School of Biological Sciences, University of Wales Bangor, Bangor LL57 2UW, UK
Author for correspondence (e-mail:
s.g.webster{at}bangor.ac.uk)
Accepted 17 June 2004
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SUMMARY |
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Key words: Carcinus maenas, Embryogenesis, Embryonic ecdysis, Quantitative RT-PCR, Crustacean hyperglycaemic hormone, Moult-inhibiting hormone, Pigment-dispersing hormone, Red pigment-concentrating hormone
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Introduction |
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An important issue concerns the hormonal control of embryonic moulting. The
fertilised egg of crustaceans contains large quantities of maternally derived
ecdysteroids and corresponding polar metabolites, which decline during
naupliar development (Goudeau and
Lachaise, 1983; Lachaise et
al., 1981
; Okazaki and Chang,
1991
; Wilder et al.,
1990
). These are presumed to be important in naupliar development.
However, during metanaupliar development, dramatic increases in ecdysteroid
levels are seen which are believed to originate via de novo ecdysteroid
synthesis by the developing Y-organ. The embryonic Y-organ has only been
observed in palaemonid shrimps during the period just prior to eye development
(Le Roux, 1983
) (A. Le Roux,
Thèse de Doctorat d'Etat, Université de Rennes, 1989).
In adult crustaceans, ecdysteroid synthesis by the Y-organ is negatively
regulated by moult-inhibiting hormone (MIH) and crustacean hyperglycaemic
hormone (CHH) (Böcking et al.,
2002; Webster,
1998
). Metanaupliar development is characterised by rapid
neurogenesis, including development of the eyestalk neurosecretory complex. We
were therefore interested in determining the development of the anatomy of
neurosecretory neurones expressing MIH and CHH during embryonic development,
since very little is known about these events during embryogenesis
(Charmantier and Charmantier-Daures,
1998
), except that an X-organ is present in the metanauplius of
Homarus gammarus (Rotllant et
al., 1995
) and that MIH neurones are evident in the first zoeal
stage of Carcinus (Webster and
Dircksen, 1991
). Additionally, since hatching is entirely
dependent on water uptake necessary to rupture the eggshell
(Saigusa and Terajima, 2000
),
we were interested in the occurrence of peripheral sources of CHH during
development. In adult Carcinus maenas, moulting involves a rapid
uptake of water just prior to ecdysis, which is necessary for rupture of the
old exoskeleton, and swelling to the subsequent postmoult dimensions. This
phenomenon involves the ephemeral release of CHH from gut endocrine cells
(Chung et al., 1999
), and
crustacean cardioactive peptide (CCAP) which is involved in stereotyped
moulting behaviour in crustaceans
(Phlippen et al., 2000
) in
ways that are reminiscent of insect eclosion
(Gammie and Truman, 1997
;
Gammie and Truman, 1999
).
Thus, we reasoned that hatching (eclosion) in crustacean embryos is analogous
to an adult moult, and that anatomical and physiological correlates might be
common to both. Secondly, it has been recognised that many neuromorphological
correlates exemplifying those of the adult are already in position at the time
of hatching (Harzsch and Dawirs,
1993
). In view of the considerable amount of information that now
exists regarding the development of the embryonic and larval nervous system of
decapods (Harzsch et al.,
1998
; Harzsch,
2003
), we were also interested in contrasting the embryonic
development of peptidergic systems involved in moulting (MIH, CHH) with those
of the classical eyestalk neurohormones and neuromodulators namely red
pigment-concentrating hormone (RPCH) and pigment-dispersing hormone (PDH). The
approach used here was firstly to relate embryonic stages where neuropeptide
gene expression could first be measured, using quantitative RT-PCR, and
secondly to correlate these results with microanatomical analyses of peptide
expression during development, particularly with regard to the identification
of embryonic neurones. Finally, correlations were made regarding quantitative
expression of peptides during hatching, to compare these with homologous
events during adult ecdysis.
