Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: davidson{at}caltech.edu)
Accepted 4 June 2003
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SUMMARY |
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Key words: Sea urchin, Pigment cells, Mesoderm specification, SMC, Notch signaling, Macroarray
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Introduction |
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SMCs give rise to four cell types: pigment cells, blastocoelar cells,
circumesophageal muscle and coelomic pouch cells
(Cameron et al., 1991;
Ruffins and Ettensohn, 1996
).
Pigment cells are the first SMC type to migrate into the blastocoel at the
early gastrula stage and by pluteus stage they are embedded in the ectoderm
(Gustafson and Wolpert, 1967
;
Gibson and Burke, 1985
;
Kominami et al., 2001
). The
pigment produced by pigment cells is a naphthoquinone called echinochrome
(McLendon, 1912
;
Kuhn and Wallenfells, 1940
;
Griffiths, 1965
). The other
SMC types generally delaminate from the archenteron tip during gastrula stage.
Blastocoelar cells are fusiform cells wandering in the blastocoel
(Burke, 1978
;
Tamboline and Burke, 1992
).
The muscle cells originating from the SMCs form the circumesophageal muscles
(Burke and Alvarez, 1988
).
Coelomic pouches derive from SMC and small micromere descendents and they
evert from the side of the archenteron tip at late gastrula
(Gustafson and Wolpert,
1963
).
SMCs are specified at the blastula stage during the eighth to tenth
cleavage stages (Horstadius,
1973; Cameron et al.,
1991
; Ruffins and Ettensohn,
1993
; Ruffins and Ettensohn,
1996
; Sherwood and McClay,
1999
; McClay et al.,
2000
). Several studies have proven that micromeres have the
ability to induce the specification of the endo-mesodermal territory
(Horstadius, 1939
;
Ransick and Davidson, 1993
;
Minokawa and Amemiya, 1999
;
Sweet et al., 1999
;
McClay et al., 2000
). Notch
(N) signaling from the micromeres to the SMC precursors has been proven to be
necessary for the differential specification of presumptive SMC and endodermal
territories (Sherwood and McClay,
1999
; Sweet et al.,
1999
; McClay et al.,
2000
). It has also been shown that LiCl treatment of embryos,
which expands both presumptive endoderm and mesoderm territories, shifts the
boundary of the N receptor localization towards the animal half of the embryo
(Sherwood and McClay, 1997
).
Moreover, the N ligand, Delta (Dl), has been recently shown to be expressed in
PMC precursors during blastula stage and to activate the N signal to the SMC
precursors (Sweet et al.,
2002
; Oliveri et al.,
2002
).
Only a few genes expressed in pigment cells have previously been isolated:
the transcription factors hmx
(Martinez and Davidson, 1997)
and not (Peterson et al.,
1999
), the actin-binding profilin
(Smith et al., 1992
;
Smith et al., 1994
) and an
uncharacterized gene, S9 (Miller
et al., 1996
). The expression of the known pigment cell genes
begins at late blastula at the earliest, and their expression is not always
restricted exclusively to the SMC lineage. In a recent study, a gene expressed
in pigment cells and in other SMCs was identified from a cDNA library from
late gastrula embryos (Shoguchi et al.,
2002
). It is not known yet if this gene is expressed earlier than
late gastrula. In other recent studies, some pigment cell specific genes were
identified through macroarray screening: the transcription factor glial
cells missing (gcm) (Ransick
et al., 2002
), a cAMP-dependent protein kinase (capk), a
dopachrome tautomerase-like (dopt) and PI103, a functionally
uncharacterized gene (Rast et al.,
2002
).
In this work, a large-scale screen to isolate genes involved in the pigment
cell specification was undertaken. To maximize the efficiency of the gene
discovery, we screened a hatched blastula cDNA macroarray library of
sufficient coverage to contain most expressed genes
(Cameron et al., 2000) and we
employed a subtractive hybridization procedure that allows the identification
of low-prevalence transcripts (Rast et
al., 2000
). About 400 cDNA clones were isolated and sequenced. A
group of these genes was characterized for temporal and spatial expression
through real time quantitative PCR (QPCR) and whole-mount in situ
hybridization. Six pigment cell specific genes were identified. Two of these
genes were functionally characterized and the results suggest that they are
required for the biosynthesis of the echinochrome pigment.
