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First published online 3 July 2008
doi: 10.1242/dev.025981
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1 European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg,
Germany.
2 Institute of Developmental Genetics, GSF-Research Center for Environment and
Health, Ingolstaedter Landstrasse 1, Munich, Germany.
Author for correspondence (e-mail:
gilmour{at}embl.de)
Accepted 19 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Lateral line primordium, Zebrafish, Collective migration, Fgf, Rosette morphogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Lecaudey and
Gilmour, 2006). A common feature of migrating epithelia is that
cells at the so-called leading edge adopt many features that are more typical
of mesenchyme, including a loss of apicobasal polarity, reduced expression of
epithelial markers, such as tight junction proteins, and increased numbers of
dynamic filopodia. This change in cell state at leading regions has been
termed a `pseudo' epithelial-mesenchymal transition (PEMT), to take into
account the fact that these cells remain in contact
(Pastor-Pareja et al., 2004).
This is a crucial step in epithelial morphogenesis, as these leading edge
cells, in many contexts, are responsible for the detection of extrinsic cues
and tissue guidance. Thus, one major aim has been to identify the signaling
pathways that select and differentiate the leading cells from the rest of the
group (Martin-Blanco and Knust,
2001). Key regulators of this decision include the c-Jun
N-terminal kinases (JNKs) and other members of the family of mitogen-activated
protein kinases (MAPKs), which are known to be required for the advancement of
the leading edge of epithelia in a number of morphogenetic contexts
(Xia and Karin, 2004). As
MAPKs act as signaling hubs downstream of a number of cell surface receptors,
the cellular responses that they elicit are highly dependent on extracellular
signals.
One large class of extracellular signaling molecules that can act through
MAPKs is the fibroblast growth factors (Fgfs). Fgfs regulate many different
types of developmental processes (Affolter
and Weijer, 2005; Thisse and
Thisse, 2005). For example, Fgf signaling has been shown to
promote the migration of mesodermal cells during gastrulation in both
invertebrates and vertebrates (Ciruna and
Rossant, 2001; Gryzik and
Muller, 2004; Yang et al.,
2002). During the development of the Drosophila
respiratory system, polarized epithelial sacs produce dynamic membrane
projections characteristic of `leader' cells in response to nearby patches of
the Fgf-ligand branchless
(Sutherland et al., 1996;
Cabernard and Affolter, 2005;
Ribeiro et al., 2002). Here,
Fgf acts as both a chemoattractant to determine the direction in which
branches migrate and as a morphogen to select leader `tip' cells from an
equivalent group of epithelial cells, with this role going to the cell with
highest levels of Fgf receptor activation
(Ghabrial and Krasnow, 2006).
How cells become arranged and organized behind the leading edge, however, is
less understood.
The zebrafish lateral line primordium is a migrating epithelial placode
whose function is to deposit a series of mechanosensory hair cell organ
progenitors, termed proneuromasts, along a stripe of the chemokine Sdf1a
(Cxcl12a - Zebrafish Information Network) that runs along each flank of the
embryo (Ghysen and Dambly-Chaudiere,
2007). Interestingly, these organ progenitors, rather than being
preassembled, form repeatedly during migration as multicellular rosette
structures that appear behind the leading edge. Here, we exploit this
interesting feature of lateral line morphogenesis to address how tissues
become organized during migration, a poorly understood subject of general
importance. Through the use of mutants and a reversible small-molecule
inhibitor, we show that the activity of two Fgf ligands, Fgf3 and Fgf10, is
required redundantly for both internal organization and tissue migration. By
performing time-lapse imaging on embryos in which Fgf signaling is acutely
inhibited or hyperactivated, we demonstrate that, surprisingly, the activation
of Fgf signaling drives cells towards a `non-leader' fate by increasing
epithelial character, essentially the opposite effect from what has been
described in other contexts. Finally, we show that the dynamic expression of
point sources of Fgf ligands controls the spatiotemporal pattern of
epithelialization underlying sensory organ formation in the lateral line.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The following mutant strains were used:
fgf3/limabsent (liat24149)
(Herzog et al., 2004);
fgf10/daedalus (daetbvbo)
(Norton et al., 2005),
cxcr4b/odyseus (odyJI049). The cldnb:gfp
transgenic line (Haas and Gilmour,
2006) and the hsp70:dn-fgfr1 transgenic line were
described previously (Lee et al.,
2005). The hsp70:fgf3-myc (hsp70:fgf3)
transgenic line was generated by placing full-length fgf3-myc
(Maves et al., 2002) under the
control of the zebrafish hsp70 promoter
(Halloran et al., 2000), using
the Tol2 Kit (Kwan et al.,
2007).
