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Development 108, 59-71 (1990)Printed in Great Britain © T h e
Company of Biologists Limited 1990
59
Immunological, biochemical and physiological analyses of
cardioacceleratory peptide 2 (CAP2) activity in the embryo of
the tobacco
hawkmoth Manduca sexta
KENDAL S. BROADIE1*, ANDREW W. SYLWESTER11, MICHAEL BATE2
and
NATHAN J. TUBLITZ1
1 Institute of Neuroscience, University of Oregon, Eugene, OR
97403, USA2 Department of Zoology, University of Cambridge,
Cambridge CB2 3EJ, UK
•Present address: Department of Zoology, University of
Cambridge, Cambridge CB2 3EJ, UKt Present address: Department of
Bioengineering, University of Iowa, Iowa City, IA 52246, USA
Summary
The cells in the embryonic CNS of the tobacco hawk-moth, Manduca
sexta, that synthesize a cardioaccelera-tory peptide 2 (CAP2)-llke
antigen were identified usingimmunohistochemical techniques. Two
distinct neuro-secretory cell types were present in the
abdominalventral nerve cord (VNC) that contain CAP2-like
immu-noreactivity during late embryogenesis: a pair of
large(diameter range 15-20 fan) cells lying along the pos-terior,
dorsal midline of abdominal ganglia A4—A8, anda bilateral set of
four smaller (diameter range 6-11 /on)neurons which lie at the base
of each ventral root inabdominal ganglia A2-A8. CAP2-like
accumulation ap-peared to follow independent patterns in the two
celltypes. CAP2-like immunoreactivity began at 60% ofembryo
development (DT) in the medial cells, accumu-lated steadily
throughout embryogenesis, and droppedmarkedly during hatching.
Lateral cells synthesized theCAP2-like antigen later in development
(70 % DT) andshowed a sharp drop in antigen levels between 75 %
and80 % of embryonic development.
Extracts from developing M. sexta embryos werefound to contain a
cardioactive factor capable of acceler-ating the contraction
frequency of the pharate adultmoth heart in a fashion similar to
CAP2. Immuno-precipitation with a monoclonal antibody that
specifi-cally recognizes the two endogenous Manduca
cardioac-celeratory peptides and purification using high
pressureliquid chromatography identified this factor as
cardioac-
celeratory peptide 2 (CAP2). Using an in vitro heartbioassay,
the levels of this cardioactive neuropeptidewere traced during the
development of the M. sextaembryo. As with the immunohistochemical
results, twoperiods during embryogenesis were identified in
whichthe level of CAP2 dropped markedly: between 75 % and80 %
development, and at hatching. Embryo bioassaysof CAP2 activity were
used to identify possible targettissues for physiological activity
during these two puta-tive release times. CAP2 was found to
accelerate contrac-tion frequency in the embryonic heart and
hindgut ofManduca in a dose-dependent fashion. Of these twopossible
targets, the hindgut proved to be more sensitiveto CAP2, having a
lower response threshold and a longerduration of response to a
given concentration of theexogenously applied peptide.
Based on these immunocytochemical, pharmacologi-cal and
biochemical results, and on a previously pub-lished detailed
analysis of Manduca embryogenesis, weconclude that CAP2 is probably
released from a specificset of identified neurosecretory cells in
the abdominalVNC to modulate embryonic gut activity at 75-80 %
ofembryo development during ingestion of the extra-embryonic
yolk.
Key words: neuropeptides, neurohormones, insectneurobiology,
developmental endocrinology, invertebrateneurodevelopment, insect
gut, invertebrate neuropeptides.
Introduction
Neuropeptides are universally recognized as an import-ant class
of intercellular messengers throughout theanimal kingdom. In mature
systems, neuropeptideshave been demonstrated to act as
neurotransmitters,
neurohormones, and paracrine factors to regulate thefunction of
most tissues including modulating CNSactivity (Pickering et al.
1987; O'Shea and Schaffer,1986; Mayeri et al. 1985; Jan and Jan,
1982; Guillemin,1978). Despite this wealth of information in
maturesystems, the functional significance of neuropeptides
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60 K. S. Broadie and others
during development has been largely ignored. In par-ticular,
little attention has been given to participation ofpeptides during
embryogenesis. To date, efforts in thisdirection have been largely
limited to tracing peptidelevels and mapping peptide distribution
using a varietyof immunological and hydridization assay
techniques(Pickering et al. 1987). In most cases, the functional
roleof these embryonic neuronal factors has proven verydifficult to
establish.
Of the few investigations concerning neuropeptidefunction in
developing systems, most have utilizedinvertebrate preparations. In
simple freshwater coelen-terates, for example, Head/Foot Activating
Peptidehas been demonstrated to possess neurotransmitterand/or
neuromodulatory properties that interact toestablish determinate
fate of regenerating and/or de-veloping tissues (Schaller, 1979).
In insects, the shed-ding of the embryonic cuticle is correlated to
a drop inthe storage levels of a neurosecretory peptide,
eclosionhormone (Truman et al. 1981). Based on this
evidence,eclosion hormone has been postulated to play a role
inembryonic moults similar to its role during larval andadult
development.
Given the diversity of neuropeptide roles post-embryonically,
coupled with the few reports in theliterature on their putative
embryonic function, it seemsplausible that peptides play a greater
variety of roles inthe developing embryo than previously thought.
