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First published online 7 February 2007
doi: 10.1242/dev.02805
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DCMB group, Biology Department, Duke University, Durham, NC 27708, USA.
* Author for correspondence (e-mail: dmcclay{at}duke.edu)
Accepted 5 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Snail, Primary mesenchyme cell, Ingression, Epithelial-mesenchymal transition, Gene regulatory network
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(reviewed by Solursh, 1986).
During EMT, the PMCs downregulate cell-cell adhesion
(Fink and McClay, 1985),
increase motility, change cell shape, and finally become mesenchymal cells
with a migratory behavior. These PMCs exclusively form the larval skeleton
later in development.
Micromeres, the PMC predecessors, appear at fourth cleavage as a result of
an unequal cleavage in the vegetal hemisphere. During early cleavage, they
become autonomously specified (Horstadius,
1973; Okazaki,
1975; Davidson et al.,
1998; Ransick and Davidson,
1993), and this specification system is coupled to the early
establishment of the animal-vegetal axis in the unfertilized egg
(Angerer and Angerer, 2003;
Brandhorst and Klein, 2002;
Ettensohn and Sweet, 2000).
Although the responsible maternal determinants initiating this event are
incompletely understood, one of the earliest known components is
ß-catenin-induced transcriptional activation at fourth cleavage
(Logan et al., 1999). The
nuclear localization of ß-catenin is required for micromere specification
and later for all endomesoderm formation, including archenteron and secondary
mesenchyme cells (SMCs) (Emily-Fenouil et
al., 1998; Logan et al.,
1999; Wikramanayake et al.,
1998). ß-catenin provides an early input into the PMC gene
regulatory network (GRN), where it activates a transcriptional repressor
pmar1, the earliest known zygotic gene expressed exclusively in the
micromere lineage (Oliveri et al.,
2002). Pmar1 protein activates the micromere specification program
by repressing an unidentified, ubiquitous repressor. As a consequence of this
derepression event, several downstream transcriptional regulators are
activated, among which are the zygotic targets, alx1
(Ettensohn et al., 2003) and
ets1 (Kurokawa et al.,
1999). When the function of either Alx1 or Ets1 is blocked, PMC
specification is disrupted. Despite the increasing knowledge of the mechanisms
of PMC specification, much less is known about what triggers the EMT process
in these cells and what molecular changes are required for the morphogenesis
of PMCs [as suggested also in Shook and Keller
(Shook and Keller, 2003)].
The Snail gene family of transcription factors has been shown to play
crucial roles in mesoderm development, cell movement and especially induction
of EMT in other systems (Barrallo-Gimeno
and Nieto, 2005; Hemavathy et
al., 2000; Nieto,
2002). Although originally identified as a mesoderm determinant in
Drosophila (Alberga et al.,
1991), the first indication that the Snail family is involved in
EMT came from Snail2 (Slug) loss-of-function studies in
chick embryos. Incubation of early chick embryos with antisense
oligonucleotides to inhibit Snail2 function led to the failure of
early mesoderm migration from the primitive streak
(Nieto et al., 1994).
Subsequent studies in cell lines and in other vertebrates later confirmed this
involvement. For example, mouse Snail (Snai1 - Mouse Genome
Informatics) is able to induce EMT when expressed in mammalian epithelial
cells (Batlle et al., 2000;
Cano et al., 2000), and
Snail-knockout mice die during gastrulation, due at least in part, to
the failure of the mesodermal cells to undergo an EMT
(Carver et al., 2001).
Furthermore, different signaling molecules, such as TGFß, FGFs, EGFs,
WNTs, BMPs and Notch, have been shown to trigger EMT processes by inducing
Snail gene expression in different cellular contexts (reviewed by
De Craene et al., 2005). The
PMC ingression of the sea urchin embryo is an excellent model for studying EMT
in vivo, and with the recently published sea urchin endomesoderm GRN
(Davidson et al., 2002a;
Davidson et al., 2002b), we
asked whether Snail has the same role in the sea urchin as in other systems
exhibiting EMTs. If so, how did it fit into the micromere GRN?
