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First published online 1 August 2007
doi: 10.1242/dev.009092
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Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
* Author for correspondence (e-mail: ettensohn{at}andrew.cmu.edu)
Accepted 15 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sea urchin embryo, Pattern formation, Gene regulatory networks, Primary mesenchyme, Transfating, Regulative development
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hörstadius, 1939).
Blastomere isolation and recombination experiments demonstrated that the fates
of cells in the early embryo are not rigidly fixed, but are influenced by
signals from neighboring cells. This developmental plasticity seems at odds,
however, with evidence that: (1) cell fates are biased at early stages; (2)
embryonic patterning is entrained by molecular asymmetries within the
unfertilized egg; and (3) distinct domains of differential gene expression
arise very early in development. The classical embryological investigations
that revealed the regulative properties of cleavage-stage embryos also
demonstrated biases in the developmental programs of blastomeres. Other
studies have shown that the unfertilized egg is polarized along the
animal-vegetal (AV) axis (reviewed by
Ettensohn and Sweet, 2000;
Brandhorst and Klein, 2002;
Angerer and Angerer, 2003) and
recent work suggests that the oral-aboral (OA) axis may also be entrained
during oogenesis (Coffman et al.,
2004). Thus, in sea urchins as in many other metazoans, the
polarity of the unfertilized egg serves as a scaffold upon which the pattern
of the early embryo is elaborated.
The partitioning of the sea urchin embryo into distinct territories of gene
expression begins during cleavage. Transcription commences immediately after
fertilization and reaches its maximum rate after only four to five cleavage
divisions (Davidson, 1986). The
first territories of differential gene expression appear by the 16-cell stage
(Di Bernardo et al., 1995;
Nasir et al., 1995;
Oliveri et al., 2002;
Wang et al., 1996;
Wikrmanayake et al., 2004). Other manifestations of molecular asymmetry are
also evident at this stage, including the polarized nuclear accumulation of
ß-catenin and SoxB1 (Angerer et al.,
2005; Weitzel et al.,
2004). These findings raise an intriguing question: how can
maternal polarity, early developmental biases, and the appearance of distinct
territories of gene expression during cleavage be reconciled with the
plasticity of cell fates that is observed during development?
Recent work concerning the early patterning of the sea urchin embryo has
focused on transcriptional gene regulatory networks (GRNs)
(Davidson et al., 2002;
Oliveri and Davidson, 2004).
One of the best-studied of these is the micromere-primary mesenchyme cell
(PMC) GRN, which underlies the development of the embryonic skeleton. An early
input into the micromere-PMC GRN is ß-catenin, a maternally supplied
protein which is stabilized preferentially in micromeres, most likely through
the polarized localization of maternal regulators of canonical Wnt signaling
(Logan et al., 1999;
Weitzel et al., 2004;
Ettensohn, 2006).
ß-catenin and its partner, LEF-TCF, are required for the expression of
pmar1, the earliest zygotically expressed component of the
micromere-PMC GRN (Kitamura et al.,
2002; Nishimura et al.,
2004; Oliveri et al.,
2002; Oliveri et al.,
2003; Yamazaki et al.,
2005). pmar1 is probably activated directly by
ß-catenin (Nishimura et al.,
2004) and appears to be the only critical target of this protein
with respect to PMC specification (Oliveri
et al., 2003).
Pmar1 is a transcriptional repressor and activates downstream genes in the
PMC GRN by a double-repression mechanism
(Oliveri et al., 2002). Early
targets include genes encoding the transcription factors Alx1
(Ettensohn et al., 2003),
Ets1/2 (Kurokawa et al., 1999)
and Tbr (Fuchikami et al.,
2002). alx1, which encodes a Paired-class
homeodomain-containing protein, is expressed in the large daughter cells of
the micromeres (the founder cells of the PMC lineage) as soon as they are
born. alx1 is required for PMC ingression and skeletogenesis, and
regulates these behaviors via downstream targets that include snail
(Wu and McClay, 2007) and
deadringer (Amore et al.,
2003). Terminal genes in the PMC network encode proteins that
directly regulate the morphogenetic behaviors of the cells, including
ingression, migration, fusion and skeletogenesis. Of these, the best
understood are biomineralization genes, many of which have now been identified
through genome-wide analysis (Livingston
et al., 2006; Wilt and
Ettensohn, 2007).
