short gastrulation
The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, a homologue of the Drosophila sog gene was isolated in the spider Achaearanea tepidariorum, and its expression and function were characterized. Expression of sog mRNA initially appears in a radially symmetrical pattern and later becomes confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene leads to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area forms normally. Furthermore, embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. It is proposed that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification (Akiyama-Oda, 2006).
The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. This study addressed how changes in this network produce evolutionary diversity in insect gastrulation. Focus was placed on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene (Goltsev, 2007).
A variety of dorsal patterning genes were examined in A. gambiae
embryos in an effort to determine the basis for the formation of distinct
ectodermal derivatives. For example hindsight (hnt; also
known as peb - Flybase) is expressed along the dorsal midline of D. melanogaster embryos, while tailup (tup) is expressed in a broader pattern that encompasses both the presumptive amnioserosa and dorsolateral ectoderm. The hnt expression pattern seen in A. gambiae is similar to that detected in D. melanogaster, although there is a marked expansion in the dorsal-ventral limits of the presumptive extra-embryonic territory. By contrast, the
tup pattern in A. gambiae is dramatically different from
that seen in D. melanogaster -- it is excluded from the prospective
serosa and restricted to the future amnion (Goltsev, 2007).
The T-box genes Dorsocross1 (Doc1) and Doc2 are
involved in amnioserosa development and expressed along the dorsal midline and
in a transverse stripe near the cephalic furrow of gastrulating D.
melanogaster embryos. The Doc1 and Doc2 orthologues in A. gambiae exhibit restricted expression in the presumptive amnion, similar to the tup pattern. The expression patterns of the two genes are identical but only Doc1 is shown. They
are initially expressed in a broad dorsal domain but come to
be repressed in the serosa. There is also a head stripe of expression
comparable to the D. melanogaster pattern. Additional
dorsal-ventral patterning genes are also expressed in a restricted pattern
within the developing amnion. Overall, the early expression patterns of tup, Doc1 and Doc2 (and additional patterning genes) foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in Anopheles (Goltsev, 2007).
In D. melanogaster, the patterning of the dorsal ectoderm depends
on Dpp and Zen, along with a variety of genes encoding Dpp signaling
components, such as the Thickveins (Tkv) receptor. Most of the corresponding
genes are expressed in divergent patterns in A. gambiae embryos. For example,
dpp and tkv are initially expressed throughout the dorsal
ectoderm, but become excluded from the presumptive serosa and
restricted to the amnion. By contrast, both genes have broad, nearly uniform expression patterns in the dorsal ectoderm of D. melanogaster embryos (Goltsev, 2007).
There is an equally dramatic change in the zen expression pattern.
In A. gambiae, expression is restricted to the presumptive serosa
territory, even at the earliest stages of development. By contrast,
zen is initially expressed throughout the dorsal ectoderm of
cellularizing embryos in D. melanogaster, and becomes restricted to the dorsal midline by the onset of gastrulation. Thus, the dpp/tkv and zen expression patterns are essentially complementary in A. gambiae embryos, but extensively overlap in Drosophila (Goltsev, 2007).
The loss of dpp, tkv, Doc1, Doc2 and tup expression in the presumptive serosa of A. gambiae embryos raises the possibility that
zen activates the expression of one or more repressors in the serosa.
It is unlikely that Zen itself is such a repressor since the expression of the
A. gambiae zen gene in transgenic Drosophila embryos does
not alter the normal development of the amnioserosa (Goltsev, 2007).
Different segmentation genes were examined in an effort to identify
putative serosa-specific repressors. For example, the gap gene
hunchback (hb) is initially expressed in the anterior
regions of A. gambiae embryos, in a similar pattern to that seen in
D. melanogaster, but by the onset of gastrulation a novel
pattern arises within the presumptive serosa.
hb expression has also been seen in the developing serosa of other insects, including a primitive fly (Clogmia) and the flour beetle, Tribolium (Goltsev, 2007).
Two additional segmentation genes behave like hb, empty spiracles
(ems) and tramtrack (ttk). ems is
involved in head patterning in D. melanogaster. Its
expression is limited to a single stripe in anterior regions of cellularizing
D. melanogaster embryos. Staining is seen in a comparable anterior region of A.
gambiae embryos, but a second site of expression (not seen in Drosophila) is also detected in the presumptive serosa (Goltsev, 2007).
Ttk is a maternal repressor that helps establish the expression limits of
several pair-rule stripes. It is ubiquitously expressed throughout the early D.
melanogaster embryo, but has a tightly localized expression pattern within the
presumptive serosa of A. gambiae embryos. Thus, novel patterns
of ems and ttk expression are consistent with the
possibility that serosa-specific repressors help subdivide the dorsal ectoderm
into separate serosa and amnion lineages in A. gambiae embryos (Goltsev, 2007).
The analysis of dorsal-ventral patterning genes identified two critical
differences between the pre-gastrular fly and mosquito embryos. First, there
are separate serosa and amnion lineages in A. gambiae, but just a
single amnioserosa in D. melanogaster. Second, there is an expansion
in the limits of the dorsal ectoderm in A. gambiae as compared with
the D. melanogaster embryo. Localized repressors might help explain
the former observation of separate lineages, but do not provide a basis for
the expansion of the dorsal ectoderm (Goltsev, 2007).
In D. melanogaster, the limits of Dpp signaling are established by
the repressor Brinker and the inhibitor Sog. Genetic studies suggest that Sog is the more critical determinant in early embryos. It is related to Chordin, which inhibits BMP signaling in vertebrates, and is expressed in broad lateral stripes encompassing the entire neurogenic ectoderm. The secreted Sog protein directly binds Dpp, and blocks its ability to interact with the Tkv receptor. However, Sog-Dpp complexes are proteolytically processed by the Tolloid (Tld) metalloprotease, which is expressed throughout the dorsal ectoderm of early Drosophila embryos. Tld helps ensure that high levels of the Dpp signal are released at the dorsal midline located far from the restricted source of the inhibitor Sog (Goltsev, 2007).
The expression patterns of the sog and tld genes in
A. gambiae are very different from those seen in D.
melanogaster. sog expression is primarily detected in the ventral mesoderm,
although low levels of sog transcripts might extend into the
ventral-most regions of the neurogenic ectoderm. This pattern is
more restricted across the dorsal-ventral axis than the D. melanogaster
sog pattern. tld expression is restricted to lateral regions of A.
gambiae embryos and is excluded from the dorsal ectoderm, which is the
principal site of expression in Drosophila. These significant
changes in the sog and tld expression patterns might
account, at least in part, for the expanded limits of Dpp signaling in the
dorsal ectoderm of A. gambiae embryos (Goltsev, 2007).
Direct evidence for broader Dpp signaling was obtained using an antibody
that detects phosphorylated Mad (pMad), the activated form of Mad obtained upon induction of the Tkv receptor. In D. melanogaster pMad expression is restricted to the dorsal midline. This is the domain where Sog-Dpp complexes are processed and peak levels of Dpp interact with the receptor Tkv. The spatial limits of the sog expression pattern are
decisive for this restricted domain of pMad activity. Just a twofold reduction
in the levels of Sog (sog/+ heterozygotes) causes a significant
expansion in pMad expression (Goltsev, 2007).
There is a marked expansion of the pMad expression domain in A.
gambiae embryos as compared with Drosophila. The domain
encompasses the entire presumptive serosa and extends into portions of the
presumptive amnion. The dpp and tkv expression patterns are
downregulated in the presumptive serosa, nonetheless, the
pMad staining pattern clearly indicates that this is the site of peak Dpp
signaling activity. The early expression of both dpp and tkv
encompasses the entire dorsal ectoderm. It would appear that peak Dpp
signaling is somehow maintained in the developing serosa even after the
downregulation of dpp and tkv expression in this tissue. A similar scenario is seen in the Drosophila embryo, in that there is downregulation of both dpp and tkv expression along the dorsal midline of gastrulating embryos (Goltsev, 2007).
To determine the basis for expanded Dpp signaling a sog enhancer was identified and characterized in A. gambiae. The D.
melanogaster enhancer is located in the first intron of the sog
transcription unit. It is ~300 bp in length and contains four evenly spaced, optimal Dorsal
binding sites. These sites permit activation of sog expression by
low levels of the Dorsal gradient; however, closely linked Snail repressor
sites inactivate the enhancer in the ventral mesoderm. A putative A.
gambiae enhancer was identified by scanning the sog locus for
potential clusters of Dorsal binding sites. The recently developed
cluster-draw program was used for this purpose since it successfully
identified a sim enhancer in the honeybee, Apis mellifera,
which is even more divergent than Anopheles. The best putative Dorsal binding cluster was identified within the first intron of the A. gambiae sog locus. Several genomic DNA fragments were tested for enhancer activity, but only this cluster was found to activate gene expression in transgenic Drosophila embryos (Goltsev, 2007).
Two different genomic DNA fragments, 3.7 kb and 1.1 kb, that encompass the
intronic binding cluster were tested in transgenic embryos. Both fragments were
attached to a lacZ reporter gene containing the core eve
promoter from D. melanogaster, and both direct lacZ
expression in the presumptive mesoderm. They exhibit the same restricted dorsal-ventral limits of expression as that seen for the endogenous sog gene in A. gambiae, although the smaller fragment produces ventral stripes whereas the larger fragment directs
a more uniform pattern. The change in the dorsal-ventral limits --
broad expression in D. melanogaster and restricted expression in
A. gambiae -- might be due to the quality of individual Dorsal binding
sites in the two enhancers (Goltsev, 2007).
Therefore, s comprehensive analysis of dorsal-ventral patterning genes in the A.
gambiae embryo reveals elements of conservation and divergence in the
gastrulation network of D. melanogaster. There is broad conservation
in the expression of regulatory genes responsible for the patterning of the
mesoderm and neurogenic ectoderm, including sequential expression of sim,
vnd and ind in the developing nerve cord. By contrast, there are
extensive changes in the expression of regulatory genes that pattern the
dorsal ectoderm. These changes foreshadow the subdivision of the dorsal
ectoderm into separate serosa and amnion lineages in A. gambiae (Goltsev, 2007).