In this study, we report the identification, characterization, and functional analysis of Lvsnail, a member of Snail family transcription factors in Lytechinus variegatus. Lvsnail mRNA is expressed dynamically in different mesodermal cell populations throughout the development of the sea urchin embryo. We show that LvSnail is required for the micromeres to undergo EMT, and ingress into the blastocoele. Moreover, LvSnail mediates the downregulation of cadherin expression and function. We further position Lvsnail in the current version of the micromere GRN, and examine its regulative relationships with several PMC genes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of Lvsnail and Lvpmar1
The coding sequence of Lvsnail was obtained by RT-PCR from a
Lytechinus variegatus gastrula cDNA library (GenBank Accession Number
DQ665364). The sequence information of Lvpmar1 (Lv_170H13; Accession
Number DQ667003) was obtained from Caltech Lytechinus variegatus
sperm genomic BAC library A, as described by Davidson et al.
(Davidson et al., 2002b), and
the full open reading frame was subsequently amplified by PCR from Lv cDNA
libraries. All PCR products were cloned into a PCS2 vector for mRNA
synthesis.
In situ hybridization
In situ hybridization was performed using standard methods
(Bradham and McClay, 2006) with
DIG-labeled RNA probes and BM purple substrate (Roche) for detection.
Hybridizations and washes were carried out at 65°C. The Lvsnail
probe corresponds to the full-length open reading frame. Other probes were
synthesized from Lvets1 (this study) and Lvalx1
(Ettensohn et al., 2003)
clones.
Morpholino antisense oligonucleotides (MASO), mRNA injections, and U0126
Two Lvsnail-specific MASOs were obtained from Gene Tools, although
Oligo2 had a higher efficiency at 1 mM, (Oligo1:
5'-AAAGACCCTCGGCATCTTCTTGATAA-3'; Oligo2:
5'-TTTTGACGAGAAAAGACCTCGGCAT-3'). Alx1MASO was injected at 2 mM
(see Ettensohn et al., 2003).
Each injected mRNA was transcribed in vitro using the mMessage mMachine Kit
(Ambion), and diluted in ddH2O. A final concentration of 25-30 ng/µL was
used for pmar1 mRNA, and 400-500 ng/µL for snail mRNA.
Double injections were performed by simultaneous injections of pmar1
mRNA plus SnaMASO or Alx1MASO plus snail mRNA at the concentrations
indicated above. CadTM-GFP was constructed by linking the transmembrane domain
of LvG-Cadherin in between two full-length GFPs. U0126 (Promega) was added to
cultures during early cleavage stages, unless otherwise noted. Treatment with
the DMSO vehicle had no effect (not shown). Doses for these reagents were
determined by dose-response experiments. U0126 was used at 10-15 µM in most
experiments.
Transplantation experiments
Animal-vegetal half transplantations were performed at the 16- or 32-cell
stage, and micromere transplantations were performed at 16-cell stage, with
L. variegatus embryos. Detailed procedures were followed as
previously described (Logan et al.,
1999).
Immunostaining
Embryos were methanol-fixed, stained with 1d5 mAb (1:200) in 4% normal goat
serum in PBS, and incubated overnight at 4°C. After washing three times in
PBS, samples were incubated with Cy3-conjugated secondary antibodies (Jackson
Immunoresearch Laboratories) for 1-2 hours at room temperature, and then
imaged as previously described (Gross et
al., 2003).