Whole-mount in situ hybridization, immunohistochemistry and hair cell labeling
In situ hybridizations (ISH) and immunofluorescence (IF) stainings were
performed according to standard procedures. The atoh1a, fgfr3 and
fgfr4 cDNA probes were cloned by RT-PCR. Primers used were as
follows:
atoh1a-F, 5'-TCCGTCCCTGTATCCATAGC-3';atoh1a-R, 5'-GTTGAATGTTCCGTCCTCGT-3';
fgfr3-F, 5'-CGGCACGAGCTTTCACACAG-3';
fgfr3-R, 5'-AAACCCTAAAGCTCCCTGTTTTT-3';
fgfr4-F, 5'-TATAATGATGCTTGGCACTATGAAA-3'; and
fgfr4-R, 5'-TGGGGAGATCTACTTGTACTCATTC-3'.
fgf10 (Ng et al.,
2002), fgfr1, fgfr2 and pea3
(Roehl and Nusslein-Volhard,
2001) RNA probes were kind gifts from Carl Neumann. For IF, the
following antibodies were used: rabbit aPKC (1/100; Santa Cruz), mouse ZO1
(1/200; Zymed), mouse acetylated-tubulin (1/1000; Sigma), rabbit GFP (1/500;
Torrey Pines Biolabs) and mouse GFP (1/500; JL8, Clontech). For labelling the
hair cells, 2- to 3-dpf embryos were incubated in FM4-64 (10 mg/ml; Molecular
Probes) in embryo medium (E3) for 2 minutes and rinsed in fresh E3.
Morpholino injection
The morpholino targeting atoh1a (MoAtoh1a,
5'-ATCCATTCTGTTGGTTTGTGCTTTT-3'; Gene-Tools, Oregon, USA) was
described previously (Millimaki et al.,
2007) and was injected at a concentration of 0.05 mM.
Heat-shock, SU5402 and DAPT treatments
hsp70:dn-fgfr1 and hsp70:fgf3 embryos (30 hpf) were
heat-shocked for 20 minutes at 37°C. For SU5402 treatments, 24-hpf
cldnb:gfp embryos were dechorionated and incubated for 6 hours in 5
to 10 µM SU5402 in E3 (Calbiochem #572630). Control embryos were treated
with E3 containing the same amount of DMSO. For time-lapse imaging during drug
treatment, pretreated embryos were mounted with agarose and E3 each containing
60 µM SU5402. For washout experiments, embryos treated with SU5402 were
washed in E3 containing 0.5% DMSO for 2 hours and mounted for imaging. For
DAPT treatment cldnb:gfp embryos at 20ss were dechorionated and
incubated in 100 µM DAPT in E3 for 10 hours.
Electron microscopy
Electron microscopy was performed as previously described
(Pouthas et al., 2008).
Mosaic analysis
Mosaic embryos were generated by transplantation as described previously
(Haas and Gilmour, 2006).
Imaging
Embryos were anesthetized in 0.01% Tricane and mounted on glass-bottom
petri dishes (Mattek) in 1% low melting point agarose. Time-lapse imaging was
performed on Perkin Elmer Spinning Disc (ERS, RS, LCI) and Olympus Fluoview
1000 confocal microscopes using 10x/NA0.3, 20x/NA0.75, 40x
W/NA1.2 or 60x W/NA1.2 objectives. Usually, z-stacks were
captured at 2-3 µm intervals and flattened by maximum projection in
ImageJ.
Image processing
Image processing and measurements were performed with ImageJ 1.38 X.
