1 The Queensland Institute of Medical Research, PO Royal Brisbane Hospital,
Herston, Brisbane 4029, Australia
2 Department of Zoology and Entomology, University of Queensland, Brisbane 4072,
Australia
3 Central Clinical School, University of Queensland, Brisbane 4029,
Australia
* Author for correspondence (e-mail: rickw{at}qimr.edu.au)
Accepted 11 February 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hemps, Ascidian Herdmania curvata, Gene expression profiling
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epidermal growth factor (EGF)-like proteins have been implicated in early
metamorphosis of three ascidian species: Ciona intestinalis
(Nakayama et al., 2001);
Boltenia villosa (Davidson and
Swalla, 2002
); and Herdmania curvata
(Arnold et al., 1997
;
Eri et al., 1999
). In
Herdmania, experimental analysis of Hemps protein function further
implicates this pathway in the initiation of metamorphosis. The Hemps
gene encodes a protein that contains four EGF-like repeats, three novel
cysteine-rich repeats and a putative secretion signal sequence
(Arnold et al., 1997
), and it
is upregulated when the Herdmania larva develops competence (i.e.
ability to initiate metamorphosis) and during the first few hours of
metamorphosis (Eri et al.,
1999
). Hemps mRNA and protein are localized to the
papillae and anterior epidermis of competent tadpole larvae, the region
previously shown to be required for induction of metamorphosis
(Degnan et al., 1997
).
Following contact with an inductive cue, Hemps protein is released from the
anterior, spreading posteriorly into the trunk and the tunic as metamorphosis
progresses. Larvae cultured in the presence of anti-Hemps antibodies do not
undergo metamorphosis, although they still retract their papillae (i.e. they
undergo the very first phase of the process). Conversely, incubation with
recombinant Hemps protein causes competent larvae to metamorphose at high
rates (Eri et al., 1999
).
These results point to a key role for Hemps in the regulation of
Herdmania metamorphosis. While EGF signalling might play a similar
role in Ciona metamorphosis
(Nakayama et al., 2001
), the
gene encoding the putative EGF protein, Ci-meta1, does not appear to
be a homologue of Hemps (Dehal et al.,
2002
). Indeed, a clear Hemps gene has not been detected
in the Ciona genome, although there are multiple genes encoding
proteins with EGF-like motifs (Dehal et
al., 2002
). Furthermore, recent data support an interaction
between noradrenaline or adrenaline and the ß1-adrenergic receptor in the
nervous system in mediating metamorphosis of Ciona saviginyi
(Kimura et al., 2003
).
The Hemps pathway appears to activate a cascade of gene expression,
starting within 3-4 hours of induction
(Eri et al., 1999). This is
consistent with data reported by Davidson and Swalla
(Davidson and Swalla, 2001
),
who showed that transcription is necessary for both acquisition of competency
and the completion of the initial events of metamorphosis in B.
villosa. Here we characterise gene expression patterns during early
Herdmania metamorphosis using a gene profiling approach
(White et al., 1999
) with a
4800 developmental cDNA clone set printed in a microarray format. Using the
anti-Hemps antibody to block Hemps, we identify genes regulated by the Hemps
pathway in early metamorphosis. Genes that are activated or repressed at 30
minutes post-induction are likely to be those that are directly regulated by
the Hemps signalling pathway.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H. curvata cDNA microarray chip construction
A partially normalised H. curvata developmental cDNA library,
representing gastrula, mid-tailbud, hatched larval, competent larval, 30
minutes post-induction (PI), 4 hours PI and 16 hours PI stages, was generated
using the Smart cDNA library construction kit (Clontech) following the
manufacturer's instructions. Prior to cDNA synthesis, 1 µg of total RNA
from each stage was combined and ethanol precipitated. A total of 4800 random
recombinant plaques were cored and the phage eluted. The insert from each of
the eluted plaques was PCR-amplified using pTripleX2 sequencing forward and
reverse primers (Clontech) and assessed by agarose gel electrophoresis. The
PCR products were precipitated with isopropanol, washed with 70% ethanol and
resuspended in 4xSSC, 0.1% sarkosyl. The 4800 DNA elements were spotted
(in duplicate) onto polylysine-coated glass slides using the GMS 417 Arrayer
robot (Genetic Microsystems). Post-processing of the slides was accomplished
according to P. O. Brown
(http://cmgm.stanford.edu/pbrown/protocols/).