Although the skeletogenic GRN is normally deployed only in the prospective
PMCs, a striking feature of sea urchin development is that every lineage of
the early embryo has the capacity to activate this GRN. A variety of surgical
and molecular manipulations have revealed this developmental plasticity. For
example, removal of micromeres at the 16-cell stage results in transformation
of macromere-derived cells to a skeletogenic fate
(Hörstadius, 1939;
Sweet et al., 1999). Even
animal blastomeres can be induced to activate the skeletogenic GRN by treating
the cells with LiCl (Livingston and Wilt,
1989), by misexpressing Pmar1 or the signaling molecule Delta
(Oliveri et al., 2002;
Sweet et al., 2002) or by
exposing animal blastomeres to inductive signals from micromeres
(Minokawa et al., 1997).
Remarkably, some populations of cells retain the capacity to activate the PMC
GRN even after the onset of gastrulation. Microsurgical removal of PMCs at the
early gastrula stage causes a subpopulation of non-skeletogenic mesoderm (NSM)
cells to switch to the PMC fate (Ettensohn
and McClay, 1988; Ettensohn,
1990; Ettensohn,
1992). Surgical removal of both PMCs and NSM cells at the
archenteron tip leads to fate-switching by presumptive endoderm cells
(McClay and Logan, 1996).
These studies indicate that many cells in the vegetal region retain the
capacity to activate the skeletogenic program long after the onset of zygotic
transcription and many hours after distinct GRNs have been activated in
various vegetal territories of the embryo.
In this study, we have taken advantage of information that has emerged concerning the architecture of the micromere-PMC GRN to begin to dissect the molecular basis of plasticity in the early embryo. Our studies reveal that many features of the micromere-PMC GRN are faithfully recapitulated in transfating NSM cells. We provide evidence, however, that different inputs activate this GRN during normal and regulative development. We demonstrate that NSM cells, which appear to deploy a basal mesodermal GRN that shares several common elements with the PCM GRN, lack only an alx1-regulated sub-program for expression of a PMC fate. One hypothesis is that the regulative deployment of the PMC GRN in NSM cells is a vestige of an ancestral program of embryonic skeletogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Microsurgery
Blastomere isolation and recombination experiments were performed as
described by Sweet et al. (Sweet et al.,
2004). Animal caps were obtained from 16-cell stage embryos. PMCs
were removed from mesenchyme blastula stage embryos following the method of
Ettensohn and McClay (Ettensohn and
McClay, 1988).
Whole-mount in situ hybridization (WMISH)
WMISH was carried out according to the method of Zhu et al.
(Zhu et al., 2001) with the
modification that formalin was typically used as the fixative instead of
glutaraldehyde. Formalin-fixed specimens were fragile but usually exhibited
little background staining. Embryos were fixed for 1 hour at room temperature
in freshly prepared 20% (vol/vol) formalin (37% formaldehyde solution) in
seawater (SW) and then permeabilized in 100% methanol for 10 minutes at
-20°C. Fixed embryos were stored overnight at 4°C in 70% ethanol
before being processed further.
Microinjection of morpholino oligonucleotides (MOs), mRNAs and fluorescent dextrans
Microinjection of MOs and mRNAs into fertilized eggs was carried out as
described by Cheers and Ettensohn (Cheers
and Ettensohn, 2004). LvAlx1 MO was described previously
(Ettensohn et al., 2003). MO
injection solution contained 2-4 mM LvAlx1 MO, 20% glycerol and 0.1% Rhodamine
dextran in water. mRNAs were injected at 0.1-4.0 mg/ml in 20% glycerol. mRNA
injection solutions were prepared using RNase-free water. Microinjection of
fluorescent dextran (5% lysine-fixable fluorescein dextran,
Mr=10,000; Invitrogen) into macromeres was carried out
according to the method of Ruffins and Ettensohn
(Ruffins and Ettensohn,
1996).