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Materials and methods |
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Immunohistochemistry
Staged embryos were either taken from ovigerous females, or for experiments
involving temporally timed staging (i.e. days after eye anlage formation, or
days before hatch), groups of embryos attached to egg strings were cultured at
18°C in 24-well tissue culture plates in sterile seawater, which was
changed daily. Under these conditions, development was identical to
corresponding events in the mother crab (at 18°C), and even under
prolonged periods of in vitro culture (3 weeks), hatching times were the
same. This allowed us to precisely stage embryo samples and estimate the
duration of each stage (Table
1). Groups of embryos were microscopically dissected in fixative
to remove the eggshell (essential to allow adequate fixation and antibody
penetration). For CHH, MIH, and PDH, Stefanini's fixative was used
(Stefanini et al., 1967
). For
CCAP and RPCH, embryos were fixed in 4% paraformaldehyde in 0.1 M phosphate
buffer (PB) containing 1% 1-ethyl 3' (3-dimethyl
aminopropyl)-cardodiimide. All fixations were for 24 hours at 4°C. Embryos
were subsequently washed for 24 hours in 0.1 M PB containing 0.1% Triton
X-100, 0.05% sodium azide (PTX). Antisera raised against Carcinus MIH
and Uca PDH have previously been described
(Dircksen et al., 1987
;
Dircksen et al., 1988
) and
were used at 1:1000 in PTX for up to 4 days at 4°C. RPCH antiserum was
raised in rabbits using an N-terminal acetylthioacetyl RPCH analogue (J.
Riehm, Pensacola) coupled to bovine thyroglobulin via
maleiamidocaproyl-N-hydroxysuccinimide (H. Dircksen, Stockholm,
Sweden). Milligram quantities of conjugate were emulsified in Freund's
adjuvant and injected at multiple subcutaneous sites into two New Zealand
white rabbits at 4-weekly intervals, followed by terminal exsanguination after
12 weeks. Principles of laboratory animal care and specific national laws were
followed. This antibody showed high specificity to RPCH and was used at 1:500
for whole mounts. Detection of primary antiserum was by goat anti-rabbit FITC
(1:50, 1-2 days RT). For double immunolabelling experiments, the second
primary antibody used (CHH) was directly coupled to Cy3, by incubating the
purified IgG (Protein A Sepharose) with excess dye in 0.1 M bicarbonate
buffer, pH 9.2 (1 hour, RT), followed by Sephadex G-25 chromatography and
concentration by ultrafiltration (Amicon PM10). Optimum working dilutions were
determined by experiment. Incubations in the Cy3-labelled antibodies were
performed for 24 hours at RT. Embryos were mounted in 50% glycerol/PTX.
Preparations were analysed by confocal microscopy using a Zeiss LSM 510
instrument. For stacked projection image analysis, proprietary instrument
software was used. In general, between 25 and 40 serial 1.5 µm images were
collected. Images were manipulated using Adobe Photoshop 7.0 and Coreldraw 8.0
software.
|
Estimation of water uptake during embryonic eclosion using [3H] water
Batches of 20 embryos, which were staged as `imminent hatch' (within 1
hour) were incubated in 10 µl crustacean saline (with/without
Ca2+) containing 1.85 MBq [3H] water for 1 hour. At the
end of the incubation period, embryos were rapidly and extensively washed with
crustacean saline, briefly blotted, transferred into tubes containing
scintillation fluid, and counted. Conversion of dpm to nanolitres (nl) gave a
measure of the net water uptake/embryo.