The major difference in the early patterning of the mesoderm in flies and
mosquitoes concerns the manner in which mesoderm cells enter the blastocoel of
gastrulating embryos. In D. melanogaster, there is a coherent
invagination of the mesoderm through the ventral furrow, much like the
movement of bottle cells through the blastocoel of Xenopus embryos. By contrast,
there is no invagination of the mesoderm in A. gambiae. Instead, the
mesoderm undergoes progressive ingression during germband elongation. This
type of ingression is seen in D. melanogaster mutants lacking
fog signaling. The A. gambiae genome lacks a clear homologue of
fog, and it is therefore conceivable that fog represents an
innovation of the higher Diptera that was only recently incorporated into the
D. melanogaster dorsal-ventral patterning network (Goltsev, 2007).
D. melanogaster is somewhat unusual in having an amnioserosa,
rather than separate serosa and amnion tissues as seen in most insects. In
certain mosquitoes the serosa secretes an additional proteinaceous membrane
that provides extra protection against desiccation.
The changes in gene expression in the D. melanogaster and A.
gambiae dorsal ectoderm provide a basis for understanding the
evolutionary transition of two dorsal tissues in A. gambiae into a
novel single tissue in higher dipterans (Goltsev, 2007).
The D. melanogaster amnioserosa expresses a variety of regulatory
genes, including Doc1/2 and tup. The expression of most of
these genes is restricted in the presumptive amnion of the A. gambiae
embryo. zen is the only dorsal patterning gene, among those tested,
that exhibits restricted expression in the serosa. Several segmentation genes
have a similar pattern, and one of these, ttk, encodes a known
repressor. Ectopic expression of Ttk causes a variety of patterning defects in
Drosophila embryos, including disruptions in head involution and
germband elongation that might arise from alterations in the amnioserosa. It is
proposed that zen activates ttk in the serosa of A. gambiae embryos. The encoded repressor might subdivide the dorsal ectoderm into separate serosa and amnion tissues by inhibiting the expression of Doc1/2 and tup in the serosa. The loss of this putative zen-ttk regulatory linkage might be sufficient to allow Dpp signaling to activate tup and Doc1/2 throughout the dorsal ectoderm,
thereby transforming separate serosa and amnion tissues into a single
amnioserosa. According to this scenario, the loss of zen binding
sites in ttk regulatory sequences might be responsible for the
evolutionary transition of the amnioserosa (Goltsev, 2007).
The formation of separate amnion and serosa tissues is not the only
distinguishing feature of A. gambiae embryos when compared with
D. melanogaster. There is also a significant expansion in the overall
limits of the dorsal ectoderm. This can be explained, in part, by distinct
patterns of sog expression. The broad expression limits of the Sog inhibitor are responsible for restricting Dpp/pMad signaling to the dorsal midline of the D.
melanogaster embryo. This pattern depends on a highly sensitive response of the sog intronic enhancer to the lowest levels of the Dorsal gradient. The Dorsal binding sites in the sog enhancer are optimal sites, possessing perfect matches to the
idealized position weighted matrix of Dorsal recognition sequences.
By contrast, the A. gambiae intronic sog enhancer
contains low-quality Dorsal binding sites, similar to those seen in the
regulatory sequences of genes activated by peak levels of the Dorsal gradient,
such as twist. The binding sites in the D. melanogaster sog
enhancer have an average score of ~10. By contrast, the best sites in the
A. gambiae sog enhancer have scores in the 6.5-7 range, typical of
enhancers that mediate expression in the mesoderm in response to high levels
of the Dorsal gradient. Although every potential
regulatory sequence in the A. gambiae sog locus was not explicitly tested, none of the putative Dorsal binding clusters in the vicinity of the gene possess the quality
required for activation by low levels of the Dorsal gradient in the neurogenic
ectoderm. Thus, the narrow limits of sog expression in A.
gambiae embryos can be explained by the occurrence of low-quality Dorsal
binding sites, along with the loss of Snail repressor sites (Goltsev, 2007).
The altered sog expression pattern is probably not the sole basis
for the expansion of the dorsal ectoderm. A. gambiae embryos also
exhibit a significant change in the tld expression pattern.
tld is expressed throughout the dorsal ectoderm in D.
melanogaster, but restricted to the neurogenic ectoderm of A.
gambiae. Tld cleaves inactive Tsg-Sog-Dpp complexes to produce peak Dpp
signaling along the dorsal midline of Drosophila embryos. It is proposed that the
altered tld pattern in combination with altered sog leads to
two dorsolateral sources of the active Dpp ligand in mosquito embryos. The sum of these sources might produce a step-like distribution of pMad across dorsal regions of mosquito embryos. This broad plateau of pMad activity might be responsible for the observed expansion of the dorsal ectoderm territory, and the specification of the serosa (Goltsev, 2007).
In Drosophila, tld is regulated by a 5' silencer element
that prevents the gene from being expressed in ventral and lateral regions in
response to high and low levels of the Dorsal gradient. This silencing activity is due to close linkage of Dorsal binding sites and recognition sequences for 'co-repressor' proteins. Preliminary studies suggest that Dorsal activates the A. gambiae tld gene, possibly by the loss of co-repressor binding sites in the 5' enhancer (Goltsev, 2007).
It is proposed that there are at least two distinct threshold readouts of Dpp
signaling in the dorsal ectoderm of A. gambiae embryos. Type 1 target
genes, such as hb, ems, ttk and zen, are activated by high
levels and thereby restricted to the presumptive serosa. Type 2 target genes,
such as tup and Doc1/2, can be activated - in principle - by
both high and low levels of Dpp signaling in the presumptive serosa and
amnion. However, these target enhancers contain binding sites for one or more
type 1 repressors expressed in the serosa. The favorite candidate repressor is
Ttk. Perhaps the type 2 tup enhancer contains optimal pMad activator
sites as well as binding sites for the localized repressor Ttk, which keeps
tup expression off in the serosa and restricted to the amnion. As
discussed earlier, the simple loss of ttk regulation by the Dpp
signaling network might be sufficient to account for the evolutionary conversion of separate serosa and amnion tissues into a single amnioserosa. Localization of this single tissue within a restricted domain along the dorsal midline would arise from concomitant dorsal shifts in the sog and tld expression patterns (Goltsev, 2007).
In the short-germ beetle Tribolium castaneum, the head gap gene orthodenticle (Tc-otd) has been proposed to functionally substitute for bicoid, the anterior morphogen unique to higher dipterans. This study reanalyzed the function of Tc-otd. A similar range of cuticle phenotypes was obtained as in previously described RNAi experiments; however, unexpected effects were noticed on blastodermal cell fates. First, it was found that Tc-otd is essential for dorsoventral patterning. RNAi depletion results in lateralized embryos, a fate map change that by itself can explain the observed loss of the anterior head, which is a ventral anlage in Tribolium. It was found that this effect is due to diminished expression of short gastrulation (sog), a gene essential for establishment of the Decapentaplegic (Dpp) gradient in this species. Second, it was found that gnathal segment primordia in Tc-otd RNAi embryos are shifted anteriorly but otherwise appear patterned normally. This anteroposterior (AP) fate map shift might largely be due to diminished zen-1 expression and is not responsible for the severe segmentation defects observed in some Tc-otd RNAi embryos. As neither Tc-sog nor Tc-zen-1 probably requires Otd gradient-mediated positional information, it is posited that the blastoderm function of Tc-Otd depends on its initial homogeneous maternal expression and that this maternal factor does not provide significant positional information for Tribolium blastoderm embryos (Kotkamp, 2010).
The rel/NF-kappaB transcription factor Dorsal controls dorsoventral (DV) axis formation in Drosophila. A stable nuclear gradient of Dorsal directly regulates ~50 target genes. In Tribolium castaneum (Tc), a beetle with an ancestral type of embryogenesis, the Dorsal nuclear gradient is not stable, but rapidly shrinks and disappears. Negative feedback accounts for this dynamic behavior: Tc-Dorsal and one of its target genes activate transcription of the IkB homolog Tc-cactus, terminating Dorsal function. Despite its transient role, Tc-Dorsal is strictly required to initiate DV polarity, as in Drosophila. However, unlike in Drosophila, embryos lacking Tc-Dorsal display a periodic pattern of DV cell fates along the AP axis, indicating that a self-organizing ectodermal patterning system operates independently of mesoderm or maternal DV polarity cues. The results also elucidate how extraembryonic tissues are organized in short-germ embryos, and how patterning information is transmitted from the early embryo to the growth zone (da Fonsaca, 2008).
Tc-Toll transcription appears to start evenly along the DV axis at the syncytial blastoderm stage but is rapidly enhanced at the ventral side, where higher levels of nuclear Tc-Dorsal accumulate. This positive feedback between Toll expression and nuclear import of Dorsal could explain an initiation of DV axis formation at ectopic positions of the embryonic blastoderm, a situation which has been observed upon experimental manipulations in beetles and various hemimetabolous insects. During normal development, however, ectopic axis formation has to be prevented, and this can be achieved by coupling positive feedback control to inhibitory processes. Linking self-enhancement to limiting mechanisms provides a general condition for pattern formation as has been shown by mathematical modeling. The Tc-Dorsal-dependent transcriptional activation of Tc-cact might provide the mechanism counterbalancing the positive feedback between Tc-Toll and Tc-Dorsal (da Fonsaca, 2008).
Within the limits of detection, Tc-cact expression appears to be restricted to the ventral side of the embryo. However, the knockdown of Tc-cact leads to nuclear import of Tc-Dorsal also at the dorsal side. To explain this long-range requirement of Tc-cact, one might speculate that detection of Tc-cact transcripts is not sensitive enough or that Tc-Cact protein is able to diffuse within the cytoplasm from ventral toward dorsal. Irrespective of the mechanism, a long-range action of Tc-Cact would meet an important prediction for pattern formation by reaction-diffusion systems, namely that the inhibitor should spread faster and thus act less locally than the activator (da Fonsaca, 2008).
Besides its potential role in pattern formation, Tc-cact activation seems also to be involved in the temporal control of the Dorsal gradient. During late blastoderm stages Tc-cact activation by Tc-dorsal is replaced through activation by Tc-twi. The Tc-twi knockdown phenotype shows that this shift is relevant to prevent Dorsal from accumulating in ventral nuclei during gastrulation. Thus, it seems that in Tribolium a Dorsal target gene is involved in terminating Dorsal function (da Fonsaca, 2008).
Collectively, these observations indicate that major evolutionary changes have occurred regarding Tc-Dorsal gradient formation and the network of downstream target genes. Nevertheless, traces of the feedback mechanisms uncovered in Tribolium have been preserved in the Drosophila lineage. Recently, zygotic enhancers of Dm-cactus and Dm-Toll were identified by ChIP-on-chip experiments and bioinformatics approaches. These enhancers contain Dm-Dorsal and Dm-twist binding sites and are active in the prospective mesoderm of Drosophila. However, the analysis of mutant phenotypes precludes an important function of these enhancers in DV patterning or cell type specification. A weak stabilizing function may explain why they were retained in evolution (da Fonsaca, 2008).
