stacks
were captured at 3- to 5-m intervals and flattened by maximum
projection. Image processing and kymographs were performed with ImageJ
1.42Q software. Data from at least two experiments were pooled. Statistical
analyses were performed using Student’s two-tailed t-test for unpaired
values where appropriate. A P-value ≤0.05 was considered statistically
significant.
Rosette number quantification using a trainable detector
The quantification of rosette numbers was based on a trainable detector.
The core algorithm was adapted from the state-of-the-art detection
methods in Computer Vision and Biomedical Image Analysis fields (Liu
et al., 2012; Leibe et al., 2008). Similar approaches have been
successfully applied to locate landmark structures like eyes/mouth in
zebrafish embryos (Ronneberger et al., 2012) and to detect mitoses
(Schlachter et al., 2010). In short, the detector uses densely computed
scale-invariant feature transform (SIFT) descriptors (Lowe, 2004) and
an efficient filtering technique (Reisert and Burkhardt, 2008) for image
patch description. The detection is carried out in a sliding-window
fashion with a linear classifier.
First, the detector was trained with manually labelled data comprising
30 non- or DMSO-treated embryos and 17 embryos treated with SU5402.
The performance of the detector was then validated by splitting the labelled
data into a training set and a test set. Each set contained ~23 images, and
the cross-validation was repeated five times with different random splitting.
This led to a 91.7% Precision/Recall at the Equal-Error-Rate point,
meaning that, with this threshold, a rosette had more than nine chances out
of ten to be detected and a detected object had more than nine chances out
of ten to be a true rosette. After training, the detector could find objects
similar to the ones that were manually labelled in the training data in any
test image, and give these objects a detection score indicating the extent of
the similarity. Using this detector, the rosette number could be counted
automatically and, thus, in an objective manner. For more detailed
explanations, we refer the readers to our supporting online material at
http://www.bioss.uni-freiburg.de/cms/assets/files/professuren/organogenesis/
rosette_detection.pdf. The trained detector (in Matlab) is available at
http://lmb.informatik.uni-freiburg.de/people/liu/rosette-detection. The full
Matlab package including the detector training algorithm is also available
upon request.
DEVELOPMENT
AC refers to a change in cell shape, whereby epithelial cells
constrict their apical side in a coordinated manner to adopt a
wedge-like shape. AC thus underlies cell and tissue shape changes
in many morphogenetic processes during development, including
gastrulation, neural tube closure, lens pit invagination and gut
morphogenesis (Pilot and Lecuit, 2005; Sawyer et al., 2010). In all
cases, apical constriction requires the activity of non-muscle
myosin (NMII), which contracts the apical meshwork of actin
leading to a decrease in apical surface (Lecuit and Lenne, 2007;
Sawyer et al., 2010). NMII comprises two heavy chains, two
essential light chains and two regulatory light chains.
Phosphorylation of the regulatory light chains by Myosin light
chain kinase or Rho-associated kinase (Rock) is the main switch
that activates NMII (Vicente-Manzanares et al., 2009). In
vertebrates, the actin-binding protein Shroom3 has been reported
to be involved in the regulation of apical constriction (Chung et al.,
2010; Haigo et al., 2003; Hildebrand, 2005; Hildebrand and
Soriano, 1999; Lee et al., 2007; Nishimura and Takeichi, 2008;
Plageman et al., 2011; Plageman et al., 2010). Shroom3 belongs to
the Shroom family of proteins, which are characterized by
conserved domains including a PDZ domain at their N-terminus,
and two Apx/Shrm-specific domains, ASD1 and ASD2, separated
by a poly-proline domain (Dietz et al., 2006; Hagens et al., 2006;
Haigo et al., 2003; Hildebrand, 2005; Hildebrand and Soriano,
1999; Lee et al., 2009). The ASD1 domain mediates direct binding
to actin but is not essential to the apical constriction activity of
Shroom3. By contrast, the ASD2 domain is essential for AC
activity and has been shown to interact physically with and to
localize Rocks (Nishimura and Takeichi, 2008), thus directly
linking Shroom3 to NMII activation and AC.
In this study, we dissected the mechanisms downstream of Fgfr1
activation that lead to proneuromast formation in the pLLP. We first
show that the actin-binding protein Shroom3 localizes in the apical
part of constricting cells. We then show that Shroom3 is essential
for the apical constriction-based assembly of proneuromasts and
that this process also requires NMII and Rho kinase activity.
Finally, we demonstrate that Shroom3 is a transcriptional target of
FGF signalling and that it mediates the activity of FGF in
proneuromast assembly.
Shroom3 in pLL morphogenesis
RESEARCH ARTICLE 4573
RESULTS
Non-muscle myosin localizes and is active in the
apical part of rosettes, and is required for their
assembly
To form proneuromasts, groups of cells in the migrating pLLP
coordinately constrict their apical side to form radially organized
clusters or rosettes. This process is controlled by FGF signalling in
the trailing cells that express fgfr1 (Lecaudey et al., 2008;
Nechiporuk and Raible, 2008). In epithelial cells, apical constriction
is generally driven by activated NMII, which contracts the apical
actin meshwork (Sawyer et al., 2010). In order to determine whether
apical constriction in the pLLP requires NMII activity, we first
determined its localization in the primordium. For this purpose, we
injected mRNAs encoding the NMII heavy chain myh9 fused to gfp
together with lynTdTomato to visualize the cell membranes. GFPmyh9 localized at the very centre of the rosettes in the pLLP (Fig.