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Results |
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MIH and CHH
Immunopositive perikarya located in the developing X-organ were first
detected at eye anlage formation. By mid-eye development, the complete
eyestalk neurosecretory system could be seen
(Fig. 2A-C). For each peptide,
four anterio-dorsal perikarya (6-8 µm) projected axon tracts
posterioventrally to the sinus gland (20 µm). Along the axon tract,
branching collaterals were observed. To eliminate the possibility (in view of
the identical numbers of CHH and MIH neurones) of colocalisation of both
peptides at this time, we performed double labelling immunohistochemistry
(Fig. 2C), which conclusively
demonstrated the absence of colocalisation. During subsequent development, no
further changes were seen for MIH, but for CHH the situation was different: At
mid-eye development, about 3-4 days after establishment of regular heartbeat,
CHH immunoreactivity (IR) was seen in the developing pericardial organs (PO)
(Fig. 2D). By full development,
fine detail could be seen including IR structures corresponding to the
segmental nerves and anastomoses between anterior and posterior bars. Although
generally discrete intrinsic perikarya could not clearly be distinguished in
the anterior and posterior bars, some preparations
(Fig. 2E) suggested that there
were 3-4 cells in the anterior bar. At this time, 4-5 pairs of CHH-expressing
cells could be observed immediately ventral to the PO
(Fig. 2E,F) and further novel
CHH-IR cells were seen (Fig.
2F). At the posterior midline of each abdominal segment, seven
pairs of tiny (6-7 µm) serially iterated cells could be seen. The position
of these cells appeared to correspond closely with the position of the
abdominal flexor muscle insertions. These cells were first observed 4 days
before hatching (Fig. 2H).
Immunolabelling was most intense just before hatching, when a further set of
dorsal, paired cells appeared (Fig.
2J), which seemed to be associated with the insertions of the
abdominal extensor muscles. The abdominal cells could be observed in the
freshly hatched zoea; an overview of all the CHH-IR structures is shown in
Fig. 2G. The fate of these
cells in subsequent zoeal life could not be followed, since the larval cuticle
becomes an overwhelming barrier to antibody penetration within a few hours of
hatching at the beginning of zoeal intermoult.
|
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Quantification of CHH and CCAP content of embryos during development
We measured whole-embryo levels of both peptides by RIA, during complete
embryonic development, to see whether events in embryos resembled those seen
during adult moulting. CHH was first detected during eye anlage development,
and remained low (0.2 fmol/embryo) until 5 days before hatching when there was
a steady increase to about 2 fmol/embryo, 1 day before hatching. During
hatching, levels increased dramatically, to over 6 fmol/embryo, and
immediately declined to less than 2 fmol/embryo within 1 hour of hatching
(Fig. 5A). CCAP was first
detected 15 days before hatching, at late eye anlage stage it is
significant to note that this stage was marked by the appearance of a regular
heartbeat (82±20 beats/minute, n=15). Peak levels of CCAP (4
fmol/embryo) were seen during hatching; these declined to intermediate levels
within 1 day post-hatching (Fig.
5A). HPLC-RIA analysis of a sample of fully developed embryos (2
days before hatching), showed the presence of immunoreactive material
corresponding to elution times of CCAP, XO-CHH, and the earlier eluting PO-CHH
(Fig. 5B).
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Discussion |
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For MIH and CHH we observed significant expression of both mRNAs at the
beginning of eye anlage formation, which rapidly increased during subsequent
development. By analogy to the detailed developmental series described by
Helluy and Beltz (1991) for
the lobster, Homarus americanus, this event could be correlated to a
period just following the naupliar moult (12% development); however, for
crabs, it seems to be somewhat later, at about 50% development
(Okazaki and Chang, 1991
),
which just precedes the rapid increases in ecdysteroid titre indicative of
premoult, and which would also correspond with a developmental stage of about
50% in the lobster. This difference is probably due to the extended
embryogenesis in the former species. The only work that has mentioned the
development of the Y-organ in embryonic crustaceans
(Le Roux, 1983
) (A. Le Roux,
Thèse de Doctorat d'Etat, Université de Rennes, 1989) indicates
that it forms just before eye development, i.e. during metanaupliar
intermoult. For Carcinus, both MIH and CHH mRNAs are clearly
expressed at the beginning of eye anlage formation, an observation that was
consistent with immunohistochemical studies. During mid-eye development, four
pairs of anterio-dorsal perikarya showing immunoreactivity to MIH or CHH were
observed in the eyes, projecting neurones to a posterio-ventral SG. While in
the adult it is well known that both neuropeptides do not colocalise
(Dircksen et al., 1988
), in
view of the very similar morphologies exhibited by both sets of neurones, we
further investigated this by double immunolabelling
(Fig. 1C). These experiments
showed conclusively that, despite the identical neuronal number and similar
position (in the developing X-organ), these peptides never colocalise. In our
previous studies on larval expression of MIH, we observed that only four
neurones (in each eyestalk) express MIH throughout larval development
(Webster and Dircksen, 1991
).
