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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02577
1 Division of Biology, 156-29, California Institute of Technology, Pasadena, CA
91125, USA.
2 Department of Biology, Duke University, Durham, NC 27707, USA.
* Authors for correspondence (e-mail: poliveri{at}caltech.edu; dmcclay{at}duke.edu)
Accepted 9 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sea urchin, foxa, Forkhead, Regulatory network, Endomesoderm, Specification
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GRN is activated by maternal inputs at the vegetal pole, first seen as
nuclearization of ß-catenin in the micromeres and in macromeres
(Logan et al., 1999
). Between
the fourth and sixth cleavage, the early signal, the molecular nature of which
is not yet known, from the micromeres provides added input to the macromeres
to accelerate endomesoderm GRN activation
(Ransick and Davidson, 1995;
Oliveri et al., 2003). At
sixth cleavage, the veg2 tier of endomesoderm cells separates from their
sister veg1 cells, the veg2 tier occupying a more vegetal position. Between
the seventh and ninth cleavages, the innermost veg2 cells receive a Delta
signal from the adjacent micromeres, and the Notch-activated cells become
secondary mesenchyme (SMC) precursors. This Notch (N) signal functionally
distinguishes SMC from endoderm specification
(Sherwood and McClay, 1999). A
second Delta signaling event later originates in the SMC precursors,
contributes to SMC specification, and is required to define the adjacent veg2
endoderm subcompartment (Sherwood and
McClay, 1999; Sweet et al.,
2002; Peterson and McClay,
2005). In the sea urchin embryo, at least 16 transcription factors
are activated specifically in cells that become endoderm, and among these is
the foxa gene.
Foxa belongs to the forkhead family of transcription factors. Orthologous
genes have been isolated in many different species, including
Drosophila (Weigel et al.,
1989), C. elegans
(Mango et al., 1994), mouse
(Ang et al., 1993), ascidians
(Corbo et al., 1997;
Olsen and Jeffery, 1997),
hemichordates (Taguchi et al.,
2000) and cnidarians
(Fritzenwanker et al., 2004;
Martindale et al., 2004). A
common feature is that Foxa factors are restricted to endodermal cells just
prior to, or during, gastrulation, and are necessary for the specification and
differentiation of endodermal structures. Later, these factors are required in
other domains and other developing structures. In Xenopus,
foxa2/hnf3ß is initially expressed at the vegetal pole of the
embryo, but is excluded from the mesoderm during gastrulation
(Suri et al., 2004). In this
embryo during gastrulation, foxa2/hnf3ß has the major role of
determining the mesodermal boundary by repressing mesoderm fate in the
endoderm.
Sea urchin foxa was originally identified in Hemicentrotus
pulcherrimus and named hnf3
(Harada et al., 1996). The
nomenclature used here is current for Forkhead transcription factors of this
class (Kaestner et al., 2000).
In situ hybridization showed Hpfoxa to be expressed initially in the
vegetal plate, then surrounding the blastopore and finally in the gut of the
embryo. Here, we characterize the functional role of foxa in the
endomesoderm regulatory network by adding a high-resolution analysis of the
foxa expression pattern in both S. purpuratus and
Lytechinus variegatus, by performing experimental perturbations and
embryological manipulations, and by using biochemical approaches to show that
foxa has at least three major roles in the embryo: it assures that
veg2 endoderm cells do not express mesoderm genes; it is required in the
stomodeal region of the oral ectoderm for the production of the mouth; and it
provides a controlling function for postgastrular development of the gut.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). An
RNA probe for foxa was synthesized in vitro from 200 ng of PCR
fragment amplified from the 224 bp cDNA clone using the primers pSport F
(5'-GTG CTG CAA GGC GAT TAA GT-3') and pSport R (5'-TGT GGA
ATT GTG AGC GGA TA-3'). For gcm probe description, see Ransick
et al. (Ransick et al., 2002),
and for gatae probe, see Lee and Davidson
(Lee and Davidson, 2004).