1A-D). The density of fusion protein increased from the most
recently formed rosette (Fig. 1C-C⬙) to the ‘oldest’ one (Fig. 1B-B⬙).
GFP-myh9 was also already detectable in the apical part of cells in
the leading region, which had not yet fully constricted their apical
membrane (Fig. 1D-D⬙). This suggests that the localization of NMII
in the apical part of pLLP cells is a very early event that prefigures
AC and rosette assembly. Using a phospho-specific antibody, we
next examined levels of phosphorylated myosin light chain 2
(pMLC2), which reflect local actomyosin activation (Amano et al.,
1996). pMLC2 signal was significantly stronger in the centre of each
rosette (Fig. 1E-G). This showed that NMII is not only localized but
also active in the centre of assembled and assembling rosettes. To
determine whether NMII was required for AC during proneuromast
assembly, we used the drug blebbistatin to block its activity (Kovács
et al., 2004). Treatment of 28 hpf embryos, in which one
proneuromast had already been deposited, with 50 M blebbistatin
led to disassembly of rosettes (Fig. 1H,I). Together, these data show
that non-muscle myosin activity is required for cells to constrict their
apical side and form radial rosettes in the trailing part of the pLLP.
Shroom3 is expressed in the rosette-forming
region of the pLLP and localizes at the apical part
of rosette cells
The actin-binding protein Shroom3 has been shown to drive AC of
epithelial cells in different biological systems by activating non-
muscle myosin (Chung et al., 2010; Haigo et al., 2003; Hildebrand,
2005; Hildebrand and Soriano, 1999; Lee et al., 2009; Nishimura
and Takeichi, 2008; Plageman et al., 2011; Plageman et al., 2010).
We therefore decided to determine whether Shroom3 plays a role
in the assembly of apically constricted rosettes in the zebrafish
pLLP. We first cloned full-length zebrafish shroom3 by RT-PCR
and 5⬘RACE. We isolated two splice variants, shr3v1 and shr3v2,
which differ in their 5⬘ end and are likely to give rise to two
different proteins, as in other vertebrates. The shorter cDNA,
shr3v2, lacks the first two exons and, as a consequence, the Nterminal PDZ domain (Fig. 2A,B). Zebrafish shr3v1 and shr3v2 are
thus orthologues of the mammalian Shroom3 isoform1 (ShroomL)
and Shroom3 isoform2 (ShroomS), respectively (Hagens et al.,
2006; Hildebrand and Soriano, 1999). Sequence analysis of the
putative proteins indicates that they contain the two Shroomspecific domains, ASD1 and ASD2, as well as a poly-proline
domain in between (Fig. 2A,B).
In order to determine whether Shroom3 could play a role in the
assembly of apically constricted proneuromasts, we analysed its
expression by in situ hybridization (ISH) between 20 and 48 hpf.
We found that shroom3 was expressed at a very high level in the
pLLP before the start of migration (not shown). Although the
expression level decreased with time, it continued during the entire
migration process (Fig. 2C,G,K). Interestingly, co-staining with a
GFP antibody in the cldnb:gfp transgenic line showed that shroom3
is neither expressed in the leading cells nor in the most trailing cells
but rather in the region where rosette assemble (Fig. 2D,H,L).
shroom3 is also expressed in additional regions of the embryo,
including the ventral part of the otic vesicle (Fig. 2E,I,M) and the
distal part of the pronephros (Fig. 2F,J,N). Using RT-PCR with
primers specific for each of the two splice variants, we showed that
shroom3 is not expressed maternally (not shown). These results
make Shroom3 a very good candidate to mediate cell shape
changes underlying rosette assembly.
We therefore wanted to determine the intracellular localization
of Shroom3 protein. For this purpose, we generated transgenic
lines expressing shr3v1 or shr3v2 fused to tagRFP under the
control of the heat shock-inducible promoter hsp70l. Upon heatshock (HS), shr3v1-tagRFP and shr3v2-tagRFP localized to the
very centre of already assembled or assembling rosettes in the
pLLP, showing a punctuate localization in the most apical part
DEVELOPMENT
Fig. 1. Non-muscle myosin is required for apical
constriction-driven proneuromast assembly. (A-D⬙) Live
images of the primordium of a 32 hpf zebrafish embryo coexpressing gfp-myh9 fusion (green) and lynTdTomato (red)
mRNAs. B-D⬙ are close-up views of the three boxed areas in
A. (E-G⬘) Side view of the primordium of a 32 hpf cldnb:gfp
embryo immunostained with Phospho-Myosin Light Chain
2 (ser19) (PMLC2) and GFP antibodies. F-G⬘ are close-up
views of the two boxed areas in G. (H,I) Live images of the
primordia of 32 hpf embryos treated with DMSO (H) or
with 50 M blebbistatin. Scale bars: 20 m in A; 10 m in
B-D⬙.