The present study also confirms this during embryonic development. In the
adult, 28-36 eyestalk neurones express MIH in comparison to 62-65 for CHH
(Dircksen et al., 1988
). In
adult Carcinus, steady-state expression of CHH mRNA is about 2
107 and for MIH 0.5-1 106 copies/cell, i.e. a
ratio of 20-40 CHH:1 MIH (Chung and
Webster, 2003
). Since both CHH and MIH perikarya have diameters of
70 µm in the adult, and 8 µm in the embryo, adult cell volumes are
about 180 pl, and for embryos, 27 pl. Thus in adults, copy number/pl are about
1 105 for CHH and 3-3.4 103 for MIH. For
embryos at mid-eye development, respective copy numbers/pl are 0.9-1.4
105 for CHH, and 4.4 103 for MIH, which are in
accordance. Thus, in the embryo, transcriptional processes are dynamically
similar to those of the adult, and steady-state ratios of CHH and MIH
transcripts are similar.
During later embryonic development, extra-eyestalk sources of CHH-like
peptides become significant: during late eye development, 3-5 days before
hatching, CHH-IR neurones became prominent in the pericardial organs (PO), and
associated peripheral neurones, and later in a set of serially iterated pairs
of lateral and dorsal cells in each segment of the abdomen. It should be noted
that with the primers used for quantitative RT-PCR, only the prototypical CHH
(XO-CHH) was measured. The PO-CHH splice variant, which is expressed by
intrinsic cells in the PO of the adult
(Dircksen et al., 2001) was not
amplified. Nevertheless, the antiserum used for ICC could detect both
translated products. We addressed this problem regarding expression of the two
CHH isoforms by measuring CHH by RIA during the days prior to eclosion. This
assay only measures XO-CHH. However, by using quantified PO-CHH, and
125-I radiolabel in conjunction with the XO-CHH antiserum, we could
detect the PO-CHH splice variant in addition to XO-CHH in HPLC-separated
peptide fractions from embryos at 3-5 days prior to hatching
(Fig. 5B). As in the adult,
both splice variants are expressed, and the PO-CHH is possibly expressed
primarily by the intrinsic neurones in the PO. For XO-CHH there is a gradual
increase in CHH levels in late embryos, culminating with a dramatic increase
prior to eclosion, and an equally impressive decline within an hour of this
event, which corresponds with shedding of the prezoeal cuticle. These events
exactly mirror those in the adult crab, where premoult is associated with a
dramatic release of CHH from gut endocrine cells
(Chung et al., 1999
). Since gut
CHH endocrine cells were absent in Carcinus embryos, we suggest that
in the embryo, the embryonic abdominal serially iterated cells may be involved
in the CHH surge seen in adult moulting. However, although CHH-IR declines
precipitously during postmoult, we were unable to record diminution in
immunoreactivity of these cells following zoeal moulting, since the cuticle
becomes completely impermeable to antibody penetration at this time. Since
rupture of the eggshell during hatching must involve significant water uptake
(Saigusa and Terajima, 2000
),
we measured net water influx in the immediate period prior to hatching, using
tritiated water. During the hour before hatching, net uptake was around 7-8 nl
(approximately 15-20% of the embryo body volume). Since this was correlated
with the peak in whole-body CHH titre, we tried to manipulate CHH release, to
see whether this would affect water uptake, in an attempt to establish a
causal relationship between water uptake and CHH release. In the adult, CHH
release from premoult (D3-4) hindgut in vitro is dramatically
diminished in nominally calcium-free conditions (J.S.C. and S.G.W.,
unpublished). In the embryo incubated in Ca2+-free medium, water
uptake was reduced compared to normal controls, and CHH release was also
impaired.