QPCR analysis
Total RNA was prepared from 10-20 embryos using Trizol (Invitrogen) with a
glycogen carrier (Ambion). The sample was used for reverse transcription (RT)
with Taqman RT-PCR kits (Applied Biosystems) after pretreatment with DNase I
(DNA-free, Ambion). QPCRs were performed using Roche LightCycler and a Fast
Start SYBR Green PCR Kit (Roche). Results were calculated by subtracting the
sample CT (crossing point threshold) from the control CT to determine
CT, and then normalized to Ubiquitin.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proteins in the snail gene family possess 4-6
C2H2 zinc-fingers
(Nieto, 2002) as the DNA
binding domain at the C-terminus. LvSnail has five zinc-fingers, and the
protein alignment from ZFII to ZFV (the first zinc-finger is missing in human,
mouse and zebrafish) shows that this region is highly conserved relative to
snail family members from other species
(Fig. 1A) [e.g. Slug (Snail2)
from vertebrates (88-89% amino acid identity) and Snail from amphioxus (87%)].
In addition, Snail family proteins are characterized by the presence of an
N-terminal SNAG domain (nine amino acids). Again, the SNAG domain of LvSnail
is almost identical to all Snail family members from other species except
Drosophila, Caenorhabditis elegans and Ciona
(Fig. 1B), which may be due to
independent losses in these lineages.
Phylogenetic analysis based on the four zinc-finger regions, using the neighbor-joining method, groups LvSnail and SpSnail together into a large clade containing snail and slug genes from vertebrates, amphioxus snail (Bf snail) and limpet snail (Pv snail2) (Fig. 1C). The topology of this phylogenetic tree, as well as the distinct protein motifs, clearly support the conclusion that Lvsnail is an ortholog of snail genes.
Lvsnail mRNA is expressed dynamically in mesoderm during gastrulation
A temporal expression profile of Lvsnail mRNA was obtained using
quantitative PCR (data not shown). Although the expression level of
Lvsnail remained relatively low throughout all stages, there was no
maternal message, and zygotic Lvsnail mRNA transcripts began to
accumulate around the hatched blastula stage, and then increased until prism
stage.
Whole-mount in situ hybridization (WMISH) showed that Lvsnail mRNA
is expressed dynamically in different territories of the embryos
(Fig. 2). No staining appears
in early cleavage stages (Fig.
2A), and expression is first detectable around the late-hatched
blastula (HB) in the thickened vegetal plate region
(Fig. 2B). At early mesenchyme
blastula (MB) stage, Lvsnail mRNA is expressed in ingressing PMCs
(Fig. 2C). The PMC expression
of Lvsnail is reduced following ingression, and staining then appears
within the SMC territory during archenteron invagination, and prior to
ingression of SMCs (Fig. 2D),
then disappears from the ingressed SMCs. Throughout gastrulation, the SMC
expression of Lvsnail persists at the tip of archenteron
(Fig. 2E), and exhibits both
oral-aboral and left-right asymmetry at the midlate gastrula stage (data not
shown). At prism stage, Lvsnail mRNA reappears in PMCs and becomes
localized to the two ventrolateral PMC clusters
(Fig. 2F,G). The expression
continues at these two sites, corresponding to the tips of the arm rods of the
pluteus larva (Fig. 2H). The
SMC expression pattern of Lvsnail is consistent with the observations
from a recent publication (Hardin and
Illingworth, 2006).
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SnaMASO-injected embryos (`Sna morphants') developed normally through the cleavage and the blastula stages, and they hatched at the same time as controls. However, when PMCs of control embryos ingressed into the blastocoele (Fig. 3A, arrow), PMCs failed to ingress in Sna morphants (>90%, Fig. 3D). The PMC ingression block continued (Fig. 3F,G; 35%, 65%, respectively) even as control siblings completed gastrulation (Fig. 3C). Although in a significantly delayed fashion, almost all Sna morphants eventually displayed a normal archenteron with no apparent phenotypic defects (see Fig. S2 in the supplementary material). Other later phenotypes were observed in these Sna morphants, including loss of pigment cells, and stunted arm rod growth (see Fig. S2 in the supplementary material), but in this study we focus only on the PMC ingression phenotype.