Overlays of visible ISH and GFP IF were obtained by inverting the NBT-BCIP
image with ImageJ.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Roehl and
Nusslein-Volhard, 2001). We analyzed the expression of a number of
Fgf ligands and receptors in order to determine which are responsible for
pea3 activation in this context. Of the four characterized Fgf
receptors, only fgfr1 is clearly expressed in the migrating
primordium (Fig. 1A, data not
shown). This approach also identified two Fgf ligands, fgf3 and
fgf10, as being candidates for mediating signaling in this context.
Interestingly, both genes are expressed within the primordium, being
upregulated in leading regions (Fig.
1B,C). In addition, expression of fgf10 can be clearly
seen in one or two cells at the centre of rosette-like proneuromasts
(Fig. 1C, arrows), whereas
fgf3 cannot be detected (Fig.
1B). Interestingly, the combined expression of these genes is
somewhat complementary to that of fgfr1 and pea3. Receptor
and target are downregulated at the leading edge and at the centre of
rosettes, both of which are sites of increased ligand transcription
(Fig. 1A,D, arrows).
We next tested whether the activity of these ligands can account for the
pea3 expression pattern, by crossing the previously identified
mutants limabsent (lia) and daedalus
(dae), which encode mutations in fgf3
(Herzog et al., 2004) and
fgf10 (Norton et al.,
2005), respectively, to our previously described lateral line
reporter, cldnb:gfp (Haas and
Gilmour, 2006). Although the expression of pea3 in the
respective mutants is indistinguishable from wild type (wt; not shown), it is
entirely absent from the migrating primordium of fgf3;fgf10 double
mutant embryos. Other embryonic expression domains, such as the somite borders
and the midbrain-hindbrain boundary, however, remain unchanged
(Fig. 1E-H). By comparison,
embryos treated with the Fgf receptor inhibitor SU5402 for five hours show a
complete absence of pea3 expression throughout the embryo
(Fig. 1I, J). We conclude that
Fgf3 and Fgf10 mediate Fgf signaling during lateral line primordium
migration.
Fgf3 and Fgf10 are required for migration and subdivision of the lateral line
We next addressed the requirement for Fgf signaling in lateral line
morphogenesis. Single mutants for fgf3 and fgf10 showed no
detectable change in lateral line development
(Fig. 2A-C), a result that is
consistent with normal pea3 expression within the primordium in these
embryos. By contrast, fgf3;fgf10 double mutant primordia showed a
number of striking defects when compared with wild-type controls
(Fig. 2A,D). The most obvious
difference was that the speed of migration was strongly reduced, with the
primordium having migrated less than half the distance of wild type by 42
hours post-fertilization (hpf) (n=18;
Fig. 2A,D,E). Furthermore,
there was a change in morphology: the migrating primordia adopted an extended
conformation, becoming twice as long as wild-type siblings
(Fig. 2F,G,J). Similarly,
blocking Fgf signaling with the Fgfr inhibitor SU5402, or using a heat-shock
inducible, dominant-negative form of Fgfr1
(Lee et al., 2005), blocked
migration and caused extended tissue morphology
(Fig. 2H-J).
Closer inspection of double mutant primordia revealed that the assembly of
neuromast progenitors was also strongly affected. Whereas two to three
rosette-like neuromast organs can be morphologically identified within
wild-type primordia (Fig. 2F),
no such organization was visible in double mutants between 28 and 36 hpf
(Fig. 2G, see also Fig. S1 in
the supplementary material). Developmental timecourses revealed that, at later
stages, organ assembly defects are less pronounced, with 71% (n=14)
of the double mutant primordia showing a normal number of rosettes. This
recovery of rosettes could be due to the fact that the fgf3 allele
used has some residual activity (Herzog et
al., 2004). Indeed, treating embryos with 5 µM SU5402 for 6
hours completely removes organ progenitors from the primordium
(Fig. 2H). Given that only
fgf10 mRNA is detected in the centre of rosettes, the finding that
rosette assembly was not affected in fgf10 single mutants was
somewhat surprising. However, expression analysis reveals that fgf3
assumes an `fgf10-like' pattern in fgf10 mutants, expanding
posteriorly in the leading region and increasing at rosette centres (compare
Fig. 2K with
Fig. 1B).