With regard to developmental expression of CCAP transcripts in embryonic
crustaceans, nothing can as yet be said, since the CCAP gene, or coding
sequences have not been determined in any crustacean (in contrast to insects,
i.e. Manduca (Loi et al.,
2001)). However, we could observe immunopositive structures
reminiscent of those seen in the adult thoracic ganglion (TG) during late eye
formation (Fig. 4D,F). Although
we could never observe serially iterated perikarya in this tissue, as shown in
the adult (Dircksen, 1998
;
Dircksen and Keller, 1988
), the
overall morphology of immunopositive structures in the thoracic ganglion and
PO was clearly reminiscent of this. Furthermore, during eclosion, CCAP levels,
measured by RIA exhibit similar patterns to those seen during adult moulting
(Phlippen et al., 2000
). It is
also notable that during eclosion, analogous stereotyped behaviours were
observed, such as dramatic increases in heart rate from 260±12,
(n=20) beats/minute in completely developed embryos to 335±14
(n=15) beats/minute just before hatching. Just after hatching,
prezoeae show stereotyped rapid, intense, but intermittent circular swimming
episodes. These phenotypes, together with those showing reduction in CCAP
content after hatching are suggestive of a large release of CCAP at eclosion,
which is analogous to events during adult ecdysis. The results obtained in
this study (for CHH and CCAP) are the first concerning the neurohormonal
control of embryonic moulting in crustaceans, and are of interest since these
events appear to be essentially the same as those seen during adult ecdysis,
but just at a very small scale.
Since expression of CHH or MIH occurred quite late in embryonic
development, we were also interested in studying the expression patterns of
the other `eyestalk' neuropeptides which are well known to have
neuromodulatory roles in addition to those first established by classical
endocrinology as `neurohormonal', i.e. the crustacean pigmentary effector
hormones, PDH and RPCH (Rao,
2001). In view of this, it seemed likely that expression might
occur earlier in embryogenesis, in comparison to MIH and CHH. The results here
were surprising. For PDH, expression of mRNA was evident throughout embryonic
development, and interestingly, maternally derived mRNA in the unfertilised
oocyte was significant. The functional significance of this observation is
unknown, but may possibly point to circadian clock-driven processes, since PDH
has been identified as an important component of the circadian clock output
pathways of Drosophila (Park et
al., 2000
; Renn et al.,
1999
). For RPCH, mRNA expression was first recorded during
naupliar development. Perhaps the most surprising observation related to the
magnitude of expression for both mRNAs, steady-state expression levels
were 10 to 100-fold greater than for CHH or MIH an observation that is
at odds with the amount of translated product in the adult. The
chromatophorotrophins are rather minor constituents of the neuropeptide
inventory of Carcinus eyestalks or sinus glands. For example, mean
total PDH content of the Carcinus eyestalk is less that 20 pmol
(Löhr et al., 1993
), for
MIH, SG levels are 36-55 pmol and for CHH 270-490 pmol
(Chung and Webster, 2003
). The
late appearance of translated peptide, despite the earlier presence of
significant numbers of transcripts was intriguing: during development,
peptides could only be detected in the metanaupliar stage, i.e. during eye
formation. For PDH peptide expression, five pairs of perikarya were first
observed 1-2 days after eye anlage formation, and following this a rapid
development of descending neurones was observed. By late eye development, a
complex arrangement of neurones, involving invariant numbers of PDH-expressing
neurones (2,5,4; Fig. 3C,D) was
seen in the eye. As has previously been mentioned
(Mangerich et al., 1987
) it
was difficult to trace individual axons, and this was also the case here,
given the small size of the developing eye (<100 µm). Notwithstanding
this, branching arborisations in the lamina ganglionaris were evident, as were
prominent axons which entered and left the SG, projecting towards the optic
nerve, and T-shaped axons from cell group 5, projecting to both the lamina
ganglionaris and the X-organ. This neuroanatomy was reminiscent of the
neuronal architecture in the adult
(Mangerich et al., 1987
).