At ingression LvSnail functions autonomously in micromeres
Chimeric embryos were generated to localize required Snail activity. To
block LvSnail function specifically in the vegetal half, we combined a control
animal half embryo (with FITC, shown in green) with a SnaMASO-injected vegetal
half (with rhodamine-conjugated dextran, shown in red)
(Fig. 4G). The resulting
embryos showed no PMC ingression (Fig.
4C,C'; 3/4, i.e. 3 out of 4 chimera embryos exhibited the
phenotype shown in the figure), similar to Sna morphants
(Fig. 4B), whereas the
reciprocal chimeric embryos (SnaMASO in animal half) developed normally
(Fig. 4D,D'; 4/4)
compared with glycerol-injected controls
(Fig. 4A). As Lvsnail
mRNA is expressed in ingressing PMCs, which are derived from the micromere
lineage, we then replaced a single micromere from a green-dyed control host
with a SnaMASO-injected micromere (in red)
(Fig. 4H). The red micromere
progeny failed to ingress (Fig.
4E', arrow), whereas the green micromere progeny (serving as
internal controls) ingressed normally and settled at the bottom of the
blastocoele (Fig. 4E,E';
arrowheads) (n=18; 5/5, 3/6, 5/7). The reciprocal experiment showed
that progeny of a single green control micromere ingressed normally even when
put onto a SnaMASO-injected host (Fig.
4F,F'; arrow). Taken together, these data show that Snail is
required in micromeres for these cells to ingress as PMCs.
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Two transcription factors in the micromere GRN, alx1
(Ettensohn et al., 2003) and
ets1 (Kurokawa et al.,
1999), are known to be essential for specifying early micromeres.
We examined the mRNA expression of these genes in the presence of SnaMASO by
WMISH and QPCR. As shown by WMISH (Fig.
5A-D) and corroborated by QPCR (data not shown), the expression
level of both genes did not change in Sna morphants. Moreover, they continued
to be expressed in the PMC precursors in the central region of the vegetal
plate (Fig. 5B,D), even though
in the absence of Snail, the PMCs failed to ingress
(Fig. 5A,C). These data suggest
that the PMCs are correctly specified at least to the level of these
transcription factors, and further suggest that snail may function
downstream of alx1 and ets1. This hypothesis was tested
next.
The mRNA expression level of Lvsnail was measured in Alx1
morphants and in embryos treated with a MEK inhibitor, U0126, which disrupts
the ERK signaling pathway and abolishes the activity of the Ets1 protein
(Rottinger et al., 2004). In
Alx1 morphants, embryos showed no PMC ingression, and exhibited reduced mRNA
expression of Lvsnail as shown by WMISH
(Fig. 6A,B). In U0126-treated
embryos, however, the expression of Lvsnail appeared to be normal
regardless of the block to PMC ingression
(Fig. 6C,D). Each of these
results was corroborated by QPCR (Table
1). Furthermore, Lvsnail expression was not affected by
injecting a dominant-negative form of Ets1 as measured by QPCR
(Table 1). These data indicate
that Lvalx1, but not Lvets1, is an upstream regulator of
Lvsnail expression in the micromere GRN.
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;
Oliveri et al., 2003).
Overexpression of Pmar1 transforms most cells of the embryo into PMCs
(Oliveri et al., 2002).
Therefore, it was hypothesized that SnaMASO would block this Pmar1
ectopic-expression phenotype, if, in fact, Snail is required for EMT.
Accordingly, Lvpmar1 was cloned and injected into eggs. Compared with
a normal control (Fig. 8A),
most cells of the Lvpmar1-injected embryo were converted into PMCs
when Pmar1 was ectopically expressed (Fig.
8B), as expected from Oliveri et al.
(Oliveri et al., 2002). In
Pmar1-overexpressing embryos, we next measured the mRNA expression of
Lvsnail, Lvalx1 and Lvmsp130 by QPCR, and as expected, the
expression level of all three genes was upregulated
(Fig. 8D). WMISH of
Lvsnail showed a ubiquitous expression, also as might be expected for
a gene downstream of pmar1 (data not shown) and consistent with the
Oliveri et al. conclusion that most or all cells were converted into PMCs.