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Lateral line phenotype cannot be explained by a defect in neurogenesis
Given that Fgfs are known to be important for neuronal differentiation in a
number of different contexts (Akai et al.,
2005; Henrique et al.,
1997; Millimaki et al.,
2007; Nechiporuk et al.,
2007), the lack of rosette assembly and aberrant migration in
fgf3;fgf10 double mutants could result from a disruption in the
neurogenesis program that is initiated within the migrating primordium.
Support for this suggestion comes from studies on the zebrafish ear, the organ
most closely related to the lateral line primordium, where it has been shown
that Fgf drives neuronal development through the activation of the proneural
gene atoh1a (Millimaki et al.,
2007). Furthermore, it has been shown that atoh1a is
expressed in the migrating primordium in a progressively restricted manner, in
a large patch at the front of the primordium that becomes focused to one or
two cells at the centre of forming rosettes
(Itoh and Chitnis, 2001). This
expression pattern, which is very similar to that of fgf10, has been
proposed to become restricted by lateral inhibition
(Itoh and Chitnis, 2001) and
is required for the differentiation of hair cells
(Sarrazin et al., 2006).
Therefore, in order to test whether Fgf controls rosette formation via the
activation of atoh1a, we analyzed the consequences of knocking down
atoh1a on rosette assembly. Injection of an atoh1a-specific
morpholino (Millimaki et al.,
2007) led to a significant broadening of its own expression domain
(Fig. 3A,B), as could be
expected from a loss of lateral inhibition. Moreover, staining these morphant
embryos with markers of hair cell differentiation, such as FM4-64 and
anti-acetylated tubulin, revealed the expected loss of hair cells
(Fig. 3G-J), confirming the
efficacy of the morpholino. Beside the absence of hair cells, atoh1a
morphant neuromasts were indistinguishable in number and morphology from wild
type (Fig. 3E-J). Furthermore,
imaging these embryos during earlier stages reveals that rosette assembly and
neuromast deposition proceeded entirely normally in atoh1a morphants
(Fig. 3C,D). We next blocked
Notch activity completely by treating embryos with the 
-secretase
inhibitor DAPT, a widely used antagonist of Notch signaling
(Geling et al., 2002).
Treatment with DAPT during lateral line migration resulted in an expansion of
atoh1a expression in the primordium (not shown) and in deposited
neuromasts (Fig. 3O,P), similar
to that observed in atoh1a morphants, thus confirming drug efficacy.
However, once again, this had no effect on the assembly of rosettes within the
migrating primordium or on the pattern of deposited neuromasts
(Fig. 3K-N). These findings
show that rosette assembly is not dependent on neurogenesis, and that
Notch-based lateral inhibition mechanisms do not play a crucial role in this
process. Therefore, we conclude that the phenotype resulting from the loss of
Fgf signaling is not a result of defective neurogenesis.
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Proneuromast assembly is a prerequisite for normal primordium migration
In addition to being less coordinated, cells in central regions of the
primordium do not become organized into proneuromasts in
fgf3;fgf10 double mutants. One possible explanation for this
phenotype is that this repeated stretching of the migrating primordium
prevents the cells from becoming organized. Alternatively, given that the
`raison d'être' of the lateral line primordium is to generate
neuromasts, an inability of cells to assemble into organ progenitors could
lead to a migration arrest through the activation of an unknown checkpoint. In
order to determine which is the primary defect, migration or rosette assembly,
we tested whether blocking stretching could rescue the rosette-loss phenotype
observed after Fgf inhibition. This was achieved by treating cxcr4b
mutant primordia, which are unable to detect and extend along the stripe of
the Sdf1a guidance cue (David et al.,
2002; Haas and Gilmour,
2006; Li et al.,
2004), with SU5402. Blocking Fgf in stationary cxcr4b
mutant primordia also led to the `melting' of formed rosettes, thus excluding
the possibility that the loss of organ assembly is a consequence of aberrant
migration (Fig. 5A,B). Further
support for the idea that loss of rosette assembly is the primary defect
resulting from loss of Fgf signaling came from examining the temporal
relationship between rosette assembly/disassembly and changes in primordium
migration. Time-lapse analyses performed on the primordia of SU5402-treated
embryos clearly demonstrate that rosettes melt before any detectable effect on
migration (Fig. 5C,D; see Movie
4 in the supplementary material). Interestingly, proneuromasts differ in their
sensitivity to chemical inhibition of Fgf signaling, becoming increasingly
resistant as they mature within the primordium. Only after all proneuromasts
have disassembled does the stretched primordium halt its migration
(Fig. 4F).