4574 RESEARCH ARTICLE
Development 139 (24)
of the cells (Fig. 2O-R). This localization was reminiscent of that
of GFP-myh9 (Fig. 1A).
Shroom3 is required for the assembly of apically
constricted rosettes in the migrating pLLP
To investigate the role of Shroom3 in LL morphogenesis, we used
a splice-blocking morpholino targeting the exon6-intron6 boundary
to simultaneously knock down both splice variants (Mo_shr3e6i6)
(Fig. 3A). RT-PCR analysis of control and morpholino-injected
embryos showed that Mo_shr3e6i6 led to an efficient retention of
intron 6 (‘mis-spliced’ in Fig. 3B). The band corresponding to the
wild-type mRNA had indeed almost completely disappeared at the
injected concentration at all three time points analysed (Fig. 3B).
The retention of intron 6 was confirmed by sequencing of the PCR
product. It leads to a premature STOP codon and, thus, to a putative
truncated protein lacking the poly-proline and ASD2 domain (Fig.
3A). This strongly suggests that Mo_shr3e6i6 leads to a dramatic
reduction of Shroom3 function.
Although the overall morphology of the embryos was
indistinguishable from injected controls (Fig. 3C), knockdown of
Shroom3 function significantly modified the internal organization
of migrating pLLP. The organization in two to three radial rosettes,
which characterize wild-type primordia, was lost in morphants
between 28 hpf and 36 hpf (Fig. 3D-G). This phenotype was
strongly reminiscent of pLLP lacking FGF activity (Lecaudey et
al., 2008; Nechiporuk and Raible, 2008). Similar to fgf3;fgf10
DEVELOPMENT
Fig. 2. shroom3 is expressed in the pLLP
and localizes at the apical part of pLLP
cells. (A) Schematic of Shroom3 showing
different conserved domains of the protein
and some of its main known interactors.
(B) Schematic of the exon-intron composition
of the two zebrafish shroom3 splice variants.
(C-N) Lateral views of embryos stained by ISH
with a shroom3 probe at 24 hpf (C-F), 36
hpf (G-J) and 48 hpf (K-N). shroom3 is
expressed in the central domain of the
migrating pLLP at all three stages (C-D⬘,GH⬘,K-L⬘). It is also expressed in the ventral
part of the otic vesicle (E,I,M) as well as in
the distal part of the pronephros (F,J,N).
(O-R⬙) Lateral views of the pLLP (O-Q⬙) or a
recently deposited neuromast (P-R⬙) in heatshocked hsp70l:shr3v1-tagRFP (O-P⬙) or
hsp70l:shr3v2-tagRFP (Q-R⬙) transgenic
embryos. P⬘,P” and R⬘,R” are close-up views
of the deposited neuromasts in P and R,
respectively. Scale bars: 200 m in C,G,K; 20
m in D-F,H-J,L-N,O-O⬙,Q-Q⬙; 10 m in P,R; 5
m in P⬘,P⬙,R⬘,R⬙.
Shroom3 in pLL morphogenesis
RESEARCH ARTICLE 4575
double mutants, the absence of radially organized rosettes became
less obvious after 36 hpf (data not shown). As a consequence, very
few neuromasts were deposited from the trailing region of such
primordia lacking apically constricted rosettes. Thus, the final
pattern of neuromasts was aberrant with hardly any organs
deposited along the first half of the migration path (supplementary
material Fig. S1, Movies 1, 2). Interestingly, the migration speed
of primordia lacking rosettes was not affected (supplementary
material Fig. S1, Movies 1-4), confirming the idea that the internal
organization of the primordium in rosettes is not a prerequisite for
coordinated migration (Aman and Piotrowski, 2008).
To characterize further the phenotype resulting from Shroom3
knockdown, we looked at the localization of molecular markers
showing a particular localization in the pLLP. The centrosome
pattern, for example, reflected the internal organization of the
primordium in rosettes. -Tubulin immunostaining indeed showed
that whereas the centrosomes appeared scattered in the leading
region, they formed organized rings around the rosette centres in
the trailing part of the primordium (Fig. 3H-I). This organization is
lost in shroom3 morphants, in which centrosomes fail to form rings
and are instead aligned along the pLLP length (Fig. 3J-K). Actin
and apical markers, such as the tight junction protein ZO-1, are
strongly concentrated at the centre of rosettes in wild-type
primordia (Hava et al., 2009; Lecaudey et al., 2008). In shroom3
morphants, by contrast, both actin and ZO1 staining were strongly
reduced at the centre of the rosettes, as shown by phalloidin
labelling and staining with a ZO1 antibody (supplementary
material Fig. S2; Fig. 3L-O). To quantify the number of rosettes
within the primordium in an unbiased manner, we developed a
trainable detector to automatically analyse pictures of cldnb:gfpexpressing primordia (see Materials and methods). The results are
displayed as detection response maps (Fig. 3D⬘-G⬘) and boxplot
diagrams (Fig. 4E,F, two left-hand boxplots). This quantification
showed that the difference in rosette numbers between injected
controls and Mo_shr3e6i6-injected embryos is highly significant.