Probe preparation and hybridisations
Labelled cDNA was prepared from 2 µg of total RNA isolated from either
normal larvae and post-larvae or anti-Hemps antibody-treated post-larvae. cDNA
was transcribed using an oligo (dT)15 primer using a dNTP mix with
a ratio of 4:1 aminoallyl-dUTP to dTTP (Sigma). Following synthesis,
aminoallyl-labelled cDNA (aa-cDNA) was purified, dried and the pellet
resuspended in 100 mM sodium carbonate pH 9. Cy3 and Cy5 reactive dyes
(Amersham) were added to their respective aa-cDNAs and the coupling reaction
allowed to proceed for 1 hour. Unincorporated dyes were removed from the
reaction by the addition of 4 M hydroxylamine. Cy3- and Cy5-labelled cDNA was
then combined and purified using a PCR cleanup kit (Qiagen). Human Cot1 DNA
(10 µg) and poly dA (2 µg) were added to the purified probe mix and
dried prior to redissolving in 4xSSC, 40% deionised formamide and 0.1%
SDS. Following incubation at 95°C for 5 minutes, and then 45°C for 90
minutes, the labelled probe mix was then loaded onto the microarray chip, and
hybridisation was carried out for 16 hours at 45°C. Following
hybridisation, the microarray chip was washed with 0.2xSSC, 0.05% SDS
followed by a 0.2xSSC wash and centrifuged at low speed to dry. Cy3 and
Cy5 fluorescence hybridised to the cDNA elements spotted onto the microarray
chip was detected with a GMS 418 Array Scanner (Genetic Microsystems) using
Imagene 4.2 software (BioDiscovery, Inc.).
In order to correct for differences between microarray chips, a reference RNA mix was used. The reference consisted of pooled RNA (10 µg) isolated from the following developmental stages: egg, gastrula, mid-tailbud, hatched, competent, 30 minutes PI, 4 hours PI, 8 hours PI and 24 hours PI. The ratio between reference and test RNA was then used to determine expression levels between different microarray chips.
Data analysis
Data generated using Imagene software for both Cy3 and Cy5 channels was
imported into Genespring 6 (Silicon Genetics) and normalised by applying a
Lowess curve to the log-intensity versus log-ratio plot. Twenty per cent of
the data was used to calculate the Lowess fit at each point. This curve was
used to adjust the control value for each measurement. If the control channel
was lower than 10, then 10 was used instead. Each measurement was divided by
the 50th percentile of all measurements in that sample. The following
filtering criteria were then applied to the normalised data: flag=0 (passed);
flag=>1 (failed). The normalised data were then filtered on the standard
deviation between individual spots. Any spots that had a standard deviation
outside the range of 0.1 to 5 were eliminated from the analysis.
DNA sequence data from the selected clones were compared (using BLAST algorithm) to known cDNA and ESTs in the NCBI and Ciona databases. BLAST analysis of both NCBI and C. intestinalis databases with a P value <1.0e-6 probability of a chance occurrence were classified significant; anything above this was considered not significant and considered unknown.
Quantitative real-time RT-PCR
Differentially expressed gene patterns identified on the arrays were
confirmed using Real-time PCR. Primer pairs were designed for the selected
genes using Taqman guidelines (PE Biosystems) such that a product of between
150 and 250 base pairs was generated in a PCR reaction (see Table S1 at
http://dev.biologists.org/supplemental/).
The annealing temperature and extension time for each of the primer pairs was
optimised using Amplitaq Gold polymerase (Roche). Following optimisation,
Quantitative real-time RT-PCR (QPCR) was performed in 15 µl volumes (cDNA;
1xQPCR master mix (Invitrogen)); 5 pmols of each primer; 1xSYBR
Green (Bioscientific) using the Rotorgene Real time PCR machine (Corbett
Research; Sydney, Australia). Each primer set was analysed against a standard
curve determined by the expression of the ascidian Enoyl-CoA-hydratase, which
was shown by array screening to be equally expressed in normal and treated
larvae (see Results).
Whole-mount in-situ hybridisation
Whole-mount in-situ hybridisation was performed as previously described
(Hinman and Degnan, 2001)
using digoxigen-labelled probes of genes of interest approximately 300 bp in
length, synthesised using T7 or T3 RNA polymerase from recombinant pBSK.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gene expression during normal metamorphosis
For each stage of metamorphosis, at least two sets of duplicate
hybridisation data were obtained and imported into Genespring 6 and grouped as
replicates. Post-larval RNA samples were obtained from individuals that had
initiated metamorphosis within 10 minutes of being subjected to the inducer
(40 mM KCl-elevated FSW). This ensured that 30-minute and 4-hour post-larval
RNAs were obtained from synchronously developing individuals. To ensure that
the 30-minute and 4-hour post-larval mRNA pools were not contaminated with
larval transcripts, post-larvae were inspected and selected manually. At 30
minutes, post-larvae were in the process of or had just completed tail
resorption. At 4 hours, post-larvae had a completely resorbed tail and were
beginning to project their ampullae
(Degnan et al., 1996).