U0126 experiments
U0126 (Calbiochem) was prepared as a 5 mM stock in DMSO and stored at
4°C. Working dilutions (6-25 µM) were prepared in SW just before use.
Embryos were placed in U0126 at the 2-cell stage or following PMC removal at
the mesenchyme blastula stage, and were raised continuously in the dark in the
presence of the drug. In control experiments, embryos were allowed to develop
in equivalent concentrations of DMSO.
RT-PCR
RT-PCR using samples of five to ten embryos was carried out according to
the method of Cheers and Ettensohn (Cheers
and Ettensohn, 2005). PCR primers for detection of
Lvpmar1 mRNA were: 5'-TGTTCAACGACAACCAGTATCCTG-3'
(forward primer) and 5'-CACGACGCCCAACTTCTTTG-3' (reverse
primer).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
To show directly that other downstream, skeletogenic genes in the PMC GRN
are activated during transfating, we analyzed the expression of Lvsm30,
Lvsm50, Lvp16 and Lvp58
following PMC removal. LvSM30 and
LvSM50 are secreted proteins occluded within the calcified spicule
(Wilt and Ettensohn, 2007).
LvP16 and LvP58 are novel, PMC-specific transmembrane proteins
(Illies et al., 2002) (C.A.E.,
unpublished observations). LvP16 plays an essential role in skeletal rod
elongation (Cheers and Ettensohn,
2005). LvP58 was identified through an in situ
hybridization screen and its function is unknown. Each of the four mRNAs is
expressed specifically by cells of the large micromere-PMC lineage during
normal development, as assessed by WMISH.
Lvsm30, Lvsm50, Lvp16 and Lvp58 mRNAs accumulated
in NSM cells during transfating (Fig.
1). Moreover, at late developmental stages, levels of Lvsm30,
Lvsm50 and Lvp16 mRNAs were spatially regulated within the
skeletogenic syncytium in a pattern that appeared identical to that normally
exhibited within the PMC syncytium (Guss et al., 1997;
Illies et al., 2002). For
example, Lvsm30 mRNA was expressed at high levels in the
ventrolateral PMC clusters at the late gastrula stage but at much lower levels
in the ventral PMC chain (Fig.
1C). Lvsm50 was expressed at high levels in the scheitel
region at the pluteus stage (Fig.
1D).
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) and
in the course of this study cloned Lvtbr and Lvets/2. Lvalx1
is expressed only by cells of the large micromere-PMC lineage
(Ettensohn et al., 2003) and
we confirmed that Lvtbr expression is also restricted to PMCs
(C.A.E., unpublished observations). ets1/2 mRNA has been reported to
be restricted to PMCs in Hemicentrotus pulcherrimus
(Kurokawa et al., 1999) but is
expressed by both PMCs and NSM cells in Paracentrotus lividus
(Rottinger et al., 2004) and
Strongylocentrotus purpuratus
(Rizzo et al., 2006). We found
that, in L. variegatus, ets1/2 mRNA accumulates in both PMCs and NSM
cells and we used a polyclonal antiserum to show that LvEts1/2 protein
accumulates in the nuclei of both cells types (data not shown). Because these
experiments showed that NSM cells normally express Lvets1/2
during gastrulation, we focused on the possible activation of the two other
genes, Lvalx1 and Lvtbr during transfating. Expression of both Lvalx1 and Lvtbr was detected in transfating cells after microsurgical removal of PMCs (Fig. 2). WMISH signal was more robust for Lvalx1 and it was therefore possible to detect Lvalx1 mRNA at much earlier stages of transfating. Lvalx1 mRNA was first detectable in cells near the tip of the archenteron 3-4 hours after PMC removal (Fig. 2C). In many embryos, it appeared that Lvalx1 expression was not radially symmetrical around the gut but was concentrated on one side (Fig. 2D,F). This was not observed in all cases, but may have been obscured in some embryos because of their orientation. Lvtbr mRNA was more difficult to detect by in situ hybridization, presumably because this mRNA was not as abundant as Lvalx1 mRNA. Nevertheless, 9-10 hours after PMC removal, it was clear that Lvtbr mRNA had accumulated in transfated mesenchyme cells that were arranged in a ring pattern characteristic of PMCs (Fig. 2H).