On an even larger evolutionary scale it is interesting to note that negative feedback control is a hallmark of NF-κB-mediated signaling. Like in Tribolium, the transcription of the Cactus homolog I-κB is activated by NF-κB in vertebrates both in the mesoderm and during innate immune response. The ensuing negative feedback loop can cause oscillatory signaling outputs or termination of signaling. Even an involvement of twist in negative feedback regulation of NF-κB has been demonstrated in vertebrate mesoderm cells. It has been proposed that the twi-NF-κB interactions represent an evolutionarily conserved regulatory module. The Dorsal/NF-κB- and twi-dependent activation of Tc-cact might be a relic of mesodermal and innate immune functions the pathway had in the common ancestor of vertebrates and arthropods. According to this scenario, the ancestral feedback mechanisms were adjusted to the needs of spatial patterning after the pathway was adopted for DV axis formation (da Fonsaca, 2008).
Classical fragmentation experiments have suggested two routes for pattern regulation along the DV axis: an early route which takes place before gastrulation and a later one which can be initiated after mesoderm internalization. Evidence has been provided for late autonomous patterning within the ectoderm that depends on the Dpp/Sog system and additional inhibitory processes. Tc-Toll knockdown embryos add additional support for this assumption. They show pattern duplications of ectodermal DV cell fates along the AP axis. This remarkable phenotype is not just restricted to the abdominal segments derived from the growth zone, but it occurs also within the anterior (thoracic) segments. Thus, it is unlikely to reflect a specific mechanism that operates only in the growth zone (da Fonsaca, 2008).
The modulation of Dpp activity underlying the periodic cell fate changes is likely to be due to periodic transcription of Tc-dpp and inhibition of Tc-Dpp diffusion or signaling along the AP axis. Since Tc-sog is not re-expressed in Tc-Toll1 RNAi embryos, the expression of other Dpp inhibitors was analyzed. Tc-bambi showed periodic expression in the same domains as Tc-dpp and thus might provide the inhibitory function. The fact that Tc-dpp is transcribed in regions of high pMAD activity suggests positive feedback control which is counterbalanced by Tc-bambi. Thus, the interaction might be similar to that described for Tc-Toll and Tc-cact (da Fonsaca, 2008).
The unusual orientation of the ectodermal patterning process might depend on the early AP asymmetry of Dpp signaling in Tc-Toll1 RNAi embryos. After Tc-Toll1 RNAi, Tc-dpp is expressed along the symmetric border between serosa and germ rudiment and in the posterior pit region (data not shown). These regions also have high levels of pMAD. Thus, the ectodermal patterning process is initiated with AP asymmetric boundary conditions after Tc-Toll RNAi. In WT embryos this process is oriented along the DV axis through the Toll-dependent activation of Tc-sog at the ventral side, which leads to a Dpp signaling gradient with peak levels along the dorsal midline (da Fonsaca, 2008).
Experiments clearly demonstrate that Tc-Dorsal is essential for establishing all aspects of normal DV polarity in Tribolium, including DV polarity of the growth zone from which the abdominal segments emerge. Thus, although DV patterning in the growth zone starts after gastrulation, when the Tc-Dorsal gradient has vanished, it is not independent of Tc-Dorsal. It is suggested that there are two ways by which early DV polarity is transmitted to the growth zone. First, distinct inner and outer cell layers are formed during gastrulation. The observation that the majority of the mesenchymal layer cells are absent in Tc-Toll RNAi embryos strongly suggests that the mesenchymal cells in the growth-zone are derived from cells internalized by ventral furrow formation in the early embryo. These cells cannot be resupplied by a growth zone-specific process of cell internalization. Thus, gastrulation-like mechanisms do not continue in the growth zone of Tribolium, as has been suggested for the tail-bud of vertebrate embryos. Second, DV patterning in the growth zone does not only depend on the generation of two separate cell layers. The ectoderm needs also to be patterned, a process which mainly depends on Dpp signaling. To a certain degree this process takes place in a Toll knockdown embryo. However, the orientation of the resulting pattern is incorrect. It is assumed that during WT development DV polarity is first established within the anterior (gnathal and thoracic) segments. Subsequently, this pattern is used as a template for the DV pattern of the abdominal segments emerging from the growth zone. This would require a process of forward-induction from differentiated to nondifferentiated tissues. Since there is no DV polarity in Tc-Toll RNAi early embryos, forward-induction cannot operate (da Fonsaca, 2008).
The loss of Toll signaling in Tribolium leads to phenotypes that are similar to those produced by the loss of the Dpp inhibitor sog. In both situations the ectoderm lacks normal polarity, the amnion and the CNS are largely deleted (the CNS is completely absent after Tc-sog RNAi and reduced to narrow periodic stripes after Tc-Toll RNAi), and the embryos form long tube-like structures. This situation is strikingly different in Drosophila. There, loss of Toll signaling leads to completely dorsalized embryos, while loss of sog causes only minor deletions in the CNS and subtle ectodermal patterning defects. These differences are due to the fact that Toll signaling in Drosophila provides functions which the Dpp/Sog system fulfils in Tribolium. For example, in Drosophila Dorsal represses dpp and activates brinker, an inhibitor of Dpp target genes, within the presumptive neuroectoderm and thereby specifies the CNS through mechanisms which act independently from and parallel to sog. These mechanisms do not exist in Tribolium. Apparently, the Dorsal gradient has a less direct role with regard to cell-type specification in Tribolium than in Drosophila, and DV patterning in Tribolium relies to higher degree on the Dpp/Sog system. Since the Dpp/Sog (BMP/Chordin) system is involved in DV axis formation in all bilaterian animals investigated so far, this is likely to represent the ancestral mode of DV axis formation. It is suggested that the trend observed by comparing Drosophila and Tribolium applies to other insect orders and that the functional shift between Dpp and Toll signaling with regard to DV axis formation will be even more prominent in basal hemimetabolous insects. Thus, the study of more basal insects groups might reveal the evolutionary path of how Toll signaling was co-opted for DV axis formation (da Fonsaca, 2008).
The evolutionary origin of the anterior–posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. This study cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral–aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral–aboral axis. It is concluded that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral–aboral body axis of Nematostella and one particular body axis of Bilateria (Rentzsch, 2006).
Nearly all metazoans show signs of bilaterality, yet it is believed the bilaterians arose from radially symmetric forms hundreds of millions of years ago. Cnidarians (corals, sea anemones, and 'jellyfish') diverged from other animals before the radiation of the Bilateria. They are diploblastic and are often characterized as being radially symmetrical around their longitudinal (oral-aboral) axis. The deployment of orthologs of a number of family members of developmental regulatory genes that are expressed asymmetrically during bilaterian embryogenesis from the sea anemone, Nematostella vectensis, have been studied. The secreted TGF-beta genes Nv-dpp, Nv-BMP5-8, six TGF-beta antagonists (NvChordin, NvNoggin1, NvNoggin2, NvGremlin, NvFollistatin, and NvFollistatin-like), the homeodomain proteins NvGoosecoid (NvGsc) and NvGbx, and the secreted guidance factor, NvNetrin, were studied. NvDpp, NvChordin, NvNoggin1, NvGsc, and NvNetrin are expressed asymmetrically along the axis perpendicular to the oral-aboral axis, the directive axis. Furthermore, NvGbx, and NvChordin are expressed in restricted domains on the left and right sides of the body, suggesting that the directive axis is homologous with the bilaterian dorsal-ventral axis. The asymmetric expression of NvNoggin1 and NvGsc appear to be maintained by the canonical Wnt signaling pathway. The asymmetric expression of NvNoggin1, NvNetrin, and Hox orthologs NvAnthox7, NvAnthox8, NvAnthox1a, and NvAnthox6, in conjunction with the observation that NvNoggin1 is able to induce a secondary axis in Xenopus embryos argues that N. vectensis could possess antecedents of the organization of the bilaterian central nervous system (Matus, 2006).
Dorsoventral patterning of vertebrate and Drosophila embryos requires bone morphogenetic proteins
(BMPs) and antagonists of BMP activity. The Drosophila gene tolloid encodes a metalloprotease similar to
BMP-1 that interacts genetically with decapentaplegic, the Drosophila homolog of vertebrate BMP-2/4.
Zebrafish embryos overexpressing a zebrafish homolog of tolloid resemble
loss-of-function mutations in chordino, the zebrafish homolog of the Xenopus BMP-4 antagonist Chordin. Zebrafish tld transcripts are detected throughout the early gastrula stage embryo. Toward the end of gastrulation expression becomes restricted, accumulating both dorsally and ventrally along the closing blastopore. Expression is also detected in the ectoderm flanking the anterior neural plate at this stage. At the 10-somite stage tld mRNA is expressed in the developing tailbud and in cells flanking the midbrain and hindbrain; these cells presumably correspond to migrating cranial neural crest.
Chordin is degraded by COS cells expressing Tolloid. These data suggest that Tolloid
antagonizes Chordin activity by proteolytically cleaving Chordin. A conserved function for zebrafish and
Drosophila Tolloid during embryogenesis is proposed (Blader, 1997).
The neuroectoderm of the vertebrate gastrula that Nieuwkoop proposed
is regionalized into forebrain,
midbrain, hindbrain and spinal cord by a two-step process.
In the activation step, the Spemann gastrula organizer
induces neuroectoderm with anterior character, followed
by posteriorization by a transforming signal. Simultaneous inhibition of BMP and Wnt signaling induces head formation in frog embryos. However,
how the inhibition of BMP and Wnt signaling pathways
specify a properly patterned head, and how they are
regulated in vivo, has not been understood.