Following normalisation and filtering, 3997 genes passed (16.7% failed). It was estimated that the expression of 49% of the clones (1957) on the microarray chip consistently changed by two-fold or greater in the post-larval stages relative to levels at the competent larval stage (Table 1). The expression of 2040 clones represented on the chip did not display this level of change. Of the clones that were altered in expression, approximately 90% showed a change of no more than 5-fold, with the remaining 10% between 5- and 10-fold. Differentially expressed genes were grouped into six different categories, based on their expression profile (Table 1). Based on transcript abundance, we estimated that about 14.4% of the genes (575 clones) were upregulated 2-fold or more 30 minutes PI and remained elevated at 4 hours PI (Table 1; Profile 1). An estimated 2.8% of the genes (112 clones) had a transient 2-fold or greater increase in expression at 30 minutes PI but had expression levels comparable to competent larvae at 4 hours PI, i.e. a less than 2-fold difference (Profile 2). An estimated 8.3% of the genes (334 clones) displayed no significant change in expression between competence and 30 minutes PI but increased significantly at 4 hours PI (Profile 3). The expression of an estimated 11.5% of the genes (461 clones) decreased by 2-fold or greater (relative to competence) at 30 minutes and 4 hours PI (Profile 4). An estimated 3% of the genes (123 clones) had reduced expression at 30 minutes PI but had expression levels comparable to competent larvae at 4 hours PI (Profile 5). An estimated 8% of the genes (320 clones) had insignificant changes in expression between competence and 30 minutes PI but then decreased by 2-fold or more by 4 hours PI (Profile 6). Profile 7 was comprised of the remaining 2040 clones (an estimated 45% of the genes), which did not change in expression (i.e. less than 2-fold) between competence, 30 minutes and 4 hours PI.
|
|
|
|
|
|
A number of genes encoding proteases and factors involved in protein
degradation and processing (Table
3, subclass AVII) were identified as being affected by the
anti-Hemps antibody treatment. This subclass includes proteins that are
implicated in innate immunity, some of which are known to be activated during
larval development and metamorphosis in other ascidians
(Davidson and Swalla, 2002).
It has been suggested that the activation of a number of immune system-related
genes during metamorphosis in B. villosa is important in the
detection of endogenous signals indicative of tissue damage rather than
deletion of foreign antigens (Davidson and
Swalla, 2002
). In this study we also report alteration in the
regulation of a number of genes involved in innate immunity. Four of these
genes, mannose-binding lectin (Hec-mbl), complement control protein,
Hikaru genki precursor and Hikaru genki type 4 (Hec-hgp1 and
Hec-hgt4, respectively) are downregulated at 30 minutes or 4 hours PI
in response to blocking of metamorphosis with anti-Hemps antibody.
In addition to genes previously shown to be involved in ascidian
metamorphosis, the treatment with the neutralising antibody identified other
genes not previously implicated in this process. These included genes encoding
structural proteins (e.g. myosins, troponins) and transcription factors
(transcription initiative factor 11A gamma chain and BTF3), a gene encoding a
heme-binding protein, which is homologous to a gene implicated in barnacle
settlement and metamorphosis (Okazaki and
Shizuri, 2000). Sixty-one of the differentially expressed genes
analysed in this study (40%) did not match with sequences in GenBank or the
C. intestinalis genome. The microarray analysis also indicated that
Hemps has its major effect at 30 minutes PI, because at 4 hours PI
approximately 50% of these genes had recovered to expression levels comparable
to normal 4-hours-PI larvae (derived from data in
Table 3).
Temporal and spatial expression of selected genes
Fifteen genes that were expressed during early metamorphosis were
classified into one of four categories based on QPCR analysis of transcript
abundance through development (Fig.
4): (1) genes predominantly or transiently expressed in larvae
and/or early metamorphosis (Hec-pnx, Hec-smdp1, Hec-mbl, Hec-meta2a,
Hec-meta2); (2) genes predominantly expressed during metamorphosis and in
the juvenile (Hec-sap, Hec-rab, Hec-cip1, Hec-cip2A, Hec-cip2B); (3)
genes predominantly expressed during embryogenesis (Hec-Hsup, Hec-lp1,
Hec-meta1); and (4) genes expressed at similar levels during
embryogenesis and metamorphosis (Hec-bcsX, Hec-shm).