In order to confirm the specificity of our morpholino knockdown
experiments, we injected Mo_shr3e6i6 in hsp70l:shr3v1-tagRFP
or hsp70l:shr3v2-tagRFP embryos and heat-shocked the embryos
at 26 hpf. Upon HS, both shr3v1-tagRFP and shr3v2-tagRFP were
able to rescue the loss of apically constricted rosettes (compare Fig.
4C,E with 4A,B). Importantly, rosettes failed to form in heatshocked, Mo_shr3e6i6-injected non-transgenic siblings (Fig. 4B),
showing that the HS does not block the effect of the morpholino.
We quantified the extent of the rescue using the rosette detector
described previously. Induction of shr3v1-tagRFP or shr3v2tagRFP in morphants significantly increased the number of rosettes
per primordium from 0.5 to almost 1.5 (Fig. 4E,F). Embryos
expressing shr3v1-tagRFP or shr3v2-tagRFP but non-injected with
the morpholino had a number of rosettes similar to wild type,
suggesting that shroom3 alone is not sufficient to induce AC and
formation of an ectopic rosette in the leading region of the pLLP
(Fig. 4E,F).
DEVELOPMENT
Fig. 3. Shroom3 knockdown blocks apical constriction and rosette assembly in the migrating pLLP. (A) Schematic showing the position of
Mo_Shr3e6i6. (B) RT-PCR with shr3- (top) and EF1-(bottom) specific primers on zebrafish embryos injected with Mo_Shr3e6i6 or with water (ctrl)
showing that Mo_Shr3e6i6 efficiently generates an alternative splice variant (mis-spliced) by blocking the excision of intron 6, potentially leading to
a truncated protein lacking the poly-proline and ASD2 domain (A). (C) transmitted light and fluorescence pictures of embryos injected either with
water (left) of with Mo_Shr3e6i6 (right). (D-G⬘) Live images of the primordium of cldnb:gfp embryos injected with water (D,D⬘) or with
Mo_Shr3e6i6 (E-G⬘) showing that knocking down Shroom3 function blocks the assembly of apically constricted rosettes (arrowheads in D). D⬘-G⬘
are the corresponding detection response images (using Jet, a blue-to-red colour map) obtained from our rosette detector (see Materials and
methods). (H-O) Side views of the primordium of 36 hpf cldnb:gfp embryos immunostained for the centrosomal marker -tubulin (H-K) or the tightjunction protein ZO1 (L-O). I, K, M and O are close-up views of the boxed areas in H, J, L⬙ and N⬙, as indicated. Scale bars: 50 m in D-G⬘; 10 m in
I,K,M,O.
4576 RESEARCH ARTICLE
Development 139 (24)
embryos with the Rock inhibitor Rockout (Provost et al., 2007).
Cldnb:gfp embryos were treated from 28 hpf onwards, once one
neuromast had been deposited, with increasing concentrations of
inhibitor for 30 minutes before image acquisition. Blocking Rock
activity led to the disassembly of radially organized rosettes in the
migrating pLLP in a dose-dependent manner (Fig. 5A-F).
Quantification of these experiments with our rosette detector
confirmed that the number of rosettes per primordium in embryos
treated with 100 and 200 M of Rockout was significantly reduced
compared with DMSO treated embryos (Fig. 5G). These results
show that Rock activity is required for the maintenance of apically
constricted proneuromasts in the migrating primordium. Together
with our previous findings, this strongly suggests that Shroom3
drives AC of the pLLP cells by activating non-muscle myosin in a
Rho kinase-dependent manner.
Altogether, this shows that Shroom3 function is required for the
assembly of pLLP epithelial cells in radially organized rosettes.
Based on the known activity of Shroom3 in other biological
systems, this strongly suggests that Shroom3 activates non-muscle
myosin activity to drive apical constriction and rosette assembly.
Rho kinase activity is required for apically
constricted rosette assembly
Activation of non-muscle myosin is regulated by phosphorylation
of either the heavy chain or the regulatory light chain (RMLC). We
have demonstrated that MLC2 is phosphorylated during rosette
assembly (Fig. 1E-G). Phosphorylation of the RMLC depends on
the activity of Myosin Light Chain Kinase (MLCK) and/or the
Rho-associated kinase (Rock) (Vicente-Manzanares et al., 2009).
As it has been shown previously that Shroom3 directly interacts
with Rock1/2 and that this interaction is necessary for AC of
neuroepithelial cells during neural tube closure (Nishimura and
Takeichi, 2008), we wanted to determine whether Rock activity
was also necessary for AC of the pLLP cells. We thus treated
Shroom3 function is required downstream of FGF
for rosette assembly
To prove that Shroom3 mediates FGF activity in assembling
apically constricted proneuromasts, we performed an epistasis
analysis. Overactivation of FGF signalling by HS treatment of
Tg(hsp70l:fgf3-Myc)zf115 embryos triggers the formation of an
ectopic radially organized cell cluster in the leading region of the
pLLP (Lecaudey et al., 2008). We have shown that shroom3
DEVELOPMENT
Fig. 4. Induction of shroom3 expression rescues rosette assembly
in Mo_Shr3e6i6-injected zebrafish embryos. (A-D⬙) Images of the
pLLP in heat-shocked non-transgenic siblings (A-B⬘), hsp70l:shr3v1tagRFP (C-C⬙) or hsp70l:shr3v2-tagRFP (D-D⬙) embryos injected with
water (A,A⬘) or Mo_Shr3e6i6 (B-D⬙). A⬘-D⬘ show the corresponding
detection response images. Scale bars: 50 m. (E,F) Boxplots displaying
the results and statistics of confidence-weighted rosette numbers from
the trained rosette detector (see Materials and methods). The red line
and black diamond represent the median and mean value of each
group, respectively. The edges of the box are the 25th and 75th
percentiles. The black dashed line extends to the most extreme data
points not considered to be outliers, and outliers are plotted individually
as red plus signs. The medians of two groups are significantly different
at the 5% significance level if their respective notch intervals do not
overlap (interval end points represent the extremes of the notches). N is
the number of independent experiments and n is the number of
embryos per condition.