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Materials and methods |
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DnN mRNA injection and LiCl treatment
DnN mRNA was synthesized in vitro from the construct LvNneg
(Sherwood and McClay, 1999)
using the mMessage mMachine kit (Ambion). Free nucleotides were removed
through Sephadex-G50 Quick Spin Columns (Boehringer Mannheim). Injection was
carried out as described previously (Mao
et al., 1996
) with 3-6 pg/zygote of mRNA
(Sherwood and McClay, 1999
).
To check for the efficiency of the N signaling block, embryos were observed at
mid-gastrula stage (36 hours), when SMCs delaminate from the archenteron tip
in normal embryos. Pigment cell number was scored on pluteus stage embryos (72
hours). LiCl treatment was performed as described
(Ransick et al., 2002
).
Embryos were collected at 19 hours (hatched blastula stage).
RNA isolation
Hatched blastula embryos were dissociated in RNA STAT-60 and total RNA was
isolated following the manufacturer's procedure (Leedo Medical Laboratories).
Polyadenylated RNA was isolated from total RNA with Poly-T(25)
magnetic beads (Dynal, Lake Success, NY).
Selectate preparation
Selectate was prepared from LiCl-treated embryos. Polyadenylated RNA was
isolated from 85 µg of total RNA with Poly-T(25) magnetic beads
(Dynal, Lake Success, NY). The mRNA isolated was used for the synthesis of
double-stranded cDNA as described by Rast et al.
(Rast et al., 2000).
A linker was ligated to the cDNA to allow PCR amplification
(Rast et al., 2000). A 600 to
800 bp cDNA was size selected by agarose gel electrophoresis. Two µl of the
size-selectate cDNA was then amplified by 20 cycles of PCR using the
biotinylated BT-LT7PRIMER and the linker primer. The (+)-strand cDNA selectate
was prepared from the amplified cDNA as described elsewhere
(Rast et al., 2000
).
Driver preparation
5862 zygotes were injected with DnN mRNA to provide mRNA for the driver
preparation and for the dnN unsubtracted probe preparation (see below).
Polyadenylated RNA was isolated from total RNA with Poly-T(25)
magnetic beads (Dynal, Lake Success, NY). Double-strand cDNA was synthesized
from the isolated mRNA as described for the selectate. To allow PCR
amplification, a linker, different from the one used in the selectate
preparation, was ligated to the cDNA according to Rast et al.
(Rast et al., 2000) but with
the following exception: the short oligonucleotide used for the linker had a
3' amino modifier C3 CPG instead of a dideoxycytidine. Half of the cDNA
was amplified by six cycles of PCR under the following conditions: 94°C
for 5 minutes followed by six cycles of 94°C for 30 seconds, 57°C for
1 minute and 72°C for 5 minutes, and a final step of 72°C for 10
minutes. The (-)-strand RNA driver was prepared from the amplified cDNA by
transcription using a Megascript T7 RNA polymerase kit (Ambion, Austin,
TX).
One quarter of the cDNA ligated to the linker was amplified by eight cycles of PCR using the same conditions described above. The amplified cDNA was used as template to synthesize the radiolabeled RNA driver probe for the macroarray filter hybridization (see below).
Subtractive hybridization
A pseudo-first order subtractive hybridization was performed with 100 ng of
(+)-strand cDNA selectate and 5 µg of (-)-strand RNA driver in a 10 µl
volume of 0.34 M phosphate buffer (PB) pH 6.8, 0.05% SDS at 65°C for 40
hours (driver C0t=2.40x102). Two bacteriophage
single-stranded DNA sequences of 500 nt (
1 and
2) were each added to the selectate at a concentration
equivalent to 5 copies/average embryo cell, as a control for the subtractive
hybridization efficiency. The same linker and T7 sequences present in the
selectate were added to the
sequences.
After subtractive hybridization, hydroxylapatite (HAP) chromatography was
performed to separate single strand sequences, unique to selectate, from
double-strand sequences (common to selectate and driver). This was performed
using a small-scale adaptation of the HAP chromatography described by Britten
et al. (Britten et al., 1974).
The chromatography bed volume was reduced to 200 µl in a water-jacketed
column with internal diameter of 0.6 cm and a reservoir volume of 2 ml. The
HAP chromatography was performed at 0.12 M PB, pH 6.8, 0.05% SDS at 60°C.
Single-stranded nucleic acid was eluted with 6 ml of 0.12 M PB, pH 6.8, 0.05%
SDS and double stranded with the same volume of 0.5 M PB, pH 6.8, 0.05% SDS.