Morpholino oligo antisense and mRNA injection constructs
A morpholino antisense substituted oligonucleotide (MASO) was synthesized
(Gene Tools) complementary to the sequence just upstream of the first possible
methionine, namely 5'-TGGGTTCCTCTTTGAAATCCACGAT-3'. A morpholino
standard control 5'-CCTCTTACCTCAgTTACAATTTATA-3' was provided by
Gene Tools. MASOs were injected in a 120 mM KCl solution, at final
concentrations of 50 mM to 300 mM. The foxa coding construct was made
using a vector derived from BlueScript (Statagene, La Jolla, CA), which
contained the 3'UTR and 5'UTR of the globin gene
(Lemaire et al., 1995). A
fragment containing the entire coding sequence of the foxa gene was
obtained by PCR, using the primers Foxa Cod F
(5'-CATACACATCAGTGGAGGCT-3') and Foxa Cod R
(5'-TCCATCTATAACTGGTCGTG-3'). The 1659 bp PCR fragment was
initially subcloned in pGEM-T easy vector (Promega). A foxa pGEM-T
positive clone was digested with EcoRI to release the 1659 bp
fragment with ends compatible with the EcoRI cloning site of the
pBlueScript derived vector. The orientation of the cloned fragments was tested
by PCR. To build the 5'foxa-GFP construct, a 540 bp fragment
containing 150 bp of 5'UTR and 390 bp coding sequence was amplified by
PCR, using the primers Foxa HindIII R
(5'-CCCCAAGCTTGATACGATCAATAGA-3') and Foxa KpnI F
(5'CCCCGGTACCAGGCTGACACTATATACT-3'). The GFP coding sequence was
subcloned in frame as described by Oliveri et al.
(Oliveri et al., 2002). The
template used for these PCR reactions was the BAC clone 41I19. Each construct
was checked by sequencing. RNA used for injection was synthesized as described
by Oliveri et al. (Oliveri et al.,
2002). The injection solutions were concentrated to 20 ng/µl,
50 ng/µl, or as indicated, and the RNA was injected together with
fluorescein dextran or rhodamine dextran (10 pg/pl; Sigma).
Embryo manipulation and imaging
Embryo cultures of L. variegatus or S. purpuratus, and
microsurgical procedures, were carried out as described earlier
(McClay, 2000;
Oliveri et al., 2003;
Sweet et al., 2004). L.
variegatus embryos were placed into Kiehart chambers in calcium-free sea
water, whereas S. purpuratus embryos were placed in calcium-free sea
water after two washes in Hyaline Extraction Medium. After surgery the embryos
were returned to sea water.
Quantitative PCR (QPCR)
Total RNA was isolated from batches (100-200) of embryos injected with
different MASOs and/or mRNA. The RNA was extracted using the RNeasy Micro Kit
(Qiagen) according to manufacturer's instructions. First-strand cDNA was
synthesized using random hexamers and the Taq Man Kit (PE Biosystems), as
described by the manufacturer. The cDNA was used directly for quantitative PCR
(QPCR) analysis. QPCR was conducted as previously described
(Rast et al., 2000). For all
QPCR experiments, the data from each cDNA sample were normalized against
ubiquitin mRNA levels, which are known to remain relatively constant during
development (Nemer et al.,
1991; Oliveri and Davidson,
2004a; Ransick et al.,
2002). The primers used can be found on the website
http://sugp.caltech.edu/resources/methods/q-pcr.psp
and have been previously published by Davidson et al.
(Davidson et al., 2002).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Sea urchin Foxa is a class A forkhead transcription factor. Predicted amino
acid sequences for the sea urchin Foxa proteins are 98.1% identical between
S. purpuratus and H. pulcherrimus, 94.5% identical between
S. purpuratus and L. variegatus, and 95% identical between
L. variegatus and H. pulcherrimus, over the whole length of
the protein. There is thus no doubt that these genes are true orthologs. The
phylogenetic relationship of Spfoxa, and thus Lvfoxa, to
other forkhead class transcription factors has been resolved by Tu et al.
(Tu et al., 2006).
Dynamic spatial expression of the sea urchin foxa gene
The analysis previously performed on H. pulcherrimus
(Harada et al., 1996) was
repeated to provide a higher resolution whole-mount in situ hybridization
(WMISH) expression of foxa (Fig.
1). At 18 hours of development, foxa is expressed at a
low level in the veg2 endomesoderm; the micromeres are devoid of expression
(Fig. 1A,B). By 21 hours,
expression of foxa becomes restricted to the endodermal ring
(Ruffins and Ettensohn, 1996).
The gene is transcribed unequally in the two sides of the endodermal ring
(Fig. 1C). At 21 hours and
beyond (asterisk in Fig. 1E-G),
the oral side of the endoderm, marked by the stomodeal expression, expresses a
higher level of foxa than does the aboral side. The stomodeal
expression domain was not identified in the previous analysis
(Harada et al., 1996).