The homeobox genes siamois and twin in
frog, and bozozok/dharma/nieuwkoid (bozozok) in zebrafish, act
downstream of beta-catenin as part of the mechanisms
establishing the dorsal gastrula (Spemann) organizer. Bozozok is distantly related to Drosophila BSH9, the Gooseberrys, Aristaless, Paired and Goosecoid. The loss of anterior neural fates observed in zebrafish bozozok (boz) mutants occurs during gastrulation due to a reduction and subsequent posteriorization of
neuroectoderm. The neural induction defect is correlated
with decreased chordino expression and consequent
increases in bmp2b/4 expression, and is suppressed by
overexpression of BMP antagonists. Whereas anterior neural marker expression is restored by ectopic BMP inhibition in early boz gastrulae, it is not maintained during later gastrulation. The posteriorization of
neuroectoderm in boz is correlated with ectopic dorsal
wnt8 expression. Overexpression of a Wnt antagonist
rescues formation of the organizer and anterior neural fates
in boz mutants. It is proposed that boz specifies formation
of anterior neuroectoderm by regulating BMP and Wnt
pathways in a fashion consistent with Nieuwkoop's two-step
neural patterning model. boz promotes neural induction
by positively regulating organizer-derived chordino and
limiting the antineuralizing activity of BMP2b/4
morphogens. In addition, by negative regulation of Wnt
signaling, boz promotes organizer formation and limits
posteriorization of neuroectoderm in the late gastrula (Fekany-Lee, 2000).
Spatial variations in the levels of bone morphogenetic protein (BMP) signaling are a critical determinant of dorsoanterior-ventroposterior
pattern in vertebrate embryos. Whereas BMP overexpression abolishes both head and trunk development, known single and double
loss-of-function mutations in BMP inhibitors have less dramatic effects. Combining mutations in the zebrafish genes
bozozok, coding for a homeodomain transcription factor, and chordino (din) causes a synergistic loss of head and trunk, whereas most cells express ventro-posterior markers and develop
into a tail. Genetic inactivation of BMP signaling fully suppresses these defects. Thus, a remarkably simple genetic mechanism, involving
a coinhibition of BMP function by the partially overlapping bozozok and chordino pathways is used to specify vertebrate head and trunk (Gonzalez, 2000).
If boz and din function redundantly in limiting BMP
signaling, then the severe morphological defects in boz;din
mutants should be correlated with greatly increased bmp
expression compared with either single mutant. During early
gastrulation, bmp4 is normally expressed
ventrolaterally and in a discrete dorsal domain. The dorsal
domain was absent in boz and boz;din mutants. Consistent with the above hypothesis, the
ventro-lateral bmp4 expression domain is more dorsally
expanded in boz;din mutants compared with either single mutant. Similarly, the ventrolateral marker eve1 is more
expanded in boz;din mutants than in either single
mutant. Later, during segmentation, the tailbud bmp4 expression
domain is almost normal in boz, somewhat expanded in
din, but dramatically expanded in boz;din embryos. Together, these morphological and gene
expression analyses indicate that most cells in boz;din mutants reside in the prospective tailbud region within a greatly expanded bmp4 expression domain.
Furthermore, boz and din function synergistically in
negative regulation of bmp4 expression (Gonzalez, 2000).
The findings described here show that several distinct effects of high
BMP activity cause the head/trunk deficiency in boz;din double
mutants. Within the ectoderm, high levels of BMP activity transform the
neuroectoderm into nonneural ectoderm.
These data suggest that almost all of the mesodermal precursors in
boz;din mutants experience very high levels of BMP activity
and, instead of contributing to head and trunk, are misallocated to the
tailbud, express ventro-posterior markers, and form a tail. This idea
is in accord with fate-mapping studies showing that ventral cells in
the zebrafish gastrula migrate to the tailbud and contribute to the
tail, and this behavior is
expanded dorsally in mutants with elevated BMP activity. Therefore, it is proposed that the high levels
of BMP signaling in boz;din embryos specify posterior
structures at the expense of anterior structures. This proposal is
consistent with the phenotype of BMP signaling mouse mutants, in which
the major defect arises in posterior structures, such as the tail and
allantois. In summary, boz and
din represent the major overlapping pathways that are absolutely essential to limit BMP activity dorso laterally and allow head and trunk formation in the vertebrate embryo (Gonzalez, 2000).
The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that
posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic
protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish
BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but
not the posterior marker, hoxb1b. Furthermore, the cells in the presumptive posterior neural region
lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD)
are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region,
counteracting the BMP signaling pathway within the target cells. The functions of Fgf3 in posterior neural development were examined. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point at which the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 has little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect is mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion is substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate (Koshida, 2002).
Reported here is the expression of the zebrafish zic1 gene, also known as opl, a homolog to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To determine whether BMP2 signaling affects the extent of zic1 expression, swirl and chordino mutant embryos were examined. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, it was found that zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. However, the most rostral expression is not affected by Shh, suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme (Rohr, 1999).
Interactions between mutations in antagonistic BMP pathway signaling components were analyzed to examine the roles
that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7,
lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, it was found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, a
mutual suppression is observed of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the
Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism
is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, it was found that the absence of Ogon or Chordin antagonism does not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly, a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype is observed when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead an apparent decrease is observed in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly ortholog of chordin, short gastrulation, these paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity (Wagner, 2002).
The zebrafish mutant ogon (also called mercedes and short tail) displays ventralized phenotypes similar to the chordino (dino) mutant, in which the gene for the Bmp antagonist Chordin is mutated. The gene responsible for ogon was isolated by a positional cloning strategy; the ogon locus encodes a zebrafish homolog of Secreted Frizzled (Sizzled), which has sequence similarity to a Wnt receptor, Frizzled. Unlike other secreted Frizzled-related proteins (sFrps) and the Wnt inhibitor Dickkopf1, the misexpression of Ogon/Sizzled dorsalizes, but does not anteriorize, the embryos, suggesting a role for Ogon/Sizzled in Bmp inhibition. Ogon/Sizzled does not inhibit a Wnt8-dependent transcription in the zebrafish embryo. ogon/sizzled is expressed on the ventral side from the late blastula through the gastrula stages. The ventral ogon/sizzled expression in the gastrula stage is reduced or absent in the swirl/bmp2b mutants but expanded in the chordino mutants. Misexpression of ogon/sizzled does not dorsalize chordino mutants, suggesting that Ogon/Sizzled requires Chordin protein for dorsalization and Bmp inhibition. These data indicate that Ogon/Sizzled functions as a negative regulator of Bmp signaling and reveal a novel role for a sFrp in dorsoventral patterning (Yabe, 2003).
The results indicate that Ogo/Szl can augment the activity of Chordin, by inhibiting an inhibitor of Chordin, by directly making Chordin more active, or by modulating the Bmp signal so that it becomes more susceptible to the Chordin-mediated inhibition. The dorsalizing activity of the Chordin protein is regulated by different mechanisms: the chordin protein level is regulated through processing by Tolloid-related metalloproteinases, and Chordin interacts physically and functionally with Bmp and Twisted Gastrulation (Tsg) to modulate Bmp activity. Tolloid-related proteins and Tsg might be involved in the function of Ogo/Szl. Alternatively, Ogo/Szl may function in parallel with Chordin. Both Ogo/Szl and Chordin are required for the formation of posterior dorsal tissues, and the loss of either Ogo/Szl or Chordin might lead to ventralization. In this scenario, the lowering of the Bmp signal by Chordin might work cooperatively with Ogo/Szl to dorsalize the embryo (Yabe, 2003).
A number of genetic and molecular studies have implicated
Chordin in the regulation of dorsoventral patterning
during gastrulation. Chordin, a BMP antagonist of 120
kDa, contains four small (about 70 amino acids each)
cysteine-rich domains (CRs) of unknown function. The Chordin CRs define a novel protein module for the binding and regulation of BMPs. The
biological activity of Chordin resides in the CRs, especially
in CR1 and CR3, which have dorsalizing activity in
Xenopus embryo assays and bind BMP4 with dissociation
constants in the nanomolar range. The activity of
individual CRs, however, is 5- to 10-fold lower than that of
full-length Chordin. These results shed light on the
molecular mechanism by which Chordin/BMP complexes
are regulated by the metalloprotease Xolloid, which cleaves
in the vicinity of CR1 and CR3 and would release CR/BMP
complexes with lower anti-BMP activity than intact
Chordin. CR domains are found in other extracellular
proteins such as procollagens. Full-length Xenopus
procollagen IIA mRNA has dorsalizing activity in embryo
microinjection assays and the CR domain is required for
this activity. Similarly, a C. elegans cDNA containing five
CR domains induces secondary axes in injected Xenopus
embryos. These results suggest that CR modules may
function in a number of extracellular proteins to regulate
growth factor signaling (Larrain, 2000).
The Chordin/BMP pathway is regulated by the zinc
metalloprotease Xolloid, a homolog
of Drosophila Tolloid that regulates the activity of Sog. The observations made in this study begin to provide a
molecular explanation for how Xolloid may regulate
Chordin. Xolloid cleaves Chordin at two sites, which had
been roughly mapped close to a region downstream of CR1 and CR3. Recently, the cleavage sites have been
sequenced and found to correspond to conserved aspartic
residues. The CR1 protein used in this
study is very similar in length (only 8 amino acids shorter)
to the fragment generated by metalloprotease cleavage in the
N-terminal site of Chordin. CR1 binds BMP4 with a lower affinity (8-fold lower), is less efficient in competing BMP4 binding to BMPR (10 times lower), and
has less biological activity (5- to 10-fold lower) than full-length
Chordin. It is conceivable that the Xolloid protease
inactivates Chordin by the generation of smaller fragments
that can still bind BMP and perhaps transport it. However,
each of these binding modules alone would not have high
enough affinity to compete with the higher affinity
of BMP for its cognate receptors, which is in the same range
as that of full-length Chordin for BMP4 (Larrain, 2000 and references therein).
In Drosophila, Sog not only
inhibits Dpp signaling but is also able to enhance it at a
distance. This enhancement of BMP signals
requires Sog diffusion (presumably carrying bound Dpp or
Screw) and the activity of the Tolloid protease. It has been suggested that the cleavage products of Sog, or Sog fragments complexed with Dpp, could augment the binding of Dpp/Screw to its receptors. None
of the Chordin constructs used in the current study, including a series of carboxy-terminal protein truncations, display
ventralizing effects as would be expected if there were
increased binding to receptors. Rather, the observation that
Chordin fragments are either weakly dorsalizing or inactive in
Xenopus assays tends to support the proposal that diffusion of Chd/Sog
complexed with BMP/Dpp contributes to the formation of
morphogen gradients in which maximal levels of signaling are
achieved by cleavage of the inhibitor and release of the active
BMP signal (Larrain, 2000 and references therein).