|
|
|
Hec-bcsX was expressed throughout development and had a significant increase in transcript abundance during larval development and then a marked reduction between 30 minutes and 4 hours PI (Fig. 4). Hec-shm transcript abundance was lowest from hatching to 4 hours PI, increasing during later metamorphosis and in the 8-day-old juvenile. Hec-Hsup and Hec-lp1 had peaks of expression during gastrulation (Fig. 4). Whole-mount in-situ hybridisation analysis of Hec-lp1 revealed that this gene is expressed in the larval palps and anterior epidermis and a subset of tunic cells in the post-larvae (Fig. 6M-O).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microarray approach to investigating transcriptional events in Herdmania metamorphosis
To investigate the importance of Hemps in the induction and regulation of
early metamorphosis, we undertook a gene profiling study using a partially
redundant (21%) microarray consisting of 4800 clones derived from a cDNA
library of mixed developmental stages from gastrula to 16-hour post-larva.
Only genes that, in a minimum of four determinations, consistently displayed
greater than 2-fold changes in transcript abundance during early metamorphosis
or in the anti-Hemps antibody treatment were scored. This conservative
approach, which has been used in a variety of microarray studies (e.g.
Butler et al., 2003;
Munoz-Sanjuan et al., 2002
),
is likely to have missed a number of genes represented on our microarray that
are differentially expressed during metamorphosis or in response to the
anti-Hemps antibody treatment. QPCR analysis of a subset of the differentially
expressed genes validated the microarray approach used in this study. Because
of the unequal redundancy of the genes represented on the 4800 clone
microarray, it is difficult to extrapolate these values to the transcriptome,
even if the Herdmania genome contains
16 000 protein-coding
genes, as is the case in C. intestinalis
(Dehal et al., 2002
).
Nonetheless, a large number of genes - probably 2000-5000 - are activated or
repressed early in metamorphosis, and approximately 20% of these genes are
regulated by the Hemps pathway. These estimations are particularly striking,
given that this period has significantly reduced overall transcriptional
activity, and in the presence of the transcriptional inhibitor actinomyosin D
tail resorption can occur (Green et al.,
2002
). This observation is similar to data reported by Davidson
and Swalla (Davidson and Swalla,
2001
), where they showed that the tail can undergo partial
metamorphosis without novel transcription in B. villosa. Combined,
these data suggest that early gene activation and repression during
metamorphosis may not be necessary for tail resorption but are involved in
downstream morphogenetic events, which include the destruction and
reorganisation of the larval body plan and formation of the juvenile plan
(Hinman and Degnan, 2000
). The
fact that treatment with anti-Hemps antibody also inhibits tail resorption
suggests that this signalling pathway also influences post-transcriptional
processes during early metamorphosis.
The Hemps pathway as a modulator of gene expression during early metamorphosis
It is very likely that the Hemps protein, a key regulator of earlier events
of metamorphosis (Eri et al.,
1999), plays a central role in the regulation of a large number of
these genes, and antibody studies, as discussed below, support this.
Alternatively, a protein such as the cornichon homologue Cnib,
described in B. villosa, might also play a role in this process by
signalling through Hemps (Davidson and
Swalla, 2001
). One approach to unravelling this complexity is to
identify and abrogate a key regulator in the pathway; in this case, we have
targeted Hemps. We have previously shown that antibodies against Hemps
abrogate normal metamorphosis and ectopic application of recombinant Hemps
acts to stimulate this process (Eri et
al., 1999
), suggesting a key role for this peptide in induction of
metamorphosis. The anterior-most cells of the trunk, which are at the base of
the palps - the PAT cells - migrate through a tunnel in the juvenile tunic to
the external environment around the time of competence in Herdmania
(Eri et al., 1999
;
Green et al., 2002
) and in
another ascidian, B. villosa
(Davidson et al., 1995
;
Davidson and Swalla, 2002
). In
Herdmania, Hemps is expressed at relatively high levels in the PAT
cells and papillae (Eri et al.,
1999
). Upon induction of normal metamorphosis, the Hemps protein
spreads to cover the anterior region of the larval trunk, suggesting that this
EGF-like peptide is signalling to cells in this region
(Eri et al., 1999
). The
anti-Hemps antibody appears to impact on metamorphosis by inhibiting this
signalling event.