From tests done earlier it was observed that about 90% of the single stranded
and double stranded nucleic acids were eluted in the first 2 ml. Therefore, 2
ml of the 0.12 M PB and 0.5 M PB elutions were desalted and concentrated with
Centricon YM-30 centrifugal filter devices (Millipore, Bedford, MA) followed
by drop dialysis on a 0.025 µm filter (Millipore, Bedford, MA). The
single-stranded fraction was amplified by nine cycles of PCR and was used to
synthesize a radiolabeled RNA probe (see below).
Probe preparation and macroarray cDNA library filter
hybridization
A 20 hour cDNA macroarray library was spotted onto Hybond N+ nylon filters
(Amersham, Pharmacia Biotech, Piscataway, NJ) by a QBot robot (Genetix, New
Milton, UK).
The subtracted radiolabeled RNA probe was synthesized by transcription from
the minimally amplified single stranded fraction using a MAXIscript T7 kit
(Ambion, Austin, TX) in the presence of [32P]UTP. The
unsubtracted RNA radiolabeled probes were prepared in the same way from 19
hour LiCl-treated and dnN-injected embryo amplified cDNA. Between 2 and 2.5
µg of radiolabeled RNA probes with a specific activity of approximately
1x108 cpm/µg were obtained. Filter hybridization was
carried out as described in Rast et al.
(Rast et al., 2000
). The same
set of filters was used for the three hybridizations to eliminate the effect
of colony growth differences between filters. Hybridized filters were exposed
to phosphor screens for about 48 hours and then scanned at 100 µm
resolution (Phosphorimager Storm 820; Molecular Dynamics, Sunnyvale, CA).
Macroarray filter analysis
Digital images of the hybridized filters were analyzed with the program
BioArray (Brown et al., 2002).
Normalization over background was calculated as the ratio between the spot
intensity quantile 80 and quantile 20 values of each block.
Sequencing
The 5'-ends of the selected cDNA clones were sequenced using ABI
prism BigDye Terminator Cycle Sequencing with an ABI 377 sequencer (Applied
Biosystems, Foster City, CA). Clone 5'-end sequences were compared to
the nonredundant GenBank using BLASTX
(Altschul et al., 1997).
Real-time quantitative PCR
Real-time quantitative PCR (QPCR) was performed using the SYBR Green system
(PE Biosystems, Foster City, CA) on an ABI 5700 Real-Time PCR machine
(Rast et al., 2000). To
measure the efficiency of the subtractive hybridization, normalized
measurements of the prevalence of the
sequences in the before and
after HAP fractions were carried out as described previously
(Rast et al., 2000
).
After the macroarray screen, QPCR analysis was performed to verify that the
positive clones were in fact differentially expressed. First strand cDNA was
synthesized from LiCl-treated, dnN-injected and normal 19 hour embryo total
RNA by random primed reverse transcription (TaqMan RT, Roche ABI). Primer sets
were designed from the clone sequences. Reactions were prepared in triplicate
using three to five embryo equivalents of cDNA per reaction. QPCR
amplification conditions were: 95°C for 30 seconds, 57°C for 30
seconds, 60°C for 1 minutes, for 40 cycles. QPCR analysis was replicated
using at least two different cDNA batches. Cycle threshold values (Ct) for
each primer set were normalized to the ubiquitin Ct for each reaction.
Ubiquitin expression is known to be approximately constant throughout gastrula
stage (Nemer et al., 1991;
Ransick et al., 2002
).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Ransick et al., 2002;
Arenas-Mena et al., 2000
). When
following the method of Arenas-Mena et al., a modified embryo fixation
solution was used consisting of 4% paraformaldehyde, 32.5% filtered sea water,
32.5 mM MOPS pH 7 and 162.5 mM NaCl (T. Minokawa and E.H.D., unpublished).
Digoxigenin (DIG)-labeled RNA probes were synthesized by transcription. The SpPks, SpFmo1, SpFmo2, SpFmo3 and SpSult DIG-labeled RNA probes were synthesized using as template the linearized plasmid of clone E62, D88, E64, E36 and B35, respectively. The resulting probe length was about 500 nucleotides for SpPks and SpSult, 1000 nucleotides for SpFmo1 and 1500 nucleotides for SpFmo2 and SpFmo3.