Alx1 is required in transfating cells
We next asked whether Lvalx1 function was required to activate the
skeletogenic GRN in NSM cells. We showed previously that injection of Alx1
morpholino (MO) into fertilized eggs completely blocked the formation of
skeletal elements (Ettensohn et al.,
2003). This indicated that NSM transfating did not occur in LvAlx1
MO-injected embryos, but did not prove that this was a direct consequence of
blocking LvAlx1 expression in NSM cells. In particular, we could not rule out
the possibility that the signal produced by large micromere descendants that
suppresses NSM transfating might still be transmitted between the two cell
populations in Alx1 MO-injected embryos.
|
). In
control experiments, we confirmed that >90% of micromere-deficient embryos
produced a normally patterned skeleton (19/21 cases;
Fig. 3F,G).
Micromere-deficient, Alx1 MO-injected embryos, however, showed no transfating
response even after extended culture periods (2-3 days). Micromere-deficient,
LvAlx1 MO-injected embryos gastrulated and gave rise to various NSM
derivatives, but almost no 6a9-positive cells formed in these embryos, which
also failed to form skeletal elements (67/70 cases)
(Fig. 3H,I). LvAlx1 MO was
present in mesomeres (in addition to macromeres) in this experiment, but
Lvalx1 is not expressed in mesomere progeny following PMC removal
(Fig. 2). Therefore, these
experiments indicate that Lvalx1 function is required autonomously in
macromere-derived cells for activation of the skeletogenic GRN.
We also tested whether Lvalx1 function was required for activation
of the micromere-PMC GRN in animal blastomeres
(Fig. 4). Mesomeres give rise
to skeletogenic cells when treated with LiCl
(Livingston and Wilt, 1989).
LiCl probably exerts this effect by stabilizing ß-catenin
(Kao and Elinson, 1998;
Logan et al., 1999). We
confirmed that animal caps isolated from 16-cell stage embryos and cultured in
seawater gave rise only to ectoderm (Fig.
4B,E), When animal caps were treated with 50 mM LiCl for 3 hours
immediately after isolation, however, they usually gave rise to gastrulae and
63% of these embryos (92/146 cases) eventually formed normally patterned
skeletons (Fig. 4C,F). LiCl
treatment also induced the expression of Lvalx1 and Lvp16, a
target of Lvalx1 (Cheers and
Ettensohn, 2005), in isolated animal caps
(Fig. 4H). In contrast, animal
caps isolated from LvAlx1 MO-injected embryos did not give rise to
6a9-positive cells or skeletal elements when exposed to LiCl under identical
conditions (56/57 cases), although LiCl still induced the formation of guts in
70% of the embryos (Fig.
4D,G). These findings demonstrate that Lvalx1 is required
for ectopic activation of the PMC GRN in animal blastomeres.
alx1 regulates PMC ingression and the activation of skeletogenic
genes (Ettensohn et al.,
2003). We asked whether the PMC-derived signal that suppresses NSM
transfating might also be regulated by alx1
(Fig. 5). Recombinant embryos
were produced by grafting micromere quartets to animal caps isolated from
16-cell stage embryos (Fig.
5A). Previous studies showed that micromeres induce animal
blastomeres to give rise to vegetal cell types, including cells that transfate
to a skeletogenic phenotype upon removal of the micromere descendants
(Minokawa et al., 1997).