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Surprisingly, closer analysis of time-lapse movies capturing SU5402 washout indicates that rosettes reappear by de novo assembly. Both the position and the temporal sequence in which these rosettes reappear were unpredictable, suggesting that the initial pattern of fgf10 foci is lost through blocking Fgf-receptor activity. Indeed, in SU5402-treated embryos and in fgf3;fgf10 double mutants, the transcription of both fgf3 and fgf10 was strongly enhanced and expanded to fill the entire primordium (Fig. 5G,H; see also Fig. S2 in the supplementary material). This reveals the existence of a negative-feedback loop in which the activation of Fgfr restricts the expression of its own ligands. This finding may explain the mutually exclusive expression of ligand and receptor observed during normal development (Fig. 1A-D).
Fgf spots increase epithelialization within the migrating primordium
One important step towards understanding how Fgf drives rosette assembly is
to define the cell-shape changes underlying this morphogenetic process.
Transmission electron microscopy reveals that proneuromasts are radially
organized assemblies of pear-shaped epithelial cells that display constricted
apical surfaces (Fig. 6A,B).
Higher magnification views of the rosette center highlighted the presence of
the three types of cell-cell junctions, tight junctions, adherens junctions
and desmosomes, arranged in the apicobasal order that is typical of epithelia
(Fig. 6B). Furthermore,
antibodies against the apical marker aPKC and the tight junction protein ZO-1
(Tjp1 - Zebrafish Information Network), a widely used marker of epithelia,
showed highly specific labelling at the centre of rosettes, confirming these
as foci of apical membrane material and intercellular adhesion junctions
(Fig. 6C,D). Both markers were
excluded from the less-organized leading region. These foci also show
increased levels of phalloidin staining
(Fig. 6E), most likely due to
the presence of the actomyosin ring responsible for apical constriction. In
order to capture this organ formation process as it happens, we performed
time-lapse imaging of embryos mounted in a `side-view' orientation that allows
the visualization of basolateral surfaces. By focusing on cells at the rear of
the leading region, we could show that proneuromasts do indeed form through
the simultaneous apical constriction of neighbouring cells
(Fig. 6F; see Movie 6 in the
supplementary material).
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In order to further test this hypothesis, we generated a stable transgenic
line expressing fgf3 under the control of the hsp70 promoter
(Halloran et al., 2000). In
situ hybridization with an fgf3 probe revealed that this ligand was
expressed at an extremely high level throughout the entire embryo 4 hours
after heat-shock induction (Fig.
7M,N). Rosettes were lost after heat-shock-induced overexpression
of fgf3 (data not shown), supporting a model in which discrete spots
are required to seed and maintain rosettes. However, by 9 hours after heat
shock, hsp70:fgf3 embryos showed a significantly increased number of
rosettes per primordium, when compared with identically treated,
non-transgenic siblings (Fig.