Overexpression of Drosophila SOG mRNA in Xenopus dorsalizes the embryo
by expanding neurogenic and dorsal paraxial tissue. When ectopically expressed on the ventral
side of the embryo, Sog induces a partial secondary axis. In addition, Sog partially rescues embryos
ventralized by ultraviolet irradiation. Since Sog induces many changes in gene expression to those
caused by truncated BMP receptors, it has been suggested that Sog functions in part by opposing
BMP-4 signaling. The recent identification of chordin, a possible Xenopus sog homolog, in
conjunction with these results supports the hypothesis that dorsal-ventral patterning mechanisms
are conserved between these two species (Schmidt, 1995).
In Xenopus, XHex, coding for a homeodomain transcription factor, and cerberus, coding for a secreted head inducing factor, are early marker genes of the anterior endomesoderm (AE), a subset of
endoderm cells fated to form the liver and foregut and implicated in head induction. Using XHex and
cerberus as markers, the signals underlying AE induction have been examined. The AE is
specified by the early blastula in the absence of mesodermal signals but cell-cell contact between
presumptive AE cells is required. In overexpression experiments maternal Wnt/beta-catenin and TGF-beta
signals (Vg1, Xnr1-2) can induce ectopic XHex and cerberus. Inhibiting these pathways with dominant
interfering signaling components block endogenous XHex and cerberus expression. The role of
signals from the organizer has been assessed. The BMP antagonists noggin and chordin are shown to be important for
maintaining XHex and cerberus expression. Ventral injection of XHex mRNA can induce ectopic
cerberus. These results indicate that endodermal and mesodermal patterning are closely coordinated and that the
AE is likely to be specified by the combined action of dorsal Wnt/beta-catenin signals and endoderm-specific
factors mediated by TGF-beta signaling. These results provide a starting point for understanding the
molecular events underlying the progressive determination of endodermally derived organs, such as the liver
and foregut (Zorn, 1999).
Anteroposterior patterning of neural tissue is thought to be directed by the axial mesoderm, which is functionally divided into head (or precordal) and trunk organizer (notochord). In Xenopus the homeobox genes goosecoid (Drosophila homolog: Goosecoid) and Otx2 (Drosophila homolog: Orthodenticle) are expressed in the precordal mesoderm; the LIM class homeobox gene Xlim-1 (Drosophila homolog: Apterous) is expressed in the entire axial mesoderm, whereas the distinct Brachyury related transcription factor Xbra (Drosophila homolog: T-related gene) is expressed in the notochord but not in the procordal mesoderm. Messenger RNA injection experiments show that Xenopus animal pole explants (caps) expressing an activated form of Xlim-1 (a LIM domain mutant named 3m) induce anterior neural markers, whereas caps coexpressing Xlim-1/3m and Xbra induce posterior neural markers. These data indicate that in terms of neural inducing ability, Xlim-1/3m-expressing caps correspond to the head organizer and Xlim-1/3m plus Xbra-coexpressing caps to the trunk organizer. Thus the expression domains of Xlim-1 and Xbra correlate with, and possibly define, the functional domains of the organizer. In animal caps Xlim-1/3m initiates expression of a neuralizing factor chordin (Drosophila homolog: Short gastrulation), whereas Xbra activates embryonic fibroblast growth factor (eFGF expression) (See Drosophila FGF homolog Branchless); these factors could mediate the neural inducing and patterning effects that are observed. A dominant-negative FGF receptor (XFD) inhibits posteriorization by Xbra in a dose-dependent manner, supporting the suggestion that eFGF or a related factor has posteriorizing influence (Taira, 1997).
In Xenopus the CNS is induced by signals emanating from
the Spemann organizer. Two proteins secreted by the organizer, Noggin (a protein that binds to and inactivates BMP-4, the vertebrate homolog of Decapentaplegic) and Follistatin, have been shown
to induce neural tissue in animal-cap assays. Chordin, the Xenopus SOG homolog, has neuralizing activity. This activity can be antagonized by Bmp-4.
Inhibition of the function of the endogenous Bmp-4 present in the animal cap leads to neural
differentiation (Sasai, 1995).
Xenopus chordin is expressed in the dorsal blastopore lip of the embryo and in
dorsal mesoderm, in particular the notochord.
Both SOG and Chordin can promote ventral development in Drosophila, in embryos given injections of messenger RNA. SOG, like Chordin,
can promote dorsal development in Xenopus. In Drosophila, SOG antagonizes the dorsalizing
effects of Decapentaplegic (DPP), a member of the transforming growth factor-beta family. One of
the DPP homologs in vertebrates, bmp-4, is expressed ventrally in Xenopus and promotes
ventral development. DPP can promote ventral fates in Xenopus. Injection
of SOG mRNA counteracts the ventralizing effects of DPP. These results suggest the molecular
conservation of dorsoventral patterning mechanisms during evolution (Holley, 1995).
Chordin antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Chordin carries out this function by binding directly to BMPS. The neural induction activity of Chordin can mimic Spemann's organizer signals at concentrations close to physiological levels. Chordin induces Neural cell adhesion molecule in Xenopus animal cap explants. Addition of equimolar amounts of BMP-4 (the Xenopus homolog of Decapentaplegic) antagonizes neural induction by Chordin. Chordin also induces mesoderm dorsalization. Induction of dorsal mesoderm is evidenced by activation of muscle actin mRNA, activation of N-CAM, and elongation of explants and formation of somites. BMP-R reverses the dorsalized phenotype caused by Chordin (Piccolo, 1996).
In a differential screen in Xenopus for downstream genes of the neural inducers, two
extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1
(Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a
Sry-related HMG factor (see Dichaete). Expression of the two genes is first detected widely in the
prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin
expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the
proneural gene Xngnr-1 (see Achaete), and initiates neural and neuronal differentiation in isolated animal
caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but
can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in
the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2
makes the ectoderm responsive to extracellular signals, demonstrating that the early phase
of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).
The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites. In biochemical experiments Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, or follistatin, and not by injection of mRNA coding for dominant-negative BMP receptor. Xolloid-treated Chordin protein is unable to antagonize BMP activity. Furthermore, Xolloid digestion releases biologically active BMPs from Chordin/BMP inactive complexes. Injection of
dominant-negative Xolloid mRNA indicates that the in vivo function of Xolloid is to limit the extent of Spemann's organizer field. It is proposed that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis (Piccolo, 1997).
In Xenopus, one of the properties defining Spemann's organizer is its ability to dorsalize the mesoderm. When placed adjacent to prospective lateral/ventral mesoderm (blood, mesenchyme), the organizer causes these cells to adopt a more axial/dorsal fate (muscle). It seems likely that a similar property patterns the primitive streak of higher vertebrate embryos, but this has not yet been demonstrated clearly. Using quail/chick chimaeras and a panel of molecular markers, it has been shown that Hensen's node (the amniote organizer) can induce posterior primitive streak (prospective lateral plate) to form somites (but not notochord) at the early neurula stage. Two BMP antagonists, noggin and chordin (both of which are expressed in the organizer), were examined for their ability to generate somites and intermediate mesoderm from posterior streak, and it was found that noggin, but not chordin, can do this. Conversely, earlier in development, chordin can induce an ectopic primitive streak much more effectively than noggin, while neither BMP antagonist can induce neural tissue from extraembryonic epiblast. Neurulation is accompanied by regression of the node, which brings the prospective somite territory into a region expressing BMP-2, -4 and -7. One function of noggin at this stage may be to protect the prospective somite cells from the inhibitory action of BMPs. These results suggest that the two BMP antagonists, noggin and chordin, may serve different functions during early stages of amniote development (Streit, 1999).
Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various
developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by
releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of
this family of developmental proteases, novel family member mammalian Tolloid-like 2 (mTLL-2) is identified and enzymatic activities and expression
domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2] are compared.
Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of
SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site,
while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and
counteract the dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic
activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and post-natal
skeletogenesis (Scott, 1999).
Patterning in the vertebrate embryo is controlled by an interplay between signals from the dorsal organizer and the ventrally expressed BMPs. The
function of Vox, a homeodomain-containing gene that is activated by the ventralizing signal BMP-4, has been examined in this study. Inhibition of BMP signaling using a dominant negative BMP
receptor (DeltaBMPR) leads to the ectopic activation of dorsal genes in the ventral marginal zone, and this activation is prevented by co-injection of Vox. chordin is
the most strongly activated of those genes that are up-regulated by DeltaBMPR and is the gene most strongly inhibited by Vox expression. This study demonstrates that Vox acts as a transcriptional repressor, showing that the activity of native Vox is mimicked by a Vox-repressor fusion (VoxEnR) and that a Vox-activator fusion
(VoxG4A) acts as an antimorph, causing the formation of a partial secondary axis when expressed on the ventral side of the embryo. Although Vox can ectopically
activate BMP-4 expression in whole embryos, no activation of BMP-4 by VoxG4A is seen, demonstrating that this activation is indirect. Using a hormone-inducible
version of VoxG4A, it has been found that a critical time window for Vox function is during the late blastula period. Using this construct, it has been demonstrated that only a subset of
dorsal genes is directly repressed by Vox, revealing that there are different modes of regulation for organizer genes. Since the major direct target for Vox repression
is chordin, it is proposed that Vox acts in establishing a BMP-4 morphogen gradient by restricting the expression domain of chordin (Melby, 1999).
The presumptive pharyngeal endoderm region of the Cynops (salamander) early gastrula induces head or trunk-tail
structures in sandwich culture. Activin-treated ectoderm can mimic this phenomenon at least at the
histological level. The patterns of expression of organizer-specific genes were examined to compare
these two inductive materials at the molecular level. A chordin cDNA clone from Cynops pyrrhogaster
(Cychd) was isolated by reverse transcription-polymerase chain reaction (RT-PCR). Cychd mRNA
is first detected in the presumptive pharyngeal endoderm and prechordal plate regions of stage 11
embryos, and is expressed continuously until stage 20. The spatiotemporal expression pattern of
Cychd is similar to that of Xenopus chordin. The patterns of expression of organizer-related genes in
the pharyngeal endoderm and activin-treated ectoderm were compared by RT-PCR analysis.
Expression of Cychd in these two materials peaks at the time when they can induce head structures
in sandwich culture. Expression of fork head and goosecoid does not change in the presumptive
pharyngeal endoderm over this period. Cychd may play a key role in head formation in the Cynops
embryo (Yokota, 1998).