Antisense morpholino injection into zygotes
The antisense morpholino oligonucleotides for SpPks and
SpFmo1 were purchased from Gene Tools, Philomath, OR. The
SpPks morpholino oligonucleotide sequence was
5'-AGCTGGTTTTATTGCTTCCCATGTT-3' and the SpFmo1 was
5'-CATGCACACGTTGCAGGAAAACTGG-3'. A random sequence morpholino
oligonucleotide was injected in parallel as control. A 200 µM solution (2-4
pl) of morpholino oligonucleotide was injected into zygotes.
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Results |
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Discussion |
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Through an antisense approach, we show that SpPks and SpFmo1 are required for the biosynthesis of the echinochrome pigment (Fig. 4; Table 3).
Glial cells missing (GCM)
gcm is a transcription factor that was first isolated from
Drosophila (Akiyama et al.,
1996). A second gcm gene, gcm/glide2,
has been recently identified in Drosophila
(Kammerer and Giangrande,
2001
). Two Gcm genes have also been isolated in mammals,
gcm1/gcmA and gcm2/gcmB
(Akiyama et al., 1996
;
Kim et al., 1998
). This
appears to be the result of separate gene duplication events in arthropods and
vertebrates (Kammerer and Giangrande,
2001
). The SpGcm identified in this work is identical to
the one isolated recently (Ransick et al.,
2002
) and so far no other gcm genes have been identified
in the sea urchin. The highest region of similarity of SpGcm to the
Drosophila and mammals gcm corresponds to the DNA-binding
domain (Ransick et al., 2002
),
which recently has been shown to be a new type of zinc-coordinating
DNA-binding domain (Cohen et al.,
2002
).
In sea urchin, SpGcm positively regulates the expression of
SpPks, SpSult and SpFmo in pigment cell precursors
(Davidson et al., 2002). In
Drosophila, gcm was shown to be required for the development of
hemocytes/macrophages (Bernardoni et al.,
1997
; Lebestky et al.,
2000
; Alfonso and Jones,
2002
) and for the regulation of the binary decision of glial
versus neuronal cell fate (Hosoya et al.,
1995
; Jones et al.,
1995
; Vincent et al.,
1996
). In mammals, Gcm genes do not seem to be involved in the
specification of glial cells, but they are required for placental development
(Schreiber et al., 2000
) and
parathyroid gland development (Gunther et
al., 2000
).
This study shows that, in the sea urchin embryo, N signaling positively
regulates SpGcm expression in pigment cell precursors, either
directly or indirectly. N signaling has been shown to regulate gcm
expression in some other developmental contexts. In Drosophila, N
signaling activates gcm in the central nervous system
(Udolph et al., 2001) and in
the dorsal bipolar dendritic sensory lineage in the embryonic peripheral
nervous system (Umesono et al.,
2002
). N signaling has instead a negative regulatory effect on
gcm in the bristle lineage of the peripheral nervous system of
Drosophila (Van De Bor and
Giangrande, 2001
). Many studies show that N signaling is a very
conserved mechanism in the development of many metazoa and it is used in
several different types of cell fate decisions. It is not surprising then that
N signaling can have either a positive or a negative regulatory effect, even
on the same target gene, depending on the combinations of regulatory factors
present in any particular developmental context.
Polyketide synthase (PKS)
Polyketide synthases are large multifunctional enzymes involved in the
biosynthesis of a wide range of polyketide compounds (for reviews, see
Hopwood, 1997;
Hopwood and Sherman, 1990
;
Staunton and Weissman, 2001
).
The sea urchin echinochrome synthesized in pigment cells is a naphthoquinone
that has the characteristics of a polyketide compound. In terms of precursors
and enzymes used, the polyketide biosynthetic pathway is very similar to the
fatty acid biosynthetic pathway. A difference is that in fatty acid
biosynthesis there is a complete reduction of the keto groups with the
production of completely saturated carbon chains, while these remain unreduced
in polyketides.
Polyketide compounds have been isolated mainly from bacteria, fungi and
plants. Several polyketides synthesized in bacteria and fungi are antibiotics,
such as erythromycin, rifamycin and actinorhodin, and several mycotoxins in
fungi. Higher plants synthesize polyketides, called flavonoids, which have
various functions: flower pigmentation, defense against pathogens
(phytoalexins), response to UV light and visible light exposure, and symbiotic
plant-pathogen interactions (Schroder et
al., 1998; Winkel-Shirley,
2002
). Polyketide compounds have also been isolated from some
marine organisms: dinoflagellate unicellular algae, macro-algae, sponges and
molluscs (Garson, 1989
). In
marine organisms, polyketides are often toxic compounds used as defense
mechanisms against predators, e.g. the brevetoxins synthesized by the
dinoflagellate Gymnodinium breve
(Garson, 1989
).