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Because many studies in cell culture of tumor cells and in mammals showed
evidence that Snail controls EMT in part by repressing E-cadherin expression
(Batlle et al., 2000;
Cano et al., 2000;
Carver et al., 2001), we next
asked if a similar repression or downregulation occurs in sea urchins. First,
although Snail family genes act as transcription repressors, to confirm that
Snail functions as a repressor in sea urchin, we converted LvSnail into an
obligate repressor by combining the Engrailed repressor domain with the
DNA-binding domain of LvSnail protein, and injected the mRNA of this fusion
construct into fertilized eggs. As expected, the observed phenotypes were the
same as seen in wild-type LvSnail-injected embryos
(Fig. 7C; data not shown).
These data support the hypothesis that LvSnail normally functions as a
repressor in the embryo. Next, to examine whether LvSnail downregulates the
expression of Cadherin, we measured and compared the mRNA expression of
LvG-cadherin in Pmar1-injected embryos and Pmar1/SnaMASO co-injected embryos
by QPCR. It was necessary to use Pmar1 as a tool because LvG-cadherin
expression is ubiquitous in the embryo, and a detectable change was not
observed by QPCR in Sna morphants, where PMCs constitute only 5% of the cells.
In Pmar1-injected embryos, the expression level of LvG-cadherin showed a
significant decrease by QPCR (a signature of mesenchyme cells), whereas an
increase of the LvG-cadherin expression was observed in embryos co-injected
with Pmar1 and SnaMASO, which is also consistent with the rescued phenotype
(Fig. 8C). This experiment
demonstrates, therefore, that Snail downregulates the expression of Cadherin
at PMC ingression in sea urchin embryos.
LvSnail positively regulates cadherin endocytosis in PMCs
At PMC ingression, the junction-associated cadherin-catenin complex is
completely endocytosed (Miller and McClay,
1997a; Miller and McClay,
1997b) and removed from the cell membrane of mesenchyme cells. As
the absence of LvSnail severely blocks PMC ingression, it is possible that
LvSnail may play a role in regulating that endocytosis process. To test this
hypothesis, a GFP reporter construct (CadTM-GFP) was used. CadTM-GFP
recapitulates the apical localization of the cadherin complex when expressed
(Fig. 8E) and also forms
punctate foci of the intracellular GFP signal when endocytosed in ingressing
PMCs (arrowheads in Fig. 8F,G).
Using this construct the following experiment was performed as shown in
Fig. 8H. Pmar1 and CadTM-GFP
were co-injected into the eggs and followed by injection at the two-cell stage
with SnaMASO (with rhodamine-dextran, shown in red) into only one blastomere
(also see Fig. 8H). In about
60% of SnaMASO two-cell injection embryos (n=70), SnaMASO
successfully blocked PMC ingression in the injected half, and most ingressing
PMCs came from the other half, where no SnaMASO was present; injecting
rhodamine-dextran only had no effect (data not shown). Examination of
mesenchyme cells in these embryos shows almost no punctate intracellular GFP
to be present in the rhodamine-marked cells (those containing SnaMASO);
punctuate intracellular GFP is also largely excluded from those few
rhodamine-marked cells that do ingress
(Fig. 8F,G; two different
embryos), whereas punctate endocytic vesicles were present in all ingressed
cells expressing the CadTM-GFP construct alone. These results indicate that
SnaMASO impairs the endocytosis of cadherin. Therefore, Snail does indeed
positively regulate the process of cadherin endocytosis, although the exact
mechanism is still not understood.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In sea urchin
embryos, EMTs occur at PMC ingression and during the formation of some SMCs.