Micromere quartets were isolated from embryos that had been injected with
fluorescent dextran alone (controls), or co-injected with fluorescent dextran
and LvAlx MO. In control embryos, fluorescent dextran-labeled micromeres gave
rise to PMCs and these formed extensive skeletal elements (16/17 cases;
Fig. 5B,E). When the micromeres
contained LvAlx1 MO, however, their descendants remained associated with the
tip of the archenteron or ingressed into the blastocoel
(Fig. 5C,F). Large numbers of
6a9-positive cells formed in these embryos and gave rise to skeletal elements
(Fig. 5D,G), but these cells
were not labeled with fluorescent dextran and were therefore derived
exclusively from the animal cap (15/19 cases). These experiments demonstrate
that the ability of micromere progeny to suppress NSM transfating is dependent
on LvAlx1 function. One possibility is that the gene encoding the PMC-derived
signaling molecule is regulated (directly or indirectly) by Lvalx1.
Alternatively, Lvalx1 may control aspects of the motility or
morphogenesis of the large micromere descendants that mediate signaling. For
example, ingression and filopodial extension by the PMCs might be required for
signaling. Although many micromere progeny ingress even when LvAlx1 expression
is blocked (Fig. 5C,F), their
migration is delayed.
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;
Rottinger et al., 2004).
Treatment of embryos with U0126, a MEK inhibitor, blocks PMC specification in
both S. purpuratus and P. lividus. Overexpression of a
dominant negative form of MEK produces a similar phenotype. We confirmed that treatment of L. variegatus embryos with U0126 suppressed PMC formation (Fig. 6A-F). Continuous exposure of L. variegatus embryos to U0126 (6-25 µM) from the 2-cell stage blocked PMC ingression and greatly reduced the numbers of 6a9-positive cells at the late gastrula stage (controls: 68.8±16.6, mean ± s.d., n=30; U0126-treated: 1.3±2.7, n=300). U0126 treatment also blocked skeletogenesis, as reported previously. Some batches of embryos treated with a relatively high concentration of U0126 (25 µM) exogastrulated, whereas at lower concentrations the archenteron invaginated, albeit in a delayed fashion. The reduction in 6a9-positive cells was not due simply to a delay in PMC specification, as numbers of 6a9-positive cells in U0126-treated embryos remained very low even when embryos were cultured for 2-3 days.
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Overexpression of LvAlx1 is sufficient to induce transfating of macromere-derived cells
Several genes in the PMC GRN are expressed by NSM cells during gastrulation
(see Discussion). Because Lvalx1 is an exception and a critical
component of the network, we tested whether ectopic expression of this gene
might be sufficient to convert NSM cells to a skeletogenic fate.
We overexpressed LvAlx1 throughout the embryo by microinjecting mRNA into fertilized eggs. Overexpression of LvAlx1-GFP confirmed that the protein was localized in the nuclei of all cells, including cells within the vegetal plate (Fig. 7A,B). Embryos injected with wild-type Lvalx1 (Lvalx1.WT) mRNA (untagged) at concentrations of 0.4-0.75 mg/ml showed a reproducible increase in numbers of 6a9-positive cells when examined at the late gastrula stage (Fig. 7C-E, Fig. 8). In some cases, very large numbers of 6a9-positive cells were observed (>100 cells). The 6a9-positive cells were usually arranged in a radially symmetrical, circumferential ring between the equator and the vegetal pole. These cells also expressed Lvp16 (Fig. 7F,G). Injection of higher concentrations of Lvalx1 mRNA (1.5 mg/ml or higher) delayed development and resulted in decreased numbers of 6a9-positive cells, whereas concentrations below 0.1 mg/ml had no effect on development. In control experiments, we injected 20% glycerol (the mRNA carrier solution) or similar concentrations of a mutant form of Lvalx1 mRNA with a stop codon introduced immediately upstream of the homeodomain (Lvalx1.STOP). This mRNA encoded a short (115 aa) N-terminal fragment of LvAlx1 that cannot bind to DNA. Neither reagent caused an increase in 6a9-positive cells (Fig. 8). Although almost all uninjected control embryos and embryos injected with Lvalx1.STOP (31/32) formed only two tri-radiate spicule rudiments, embryos injected with Lvalx1 mRNA usually produced supernumerary tri-radiate spicules (average=4.6/embryo, n=28).