7O-Q). Interestingly, the size of the rosette-free leading region
was strongly reduced or even absent in hsp70:fgf3 embryos, as
confirmed by measuring the distance from the centre of the first rosette to
the tip (Fig. 7R). Therefore,
the misexpression of Fgf ligands causes a shift in the composition of the
primordium, such that there is an increase in epithelialization at the expense
of the mesenchyme-like leading region.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Here, we show that Fgf signaling plays a complementary role
during lateral line development, that is, it allows cells of the primordium to
adopt a fate other than that of the leading edge. As cells pass from the
leading to the organ-forming region, they become responsive to Fgf signaling
and adopt a textbook, epithelial morphology, resulting in increased cell
packing and order. At what level does Fgf act to drive this change in cell
phenotype? Loss of Fgf signaling results in a number of deviations from normal
cell shape and behaviour, including the loss of apical constrictions and
coordinated migration, which could potentially explain the inability of the
cells to become assembled into rosette-like proneuromasts. However, the length
of time required to either induce the phenotype with SU5402, or reinstate
normal morphology by SU5402-washout, is in the range of hours. Such a sluggish
response would not be expected if Fgf signaling were to act specifically as an
internal guidance cue or drive apical constriction through the direct
activation of the actomyosin system. Furthermore, Fgf-deficient primordia show
an absence of the tight junction protein ZO-1, which is unlikely to be due to
a concentration effect but rather to reduced expression of this epithelial
marker. Indeed, that Fgf signaling drives fate decisions in this context is
already apparent from our finding that active signaling through Fgfr is
required to restrict the pattern of transcription of fgf3 and
fgf10 (discussed below). These and other findings suggest that
patterned Fgf signaling leads to a switch in local cell state, such that cells
increase their epithelial character. Although this does not exclude that Fgf
signaling additionally regulates cell movement and apical constriction
directly, we would imagine that the loss of these activities alone would be
insufficient to explain the observed phenotype.
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;
Dubrulle et al., 2001;
Dubrulle and Pourquie, 2004).
As both systems generate a series of repetitive epithelial packets from
less-ordered mesenchymal cells, the similarities are striking, and it is
therefore intriguing that Fgf signaling promotes EMT in one context but MET in
another.
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). It was therefore surprising that perturbing
Notch-based lateral inhibition, either by atoh1a knockdown or by
treatment with the Notch antagonist DAPT, had no clear effect on rosette
assembly or on the final pattern of neuromasts. This is consistent with the
finding that mindbomb mutants have reduced Notch activity but a
seemingly normal pattern of deposited neuromasts
(Itoh and Chitnis, 2001). So
how does fgf10 expression get restricted to dots? Interestingly,
fgf10 and fgfr1 show mutually exclusive expression during
organ assembly, suggesting that fgf10 expression may become
restricted via a negative-feedback loop. Furthermore, the expression of both
ligands expands throughout the entire primordium in fgf3;fgf10 double
mutants and SU5402-treated embryos, giving credence to the idea that Fgfr1
activity directly restricts Fgf ligand expression, possibly through a lateral
inhibition-type mechanism. Interestingly, inactivating fgf10 alone,
through the use of fgf10 mutants, leads to the formation of rosettes
that are nucleated by dots of fgf3, a clear demonstration of the
robustness of this pattern forming system.
A simple mechanism for generating rosettes within epithelia
In addition to determining the spatiotemporal pattern of epithelialization
within the migrating primordium, Fgf signaling leads to the formation of
rosette assemblies. The teleological reason for rosettes in this context is
obvious, as these are the forerunners of neuromasts, a series of volcano-like
organs that penetrate the skin to project mechansensory hairs cells into the
surrounding water. Similar multicellular rosettes have recently been shown to
arise in other epithelial tissues, suggesting that they could represent a
common functional unit of epithelial morphogenesis
(Blankenship et al., 2006;
Brown et al., 2006;
Wagstaff et al., 2008). They
have been particularly well described in the context of Drosophila
germband extension, where these transient rosettes directly participate in
tissue elongation by forming and resolving in a directional manner that is
influenced by embryonic anteroposterior patterning cues. Despite superficial
similarities between these rosettes and those in the primordium, there are
fundamental differences in their three-dimensional organization. The rosettes
in the Drosophila blastoderm form when two rows of cells that are in
contact with each other constrict their shared interface, that is, one side of
their apical surface, with the resultant rosette resembling a pie where each
slice is a cell (Blankenship et al.,
2006; Zallen and Blankenship,
2008). The rosettes of the lateral line primordium, by contrast,
assemble by bunching together cells whose entire apical surface is tightly
constricted, with the resultant rosette resembling a garlic bulb where each
clove is a cell. Another type of rosette that forms through the constriction
of interfacial surfaces, rather than the entire apical surface, appears in the
wake of the morphogenetic furrow that patterns the Drosophila retina.