Following primary mesenchyme cell (PMC) ingression
(Fig. 1D), the endoderm
expresses the gene at a high level. Thus, initially there is a transient
expression in the endomesoderm, some of which will be specified as muscle and
coelomic pouch SMCs, then, later, foxa is exclusively an endodermal
and stomodeal gene.
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Conversion of endoderm to mesoderm on interference with foxa expression
A morpholino antisense substituted oligonucleotide (MASO) complementary to
the translational start site of both the S. purpuratus and L.
variegatus foxa mRNA was tested for efficacy in arresting translation
using a GFP construct (5'foxa-GFP), which contained in-frame
the target site sequence of the gene. As illustrated in
Fig. 3J,K, the foxa
MASO very effectively blocks the translation of RNA containing the initial ATG
of the foxa message, but fails to block the translation of an altered
form (5 base changes) that no longer recognizes the MASO (data not shown). Two
different concentrations of foxa MASO were then injected into
fertilized eggs. The embryonic phenotypes observed were of increasing severity
at higher concentrations, and were equivalent in both species. Up to PMC
ingression, the foxA MASO-injected embryos appeared normal, but at
gastrulation MASO treatment caused a delay of gut invagination, and, at
increased concentrations, a complete failure of invagination
(Fig. 3A,B). If invagination
occurred in MASO-treated embryos, the foregut of the embryonic archenteron was
truncated or missing altogether (Fig.
3E,H). This region of the archenteron is most sensitive to the
absence of Foxa, consistent with the hypothesis that the high level of
foxa expression in the foregut region during gastrulation is crucial
for specification (Fig. 1I,J).
At higher concentrations of MASO the whole gut is absent, and in its place is
a small, everted, scar-like structure (Fig.
3D,G). In both species, the overall effect of foxa MASO
treatment is a reduction in the mass of endoderm. Even if invagination occurs,
the gut fails to connect with the oral ectoderm and a mouth never forms
(Fig. 3P). In addition,
MASO-treated embryos produce excess numbers of pigment cells
(Fig. 3M,N; an average increase
of 40-70%, three experiments, n=10 experimental and 10 control
embryos counted at the same stage in each experiment). These data suggest that
an absence or reduction of Foxa leads to a diversion of presumptive endodermal
cells to mesodermal fate, and a reduction in endoderm specification. The MASO
phenotypes are consistent with the expression data shown in
Fig. 1, and together with that
data confirm foxa as a key regulatory gene of the endomesoderm
GRN.
The foxa gene and the endomesoderm GRN
Table 1 shows the effects,
as reflected by QPCR, on genes that execute functions essential to
endomesoderm specification. Absence of Foxa function has very specific
effects, and the expression of the large majority of genes tested was not
quantitatively perturbed in the timeframe studied. The level of foxa
transcript itself increases sharply when foxa mRNA translation is
inhibited. Thus, the foxa gene is subject to repression by Foxa
protein. This result at least partially explains the oscillatory character of
the temporal expression profile observed for the foxa gene
(Fig. 2). An early repressive
effect was also seen on gatae at the blastula stage, when
foxa is still transiently expressed in the endomesodermal territory
and gatae is expressed in territory that will become late SMC
mesoderm (Fig. 1)
(Lee and Davidson, 2004). By
the time the expression of gatae occurs in definitive endoderm at
mesenchyme blastula stage, there is no evident foxa repression of
gatae. Expression of both gatae and endo16, a
well-known endodermal marker gene, are controlled at this stage by other known
endodermal regulators (Yuh et al.,
1994) (Table 1). At
late gastrula stage in foxa MASO-treated embryos, the gatae
and endo16 genes show a strong decrease in the level of expression,
an obvious consequence of the prior failure of endomesoderm specification
(Fig. 3). These last results
are not interpreted as evidence for direct regulatory gene interactions in
late gastrula, but as a consequence of the earlier failure of Foxa to
contribute to endoderm specification. Foxa is also an important regulator of
hedgehog (hh) expression, which is known to be expressed in
endoderm beginning just before mesenchyme blastula stage
(Walton et al., 2006).
|
) is
revealed to be a target of foxa repression in a time window that
begins after 24 hours and extends at least until 34 hours. This result
provides a direct explanation for the essential role of foxa in
confining endodermal cells to their appropriate fate, as we demonstrate in the
following experiments.
foxa repression is required to exclude mesoderm fate in endoderm cells
foxa MASO reduces gut size or eliminates it, and additional
pigment cells are observed (Fig.