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Lvpmar1 does not activate the skeletogenic GRN network during NSM transfating
pmar1 is a critical, upstream component of the large micromere-PMC
GRN (Kitamura et al., 2002;
Nishimura et al., 2004;
Oliveri et al., 2002;
Oliveri et al., 2003;
Yamazaki et al., 2005).
Because we found that at least two genes encoding transcription factors of the
PMC GRN, Lvalx1 and Lvtbr, are expressed ectopically in
macromere-derived cells during transfating, we asked whether these genes might
be activated by pmar1.
Preliminary studies confirmed that pmar1 plays a role in PMC specification in L. variegatus similar to that observed in other species. Analysis of a BAC clone containing the Lvpmar1 locus (GenBank accession no. AC131562, Sea Urchin Genome Project BAC Clone #170H13) showed that in L. variegatus, as in other species, the pmar1 locus consists of several tandem copies of the gene (Fig. 10A). At least ten repeats of the gene are present in L. variegatus, and possibly more, as one end of the BAC insert lies within the tenth repeat. The coding sequences of the tandem copies of Lvpmar1 are very similar at the nucleotide level and we designed a single pair of PCR primers that would recognize transcripts from any of the ten genes. These primers spanned a conserved intron, thereby allowing us to unambiguously distinguish mRNA-based amplification from amplification driven by contaminating genomic DNA. PCR amplification using these primers yielded a single product of the expected size. This PCR product was cloned and sequenced, confirming that it corresponded to Lvpmar1 (data not shown).
|
). We next explored whether Lvpmar1 expression was activated in NSM cells during transfating (Fig. 10D). PMCs were removed from mesenchyme blasula stage embryos and embryo lysates were prepared 0, 3, 6 and 9 hours after surgery. Lvalx1 and Lvp16, both of which are downstream of pmar1 in the micromere-PMC GRN, are expressed at high levels in transfating cells within 9 hours after PMC depletion (Fig. 2C-G, Fig. 10D).
In three independent RT-PCR experiments carried out on different batches of PMC-deficient embryos, we were unable to detect Lvpmar1 expression (Fig. 10D). To assess the sensitivity of our RT-PCR analysis, we prepared cell lysates from normal 16-cell stage embryos, when Lvpmar1 is expressed at relatively low levels, and prepared serial dilutions of these cell lysates. We could reliably detect Lvpmar1 expression using quantities of cell lysate that were equivalent to 1/100 embryo (three independent replicates are shown in Fig. 10D). We cannot exclude the possibility that Lvpmar1 is expressed at very low levels in transfating NSM cells. These experiments indicate, however, that if pmar1 mRNA is present, it is expressed at a level less than 1% of that normally observed at the 16-cell stage.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Blastomere isolation and transplantation experiments show that many aspects of
the specification and early differentiation of micromeres, including the
activation of several terminal skeletogenic genes in the PMC GRN, are
independent of signals from other cells
(Stephens et al., 1989;
Stephens et al., 1990)
(reviewed by Ettensohn et al.,
1997). Several workers have reported that micromeres isolated at
the 16-cell stage and cultured in plain seawater (i.e. without serum or other
supplements) execute many features of their normal morphogenetic program,
including migration, fusion and the secretion of small calcareous granules
(Hodor and Ettensohn, 1998;
Okazaki, 1975;
Page and Benson, 1992). It was
therefore surprising when recent studies showed that the MAPK pathway plays an
essential, positive role in the deployment of the PMC GRN, probably through a
requirement for ERK-mediated phosphorylation of Ets1/2
(Fernandez-Serra et al., 2004;
Rottinger et al., 2004). Two
possible interpretations of these findings are that micromere descendants
activate ERK via autocrine signaling or by a ligand-independent mechanism.