FGF signalling is necessary and sufficient for
expression of shroom3 in the pLLP
The FGF signalling pathway is required for cells of the pLLP to
assemble into radially organized and apically constricted clusters
that prefigure the neuromasts (Lecaudey et al., 2008; Nechiporuk
and Raible, 2008). Our findings suggested that Shroom3 could act
downstream of FGF signalling to drive AC in the trailing cells of
the primordium. In order to test a potential link between FGF
signalling and Shroom3, we checked whether FGF activity
controlled shroom3 expression. We first analysed shroom3
expression in embryos treated with the FGFR inhibitor SU5402.
After treatment with 10 M SU5402, the expression of shroom3 in
the pLLP was completely abolished (compare Fig. 6C,D with
6A,B). By contrast, the expression of shroom3 in the otic vesicle
and in the pronephros was not significantly affected (not shown).
Similar results where obtained using the Tg(hsp70l:dnfgfr1EGFP)pd1 transgenic line (Fig. 6E-J). Thus, FGF signalling is
necessary for the expression of shroom3 in the pLLP. We then
tested whether, conversely, FGF signalling was sufficient to induce
shroom3 expression. Overactivation of FGF signalling using the
Tg(hsp70l:fgf3-Myc)zf115 line led to increased shroom3 expression
and an expansion of its expression domain to the leading region of
the primordium (Fig. 6K-N). In addition, shroom3 was now
expressed in the already deposited neuromasts in which it is
normally downregulated (Fig. 6M, arrowheads). The expression,
however, remained restricted to forming rosettes in the pLLP and
was not induced in any ectopic region, suggesting that other factors
restrict the competence to express shroom3 downstream of FGF.
Here again, the expression in the otic vesicle and pronephros was
not significantly affected (not shown). We quantified the expansion
of shroom3 expression by measuring the size of the ‘shroom3-free’
domain in the leading region (Fig. 6O). The ‘shroom3-free’ domain
was reduced by half in embryos overexpressing fgf3 compared with
wild-type siblings. In conclusion, FGF signalling is both necessary
and sufficient to activate shroom3 expression in the pLLP. This
strongly supports a role for Shroom3 in translating FGF activity
into AC and proneuromast formation.
Shroom3 in pLL morphogenesis
RESEARCH ARTICLE 4577
Fig. 5. Rho kinases are also required for the assembly of pLLP in apically constricted rosettes. (A-F) Live images of the pLLP of zebrafish
embryos treated with DMSO (A), or 50 M (B), 100 M (C,D) or 200 M (E,F) of the Rho kinase inhibitor Rockout. (G) Boxplot of the quantification
of the Rockout treatment experiments. Scale bars: 50 m.
that the HS does not block the effect of the morpholino. We
quantified the phenotype by measuring the distance between the tip
of the primordium and the first rosette centre (Fig. 7, white
arrowheads). This distance was significantly smaller in heatshocked, water-injected Tg(hsp70l:fgf3-Myc)zf115 embryos (dark
blue) compared with non-heat-shocked embryos (red). However,
the same distance in heat-shocked Mo_shr3e6i6-injected
Tg(hsp70l:fgf3-Myc)zf115 embryos (purple) was significantly
increased compared with water-injected embryos (blue and red).
The difference between heat-shocked and non-heat-shocked
Mo_shr3e6i6-injected embryos was not significant (compare
turquoise bars with purple bars) showing that the Mo_shr3e6i6 was
able to totally block the strong FGF overactivation induced in
Tg(hsp70l:fgf3-Myc)zf115 embryos. In conclusion, this experiment
demonstrates that Shroom3 is required downstream of FGF to
assemble apically constricted rosettes.
Fig. 6. FGF signalling is necessary and sufficient for activating shroom3 expression in the pLLP. (A-N⬘) Lateral views of zebrafish embryos
stained by ISH with a shroom3 probe. Treatment of embryos with 10 M of SU5402 (C-D⬘), or overexpression of dn-fgfr1 (H-J) totally inhibits
shroom3 expression in the pLLP (compare C-D⬘ with A-B⬘, and H-J with E-G). Overexpression of fgf3 after heat-shock of Tg(hsp70l:fgf3-Myc)zf115
embryos leads to an increase and expansion of shroom3 expression (compare M-N⬘ with K-L⬘). (O) Quantification of the length of the ‘shroom3free’ zone in the leading region in heat-shocked Tg(hsp70l:fgf3-Myc)zf115 (red) and non-transgenic siblings (blue). Statistical analyses were
performed using the t-test. Error bars represent s.e.m. Scale bars: 200 m in A,C,E,H,K,M; 20 m in B,B⬘,D,D⬘,F,G,I,J,L,L⬘,N,N⬘.