3N,P). Because in normal embryos foxa is expressed
exclusively in veg2 endoderm after very early stages of development, the
implication is that a specific function of this gene is to prevent a number of
veg2 progeny from executing mesodermal fates. To test this hypothesis
directly, a mosaic analysis was performed (see
Fig. 4).
The foxa MASO was injected into a group of eggs along with Rhodamine dextran. At the 60-cell stage, two fluorescent, MASO-bearing cells from each embryonic tier were transplanted to equivalent positions in place of two normal cells in an unlabeled host embryo, as shown in the diagrams to the left in Fig. 4. These mosaic embryos were then compared with the respective mosaic control embryos, in which the fluorescent transplanted blastomeres contained no MASO. As shown in Fig. 4, control and foxa MASO animal tier, veg1 and micromeres cells behaved identically in the host embryos. However, the fate of foxa MASO-injected veg2 cells was very different from that of their controls. Transplanted veg2 blastomeres unable to generate the Foxa protein produced only dispersed mesodermal cell types, whereas the control dye-injected veg2 cells became normal gut endoderm, as well as mesodermal cells. This same outcome was observed both in embryos of S. purpuratus and L. variegatus; 24 cases of each tier in each species were scored. Some experimental veg2 transplant embryos had a few fluorescent cells in the gut, but these were always far fewer in number than in control archenterons, and these often differentiated as ectopic pigment cells or ectopic coelomic pouches, both SMC derivatives. Thus, one function of the foxa gene is to repress mesodermal specification in the subset of veg2 cells normally fated to generate gut endoderm.
Further evidence of mesoderm fate exclusion: foxa MASO effects on spatial gene expression patterns
WMISH was carried out on foxa or control MASO-injected embryos at
blastula (data not shown), mesenchyme blastula, early gastrula and late
gastrula stages (18 hours, 23-24 hours, 30-34 hours and 48 hours
postfertilization for S. purpuratus), along with non-injected
controls. The genes targeted in this series of experiments were those
implicated as being downstream targets of foxa in the QPCR
experiments (see Table 1),
namely foxa itself and the mesoderm regulator gcm
(Ransick et al., 2002). In
addition, we looked at gatae expression as an indicator of endoderm
regulatory state. Representative WMISH results are reproduced in
Fig. 5. As expected
(Table 1), gatae
expression appeared normal until the onset of gastrulation (data not shown).
As the MASO phenotype indicates, the foxa gene is required as a
positive regulator of later endoderm development
(Fig. 5C,F). Even though
gastrulation of foxa MASO-treated embryos was delayed or did not
occur at all, they produced a higher level of foxa transcripts than
did controls (Fig. 5G-L), just
as had been demonstrated by the QPCR data
(Table 1). As indicated
earlier, one MASO effect is the maintenance of a high level of foxa
transcripts in the veg2 cells, by cancellation of the foxa
autorepression circuit.
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;
Sherwood and McClay, 1999;
Sweet et al., 2002). The Delta
signal is transduced by the Notch (N) receptor in these cells. For early SMC
specification, a primary regulatory gene target of N signaling is
gcm. The genomic interaction is direct, as Su(H) target sites that
mediate these N effects have been demonstrated in the cis-regulatory apparatus
of the gcm gene (Ransick and
Davidson, 2006). At the beginning of the mesenchyme blastula
stage, the delta gene is expressed in the SMCs, and this later signal
is received by both late-specified SMCs
(Sweet et al., 2002) and veg2
endoderm (Peterson and McClay,
2005). It is reasonable to suppose, therefore, that foxa
repression of mesoderm fates in the endoderm is required to preclude the same
response to N signaling in the endoderm. Embryo recombinants were designed as
tests of this proposition. We investigated whether the foxa MASO could produce a transformation to mesodermal fates in veg1 endoderm, as these cells are not exposed to Delta-Notch signaling. At the 60-cell stage, fragments from embryos injected with Rhodamine dextran and foxa MASO, were recombined with control embryo fragments, labeled with fluorescein (Fig. 6A,D). Veg1 plus the animal hemisphere of foxa MASO embryos were combined with control veg2 plus micromere fragments, and the reciprocal recombinations were also generated. When the veg2 tier contained the foxa MASO, excess SMCs were produced by this tier, and there was very little veg2 contribution to the gut (Fig. 6A-C). Instead, the control veg1 cells made up almost all of the gut, even the foregut, which normally receives almost no contribution from the veg1 descendants. The foregut patches in Fig. 6B,C (red) were observed frequently, and much of that tissue became mesodermal coelomic pouches. Essentially, this is the same as the result shown in the mosaics (Fig. 4). In the reciprocal combination (Fig. 6D-F), in which all veg1 cells contained the foxa MASO, most of the gut is derived from the control veg2 cells (green). Thus, there is a subnormal contribution of veg1 cells (red) to the gut. The foxa MASO-injected veg1 cells contributed a small amount of gut in some cases (the MASO-treated cells produced a subnormal amount of hindgut, see Fig. 6E,F), but in each case the control veg2 cells regulated to compensate for the reduced endoderm formed by foxa-MASO veg1 cells. It follows that, in the veg1 cells, the foxa gene contributes to postgastrular specification of the endoderm, as, in its absence, far fewer veg1 cells become hindgut and midgut endoderm. Notably, no excess SMC types of veg1 origin were observed. Veg1 cells do not receive the Delta-Notch signal, and therefore one function employed by Foxa in veg2 cells (gcm repression) is not detectable in veg1 cells.