The cAMP signaling system has been postulated to be involved in the embryogenesis of many animal species, however, little is known about
its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the
Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT1a R),
which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos,
ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induces a secondary axis. The secondary axis induced by ventral stimulation of the 5-HT1a R is usually incomplete, with no head structure or much reduced anterior structure. At the molecular level, 5-HT1a R stimulation induces expression of the dorsal mesoderm marker genes, and down-regulates expression of the ventral markers but has no effect on expression of the pan mesodermal marker gene in ventral marginal zone
explants. Stimulation of 5-HT1a R at stage 8 induces the
expressions of noggin and chordin, but not siamois in the
gastrula (stage 11.5) VMZ explants. In contrast, the expression of ventral-specific marker, BMP-4, is significantly reduced in the stage 11.5 VMZs that had been stimulated with 5-HT. The expressions of
Xvent-2 and PV.1 are also reduced but not as greatly as
BMP-4. The expression of Xbra, a generic
mesoderm marker, did not change in both DMZ and VMZ explants, indicating that the formation of mesoderm is not affected by the receptor activation. Ventral expression and stimulation of the receptor partially restores dorsal axis of UV-irradiated axis deficient embryos. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in the
patterning of early embryos (Kim, 1999).
Not surprisingly, given the number of proteins associated with the organizer, Noggin also binds to and inactivates BMP-4. This is in addition to the known binding and inactivation of activin (a potent mesoderm inducer and homolog of TGFß) by follistatin. BMP-4 and BMP-2 effectively compete for binding to Noggin, while BMP-7 binds less tightly and TGFß not as all. Noggin binding prevents BMP-4 from binding to its receptor (Zimmerman, 1996 and references).
BMP activity is controlled by several
secreted factors including the antagonists chordin and Short gastrulation (Sog). A second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signaling using anti-phospho-Smad staining shows that the tsg and sog
loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, Tsg and Sog together make a more effective inhibitor of BMP signaling than either
of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants.
Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype.
Tsgs from different species are functionally equivalent, and it has been concluded that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signaling (Ross, 2001).
Since the phenotypes of tsg and sog mutants are similar, attempts were made to determine whether Tsg can enhance the binding of Sog to ligand. Co-immunoprecipitation of DPP by Sog is greatly enhanced when these two factors are coexpressed in S2 cells along with Tsg. To test whether the combination of Sog and Tsg blocks Dpp signaling better than Sog alone, an S2 cell-culture assay was developed for Dpp signaling. At high concentration Tsg alone can block Dpp signaling; however, at lower concentration, the combination of Tsg and Sog together dramatically reduces the Dpp-dependent accumulation of P-MAD much more efficiently than either can alone. In vivo overexpression of sog and tsg together can completely reverse the phenotype of ectopic dpp expression in the wing, whereas the expression of either alone has no effect. It is concluded that a complex of Tsg and Sog is an efficient antagonist of Dpp signaling (Ross, 2001).
To determine whether Tsg is conserved among other species, genes in the database related to Drosophila Tsg were sought and found in human, mouse, zebrafish and Xenopus. In addition, a second tsg-related sequence was found in Drosophila (tsg2) and a second zebrafish tsg (tsg1) was obtained using degenerate polymerase chain reaction (PCR) methods. The protein products show extensive similarity with about 50% of 202 amino-acid residues matching in all four species. The pairs of tsg genes in fly and fish are closer to each other than to tsg in any other species, suggesting independent gene-duplication events in these two species. The human, mouse and zebrafish (tsg1) genes were mapped by a combination of fluorescence in situ hybridization (FISH) or radiation hybrid mapping. The mouse gene maps to 17E1.3–E2, a region that is syntenic to 18p11.2–3 where the human homologue resides. In zebrafish, tsg1 is located at linkage group 24-74.5, which is syntenic to the human locus and indicates that all three genes are probably functional orthologues (Ross, 2001).
The zebrafish tsg1 gene is expressed uniformly in early embryos, whereas zebrafish tsg2 is only expressed at later stages. Hence, the analysis was focused on zebrafish tsg1 and morpholino oligonucleotides were used to reduce the function of this gene in early zebrafish development. Injection of a tsg1 morpholino oligonucleotide (ztsg-MO: see Taylor, 1996 for a description of the morpholino modification) produces a phenotype characteristic of expanded BMP signaling. Using morphological criteria and fluorescent red blood cells, it was found that embryos develop expansions of the ventral fin region that correspond to ectopic blood islands, a tissue derived from ventral mesoderm. Injected embryos also show an expansion of GATA2, loss of paraxial mesoderm (visualized with the marker myoD), and a mild reduction of anterior ectodermal tissues (detected by staining for krox20). Caudal expression of bmp4 is also expanded in these embryos, while the anterior ectodermal marker otx2 is reduced. Treated embryos also exhibit an expansion in apoptotic cells ventral to the yolk extension, similar to dino and mercedes mutants. Overall, this phenotype is very similar to that of ogon/mercedes mutants and moderate chordin loss-of-function mutants, and represents a modest ventralized phenotype (Ross, 2001).
Since Drosophila data suggest that one function of Tsg is to cooperate with Sog to inhibit BMP signaling, it was asked whether the same relationship is true in vertebrates by determining whether a modest reduction of zebrafish chordin activity can enhance the effect of a moderate reduction in tsg1 activity. Sub-inhibitory levels of a zebrafish chordin morpholino oligonucleotide and tsg1-MO were injected into wild-type embryos, and the effect on ectopic blood island development was scored. These two morpholino oligonucleotides synergistically enhance blood island expansion, supporting the view that both of these gene products co-operatively inhibit BMP signaling. As with the Drosophila components, it was found that the combination of purified mouse chordin and Tsg is better able to inhibit mouse BMP-stimulated phosphorylation of Mad in S2 cells than either can alone (Ross, 2001).
A test was performed for synergy between Tsg and chordin mRNA in Xenopus embryos by co-injecting their mRNAs and scoring for enhancement of secondary axis formation. Co-injection of Xenopus Tsg and chordin reveals a dose-response optimum. When a sub-inhibitory dose of chordin mRNA is supplemented with increasing levels of Tsg mRNA, the fraction of embryos exhibiting a secondary axis increases up to 4.5-fold over chordin alone at a 1/5 ratio of Tsg/chordin mRNA. However, if the Tsg/chordin ratio is increased to 1:1 or higher, the number of secondary axes is reduced to basal levels and the resulting tadpoles have normal morphology. Injection of 150 pg Tsg alone (the highest concentration of Tsg mRNA used in these experiments) has no effect on embryonic development. Notably, if the level of Tsg relative to chordin is increased in the S2 experiments, no reversal of the inhibition phenotype is seen, suggesting that additional factors probably modulate the in vivo response. Taken together, it is concluded that, like Drosophila Tsg, vertebrate Tsg can co-operate with chordin to inhibit BMP signaling (Ross, 2001).
As a final test of the functional equivalence of the vertebrate and invertebrate tsg genes, the human and mouse genes were expressed under the control of the UAS promoter in flies, and Drosophila Tsg mRNA was injected into zebrafish embryos. The phenotype of animals expressing human Tsg and Drosophila sog in wing discs resembles that of dpp shortvein alleles and is very similar to that produced by coexpression of the Drosophila tsg and sog genes. When injected into zebrafish, Drosophila tsg produces a dorsalized phenotype equivalent to that produced by zebrafish tsg1, which includes reduced axial length and expansion of krox20 (Ross, 2001).
These experiments suggest that Tsg has three molecular functions. (1) It can synergistically inhibit Dpp/BMP action in both Drosophila and vertebrates by forming a tripartite complex between itself, Sog/chordin and a BMP ligand. (2) Tsg seems to enhance the Tld/BMP-1-mediated cleavage rate of Sog/chordin and may change the preference of site utilization. (3) Tsg can promote the dissociation of chordin cysteine-rich (CR)-containing fragments from the ligand. Different organisms may exploit each of these properties to different degrees during development depending on the relative in vivo concentrations of each molecule. It is proposed that in Drosophila and zebrafish the primary function of Tsg is to form a tripartite complex between itself, Sog/chordin and a BMP ligand. In Drosophila, this complex acts to redistribute a limiting amount of Dpp, such that activity is elevated dorsally at the expense of being lowered laterally. The net driving force for this redistribution is likely to be diffusion of Sog from its ventral source of synthesis. This is consistent with the finding that Sog diffusion is essential for activation of genes such as race that require high levels of Dpp/SCW signaling. In this model Tld would serve to modulate both the net movement of Dpp and its release from the inhibitory complex by cleaving Sog. The ability of Tsg to enhance the rate of Sog cleavage may also be an important aspect of this model in that it helps ensure the proper timing of these rapid developmental events. It seems unlikely that Tsg is needed to remove an inhibitory CR-containing fragment from Dpp, since the affinity of full-length Sog for Dpp in the absence of Tsg seems to be low. Likewise, in zebrafish the phenotype of reduced Tsg function is ventralized and not dorsalized as would be predicted if Tsg were primarily needed to release inhibitory CR fragments from ligand. In Xenopus, however, perhaps the endogenous levels of full-length chordin and CR fragments are higher than in zebrafish, thereby making the CR displacement activity of Tsg the more important biological function. Determination of the in vivo levels of these proteins, along with a more careful analysis of the concentration optima for each type of reaction involving Tsg function, will be required before all of its in vivo activities can be understood (Ross, 2001).
The characterization of
the vertebrate Tsg homologs is reported. Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient
inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late
developmental stages. These data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis (Chang, 2001).
Human Twisted gastrulation (TSG) was isolated in a screen for secreted factors, and mouse and Xenopus Tsg were isolated by low-stringency hybridization using human TSG as the probe. These vertebrate Tsgs have a high sequence homology to one another (more than 80% identical) and are about 30% identical to Drosophila Tsg at the amino-acid level. Tsg is expressed maternally and in all developmental stages in Xenopus, and at least from gastrula stages onward in
mouse. Expression of Tsg is also detected in a variety of adult tissues in both mouse and human (Chang, 2001).
To study the function of Tsgs, their activities were examined in Xenopus ectodermal explants (animal caps). Human, mouse and Xenopus
Tsg induce the cement gland and the neural markers XAG-1, OtxA and NRP-1 with comparable efficiency, suggesting that these vertebrate Tsgs function
similarly in Xenopus. The induction of cement gland and neural markers in animal caps in the absence of mesoderm is normally associated with inhibition of the
BMP signaling, so whether Tsg could directly block the activity of BMP was addressed. The effect of Tsg on ventralization of
the ectodermal cells by BMPs was examined. Intact animal caps express high levels of epidermal keratin. This expression is suppressed when caps
from blastula stages are dissociated for 4 h. Cement gland and neural markers are turned on in these dissociated samples. While Bmp2
restores the transcription of epidermal keratin and inhibits the expression of neural markers, it cannot do so in the presence of Tsg.