DEVELOPMENT
expression is ectopically induced in this additional proneuromast
(Fig. 6N). We therefore reasoned that, if Shroom3 is mediating
FGF activity, blocking Shroom3 activity in Tg(hsp70l:fgf3Myc)zf115 should revert this phenotype. As expected, whereas
control-injected Tg(hsp70l:fgf3-Myc)zf115 embryos had a normal
pattern of rosettes in the absence of HS (Fig. 7A), they assembled
an ectopic rosette in the leading region of the pLLP upon HS (Fig.
7B, white arrowhead). Formation of this ectopic rosette was
blocked in heat-shocked Tg(hsp70l:fgf3-Myc)zf115 injected with the
Mo_shr3e6i6 (Fig. 7C,D). This shows that Shroom3 is specifically
required for the assembly of the ectopic rosette in the leading
region following overactivation of FGF signalling. As a control,
non-heat-shocked Mo_shr3e6i6-injected Tg(hsp70l:fgf3-Myc)zf115
embryos, as expected, failed to assemble any rosette (Fig. 7E).
Importantly here again, rosettes also failed to form in heat-shocked,
Mo_shr3e6i6-injected non-transgenic siblings (Fig. 7F), showing
4578 RESEARCH ARTICLE
Development 139 (24)
Conversely, we wanted to test whether exogenously overexpressed
shroom3 is sufficient to block the loss of rosettes resulting from
inhibition of FGF activity. For this purpose, we first induced
shroom3
expression
using
hsp70l:shr3v1tagRFP
or
hsp70l:shr3v2tagRFP
transgenic
embryos
and
then
pharmacologically blocked FGF signalling using the FGF inhibitor
SU5402. As expected, non-transgenic siblings treated with 5 M
SU5402 lost their apically constricted rosettes (compare Fig. 8B,F
with 8A,E). By contrast, shr3v1 or shr3v2 overexpression in heatshocked transgenic embryos was sufficient to block the loss of
rosette formation following SU5402 treatment (Fig. 8D,H). In
addition to the loss of rosettes, we checked the efficiency of the
SU5402 treatment by ISH with the FGF target gene pea3. pea3
expression was very strongly reduced or totally absent upon SU5402
treatment and after HS (supplementary material Fig. S3). Together,
these experiments show that Shroom3 acts downstream of FGF
signalling to induce the assembly of apically constricted rosettes.
In conclusion, we have uncovered the mechanism by which FGF
signalling translates into the formation of apically constricted
rosettes in the pLLP. FGF signalling is necessary and sufficient to
induce the expression of shroom3. In turn, Shroom3 drives the
assembly of apically constricted rosettes. We show that AC in the
pLLP also requires the activity of non-muscle myosin and Rock.
This strongly suggests that Shroom3 recruits Rho kinases to the
apical side of the cells where they can activate non-muscle myosin
to constrict the apical actin meshwork (Fig. 9).
DISCUSSION
Shroom3 drives apical constriction and
proneuromast formation in the pLLP
Our results indicate that Shroom3 is required for the assembly of
apically constricted epithelial clusters that prefigure the sensory
organs in the zebrafish pLL downstream of the FGF signalling
pathway. Shroom3 has been shown to interact physically with
Rock1/2 via its ASD2 domain and to localize Rock1/2 to the apical
side of epithelial cells where it can phosphorylate and activate
NMII (Nishimura and Takeichi, 2008). Using pharmacological
inhibitors, we showed that blocking the activity of NMII or Rock
leads to a loss of apically constricted rosettes similar to what is
occurring in the absence of FGF or Shroom3 activity. It is therefore
reasonable to speculate that Shroom3 regulates AC in the pLLP in
a Rock- and NMII-dependent manner. We therefore propose a
model in which activation of Fgfr1 in the trailing part of the
primordium induces expression of shroom3. Shroom3, in turn,
would physically interact with, and localize, Rho kinases to the
apical part of the cells where they phosphorylate and activate nonmuscle myosin leading to AC of the cells and rosette formation
(Fig. 9).
How Shroom3 itself gets localized to the apical part of the cells
is still unclear. Shroom3 has been shown to bind actin directly via
its ASD1 domain and a number of studies suggest that this
interaction plays a role in Shroom3 apical localization (Dietz et al.,
2006; Hildebrand and Soriano, 1999). Furthermore, the localization
of dShrmA, the Drosophila Shroom variant that is most similar to
Shroom3, has also recently been shown to require intact actin, as
treatments with actin inhibitors lead to a mislocalization of
dShrmA (Bolinger et al., 2010). These data suggest that the actin
meshwork itself recruits the machinery that is necessary for its
contraction and, thus, for cell shape changes. However, a number
of other studies suggest, by contrast, that Shroom3 recruits actin
(Haigo et al., 2003; Lee and Harland, 2007; Plageman et al., 2010).