In this study, we have characterized a Snail gene in the sea urchin embryo,
and show that Snail is required for PMC ingression, which is consistent with
Snail being an evolutionarily conserved modulator of cell movements, rather
than determining cell fates
(Barrallo-Gimeno and Nieto,
2005). Moreover, with the micromere GRN, perturbation studies
place Snail as a link between specification and morphogenesis of PMCs. Snail
is expressed late in specification of micromeres, just before ingression is
launched.
Sea urchin Snail function is required for PMC ingression
The conclusion that Snail is required for PMC ingression is supported by
several independent observations. First, functional knockdown of Snail with
SnaMASO blocks the first event in PMC morphogenesis (EMT/ingression)
(Fig. 3), as well as the
expression of several PMC differentiation genes
(Fig. 5G). Second, the SnaMASO
chimeric experiments demonstrate that Snail function is necessary in
micromeres for ingression to occur (Fig.
4), as Snaildeficient micromere descendants do not migrate into
the blastocoele of a normal host embryo, and stay at the tip of the
archenteron (Fig. 4E,E');
absence of Snail everywhere else in the embryo has no effect on the
Snail-expressing micromere, which retains its ability to ingress
(Fig. 4F,F'). Taken
together, these data clearly indicate that Snail is involved in control of the
EMT process, particularly during PMC ingression.
Although Snail family genes are known to be involved in EMTs in metastatic
progression of tumors, mesoderm development, and neural crest cell migration
in vertebrates (for reviews, see
Barrallo-Gimeno and Nieto,
2005; Hemavathy et al.,
2000; Nieto,
2002), our finding here is nonetheless intriguing as this is the
first report to date showing that an invertebrate Snail gene controls a bona
fide EMT (i.e. PMC ingression), which is characterized by cells migrating as
individual cells through the extracellular matrix. Snail also has been shown
to be involved in cell movements that do not require a full EMT in different
experimental models. During mesoderm formation in Xenopus embryos,
for example, individual mesenchymal cells are not formed, but rather a mass of
sheet-like epiblast cells penetrate the blastocoele (i.e. involution), during
which cells maintain contact with each other while migrating
(Keller et al., 2000).
Likewise, during gastrulation in Drosophila, the cells of the
invaginating ventral furrow give rise to mesoderm. This migration of the
presumptive mesoderm occurs as a group of cells, where cell-cell adhesion is
reduced but maintained due to a switch in expression from E- to N-Cadherin
(Oda et al., 1998). Thus, the
cell population remains adherent enough to move as an intact sheet. Other
similar processes can be observed in hair bud formation
(Jorda et al., 2005), or wound
healing (Savagner et al.,
2005) in mice. Hence, Snail genes are involved not only in full
EMTs in most deuterostome lineages, but also in various types of cell
movements throughout the animal kingdom
(Barrallo-Gimeno and Nieto,
2005; De Craene et al., 2005b).
Downregulation of cadherin expression by Snail is conserved in sea urchins
Drosophila and mouse Snail mutants fail to downregulate the
expression of the cell adhesion molecule E-cadherin
(Carver et al., 2001;
Oda et al., 1998) during
gastrulation, and, in mammalian epithelial cells, Snail protein has been shown
to bind to the promoter region of E-cadherin, and repress its
transcription (Batlle et al.,
2000; Cano et al.,
2000). In sea urchin Sna morphants, a failure to downregulate
cadherin expression occurs and this, in part, may explain the inability of
PMCs to ingress when Snail is eliminated. This hypothesis is strongly
supported by the outcome from the Pmar1/SnaMASO co-injection rescue experiment
(Fig. 8), which allowed us to
examine the repression (directly or indirectly) of cadherin by Snail in vivo,
under conditions where all or most cells of the embryo were converted to
micromeres. Thus, the downregulation (or repression) of cadherin expression by
Snail in association with cell movement appears to be well conserved in
insects, sea urchins and vertebrates (this study)
(Oda et al., 1998;
Carver et al., 2001;
Jamora et al., 2005;
Yamashita et al., 2004).