These results indicate that Tsg directly antagonizes the neural inhibition and epidermal induction activity of BMPs in dissociated animal caps (Chang, 2001).
The effect of Tsg on ventralization of the mesodermal explants by BMPs was examined. Bmp4 inhibits dorsal and induces ventral gene expression in dorsal
marginal zone (DMZ) explants. Coexpression of Xenopus Tsg with Bmp4 re-establishes dorsal marker expression and reduces the ventral gene
induction in these explants, indicating that it also inhibits BMP activity in the mesodermal cells. Notably, a reduction in the level
of OtxA (a marker for anterior neural tissue at tailbud stages) was detected when Tsg alone was expressed in the DMZ. This phenotype has not been observed
with other BMP antagonists, and suggests that Tsg may be involved in processes other than inhibition of BMP signaling. To determine whether Tsg also blocks
other signal transduction pathways, the effect of Xenopus Tsg on marker induction by activin, basic fibroblast growth factor (FGF) and Wnt8 was examined.
Tsg specifically inhibits BMP-dependent mesoderm induction, but does not interfere with the expression of the markers induced by the other signaling molecules. These results demonstrate that Tsg is specific for a BMP pathway and does not block activin, FGF or Wnt signaling (Chang, 2001).
The in vivo function of Tsg was examined by gain-of-function studies. Injection of Tsg RNAs from different vertebrates in early embryos induces a similar phenotype, indicating that they have similar activities in vivo. The embryos injected with Tsg RNA exhibit several developmental defects at tadpole
stages, such as an enlargement of the dorsal fin, malformation of the proctodeum, and a reduction of the head. Lineage tracing with nuclear
ß-gal shows that the effect induced by Tsg is non-cell autonomous. The phenotype of Tsg-expressing embryos is unique and does not
resemble the phenotypes induced by overexpression of other BMP antagonists. Therefore, whether Tsg affects the cells and tissues that rely on the
BMP signaling was addressed. The formation of the blood cells, which are derived from ventral tissues and require active BMP signals, was examined. In control embryos, the blood cells stained with benzidine were localized at the ventral side of the embryos; however, this staining was not observed in embryos injected with Xenopus Tsg RNA. Furthermore, in situ hybridization with a blood-specific anti-T1-globin probe reveals that blood cells are absent in the Tsg-expressing
embryos. The formation of the heart, which has been reported in both the chick and the frog to require BMP signaling, was examined. In situ
hybridization with a heart marker, Nkx2.5, shows that the Tsg-expressing embryos have reduced transcription of Nkx2.5. In contrast, the expression
of a dorsal mesoderm marker in muscle is not decreased by Tsg. These data show that Tsg interferes with the development of several tissues that require
BMP signaling (Chang, 2001).
To further analyse the effects of Tsg, expression of both dorsal and ventral genes was assayed in Tsg-injected embryos at different developmental stages. In
gastrula embryos, several ventral markers, such as Xhox3, Vent1 and Msx1, are downregulated by Xenopus Tsg, whereas dorsal markers, including Sox2,
goosecoid and Hex, are not much affected. The expression of the dorsal gene chordin also remains the same in most cases, although a slight reduction of its level was occasionally seen. The pattern of gene expression changes during development, so that at later stages, Xhox3 and Msx1 are also expressed
in the neural tissue and neural crest cells. The reduction of their expression is less profound, and at tailbud stages Xhox3 expression is normal. In agreement with whole-mount staining results, the transcripts of both globin and Nkx2.5 are reduced by Tsg, whereas the muscle actin remains intact at tailbud stages. Unexpectedly, the reduction of several anterior genes, such as Hex (a marker for anterior endoderm) and goosecoid (a prechordal mesoderm
gene), by Xenopus Tsg is seen from neurula stages onward. Sox2, whose expression domain includes the anterior neural region, is also slightly downregulated. These
data demonstrate that Tsg reduces BMP signaling at early developmental stages; furthermore, Tsg may have one or more additional functions in anterior
embryonic development at a stage after the onset of gastrulation (Chang, 2001).
Although Tsg can inhibit BMP function, it does not induce a partial secondary axis when injected into the ventral side of early Xenopus embryos. One possible
explanation for this is that Tsg may be a highly diffusible molecule, as has been observed in Drosophila, and does not accumulate to high concentrations at the
ventral side to establish an organizer-like activity. To test this hypothesis, a membrane-tethered form of Tsg was constructed by fusing the full-length Xenopus
Tsg in frame with an integral membrane protein CD2. The chimaeric protein, Tsg-CD2, induces cement gland and neural markers in animal caps to the
same extent as wild-type Xenopus Tsg, suggesting that the two proteins have similar activities. Injection of the CD2 RNA into early frog embryos does
not induce any phenotype, but ventral injection of Tsg-CD2 RNA leads to embryos with partial secondary axes. These results indicate that localized Tsg behaves like other BMP antagonists and can induce ectopic dorsal structures on the ventral side of frog embryos (Chang, 2001).
To investigate whether Tsg modifies the BMP inhibitory activity of chordin, the effect of coexpression of chordin and Xenopus Tsg in both
ectodermal explants and whole embryos was examined. In animal caps, a low dose of either chordin or Tsg does not efficiently block Brachyury (Bra) induction by Bmp4; coexpression of both genes leads to a more effective inhibition of the BMP activity. In whole embryos, chordin induces a partial secondary axis when
expressed at the ventral side; coexpression with Xenopus Tsg at a ratio of Tsg/chordin below 1/1 does not prevent the ectopic axis-induction by chordin.
Instead, many embryos show a dorsalized phenotype with reduced trunk and tail. Notably, with an increasing Tsg/chordin ratio (2/1 or higher), the
secondary axis disappears in an increasing number of embryos, and these embryos resume a typical Tsg phenotype. Tsg and
chordin still inhibit Bra induction by Bmp4 in animal caps at these high ratios. The data suggest that although Tsg and chordin expression together
lead to a more efficient BMP inhibition in ectodermal explants regardless of the amounts of RNA injected, the in vivo phenotypes depend on the relative
abundance of the two genes. The exact mechanism underlying this observation remains unclear. There are at least two possibilities that are not mutually
exclusive. The highly diffusible Tsg may help to diffuse the chordin protein through complex formation, which may result in dilution of the chordin function at the ventral side and inhibition of the secondary axis induction by chordin. Alternatively, other endogenous factors may interact with chordin and/or Tsg and modify their activities and the phenotypes of coexpression of chordin and Tsg (Chang, 2001).
Tsg, first identified in Drosophila and shown to have a function in development of the dorsal midline, was originally proposed to potentiate DPP activity.
This hypothesis, however, has recently been challenged by the observation that a processed SOG product, which has a broader inhibitory spectrum towards BMP
members, rescues the tsg mutant phenotype, whereas Dpp does not. This result indicates that Tsg may participate in alternative processing of Sog to
generate a 'supersog' to block the DPP signaling. Tsg may therefore be involved in inhibiting, rather than enhancing, the Dpp activity. A similar mechanism
suggesting that Tsg may participate in inhibition of the BMP signaling has now been proposed to also work in vertebrates. Tsg stimulates chordin cleavage
at a unique site, and Tsg enhances chordin activity in both Xenopus and zebrafish. This study shows that Tsg can function as a BMP antagonist in embryonic
explants and interfere with BMP-dependent tissue formation in frog embryos. The activity of Tsg, however, is different from that of other BMP antagonists, since
Tsg also induces defects in the anterior tissues, such as the anterior endoderm expressing the Hex gene and the prechordal mesoderm that expresses goosecoid.
This phenotype may underlie the recent interpretation that vertebrate Tsg acts to promote BMP signaling, since it has been reported that elevated BMP
expression leads to reduction of dorsal markers at the gastrula stages, which results in truncation of anterior and dorsal tissues at late stages. It is currently unclear, however, whether the defects induced by TSG truly reflect an enhancement of BMP signaling. Although the possibility cannot be ruled out that Tsg can function both as a BMP agonist and antagonist depending on the presence or absence of other factors in specific regions of the embryos at particular
developmental stages, it is also possible that the late effects are still mediated by inhibition of a BMP pathway. Several BMPs are expressed in the anterior endoderm and/or prechordal mesoderm in chick and fish, and in chick, BMPs may be involved in specification of prechordal mesoderm. It is possible that
Tsg inhibits a different spectrum of BMPs from other BMP antagonists, either alone or by alteration of chordin specificity, thereby leading to the unique
phenotype in the anterior tissues. Another possibility is that Tsg may have one or more additional functions independent of BMP signaling. Further investigation
is required to understand the in vivo function of Tsg (Chang, 2001).
The Chordin requirement in Xenopus development has been analyzed. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreases Chordin production. Embryos develop with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations: (1) dorsalization by lithium chloride treatment is completely blocked by Chd-MO; (2) Chd-MO inhibits elongation and muscle differentiation in Activin-treated animal caps; (3) Chd-MO completely blocks the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation (Oelgeschläger, 2003a).
The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. The function of Tsg in embryonic patterning was investigated by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain are unable to bind BMP, yet ventralize the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity requires an intact C-terminal domain and can block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity is specific for CR-containing proteins since it does not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants are stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos (Oelgeschläger, 2003b).
The ectoderm gives rise to both neural tissue and epidermis. In vertebrates, specification of the neural plate requires repression of bone morphogenetic protein (BMP) signaling in the dorsal ectoderm. The extracellular BMP antagonist Chordin and other signals from the dorsal mesoderm play important roles in this process. Zebrafish mutant combinations that disrupt Chordin and mesoderm formation were used to reveal additional signals that contribute to the establishment of the neural domain. Fibroblast growth factor (FGF) signaling accounts for the additional activity in neural specification. Impeding FGF signaling results in a shift of ectodermal markers from neural to epidermal. However, following inhibition of FGF signaling, expression of anterior neural markers recovers in a Nodal-dependent fashion. Simultaneously blocking, Chordin, mesoderm formation, and FGF signaling together eliminates neural marker expression during gastrula stages. FGF signaling is required for chordin expression but it also acts via other mechanisms to repress BMP transcription during late blastula stages. Activation of FGF signaling is also able to repress BMP transcription in the absence of protein synthesis. These results support a model in which specification of anterior neural tissue requires early FGF-mediated repression of BMP transcript levels and later activities of Chordin and mesodermal factors (Londin, 2005).