Formation of the mouth requires foxa gene expression in the oral ectoderm
The foxa gene is expressed in the oral ectoderm region, beginning
at about 24 hours after fertilization (Fig.
1), and, in the whole embryo, foxa MASO precludes
formation of the mouth (Fig.
3). To confirm directly that oral expression of foxa is
necessary for mouth formation, chimeric recombinations were generated (see
Fig. 7). Recombinants were
produced at the 32- to 60-cell stage. Uninjected halves (green) were
reciprocally recombined with foxa MASO-injected halves (red; see
Fig. 7A,D). The embryos were
imaged at 40 hours. Embryos with control (green) animal halves had a normal
mouth (Fig. 7C). Embryos
without Foxa in the animal half (Fig.
7E,F) make a normal gut but no mouth. Thus, the oral territory of
foxa expression is indeed required for production of the mouth.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The participation of a given regulatory gene in multiple developmental
processes, controlled by separate GRNs, is an emergent theme in regulation
molecular biology. From an external point of view, this is an obvious and
general requirement stemming from the fact that all bilaterians use
essentially the same regulatory gene toolkit, so that in their immensely
diverse developmental processes the same genes have to be deployed over and
over again for different purposes (for reviews, see
Davidson, 2006;
Erwin and Davidson, 2002).
Furthermore, in the sea urchin embryo, 80% of all regulatory genes are used
just to get to the late gastrula stage
(Howard-Ashby et al., 2006),
and multiple re-use of these genes is therefore an inescapable inference. Many
particular examples of regulatory genes that execute totally distinct
functions during sea urchin embryogenesis are indeed already in hand, for
example hnf6 (Otim et al.,
2004), otx (Li et
al., 1997; Yuh et al.,
2002), deadringer
(Amore et al., 2003),
gsc (Angerer et al.,
2001), and blimp1/krox
(Livi and Davidson, 2006a;
Livi and Davidson, 2006b). The
C. elegans ortholog of the foxa gene, pha4,
regulates different gene batteries at different times
(Ao et al., 2004;
Gaudet and Mango, 2002;
Gaudet et al., 2004), and a
particular aspect of its multiple capabilities is that the Foxa transcription
factor recognizes high and low affinity target sites according to its
concentration. That the same is likely to be true for sea urchin foxa
is implied by the oscillatory time course shown in
Fig. 2. This is clearly due to
foxa autorepression (Table
1). But when the autorepression is blocked from occurring by the
introduction of foxa MASO, there is no change in the location of
expression (Fig. 5), so the
only significance of the normal oscillation is to alter the concentration of
the foxa gene product over time within the cells of the endoderm.
Where the specific targets of foxa are known, i.e. in the early phase
of its function, we can associate the level of foxa expression with a
given function, and from the time course of the oscillation phase we can
determine the temporal duration of that function.
The foxa gene in the endomesoderm GRN
During the blastula stage, foxa has no input into any known
portion of the regulatory apparatus controlling endoderm specification
(Table 1; genes not affected by
foxa MASO), nor does the blockade of Foxa translation cause any
digression from normal developmental morphology up to gastrulation. But
nonetheless, this gene has a function that is essential for endoderm
specification, namely, to permit this specification to occur at all.