These arc-like rosettes, which are the forerunners of the ommatidia, are built
around atonal-expressing R8 cells that `recruit' surrounding cells in
a stepwise fashion through the activation of the Egf-like ligand
spitz (Brown et al.,
2006). While the parallels with the lateral line system are
clearly more obvious in this case, our demonstration that Notch signaling or
Atoh1a is not required for rosette assembly suggests that there may be
differences in the gene regulatory networks involved.
Collectively, our data lead us to propose the following model for rosette
morphogenesis in the lateral line. An Fgf-based lateral inhibition-type
system, like that suggested above, generates a single cell that expresses
significantly higher levels of Fgf ligand and activates Fgfr1 in surrounding
cells, as supported by upregulated pea3 expression. As fgf10
forms a point source, it induces the radial epithelialization of surrounding
cells, which rise-up apicobasally and form tight junctions, as we have
demonstrated. However, the central cell does not signal in an autocrine
manner, as confirmed by the absence of pea3 transcription, and is
thus refractory to induction and maintains its mesenchyme-like state. This
would result in a ring of heightened epithelial cells that has a flatter cell
at its centre (Fig. 8). There
are alternative scenarios for how this situation could be resolved to form a
rosette. The most likely invokes the activation of a dedicated cell signaling
system that leads to increased cohesion between the nucleating cell and the
neighbouring epithelia, causing the latter to apically constrict and cover the
former. As this apical constriction occurs over several cell diameters, the
central cell may secrete a signal that ensures directionality, a function that
could be fulfilled by fgf10 itself. Alternatively, if the centre cell
simply maintains its leading-edge characteristics, it may initiate rosette
formation by triggering a response similar to wound healing in surrounding
cells. This second scenario is somewhat simpler, as it generates rosettes by
exploiting an intrinsic property of epithelium, which is to undergo
coordinated apical constrictions whenever its integrity is disrupted
(Jacinto and Martin, 2001;
Martin and Parkhurst, 2004).
Indeed, the rosettes that form within the lateral line are, at their core,
more similar to those generated by wounding an epithelium than either of the
developmental examples given above (Martin
and Parkhurst, 2004). However, in the case of the lateral line,
cells at the centre of the rosettes are not extruded and discarded from the
epithelium, as damaged cells are during wound healing, but are rather
encapsulated through this apical constriction process. These are most likely
the cells that give rise to sensory hair cell progenitors within a few hours
of this morphogenetic event.
Tissue compartmentalization is required for efficient migration
One of the surprising findings of this work is that a defect in the
internal organization of the primordium has such a strong detrimental effect
on its migration. This finding is perhaps counterintuitive, as increasing the
relative proportion of cells with mesenchyme-like, leading edge fate could be
expected to improve migration efficiency rather than decrease it. Given the
numerous examples in which Fgf ligands have been shown to act as
chemoattractants during cell migration
(Affolter and Weijer, 2005),
one alternative explanation for this result is that Fgf3/Fgf10 act as
diffusible guidance cues that regulate directionality within the migrating
primordium. However, several findings presented here make it unlikely that
this is the primary role of internal Fgf signaling during primordium
migration. Not only does directional migration continue normally for several
hours after blocking Fgf signaling with SU5402, but a change in rosette
assembly always prefigures a change in migration behaviour. Furthermore,
ubiquitous, high-level expression of fgf3 does not lead to a rapid
change in tissue migration behaviour or direction, which would be expected if
it were to act as a chemoattractant. Rather, we propose that the primordium
possesses an internal checkpoint that halts migration whenever there is a
problem with rosette assembly. Of course, a mechanism that couples migration
behaviour to organ assembly makes biological sense, as a migrating primordium
that does not deposit organs is of little use. As with rosette assembly, this
checkpoint could be mediated through the activation of a dedicated signaling
system or it may exploit intrinsic mechanical properties of the tissue.
Indeed, rosette formation may facilitate migration simply by virtue of the
fact that it converts a large number of migrating mesenchyme-like cells into
two or three packets that could be controlled as single units, thus reducing
the complexity of the system. Answers to such questions will come from a
deeper understanding of the respective mechanical properties of epithelial and
mesenchyme-like domains within migrating tissues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2695/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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