Microarray analysis of larvae treated with the anti-Hemps antibody indicates that genes that displayed a significant increase or decrease in transcript abundance at 30 minutes PI are probably directly regulated by the Hemps pathway. Genes affected by the anti-Hemps antibody treatment at 4 hours might be indirectly regulated by this pathway. Interestingly, most of the genes affected by the knockdown of Hemps activity are differentially expressed to a greater extent in the anti-Hemps antibody treatments than in normal development. For example, many of the genes that are normally upregulated in 30-minute PI post-larvae (i.e. normal expression profiles 1 and 2) are expressed at even higher levels in larvae treated with the anti-Hemps antibody. These data indicate that the knockdown of the Hemps signalling pathway does not result in maintenance of the larval gene expression profile (i.e. a large majority of the genes that are normally differentially expressed in post-larvae compared with competent larvae are not scored as equally expressed in the anti-Hemps antibody knockdown experiment). Since the knockdown of Hemps activity affects the gene expression in the 30-minute PI post-larvae to a greater extent than the 4-hour post-larvae, it appears that the Hemps pathway directly regulates early response genes at metamorphosis. Combined, these observations suggest that the Hemps signalling pathway, while crucial for the induction of metamorphosis, contributes more significantly to the modulation of early gene expression, rather than initial gene activation or repression. The extent of activation or repression of many genes during normal early metamorphosis appears to be dampened by the Hemps pathway. In the case of these genes, experimental knockdown of Hemps results in a far greater increase or decrease in transcript abundance than observed during normal metamorphosis.
Analysis of larvae treated with the anti-Hemps antibody has led to the
identification of a large number of genes that are directly or indirectly
regulated at the transcriptional level by the Hemps signalling pathway. Of the
genes significantly affected by the knockdown of Hemps activity by the
neutralising antibody during the first 4 hours of metamorphosis (384 clones),
20% show significant difference in expression levels at 30 minute PI. These
genes include homologues to genes already shown to be involved in marine
metamorphosis (Nakayama et al.,
2001; Okazaki and Shiruri,
2000
; Davidson and Swalla,
2002
). These include Hec-meta1, -meta2 and
-meta3, which are homologues to genes involved in cell migration,
adhesion and specialised secretory roles (Nakayama et al.,
2001
,
2002
;
Noh et al., 2003
;
Shao and Haltiwanger, 2003
).
Genes implicated in innate immunity were also identified among the genes
regulated by Hemps and include Hec-mbl, Hec-hgp1 and Hec-hgp4.
Homologues of the latter genes are implicated in metamorphosis in B.
villosa (Davidson and Swalla,
2002
). Genes affected by the anti-Hemps antibody treatment at 4
hours PI might either be regulated by the Hemps pathway or reflect differences
in larval and post-larval expression profiles.
Ascidian metamorphosis: conserved and species-specific components
Ascidians have a pelagobenthic biphasic life cycle with morphologically
distinct larval and adult body plans. While the biphasic life cycle is the
most common form of development among metazoans
(Brusca and Brusca, 2003),
molecular phylogenetic analyses indicate that the ascidian life cycle might be
derived from a worm-like deuterostome ancestor
(Cameron et al., 2000
; Swalla
et al., 2000). Recently, the molecular basis of metamorphosis has been
investigated in the aplousobranch C. intestinalis (Nakayama et al.,
2001
,
2002
) and two stolidobranchs,
B. villosa (Davidson and Swalla,
2002
) and H. curvata (reviewed in
Degnan, 2001
). Progression
through metamorphosis appears to be very similar in these disparate ascidians,
including the ordered autolysis of larval axial structures, and proliferation
and morphogenesis of larval endoderm and mesenchyme rudiments to form the
adult body wall musculature, haemopoietic system and functional gut (e.g.
Cloney, 1982
; Hirano and
Nishida, 1997
,
2000
). Given the shared
external characteristics of metamorphosis and evolutionary history of these
ascidians, it might be expected that related molecular mechanisms would
underlie metamorphosis in these ascidians and that these might be different
from those observed in other metazoans.
Interestingly, 40% (61 of 151 genes) of the sequenced genes that were
differentially expressed when incubated with the neutralising antibody did not
match significantly with any genes in the C. intestinalis genome
(Dehal et al., 2002) or
cDNA/EST database (Satou et al.,
2001
), or any other sequences in the GenBank database. While this
observation might be related to the relatively large distances between
Herdmania and Ciona, it does indicate that there are
significant differences in the genetic networks operational at metamorphosis
in these ascidians. This is further substantiated by the failure to identify a
Hemps homologue in the Ciona database. In addition, recent data
demonstrate that the noradrenaline and ß1-adrenergic receptor system
triggers early metamorphosis in C. savignyi
(Kimura et al., 2003
),
pointing to different control pathways in the different ascidians. Comparison
of these unknown Herdmania ESTs with the sequences of four other more
closely related stolidobranch ascidians in GenBank - B. villosa (94
sequences), Halocynthia roretzi (4423), Polyandrocarpa
misakiensis (501) and Botryllus schlosseri (244) failed to
identify other ascidian-specific sequences.