The S. purpuratus sequences isolated in this work showed the highest similarity to bacterial and fungal PKSs (see Table S1 at http://dev.biologists.org/supplemental/). A lower sequence similarity of the S. purpuratus genes was observed to the human and C. elegans fatty acid synthases (FAS; NCBI Accession Numbers NP_004095 and NP_492417) and to C. elegans PKS (NCBI Accession Number NP_508923). The S. purpuratus PKS sequences also showed lower similarities to some uncharacterized Drosophila proteins (CG3524, CG17374, CG3523) that are similar to the vertebrate FASs and fungal PKSs (NCBI Accession Number AAD43562, AAB08104). A pks gene cluster has not been identified in human.
Generally the Pks genes are organized in clusters and they contain very
large ORFs. The different ORFs either together encode a very large
multifunctional protein (type I PKS), or they encode separate proteins (type
II PKS). The cDNA sequence contigs of the isolated clones show that the sea
urchin SpPks belongs to the type I class. In most PKSs, the series of
ORFs each encode domains with a unique catalytic function. The S.
purpuratus PKS sequences are highly similar to the ketosynthase (KS), the
acyltransferase (AT) and the alcohol dehydrogenase-zinc-dependent domains
(ADH-zinc). This specific combination of catalytic domains is present in
several PKSs, e.g. in the bacteria Streptomyces coelicolor, Stigmatella
aurantica and Nostoc (NCBI Accession Number NP_ 630898,
CAD19090, NP_486720). The PKS domains cooperatively catalyze the condensation
of simple carbon units (acetyl, propionyl or butyryl units) to produce the
polyketone linear chain, which is then further modified by other PKS domains
and cyclized. A biochemical study by Salaque et al.
(Salaque et al., 1967) showed
that acetic acid molecules are used as precursors in the biosynthesis of
echinochrome A in sea urchin. Thus, the SpPKS sequence similarity data
together with the chemical structure of the echinochrome and the simple carbon
units used for its biosynthesis suggest that SpPKS is directly involved in the
biosynthesis of this pigment.
The function of sea urchin embryonic pigment cells is not completely
understood. Considering that pigment cells are embedded in the epithelium of
the larva and that polyketides are also photoactive compounds, it is possible
that pigment cells have a role in photoreception. In support of this
hypothesis are observations of light-induced alterations of pigment cell shape
and pigment granule displacement within the pseudopodia in the sea urchin
Centrostephanus longispinus
(Weber and Dambach, 1974;
Gras and Weber, 1977
;
Weber and Gras, 1980
). Pigment
granule translocation accompanied by cell shape changes have also been
observed in dermal photoreceptors (melanophores) of amphibians and fish
(Wise, 1969
;
Schliwa and Bereiter-Hahn,
1973
). By considering the echinochrome chemical structure, it
might have antibiotic properties as many polyketide compounds. Some evidence
supporting this hypothesis has been provided by Service and Wardlaw
(Service and Wardlaw, 1984
).
In addition, the morphology and behavior of pigment cells are, to some extent,
similar to those of macrophages. Pigment cells have a stellate shape with two
or three pseudopodia, which can be rapidly extended and contracted, and they
have the ability to migrate within the larval epithelium and the basal lamina
(Gibson and Burke, 1987
).
Flavin-containing monooxygenases (FMOs)
Generally FMOs are NADPH-dependent flavoproteins that catalyze the
oxidation of a wide variety of compounds containing nucleophilic heteroatoms.
FMOs are involved in the detoxification of several xenobiotics and in the
molecular activation of different kinds of metabolites.
FMOs are found in bacteria, higher metazoa
(Hines et al., 1994;
Gasser, 1996
;
Schlenk, 1998
;
Cashman, 2000
;
Ziegler, 2002
) and in plants
(Zhao et al., 2001
;
Tobena-Santamaria et al.,
2002
). In this work three different sea urchin Fmo genes have been
isolated. Five members of the Fmo multigene family have been identified so far
in mammals (for reviews, see Hines et al.,
1994
; Gasser,
1996
; Cashman,
2000
).