In support of this hypothesis, Shroom3 can, in addition to binding
actin directly, interact with and apically localize proteins of the
MENA/VASP family via its poly-proline domain (Plageman et al.,
2010). MENA/VASP proteins are involved in apical accumulation
and organization of actin (reviewed by Bear and Gertler, 2009;
Krause et al., 2003), and thus could indirectly mediate the
localization of actin by Shroom3. This shows that the exact cellular
mechanism underlying Shroom3 localization and more generally
Shroom3-mediated AC is still unclear. Whether MENA/VASP
proteins are involved in lateral line morphogenesis is unknown.
Finally, our data show that, like in other vertebrates (Haigo et al.,
2003), the N-terminal PDZ domain of Shroom3 is neither required
for its localization nor for its function as we showed that Shr3v2,
which lacks this domain, shows the same localization and function
as Shr3v1.
shroom3 is expressed specifically in the central part of the pLLP
and gets downregulated in assembled proneuromasts before
deposition. This suggests that Shroom3 function is only required to
set-up and activate the AC machinery at the apical side of the cells
and that once the apical surface is constricted, Shroom3 seems not
to be required anymore for its maintenance in mature rosettes.
DEVELOPMENT
Fig. 7. Shroom3 mediates FGF activity during
assembly of apically constricted rosettes.
(A-F) Pictures of the primordium of cldnb:gfp
zebrafish embryos carrying a hsp70l:fgf3-Myc
transgene (A-E) or of their non-carrier siblings (F).
Embryos have been heat-shocked for 15 minutes
(B-D,F) or not heat-shocked (A,E) and either
injected with Mo_Shr3e6i6 (C-F) or injected with
water (A,B). All embryos were kept at 25°C
before and after heat-shock. Seven hours after
HS, an ectopic rosette has formed in the leading
region of Tg(hsp70l:fgf3-Myc)zf115 (B), but not in
Mo_Shr3e6i6-injected siblings (C,D).
(G) Quantification of the distance between the tip
of the pLLP and the first rosette centre. Statistical
analyses were performed using the t-test and
error bars represent s.e.m. Scale bars: 50 m.
Shroom3 in pLL morphogenesis
RESEARCH ARTICLE 4579
Pharmacological treatment experiments support this idea as FGF,
Rock and myosin inhibitors disassemble already-formed rosettes
within the migrating pLLP, but not already-deposited proneuromasts
(data not shown). This supports a model in which the AC module is
required for rosette assembly and maintenance within the pLLP, but
is dispensable after deposition. It is likely that cell-cell adhesion
molecules including E- and N-cadherin or ZO-1, which are
concentrated in the rosette centres, play an important role in this AC
maintenance phase (Hava et al., 2009; Kerstetter et al., 2004;
Lecaudey et al., 2008; Liu et al., 2003; Matsuda and Chitnis, 2010).
Internal organization of the pLLP in rosettes is not
required for migration
We show here that Shroom3 function is required downstream of
FGF to drive AC. Although the loss-of-rosettes phenotypes are
very similar in Shroom3 and FGF loss-of-function conditions, the
migration phenotypes are totally different. Whereas FGF-deficient
primordia migrate in a very slow and uncoordinated manner
(Lecaudey et al., 2008; Nechiporuk and Raible, 2008), loss of
Shroom3 function does not affect migration. The migration defect
in FGF-deficient primordia has been proposed to be a secondary
consequence of the absence of rosettes, suggesting that the internal
organization of the primordium is a prerequisite for proper
migration (Lecaudey et al., 2008). However, a more recent study
showed that the expression of the G protein-coupled receptorencoding gene cxcr7b is downregulated in the absence of FGF
signalling. This is likely to be the cause, at least in part, of the poor
migration of FGF-deficient primordia (Aman and Piotrowski,
2008; Nechiporuk and Raible, 2008). Our results strongly support
this model in which the internal organization in radially organized
rosettes is not required for proper migration.
Shroom3 translates FGF signalling activation into
apical constriction
We show that Shroom3 is required for the induction of an ectopic
rosette in the leading region upon overactivation of the FGF
DEVELOPMENT
Fig. 8. Shroom3 overexpression counteracts the loss of rosettes induced by FGFR inhibitor treatment. (A-H⬙) Images of the pLLP in heatshocked non-transgenic siblings (A-B⬘,E-F⬘), hsp70l:shr3v1-tagRFP (C-D⬙) or hsp70l:shr3v2-tagRFP (G-H⬙) zebrafish embryos treated with DMSO
(A,C,E,G) or with 5 M SU5402 for 4 hours (B,D,F,H). Treatment with 5 M of SU5402 for 4 hours leads to a loss of apically constricted rosettes
(compare B,F with A,E). Induction of shr3v1 (C,D) or shr3v2 (G,H) expression upon heat-shock blocks the loss of rosettes induced by SU5402
treatment (D,H). (I,J) Boxplots displaying the quantification of this experiment by the rosette detector for shr3v1 (I) and shr3v2 (J). Arrowheads
indicate centres of apically constricted rosettes. Scale bars: 50 m.