An H. curvata homologue of the C. intestinalis
metamorphosis gene Ci-meta1 is downregulated in response to
anti-Hemps treatment following an inductive cue. Ci-meta1 was
identified by differential screening of cDNA libraries of swimming larvae and
metamorphosing C. intestinalis juveniles
(Nakayama et al., 2001) and is
not expressed at the larval stage but is expressed immediately after
initiation of metamorphosis. Hec-meta1 encodes a 297 amino acid
protein, containing two calcium-binding EGF-like domains and a secretion
signal peptide. Ci-meta1 is three times larger than
Hec-meta1 and contains 13 calcium-binding EGF-like repeats. While
both these proteins are implicated in metamorphosis, possession of common
EGF-like repeat domains does not imply that they are homologous and play the
same role in metamorphosis. Indeed, whole-mount in-situ hybridisation analysis
shows that Ci-meta1 expression is localised to the adhesive organ and
anterior epidermis of C. intestinalis
(Nakayama et al., 2001
),
whereas Hec-meta1 is predominantly expressed in the posterior trunk
of H. curvata. Prior to metamorphosis, Hec-meta1 expression
is in the epidermis of the trunk. Ci-meta1 expression is also
localised to the anterior end of the larval trunk, a region in which the
signal that initiates ascidian metamorphosis originates and in which Hemps
protein is also localised (Degnan et al.,
1997
; Eri et al.,
1999
). In addition, both Hemps and Ci-meta1 are activated
immediately at the beginning of metamorphosis and are both putative secretory
proteins containing common EGF-like domains
(Arnold et al., 1997
;
Eri et al., 1999
;
Nakayama et al., 2001
). Thus
the two genes may have similar roles in these ascidians. The identification of
homologues of EGF signalling proteins implicated in B. villosa
metamorphosis (Davidson and Swalla,
2001
) adds further support for a central role for EGF-like
proteins in ascidian metamorphosis.
We have also identified three genes that are homologues of
Ci-meta2 (Nakayama et al.,
2001). Two of these genes are upregulated at 30 minutes PI in
normal metamorphosis and are inhibited in their response by anti-Hemps
antibody. Ci-meta2 encodes a protein with a putative secretion signal
and three thrombospondin repeats. This gene is upregulated in the
metamorphosing juveniles and is expressed in the larval adhesive organ, neck
region and dorsal trunk (Nakayama et al.,
2001
). The function of the protein remains unknown but is
structurally related to proteins involved in cell adhesion, cell migration and
those with specialised secretory roles (Nakayama et al.,
2001
,
2002
;
Noh et al., 2003
;
Shao and Haltiwanger, 2003
;
Tomley et al., 2001
).
Another gene, Hec-bcsx1, is upregulated late in metamorphosis and
in the Herdmania juvenile is homologous to a barnacle metamorphosis
gene (Okazaki and Shizuri,
2000). The expression of this gene is not appreciably affected by
anti-Hemps antibody treatment. This gene does not change significantly in its
expression until later in metamorphosis, possibly after the period the Hemps
signalling pathway has greatest effect. Both array screening and QPCR revealed
significant downregulation of Hec-mbl. This gene is homologous to the
B. villosa mannose specific lectin (MBL) gene
(Davidson and Swalla, 2002
).
MBL normally activates a serine protease and downstream complement signalling
(Matsushita et al., 1998
;
Sekine et al., 2001
). In
addition, other genes homologous to innate immunity genes are also
downregulated in response to anti-Hemps antibody. Davidson and Swalla
(Davidson and Swalla, 2002
)
have described the differential expression of several immune system-related
genes during ascidian metamorphosis. They suggested that alterations in these
genes might not involve an immune response per se but that the immune
system-related proteins might function in the developmental regulation of cell
adhesion and migration.
In summary, we have abrogated the function of Hemps, a key regulator of
metamorphosis, to identify downstream genes implicated in this process.
Alterations in expression were confirmed by QPCR, and the localisation of
expression of these genes was compatible with a role in metamorphosis. Using a
microarray gene profiling approach, we have found that a significant portion
of the genome was activated or repressed immediately upon the larva coming in
contact with a cue that induces metamorphosis and that the Hemps signalling
pathway affected the expression of approximately 17% of these genes, and 11%
of genes that normally have no change in expression throughout metamorphosis.