The function has been investigated of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and this study shows that Smicl is involved in a two-step process that is necessary for this activation. In the first step, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induce expression of Chordin. As well as revealing the function of Smicl in the early embryo, this work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo (Collart, 2005).
An unexpected role is reported for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10-8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibits Xlr, suggesting a wider role for this molecular mechanism (Lee, 2006).
Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. It was found, through embryological and biochemical experiments, that (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo, (2) CV2 complexes with Twisted gastrulation and BMP4, (3) CV2 is not a substrate for tolloid proteinases, (4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range), (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes, and (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. It is proposed that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels (Ambrosio, 2008).
Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. A strong genetic interaction has been reported between CV2 and Tsg, and a role for this interaction has been proposed in the shaping of the BMP morphogenetic field during vertebral development. The present study investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. Immunostainings were performed for CV2 and Chd protein on wild-type, CV2-/- or Chd-/- mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations it was found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2-/- mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2-/- embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2-/- vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields (Zakin, 2010).
In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. A new BMP1/Tolloid assay was developed using a fluorogenic Chordin peptide substrate, and an unexpected negative feedback loop for BMP4 was identified, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin (Lee, 2009).
The differentiation of cell types along the vertebrate D-V axis is regulated by an extracellular network of BMPs and their regulators, such as Chordin, BMP1/Tolloid, Tsg, and Crossveinless-2, in animals as diverse as Xenopus, Drosophila, zebrafish, amphioxus, hemichordates, and spiders. In addition, in the vertebrates, additional extracellular BMP antagonists such as Noggin and Follistatin cooperate with the anti-BMP activity of Chordin. The complexity of this biochemical pathway raises the question of why so many components and regulatory interactions are required to establish a simple gradient of BMP signaling through the transcription factors Smad1/5/8. One reason is that a stable gradient must be robustly maintained through many hours of development (from blastula until the end of gastrulation) at a time during which the three embryonic germ layers are undergoing massive morphogenetic movements. In addition, the frog embryo must have the ability to adapt to changes in temperature in its environment (Lee, 2009)
The patterning system must be resilient, given the self-regulating nature of development. When Xenopus embryos are cut in half, they will attempt to regenerate an embryo as perfect as possible, producing in some cases identical twins. This implies that cells in the dorsal and ventral poles of the early embryo communicate with each other, forming a self-regulating embryonic field. At a molecular level, these cell-cell communications can be explained by a pathway in which dorsal BMPs (ADMP and BMP2) and their antagonist, Chordin, are repressed at the transcriptional level by BMP signaling, while on the ventral side, BMP4/7 and CV2 are activated by the same signal, providing a self-regulating system. The key controlling element in this D-V conversation is provided by BMP1/Tolloid enzymes that degrade Chordin/BMP complexes releasing active BMP that are regulated by the Sizzled/Ogon-secreted competitive inhibitor. In this study, a novel regulatory node in the D-V patterning pathway, in which BMP4 serves as a feedback inhibitor of the BMP1 and Tolloid-related enzymes, was introduced (Lee, 2009).
A synthetic Chordin octapeptide spanning the C-terminal cleavage site that fluoresces when cleaved by Tolloids provided a quantitative enzymatic assay. This new assay was essential to the work, because both Chordin and Sog become better substrates for Tolloids when bound to BMP. It is therefore not possible to conduct a biochemical study on the digestion of full-length Chordin/Sog plus or minus BMP, because BMP affects both the substrate and the enzyme. The conformational change in the Chordin/Sog substrate would have precluded the discovery of the inhibition of enzyme activity by BMP4 (Lee, 2009).
Inhibition of BMP1/tolloids by BMP4 was specific, because it was not observed with other proteins such as Activin A, Tsg, Follistatin, and Noggin. The kinetics followed those of a Michaelis-Menten noncompetitive inhibition. This meant that BMP4 affected the activity of the enzyme by binding to a site distinct from the catalytic center. BMP4 was found to bind directly to CUB domains with high affinity. The Ki or inhibition constant (concentration at which half of the enzyme is bound to the inhibitor) for BMP1 was in the 40 nM range, and in the 14-20 nM range when measured by direct binding. This is within physiological levels, since the Km (Michaelis constant or affinity of the enzyme for its substrate) of BMP/Tolloids for Chordin substrate was between 17 and 25 nM, and of 96 nM for its BMP1/PCP activity (Lee, 2009).
The ventral center of the Xenopus gastrula expresses a chordin-like protein called CV-2 that strongly binds Chordin/BMP complexes transported from more dorsal regions of the embryo and facilitates BMP signaling through its cognate receptors after cleavage of Chordin by BMP1/tolloids. This suggests that in vivo free BMP is locally concentrated at sites of high CV2 and chordinase activity; it is in these regions that the negative feedback loop should be most effective. Not only will the BMP levels be highest, but also the Chordin levels will be lowest. The affinities of the interaction between BMP4/Tsg/Chordin and Tolloid may also be enhanced by the recently described Olfactomedin-related adaptor protein Ont-1, which brings together Chordin and tolloids (Lee, 2009).
The importance of the interaction between Tolloid and BMPs for developmental patterning in vivo is suggested by Drosophila genetics. A very large allelic series of tolloid mutants has been obtained that display a graded series of patterning defects along the D-V axis in Drosophila. This suggests that Tolloid provides a rate-limiting step during patterning. Therefore, any decrease in its activity caused by binding of BMPs would be expected to regulate the signaling gradient. The antimorphic tolloid mutations, which are proteolytically inactive but display anti-BMP effects, demonstrate that endogenous Tolloid enzyme is expressed at high enough levels to function antagonistically toward Dpp in vivo. Thus, at least in Drosophila, the interactions between Tolloid and BMPs discovered in this study function at physiological concentrations of D-V pathway components (Lee, 2009).
There previously had been isolated reports showing that DN-BMP1/tolloids dorsalized Xenopus ventral mesoderm, which should lack Chordin. One possible interpretation for these results was the presence of a Chordin counterpart, such as CV2, expressed in the high-BMP regions of the embryo. However, it was later found that CV2 is resistant to degradation by tolloids/BMP1. Instead, it was found that the anti-BMP effect of DN-tolloids, which can take place in Chordin-depleted embryos, are due to the sequestration of BMP ligands through direct binding to CUB domains (Lee, 2009).
It was initially hoped that the second site mutations described in Drosophila Tolloid CUB domains would point to amino acid residues critical for Tolloid binding of BMP4. Instead, all second site mutations affected Tolloid/BMP1 protein secretion. These second site antimorphic revertants behave essentially as null mutations of tolloid because they are not secreted. It is likely that the original antimorphic mutants displayed anti-Dpp effects because they bound BMPs in the Drosophila embryo (Lee, 2009).
CUB domains are also required for enzymatic activity. In the case of BMP1/PCP, it has been shown that the procollagen substrate is not efficiently recognized when CUB2 of BMP1 is deleted. However, the protease domain plus CUB1 is sufficient for BMP1 chordinase activity. In the case of Drosophila Tolloid, CUB4 and CUB5 are required to cleave Sog, and for Xolloid, CUB1 and CUB2 are required for recognition and cleavage of Chordin. Thus, CUB domains in Tolloid/BMP1 have specific functions in substrate recognition. CUB domains are also required for secretion, in addition to serving as inhibitory BMP-binding sites. As an interaction between the BMP1 prodomain and BMP4 has also been reported, it should be noted that the prodomain was lacking in all the CUB domain constructs used in the present study (Lee, 2009).
CUB domains are present in many secreted or transmembrane proteins, but their biochemical function remains unknown. The human genome contains 56 different loci encoding CUB domain-containing proteins. The finding that the CUB domains of BMP1 and Tolloid bind BMP4 suggests the exciting possibility that CUB domains may serve as binding modules for TGF-β superfamily ligands in other extracellular proteins as well. In the future it will be interesting to investigate, for example, the binding properties of the CUB domains found in Complement components C1r and C1s, which function in the opsonization of antigens. Another interesting protein is CUB domain-binding protein 1 (CDCP-1), a transmembrane receptor with three CUB domains that activates the Src tyrosine protein kinase and promotes metastases in human cancers; TGF-β also promotes metastases. Other CUB domain-containing proteins include membrane frizzled-related protein (MFRP), in which mutations in CUB domains cause nanophthalmos; procollagen C-peptidase enhancer (PCPE), known as a potent enhancer of BMP1/PCP activity in procollagen processing; the WNT transmembrane coreceptor Kremen; and many other extracellular or transmembrane proteins (Lee, 2009).
The effects of enzymatic inhibition -- in this case, noncompetitive inhibition by BMP4 -- were integrated into a reaction-diffusion model to understand its effect on the BMP morphogen gradient of the early Xenopus embryo. This mathematical modeling predicted that Tld activity will be inhibited in ventral regions in which BMPs are present in high concentrations. An unexpected finding was that Chordin itself is a major regulator of BMP/Tolloid activity. At high concentrations, such as in the dorsal side of the frog gastrula and likely in the fly ventral blastoderm, Chd/Sog complexed with Tld is predicted to decrease the availability of free (active) BMP/Tolloid. This will inhibit degradation of Chordin-BMP complexes, preventing local BMP release and signaling, enabling the complex to diffuse further (Lee, 2009).
These observations suggest that the Tolloid inhibition by BMP also takes place in fruit flies, which provide a system much more amenable to the visualization of gradients, and for which sophisticated mathematical modeling already exists. In the future, it will be interesting to investigate whether CUB domains generally serve as BMP or TGF-β superfamily-binding modules. This approach has been productive in the case of the CR/vWFc domains of Chordin, which function as BMP-binding modules in many proteins (Lee, 2009).
The present study suggests that the antimorphic revertant mutations, were based on direct Dpp-Tolloid associations and were indicators of a crucial step in the formation or maintenance of the self-adjusting D-V morphogen gradient. The findings in Drosophila and Xenopus also suggest that this extracellular negative feedback regulation was already present in the patterning system of Urbilateria, the last common ancestor of all bilateral animals that lived more than 535 million years ago. Finally, the direct binding of BMP4 to BMP1 explains why highly purified bone-inducing protein preparations contained BMP1/Tolloid in addition to BMP2-7. It may be worthwhile to explore the value of BMP1 or its CUB domains as a delivery system for BMPs in therapeutic interventions, such as the repair of bone fractures (Lee, 2009).
Continued: Evolutionary Homologs part 2/2
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