Fig. 9. Model of the Shroom3-driven apical constriction
mechanism leading to rosette assembly in the pLLP. The top panel
represents the pLLP with turquoise nuclei corresponding to cells
expressing Fgf ligands and dark blue nuclei corresponding to cells
expressing fgfr1 [adapted from Lecaudey et al. (Lecaudey et al., 2008)].
fgf10-expressing cells signal to their fgfr1 expressing neighbours
leading them to apically constrict and form a rosette. The bottom panel
is a model for the underlying mechanism. Upon Fgf10 binding, Fgfr1 is
activated and then in turn activates the expression of shroom3.
Shroom3 localizes to the apical part of the cells, possibly via its
interaction with actin or an apical junctional complex (AJC) protein.
Shroom3 also interacts and localizes with Rho-associated kinases,
which, once at the apical side, phosphorylate and activate non-muscle
myosin, leading to actin contraction and AC of the cell.
signalling pathway. We could, however, not induce such an ectopic
rosette by overexpressing shroom3 alone either by mRNA injection
or in our HS lines. One reason for that might be that we do not
reach a high enough level of expression. Alternatively, Shroom3
might not be sufficient to induce the formation of a rosette de novo
in the leading region of the pLLP. Shroom3 is indeed sufficient to
induce AC in epithelial cells (Haigo et al., 2003; Hildebrand,
2005). The leading region of the pLLP, however, has rather
mesenchymal characteristics (Lecaudey et al., 2008). This strongly
suggests that a prerequisite for AC is the acquisition of epithelial
properties, including apico-basal polarity. For this reason, we also
think that the activation of shr3v1/v2 expression in embryos
previously treated with SU5402 (similar to the experiment
presented in Fig. 8 but the other way around) would not be
sufficient to re-establish rosettes because the cells would have lost
their epithelial properties. We have previously proposed that an
important function of FGF is to trigger such a mesenchymal-toepithelial switch at the transition between the leading and the
Development 139 (24)
trailing pLLP domains (Lecaudey et al., 2008). Our results here
fully support this model. We propose that, in addition to shroom3,
FGF signalling activates (or represses) the expression of additional
factors that are required first for epithelial fate acquisition (or to
block mesenchymal fate), and subsequently for Shroom3-mediated
AC.
It might be surprising that the signalling cascade linking the
activation of Fgfr1 to AC involves transcriptional regulation and
not a faster post-translational regulation, such as phosphorylation.
However, this result is in agreement with previous findings
showing that it takes several hours for the FGF inhibitor SU5402
to ‘disassemble’ rosettes. It also takes several hours after its
washout for apically constricted rosettes to reform (Lecaudey et al.,
2008; Nechiporuk and Raible, 2008). Thus, by inducing the
expression of shroom3, and potentially other epithelialization
factors, FGF is priming the cells for the AC process, which can be
induced by Shroom3 as the main organizer through activation of
NMII.
While our manuscript was under revision, an interesting report
was published in Development showing that activated Fgfr signals
through Ras-MAPK to induce apical localization of Rock2a, thus
promoting apical constriction and rosette formation in the pLLP
(Harding and Nechiporuk, 2012). Our data both support and
complement these findings by identifying Shroom3 as the
mechanistic link between FGF signalling and apical constriction.
Together, these data suggests a model in which shroom3 expression
is activated downstream of Fgfr/Ras/MAPK to recruit Rock2a to
the apical side of the cells. Rock2a would in turn activate nonmuscle myosin to constrict the apical actin meshwork, leading to
rosette formation.
In conclusion, FGF independently controls at least three different
cell responses in the pLLP: (1) cell migration by, at least in part,
preventing the repression of cxcr7b by Wnt (Aman and Piotrowski,
2008); (2) cell fate by activating atoh1a expression in the leading
region (Lecaudey et al., 2008; Matsuda and Chitnis, 2010;
Nechiporuk and Raible, 2008); and (3) cell shape by activating
shroom3 expression (this study). FGF signalling, therefore, has the
potential to coordinate different cellular responses within individual
cells and between neighbouring cells within the pLLP. Thus, we
provide here the first example of a mechanistic link between
patterning information and the cytoskeletal machinery during
proneuromast morphogenesis.
Acknowledgements
We thank Darren Gilmour for initial support and for the
Tg(−8.0cldnb:lynEGFP)zf106 and Tg(hsp70l:fgf3-Myc)zf115 lines; Erez Raz for the
gfp-myh9 construct; Caren Norden for advice on the pMLC2 staining protocol;
and Kristen Kwan and Chi-Bin Chien for the Tol2kit. We are grateful to S.
Goetter for fish care; Lisa Schmunk for help in identifying transgenics; and the
Life Imaging Centre (LIC) for imaging advice. We especially thank Stefan Eimer
for critical reading of the manuscript.
Funding
This work was supported by the German Research Foundation (DFG) [EXC
294-BIOSS to S.E., K.L., O.R. and V.L.; SFB 850-TP A2 to M.E.A. and V.L; GRK
1104 to S.A. and V.L.], the ‘Juniorprofessuren Programm’ of the Ministry of
Science, Research and Art of Baden-Württemberg (fellowship to N.M.) and a
Marie Curie Reintegration Grant [PERG06-GA-2009-256292 Morphogenesis to
V.L.].
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.083253/-/DC1
DEVELOPMENT
4580 RESEARCH ARTICLE
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DEVELOPMENT
Shroom3 in pLL morphogenesis