The mammalian FMO enzymes exhibit different species-, developmental- and
tissue-specific expression, and different substrate specificity
(Dolphin et al., 1996;
Dolphin et al., 1998
;
Koukouritaki et al., 2002
). In
this work, we show evidence that supports an involvement of SpFmo1 in
echinochrome biosynthesis. Taking into account that the FMOs show different
substrate specificity in other organisms, it could also be that SpFmo1,
SpFmo2 and SpFmo3 are involved in different catalytic steps of
echinochrome biosynthesis.
The human FMO1, FMO2, FMO3 and FMO4 are all localized on
the same chromosome arm (Dolphin et al.,
1991; Shephard et al.,
1993
; McCombie et al.,
1996
). This indicates a possible co-regulation of the Fmo genes.
The three Fmo genes isolated in this work are expressed in the same cell type,
the pigment cells, and their expression starts at the same time (at about 15
hours; C.C. and E.H.D., unpublished). These results suggest that the three
SpFmo genes might also be co-regulated.
Sulfotransferase (SULT)
Sulfotransferases catalyze the sulfate conjugation of a broad range of
substrates leading either to their detoxification or bioactivation.
Sulfotransferases also show different developmental and tissue-specific gene
expression (Her et al., 1997;
Dunn and Klaassen, 1998
).
Sulfotransferases are present from bacteria to higher eukaryotes. In rats
and humans they catalyze the sulfate conjugation of hormones,
neurotransmitters and various drugs and xenobiotics (for reviews, see
Falany, 1997;
Weinshilboum et al., 1997
).
The sult gene identified in this work is expressed exclusively in
pigment cells, suggesting that it could be involved in a specific function of
pigment cells.
New insight into the pigment cell specification process
The molecular basis of pigment cell specification is largely unknown. N
signaling at blastula stage was shown to be necessary for pigment cell
specification as well as for the other SMC types
(Sherwood and McClay, 1999;
Sweet et al., 1999
;
McClay et al., 2000
). The
large-scale screening for SMC-specific genes carried out in this work allowed
the isolation of six different pigment cell specific genes downstream of N,
one transcription factor SpGcm and five enzymes, SpPks, SpFmo1,
SpFmo2, SpFmo3 and SpSult.
Pigment cells might be the only SMC type exclusively regulated by the N
pathway through the Dl-N signaling at seventh to ninth cleavage. A study on
the role of the N ligand Dl in mesoderm specification showed that Dl depletion
in micromeres completely eliminates pigment cells, while muscle cells were
normal and blastocoelar cells decreased by about 50%
(Sweet et al., 2002). Muscle
cell specification does not require micromere descendant Dl expression, but it
does require mesenchyme-blastula Dl expression in the SMC precursors
(Sweet et al., 2002
).
Blastocoelar cell specification requires Dl expression in both micromere
descendants and mesenchyme-blastula SMC precursors, and probably also
additional genetic inputs (Sweet et al.,
2002
). DnN-injected embryos are completely depleted of pigment
cells but they show only about 40% depletion of blastocoelar, muscle and of
coelomic pouch cells (Sherwood and McClay,
1999
). It is possible that the dnN receptor is not sufficient to
eliminate N signaling completely through the endogenous N receptor and that
the alternative SMC type specification processes are differentially sensitive
to N signaling, probably having partially different N downstream effectors.
Another possibility is that the non-pigment SMC types are specified by a
combination of N signaling and other genetic pathway inputs.
Current gene expression profiles suggest a relatively shallow regulatory
pathway for pigment cell specification. The N ligand Dl is expressed in 7th
cleavage (8 hours in S. purpuratus) micromere descendants
(Sweet et al., 2002;
Oliveri et al., 2002
), about 2
or 3 hours before SpGcm starts to be expressed. In addition, recent
data show that SpGcm is a direct target of N (A. Ransick and E.H.D.,
unpublished). It is also known that SpGcm positively regulates the
expression of SpPks, SpSult and SpFmo1
(Davidson et al., 2002
).
SpGcm begins to be expressed between 10 and 12 hours
(Ransick et al., 2002
), while
SpPks, SpFmo and SpSult gene expression begins at about 15
hours (C.C. and E.H.D., unpublished). Further studies could soon bring to
light all the genetic components of the pigment cell specification pathway.
The differentiation gene battery at the end of this pathway should include the
genes discovered here, SpPks, SpFmo1, SpFmo2, SpFmo3 and
SpSult.
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ACKNOWLEDGMENTS |
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