Indeed, E-cadherin loss of expression leads to tumor progression
(Perl et al., 1998), and the
transcriptional repression of cadherin expression by Snail plays a major role
in the EMTs (Batlle et al.,
2000). However, cadherins can be functionally inactivated by other
different mechanisms, such as post-translational control
(Cavallaro and Christofori,
2004). Moreover, a recent publication has shown that p38
downregulates E-cadherin post-transcriptionally during mouse gastrulation
(Zohn et al., 2006), although
in sea urchin, loss of p38 activity affects neither the ingression of PMCs nor
SMCs (Bradham and McClay,
2006).
|
;
Lu et al., 2003;
Janda et al., 2006). In the
sea urchin embryo, cadherin endocytosis occurs during PMC ingression
(Miller and McClay, 1997a),
and this appears to be positively regulated by Snail (as shown in
Fig. 8E-G). Thus, Snail
regulates cadherin removal from cell membranes via endocytosis, and it also
regulates termination of cadherin transcription, both of which enable the
transformation and maintenance of the mesenchyme cell phenotype. Snail
therefore orchestrates a double mechanism to eliminate cadherin during the EMT
process. Lvsnail is expressed not only in PMCs but in other mesodermal cell populations as well. Some secondary mesenchyme cells (e.g. pigment cells and blastocoelear cells) also undergo a similar EMT process prior to their migratory behaviors. Preliminary data from SnaMASO perturbation studies suggest that Snail is involved in those EMTs as well (see Fig. S3 in the supplementary material). Thus, it is highly possible that Snail plays an essential role upstream of each EMT event during sea urchin embryogenesis. In addition Snail might also be involved in other aspects of development, given its dynamic expression pattern. These potential functions were not explored here, but warrant further analyses.
Snail acts downstream of Pmar1 and Alx1 in the micromere-PMC GRN
Our results show that Pmar1 and Alx1 are positive regulators of Snail mRNA
expression, and the ability of both transcription factors to influence PMC
ingression operates through Snail (see Fig.
9).
Pmar1 responds to the maternal ß-catenin signal and initiates the
entire PMC GRN (Oliveri et al.,
2003). Overexpression of Pmar1 significantly elevates the
expression level of Snail (Fig.
8D), as expected if Pmar1 is upstream of Snail. Snail is also
downstream of Alx1, an important PMC specifier, known to be under control of
the Pmar1 de-repression system (Ettensohn
et al., 2003). In the absence of Alx1, Snail expression decreases
significantly (Fig. 6A,B),
whereas Alx1 expression is unaffected in Sna morphants
(Fig. 5A,B). Further, Snail
expression rescues Alx1 knockdown, at least partially. These results strongly
support the notion that Snail acts downstream of micromere specification, but
upstream of, and is required for, PMC ingression. Snail mRNA injection rescues
the ingression of Alx1-depeleted micromeres in about 60% of the chimera
embryos (Fig. 7). This partial
rescue suggests that Alx1 regulates PMC ingression partly through Snail but
may require other unknown gene targets, thereby preventing a full rescue
phenotype. Nevertheless, the rescue observed here further validates the
designated position of Snail in the PMC GRN
(Fig. 9).
Ets1 has been shown to impact PMC ingression when perturbed
(Kurokawa et al., 1999;
Rottinger et al., 2004). Our
data show that Snail is neither upstream nor downstream of Ets1. Ets1 mRNA
expression is unaffected in the absence of Snail
(Fig. 5C,D), and Snail mRNA
expression is also unaltered in U0126-treated embryos
(Fig. 6C,D). These results lead
us to conclude that, even though Ets1 is an important upstream regulator of
PMC formation, the effect of Ets1 on PMC ingression does not function through
Snail, but likely through other unidentified factors, which are also involved
in the EMT process of PMCs (denoted as Y in
Fig. 9).