The regulation of the skeletogenic GRN in NSM cells during transfating
appears different from the normal pathway in several respects. First, in NSM
cells, the skeletogenic network is tightly regulated by extrinsic signals.
These signals emanate from a different population of cells (PMCs) and, in
contrast to possible homotypic interactions within the PMC lineage
(Rottinger et al., 2004), they
act in a negative fashion to block the deployment of the skeletogenic GRN. In
NSM cells, the skeletogenic GRN remains subject to cell signaling even late in
development, after maternal regulators have presumably disappeared. The most
direct evidence that control of the GRN differs during normal and regulative
development comes from our observation that pmar1, a key activator of
the micromere-PMC GRN, is not expressed at detectable levels by NSM cells
during transfating. Our findings therefore argue that different upstream
mechanisms activate the skeletogenic GRN in micromeres and NSM cells, but
result in the same output.
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), which
has recently been shown to be encoded by the hesC gene
(Revilla-i-Domingo et al.,
2007). It will be important to determine whether hesC is
normally expressed by NSM cells at the gastrula stage. If not, then this would
imply that alx1 is normally repressed in the NSM territory by a
hesC-independent mechanism or is no longer regulated by repression.
If hesC is normally expressed by NSM cells and repressed during
transfating, then our findings suggest that such repression occurs by a
pmar1-independent mechanism, in contrast to the normal pathway.
Regulative deployment of the PMC GRN in different lineages is likely to be context dependent
Several genes in the PMC GRN are expressed by NSM cells during normal
development, including ets1/2
(Rottinger et al., 2004;
Rizzo et al., 2006),
snail (Wu and McClay,
2007) and delta
(Sweet et al., 2002). NSM
cells, like the presumptive PMCs, contain phosphorylated ERK
(Fernandez-Serra et al., 2004;
Rottinger et al., 2004);
therefore, Ets1/2 is also likely to be phosphorylated in NSM cells. Other
transcription factors have been identified that show restricted expression in
PMCs and NSM cells (e.g. erg), and a recent in situ hybridization
screen has identified new examples of genes expressed only by these cell
populations (Zhu et al., 2001)
(C.A.E., unpublished observations). The cyIIa gene is expressed by
PMCs and NSM cells (as well as endoderm cells) and a pan-mesodermal regulatory
element has been identified (Martin et
al., 2001). These observations suggest that NSM cells normally
deploy many elements of the PMC GRN but lack certain key components that
direct PMC fate specification.
One critical component of the skeletogenic GRN not normally expressed by
NSM cells is alx1 (Ettensohn et
al., 2003). Our experiments show that expression of
Lvalx1 is sufficient to induce macromere descendants to activate
downstream skeletogenic genes, including Lvp16 and genes of the
msp130 family, and renders the cells responsive to PMC-specific
guidance cues. Macromeres give rise to several cell types and we do not know
which cells within this population respond to Lvalx1 misexpression.
It is clear, however, that ectopic expression of Alx1 does not cause the kind
of global transfating response produced by Pmar1
(Oliveri et al., 2002;
Oliveri et al., 2003). We
therefore propose that in specific cellular contexts (e.g. perhaps in the
context of a `mesenchymal' GRN already deployed in NSM cells), alx1
functions as a critical regulator of a molecular subroutine that provides
inputs into several cell behaviors and activates the complete battery of
skeletogenic genes.
Regulative deployment of the PMC GRN can occur in territories of the embryo
other than the NSM. Mesomeres can be induced to activate the pathway by
treating the cells with LiCl (Livingston
and Wilt, 1989) or by ectopic activation of Notch-Delta signaling
(Sweet et al., 2002). LiCl
stabilizes ß-catenin by inhibiting GSK3ß and may activate
pmar1 and the remainder of the PMC GRN by mechanisms that essentially
mimic the normal upstream activation seen in the micromere-PMC lineage. This
is consistent with the finding that misexpression of pmar1 alone in
animal cells is sufficient to activate the PMC GRN
(Oliveri et al., 2002;
Oliveri et al., 2003).