Based on sequence similarity of genes involved in metamorphosis in other
ascidians (Nakayama et al.,
2001; Davidson and Swalla,
2002
) and expression patterns, it seems likely that a number of
the H. curvata genes identified in this study play a conserved role
in ascidian metamorphosis as well as key roles in embryogenesis. Specific
disruption of these genes will allow for further dissection of the pathways
involved in metamorphosis and for the assignation of functional activity to
these genes.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnold, J. M., Eri, R., Degnan, B. M. and Lavin, M. F. (1997). Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae. Dev. Dyn. 210,264 -273.[CrossRef][Medline]
Brusca, R. C. and Brusca, G. L. (2003).Invertebrates. 2nd edn. Sunderland, MA: Sinauer Associates.
Butler, M. J., Jacobsen, T. L., Cain, D. M., Jarman, M. G.,
Hubank, M., Whittle, J. R., Phillips. R. and Simcox, A.
(2003). Discovery of genes with highly restricted expression
patterns in the Drosophila wing disc using DNA oligonucleotide microarrays.
Development 130,659
-670.
Cameron C. B., Garey, J. R. and Swalla, B. J.
(2000). Evolution of the chordate body plan, new insights from
phylogenetic analyses of deuterostome phyla. Proc. Natl. Acad. Sci.
USA 97,4469
-4474.
Chambon, J. P., Soule, J., Pomies, P., Fort, P., Sahuquet, A.,
Alexandre, D., Mangeat, P. H. and Baghdiguian, P. (2002).
Tail regression in Ciona intestinalis (Prochordate) involves a
caspase-dependent apoptosis event associated with ERK activation.
Development 129,3105
-3114.
Cloney, R. A. (1982). Ascidian larvae and events of metamorphosis. Amer. Zool. 22,817 -826.
Cloney, R. A. (1990). Urochordata-Ascidiacea. In Reproductive Biology of Invertebrates. Vol.4 , Part B (ed. K. G. Adiyodi and R. G. Adiyodi), pp.391 -451. New Delhi: Oxford and IBH Publishing.
Corbo, J. C., DiGregorio, A. and Levine, M. (2001). The Ascidian as a model organism in developmental and evolutionary biology. Cell 106,535 -538.[Medline]
Davidson, B. and Swalla, B. J. (2001). Isolation of genes involved in ascidian metamorphosis, epidermal growth factor and metamorphic competence. Dev. Genes Evol. 211,190 -194.[CrossRef][Medline]
Davidson, B. and Swalla, B. J. (2002). A molecular analysis of ascidian metamorphosis reveals activation of an innate immune response. Development 129,4739 -4751.[Medline]
Davidson, E. H., Peterson, K. J. and Cameron, R. A. (1995). Origin of bilaterian body plans, evolution of developmental regulatory mechanisms. Science 270,1319 -1325.[Abstract]
Degnan, B. M. (2001). Settlement and metamorphosis of the ascidian Herdmania curvata. In Biology of Ascidians (ed. C. C. Lambert, H. Yokosawa and H. Sawada), pp.258 -263. Tokyo: Springer-Verlag.
Degnan, B. M., Rhode, P. R. and Lavin, M, F. (1996). Normal development and embryonic gene activity in the ascidian Herdmania momus. Marine. Fresh Water Res. 47,543 -551.
Degnan, B. M., Souter, D., Degnan, S. M. and Long, S. C. (1997). Induction of metamorphosis with potassium ions requires development of competence and an anterior centre in the ascidian Herdmania momus. Dev. Genes Evol. 206,370 -376.[CrossRef]
Dehal, P., Satou, Y., Campell, R. K., Chapman, J., Degnan, B.
M., De Tomaso, A., Davidson, B., DiGregorio, A., Gelpke, M., Goodstein, D. M.
et al. (2002). The draft genome of Ciona
intestinalis, insights into chordate and vertebrate origins.
Science 298,2157
-2167.
Eri, R., Arnold, J. M., Hinman, V. F., Green, K., Jones, M.,
Degnan, B. M. and Lavin, M. F. (1999). Hemps, a novel
EGF-like protein, plays a central role in ascidian metamorphosis.
Development 126,5809
-5818.
Green, K. M., Russell, B. D., Clark, R. J., Jones, M. K., Garson, M. J., Skilleter, G. A. and Degnan, B. M. (2002). A sponge allelochemical induces ascidian settlement but inhibits metamorphosis. Mar. Biol. 140,355 -363.[CrossRef]
Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. and Krasnow, M. A. (1998). Sprouty encodes a novel anatgonist of FGF that patterns apical branching of the Drosophila airways. Cell 92,253 -263.[Medline]
Hinman, V. F. and Degnan, B. M. (2000). Retinoic acid perturbs Otx gene expression in the ascidian pharynx. Dev. Genes Evol. 210,129 -139.[CrossRef][Medline]
Hinman, V. F. and Degnan, B. M. (2001). Homebox genes, retinoic acid and the development and evolution of dual body plans in the ascidian Herdmania curvata. Amer. Zool. 41,664 -675.