There are several additional transcription factors already identified in
the PMC GRN, including dri (Amore
et al., 2003), hnf6
(Otim et al., 2004) and
tbr (Croce et al.,
2001; Fuchikami et al.,
2002). Although these genes are expressed earlier than Snail, they
cannot be upstream regulators of Snail, as perturbation of these genes fails
to show any effect on PMC ingression. Instead these transcription factors
primarily affect the skeletogenic differentiation of PMCs. It is clear that
many of the PMC components are specified in a pathway that is independent of
Snail expression so it is not surprising that a substantial number of
micromere transcription factors operate independently of Snail and do not
require Snail for their function.
Three PMC terminal differentiation genes were examined in this study. Our
data show that Snail positively influences sm30, sm50 and
msp130 expression. Given the fact that Snail itself is a
transcriptional repressor, Snail must indirectly regulate these genes by
repressing an (or some) unknown repressor(s), a regulatory device that is
similar to the Pmar1 de-repression system. The initiation of sm30
expression may be relatively proximal to Snail function, as sm30
transcripts accumulate immediately after PMC ingression
(Guss and Ettensohn, 1997),
and Snail is expressed in PMC clusters
(Fig. 2F,G), where
sm30 is also highly expressed later at prism stage
(Guss and Ettensohn, 1997). On
the other hand, both sm50 and msp130 expression are
initiated earlier than, and therefore independently of, Snail expression
(Guss and Ettensohn, 1997)
(data not shown). Thus it is likely that Snail regulates the maintenance of
expression of these genes in PMCs at ingression. Further identification of
Snail target genes will help elucidate relationships between Snail and the PMC
differentiation program, and also help unravel the connection between pre- and
post-EMT gene network states in PMCs.
A subnetwork of EMT genes controls PMC ingression
The Pmar1>Alx1>Snail>Cadherin hierarchical regulatory relationship
we show here reveals one trajectory through the micromere GRN. That trajectory
is necessary for ingression, but it is not the exclusive pathway required, as
evidenced by the Ets1 data and by the incomplete rescue of Alx1 morphants.
Even so, the functional analyses of Snail and perturbation studies establish a
tight link between early micromere specification and PMC ingression
(Fig. 9). Previous studies of
several PMC regulatory genes in the GRN, including pmar1, alx1 and
ets1, all focused on their functions on specifying micromeres in
advance of the differentiation of PMCs. Here, we show that in addition to
preparation for differentiation, the specification through Snail enables the
cells to transit into morphogenesis. Other transcription factors govern
various mechanisms of differentiation independently of the pathway through
Snail.
With Snail occupying an important role in regulating PMC ingression, this
study provides groundwork for investigating the molecular basis of EMT in
PMCs, and further strengthens the hypothesis that a group of genes controls
the EMT of PMCs in the sea urchin embryo [as previously suggested in
Fernandez-Serra et al. (Fernandez-Serra et
al., 2004)]. We propose that this subnetwork of EMT genes
(including snail), which functions downstream of the micromere-PMC
specification program, attenuates cell-cell adhesion
(Fink and McClay, 1985;
Hertzler and McClay, 1999),
and upregulates molecules associated with cell motility changes, such as Rho
GTPases (Liu and Jessell,
1998) and metalloproteinases (MMPs)
(Yokoyama et al., 2003;
Miyoshi et al., 2004;
Jorda et al., 2005;
Ingersoll and Pendharkar,
2005). Eventually, this complex subnetwork orchestrates an EMT
event by summing up the spectrum of molecular and cellular changes, and then
triggers PMC ingression.
Future investigations both in PMC formation in the sea urchin and in comparative studies, will be of value to further understand how other transcription factors operate with Snail to engage the EMT mechanism. Thus, construction of a more complete pre-EMT subnetwork will indeed contribute to the understanding of the mechanism(s) controlling PMC ingression in the sea urchin, and also provide useful insight into the complex molecular strategies that regulate EMT events in other organisms.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1061/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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