Fig. 8A shows the inputs that
activate foxa in the veg2 endoderm. Following mesoderm specification,
the delta gene becomes active in mesoderm cells
(Fig. 8C), although this second
phase of Delta expression is independent of the first PMC Delta signal
(Sweet et al., 2002). The
second signal is received in the adjacent veg2 cells, where it is essential
for activation of the essential endoderm regulatory gene gatae. This
has been shown to be a direct cis-regulatory input mediated by the Su(H)
transcription factor (P. Y. Lee and E.H.D., unpublished). As the gcm
gene is directly activated by N signaling via Su(H) as well (op cit),
and because foxa expression in the endoderm normally represses
gcm (Table 1,
Fig. 5), the effect of
preventing foxa expression is to promote ectopic gcm
expression in cells that normally would become endoderm. These cells now
become respecified as mesodermal cells (Figs
4,
6). In other words, a means of
preventing gcm expression in endoderm is essential as a device to
permit endoderm specification resulting from the N input.
Seen in this light, the fine-tuned elegance of the foxa regulatory
architecture (Fig. 8) is
thought provoking when viewed in evolutionary terms. Both PMCs and
precociously specified pigment cells are echinoid specialties, and so at least
parts of this architecture are likely to have been installed since the
divergence of euechinoids. In starfish, there is also a N input into
gatae in the endoderm at the equivalent stage, and foxa also
represses itself, but gcm is expressed quite differently and in cells
arising elsewhere (Hinman et al.,
2003) (V. F. Hinman and E.H.D., unpublished). The foxa
repression function has different targets in the starfish, including
gatae in the mesoderm (Hinman et
al., 2003). Since divergence, not only has foxa
repression of gcm in the endoderm been inserted in the euechinoid
lineage, but its operation is temporally regulated by its own autorepression
so as to operate at just the right time to control N signal effects; high
levels of foxa gene product are apparently required for both
gcm repression and foxa autorepression.
Notch signaling is directly or indirectly required for specification of the
late delaminating SMC derivatives, muscle and coelomic pouches
(Sweet et al., 2002;
Oliveri et al., 2002;
Peterson and McClay, 2005;
Sherwood and McClay, 1999).
The precursors of these cells are sorted out within the SMC domain during the
blastula stage (Ruffins and Ettensohn,
1996), when foxa is transiently expressed in an
overlapping domain with gcm. Because N signaling activates the
gcm gene, and indeed is the necessary input required earlier to
activate gcm in the mesoderm cells in response to the initial Delta
signal received from the PMCs (Ransick and
Davidson, 2006), and because Foxa represses gcm, the
early phase of foxa expression could affect fate allocation among
SMCs as well. In this case, some of the excess pigment cells produced by
foxa MASO treatment would reflect an alteration of SMC fate
balance.
Later functions of foxa
Only phenotypical evidence suggests later roles of foxa, and no
direct gene targets in either the archenteron or the stomodeal area are
demonstrated. Furthermore, it must be considered whether the effects of
foxa MASO treatment on development of the archenteron (see
Fig. 3A-I,
Fig. 5A-F) could, at least in
part, just be secondary effects of the conversion of veg2 cells to SMC fates.
Alternatively, convincing arguments suggest that foxa directly
promotes the gene expression required for further development of the
archenteron. The postgastrular roles of this gene are most simply interpreted
as the provision of positive inputs into gut and stomodeal genes, even though
the pregastrular role, the only one of which we know the mechanism, is a
repressive one. It is possible that the amplitude of expression seen in
Fig. 2 is permanently damped
after gastrulation gets underway, and, as we speculate above, perhaps Foxa
acts as a repressor only when expressed at high levels. This would explain the
autorepression revealed by increased foxa production in the presence
of foxa MASO, and the repression effect on gcm expression
only at a time when endogenous foxa is at its highest levels.
The conversion to an SMC fate affects only veg2 and not veg1 endoderm. In
the absence of veg2 endoderm, or if veg2 is unable to express foxa,
veg1 endoderm can form the entire gut. The chimera experiments
(Fig. 6) show clearly that when
veg1 contains the foxa MASO it fails to generate hindgut endoderm or
contribute at all to the midgut, as it does in normal embryos
(Logan and McClay, 1997;
Ransick and Davidson, 1998).
But because veg1 is not subject to conversion to SMCs (because it has not
received the Notch signal), the foxa MASO must interfere with other
functions of foxa needed in the postgastrular endoderm of the
hindgut. Note that foxa is normally expressed in the late gastrula in
all of the gut (Fig. 1). It is
likely that the MASO effects on foregut development are also due to the
failure of regulatory interactions needed for that aspect of gut morphogenesis
and differentiation, this time in veg2 derivatives. These deductions predict
postgastrular GRN subcircuits in which the foxa gene, no doubt
together with other regulators, operates batteries of downstream genes that
are required in the anterior and posterior domains of the gut,
respectively.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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