Activation of the skeletogenic GRN in mesomeres by ectopic activation of
Notch-Delta signaling has not been investigated in detail. Activation of the
skeletogenic GRN by presumptive endodermal cells during gastrulation
(McClay and Logan, 1996) may
occur by yet another mechanism, as these cells have their own distinctive
developmental history. Further investigations of each of these regulative
pathways will be informative and may reveal common features.
The PMC GRN and the evolution of regulative processes
It is widely believed that micromeres and an early-ingressing, skeletogenic
mesenchyme are relatively recent evolutionary innovations. All adult
echinoderms have a calcified endoskeleton, suggesting that this is an ancient
feature of the phylum. Among echinoderms, however, only echinoids (sea urchins
and sand dollars) and ophiuroids (brittle stars) form an extensive embryonic
skeleton, and only echinoids form micromeres. Furthermore, the development of
cidaroid urchins (subclass Cidaroidea), a basal group within Echinoidea
(Smith et al., 2006), is
characterized by the formation of variable numbers of micromeres and a
late-ingressing skeletogenic mesenchyme
(Schroeder, 1981;
Wray and McClay, 1988).
These observations suggest that the ancestral echinoderm lacked micromeres, early-ingressing mesenchyme and an embryonic skeleton (Fig. 12, top row), but had NSM and a program of adult skeletogenesis. Early in echinoid evolution, the adult skeletogenic GRN, which probably included alx1 and many of the known biomineralization genes, was co-opted by a sub-population of NSM cells, thereby creating an embryonic skeletogenic mesenchyme (Fig. 12, middle row). At this time, the formation of embryonic skeletogenic mesenchyme was not linked to micromere formation. Subsequently, in the lineage that gave rise to euechinoids, the skeletogenic program again shifted earlier in development (Fig. 12, bottom row). At this time, activation of the GRN became tightly coupled to a strict system of unequal cleavage in the vegetal hemisphere (micromere formation). At the molecular level, new linkages (including pmar1) coupled the existing skeletogenic GRN to an ancient, maternally based system of embryo patterning (the ß-catenin system). According to this scenario, the micromere-PMC GRN was established by forging new connections between these two pre-existing molecular programs.
We speculate that as this system evolved, a mechanism also arose for suppressing the skeletogenic potential of non-micromere-derived cells, thereby restricting skeletogenic differentiation to the early-ingressing mesenchyme (Fig. 12, bottom row, white arrows). The existence of this suppressive interaction is apparently ancient and widespread within the euechinoid urchins, as it has been observed in every species that has been carefully examined (at least seven species to date). According to this hypothesis, then, the regulative transfating of NSM cells is a vestige of an ancient program of skeletogenesis. If true, then the regulation of the skeletogenic GRN in transfating NSM cells may more closely resemble the pathway that operates in the late-ingressing, skeletogenic mesenchyme of cidaroid urchins than the micromere-based system seen in modern euechinoids.
Plasticity of early patterning in other species
The co-existence of early molecular asymmetries and developmental biases on
the one hand, and plasticity/regulative potential on the other, is not unique
to sea urchin development. Indeed, it seems likely that this is a universal
feature of metazoan embryos. All animal embryos that have been well-studied
exhibit characteristics of both mosaic and regulative development, and it has
been argued that these terms have lost their utility
(Lawrence and Levine, 2006).
Recent work on early mouse development has provided strong evidence of
developmental biases and molecular specialization at the earliest stages of
cleavage (Zernicka-Goetz,
2006). These findings have sparked controversy, in part, because
of the long history of work documenting the extensive regulative properties of
mouse embryos. They are completely consistent, however, with the current
picture of early sea urchin development. As the molecular and genetic
mechanisms of early embryo patterning are elucidated we will gain a better
understanding of how these processes respond to perturbations and underlie
regulative phenomena.
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ACKNOWLEDGMENTS |
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