During later development, axons ran throughout the thoracic ventral nerve
cord, and eventually the abdomen. With regard to the thoracic projections
(Fig. 3E), it was interesting
to note that eight clusters of dendrites, which presumably correspond to the
developing segmental ganglia were evident. With reference to the detailed
description of the development of the post-mandibular and thoracic neuromeres
of decapod crustaceans (Harzsch,
2003
) there is some correspondence. If the first developing
post-oesophageal dendrites correspond to the anterior mandibular ganglion, and
the second (where the median nerve arises) is the posterior mandibular
ganglion, then subsequent dendritic masses would correspond to maxilla 1, 2
and the first 4 thoracic neuromeres, notwithstanding the absence of
immunoreactive structures in thoracic neuromeres 5-8. It should also be noted
that at this time, single fibres, presumably projecting along the
stomatogastric nerves, arise from the developing ganglion in the
circumoesophageal connectives, which suggest that the beginnings of a PDH
phenotype of the adult somatogastric ganglion (STG)
(Mortin and Marder, 1991
)
develops at this time.
For RPCH, significant levels of transcript were first observed during
naupliar development, which then increased dramatically. An important
correlative event concerned the appearance of small red chromatophores
coincident with eye anlage development, as reported for Homarus
americanus (Helluy and Beltz,
1991). This correlated with a 5 to 10-fold increase in expression
of RPCH mRNA. However, peptide immunoreactivity was first detected 5-6 days
after eye anlage formation (Fig.
4A). While initial expression of RPCH was restricted to the XO-SG
axis at this time, further development revealed a notable complexity of RPCH
immunopositive neurones (Fig.
4B,C). The salient features here concern the appearance of three
pairs of perikarya in the posterior protocerebrum 6-7 days before hatching,
which project descending axons, contra- and ipsilaterally, around the
oesophagus to the post-commissural nerve, and then project anterio-dorsal
digitate projections which are undoubtedly release sites
(Fig. 4C) and which probably
correspond to the somewhat enigmatic post-commissural organs (PCO). Although
described over 50 years ago (Knowles,
1953
; Maynard,
1961
) in adult crustaceans, their structure and function has been,
rather surprisingly, overlooked. Excepting early observations
(Carlisle and Knowles, 1953
;
Knowles, 1953
) suggesting that
they are a source of pigment-concentrating hormone activity, there appear to
have been no other studies upon this neurohaemal tissue. Given the large
amount of RPCH stored in these structures, which seem (from intensity of
immunoreactivity) to far exceed that in the SG, the PCOs may have some
particular significance in embryonic life.
In summary, the present study has described the fine detail of developmental expression of neurohormones and their transcripts involved in embryonic moulting in a crab model, and contrasts these with expression patterns of the chromatophorophins, which have roles as neurotransmitters as well as classical neurohormones. This study additionally details embryonic expression of novel peptide-producing cells and neurones. Now that normal developmental expression patterns are known, the next appropriate and exciting step must be to use gene-silencing technologies, to knockdown expression of each of these neuropeptides during embryonic development, and observe subsequent phenotypes. Indeed, the small size of crustacean embryos, coupled to their rapid development and the relatively simple anatomy of neuropeptide-expressing neurones, makes this an attractive prospect.
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ACKNOWLEDGMENTS |
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Footnotes |
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