Hirano, T. and Nishida, H. (1997). Developmental fates of larval tissues after metamorphosis in ascidian Halocynthia roretzi. I. Origin of mesodermal tissues of the junenile. Dev. Biol. 192,199 -210.[CrossRef][Medline]
Hirano, T. and Nishida, H. (2000). Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi. II. Origin of endodermal tissues of the juvenile. Dev. Genes Evol. 210, 55-63.[CrossRef][Medline]
Jackson, D., Leys, S. P., Hinman, V. F., Woods, R. G., Lavin, M. F. and Degnan, B. M. (2002). Ecological regulation of development, induction of marine invertebrate metamorphosis. Int. J. Dev. Biol. 46,679 -686.[Medline]
Kimura, Y., Yoshida, M. and Morisawa, M. (2003). Interaction between noradrenaline or adrenaline and the beta 1-adrenergic receptor in the nervous system triggers early metamorphosis of larvae in the ascidian, Ciona savignyi. Dev. Biol. 258,129 -140.[CrossRef][Medline]
Lee, Y-H., Huang, G. M., Cameron, R. A., Graham, G., Davidson,
E. H., Hood, L. and Britten, R. J. (1999). EST analysis of
gene expression in early cleavage-stage sea urchin embryo.
Development 126,3857
-3867.
Matsushita, M., Endo, Y. and Fujita, T. (1998). MASP1 (MBL-associated serine protease 1). Immunobiology 199,340 -347.[Medline]
Munoz-Sanjuan, I., Bell, E., Altmann, C, R., Vonica, A. and
Brivanlou, A. H. (2002). Gene profiling during neural
induction in Xenopus laevis, regulation of BMP signaling by
post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein.
Development 129,5529
-5540.
Nakayama, A., Satou, Y. and Satoh, N. (2001). Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis. Dev. Genes Evol. 211,184 -189.[CrossRef][Medline]
Nakayama, A., Satou, Y. and Satoh, N. (2002). Further characterization of genes expressed during Ciona intestinalis metamorphosis. Differentiation 70,429 -437.[CrossRef][Medline]
Nishida, H. (2002). Specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and molecules. Int. Rev. Cytol. 217,227 -276.[Medline]
Noh, Y. H., Matsuda, K., Hong, Y. K., Kunstfeld, R., Riccardi,
L., Koch, M., Oura, H., Dadras, S. S., Streit, M. and Detmar, M.
(2003). An N-terminal 80 kDa recombinant fragment of human
thrombospondin-2 inhibits vascular endothelial growth factor induced
endothelial cell migration in vitro and tumor growth and angiogenesis in vivo.
J. Invest. Dermatol.
121,1536
-1543.
Okazaki, Y. and Shizuri, Y. (2000). Structures of six cDNAs expressed specifically at cypris larvae of barnacles, Balanus amphitrite. Gene 250,127 -135.[CrossRef][Medline]
Satoh, N. (1994). Developmental Biology of Ascidians. Cambridge, UK: Cambridge University Press.
Satoh, N. (2001). Ascidian embryos as a model system to analyze expression and function of developmental genes. Differentiation 68,1 -12.[CrossRef][Medline]
Satou, Y., Takatori, N., Yamada, L., Mochizuki, Y., Hamaguchi,
M., Ishikawa H., Chiba S., Imai, K., Kano S., Murakami S. D. et al.
(2001). Gene expression profiles in Ciona intestinalis
tailbud embryos. Development
128,2893
-2904.
Sekine, H., Kenjo, A., Azumi, K., Ohi, G., Takahashi, M.,
Kasukawa, R., Ichikawa, N., Nakata, M., Mizuochi, T., Matsushita, M. et
al. (2001). An ancient lectin-dependent complement system in
an ascidian novel lectin isolated from the plasma of the solitary ascidian
Halocynthia roretzi. J. Immunol.
167,4504
-4510.
Shao, L. and Haltiwanger, R. S. (2003). O-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats, unusual modifications in unusual places. Cell Mol. Life Sci. 60,241 -250.[CrossRef][Medline]
Tomley, F. M., Billington, K. J., Bumstead, J. M., Clark, J. D. and Monaghan, P. (2001). EtMIC4, a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats. Int. J. Parasitol. 31,1303 -1310.[CrossRef][Medline]
White, K., Scott, A., Rifkin, T., Hurban, P. and Hogness, D.
(1999). Microarray analysis of Drosophila development
during metamorphosis. Science
286,2179
-2184.