Oneapproach to the study of peptides during developmentis to
investigate the embryonic role of peptides that arepresent and
functionally important in the mature ner-vous system. Such studies
are best accomplished in adeveloping system that is relatively well
characterizedand experimentally tractable. One preparation
thatmeets these criteria is the tobacco hawkmoth, Manducasexta,
whose CNS has been the subject of intensivestudy (Weeks, 1987;
Levine, 1986; Tublitz et al. 1986;Truman, 1985).
Our focus in the present study is two
cardioregulatoryneuropeptides, cardioacceleratory peptide 1 and
2(CAP1 and CAP2), that were originally isolated in thepharate adult
stage of M. sexta (Tublitz and Truman,1985a). Using in vitro and in
vivo heart bioassays, thesepeptides were shown to be released into
the haemo-lymph from individually-identified neurosecretory cellsin
the CNS, accelerating heart contraction frequency ina
dose-dependent manner (Tublitz and Truman,1985a, b). Biochemical
and physiological studiesdemonstrated that both peptides play
important physio-logical roles in the adult moth, acting as
cardioexcit-atory hormones during wing-spreading behavior andflight
(Tublitz and Truman, 1985b; Tublitz and Evans,1986; Tublitz, 1989).
Further work has recently shownthat CAP2 strongly excites the
hindgut in fifth instarlarvae, apparently to aid gut emptying
during thisdevelopmental stage (N.J. Tublitz, unpublished
obser-vations). The CAPs thus have several different stage-specific
functions.
Since the CAPs perform several different rolesthroughout
post-embryonic life, we were interested inidentifying other CAP
roles even earlier in develop-
ment, during embryogenesis. Results from
preliminaryimmunocytochemical studies using an anti-CAP anti-body
identified several sets of CAP-immunopositiveneurons in the latter
stages of embryogenesis (Broadieet al. 19896). In the present
study, we use pharmaco-logical, biochemical, and immunocytochemical
tech-niques to trace the acquisition and distribution of theCAP
peptides during embryo morphogenesis. Ourresults provide several
lines of evidence strongly sup-porting a functional role for one of
the CAPs duringembryo formation.
Materials and methods
AnimalsTobacco hawkmoths, M. sexta, were reared on an
artificialdiet (Bell and Joachim, 1978) in a controlled
temperature(27°C [light], 25°C [dark]) room with a 17h
photoperiod.Humidity was kept above 50% at all times. Under
thesestandard conditions, the time duration from oviposition
tohatching was 95±3h (mean±s.E.M.; ^=100). For this study,embryonic
development is expressed as a percentage of thetotal development
time (DT) using morphological develop-ment characteristics as
observed in our laboratory (Broadie etal. 1989a) and elsewhere
(Dorn et al. 1987).
Antibody production and specificityA mouse monoclonal antibody,
6C5, shown to be highlyspecific for both CAP, and CAP2 (Tublitz and
Evans, 1986),was used in all trials. 6C5 isolation and preparation
has beendescribed previously (Taghert et al. 1983, 1984). In short,
thecrude extract of 3000 pharate adult Manduca perivisceralorgans,
the neurohaemal release site in the VNC for CAPs,was used as the
inoculum. Antibody specificity was estab-lished by several
independent criteria including morphologi-cal staining of known
CAP-containing cells in pharate adultabdominal ganglia, in vivo
immunoneutralization, in vitroELISA assays and immunoprecipitation
of both CAPs (Tub-litz and Evans, 1986: Taghert et al. 1983, 1984).
6C5 was alsoshown to bind specifically both CAP! and CAP2 that had
beenpurified to homogeneity using high pressure liquid
chroma-tography (HPLC; Tublitz and Evans, 1986).
To reconfirm the specificity of our anti-CAP antibody 6C5,a
series of incubations was performed to establish the natureof the
antigen(s) capable of immunoprecipitating the anti-body. Both the
suspect neuropeptide antigen, CAP2, and arange of other known
invertebrate cardioexcitatory neuro-peptides were incubated with
6C5 (diluted 1:1000 in 0.4%saponin-PBS+1.0% BSA) for 30min prior to
applicationonto a fixed embryo. Neuropeptides used were as
follow:HPLC-purified CAP2, peptide F
(thr-asn-arg-asn-phe-leu-arg-phe-amide) kindly supplied by Dr B. A.
Trimmer (Trim-mer etal. 1987), and two molluscan cardioexcitatory
peptides;FMRFamide (phe-met-arg-phe-NH2; Greenberg and Price,1979),
and one of the small cardioactive peptides (SCPD,
met-asn-tyr-leu-ala-phe-pro-arg-met-NH2; Lloyd, 1978). Thestaining
protocol used with these putative 6C5-antigen com-plexes was in all
other ways identical.
ImmunohistochemistryEmbryos were dissected free of the chorion
and yolk with fineforceps and the ventral CNS exposed. This
dissection wasaccomplished by securely fastening the specimen in a
Sylgard(Dow-Corning) dish containing Manduca saline, making an
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Peptidergic modulation of the insect embryonic gut 61
anterior-directed incision through the dorsal body wall fromtail
horn to head capsule, pinning open the body wall, andremoving the
embryonic gut to expose the ventral nerve cord(VNC). Dissected
embryos were incubated at 4°C with gentleagitation in a modified
Bouin's/glutaraldehyde fixative (2 %glutaraldehyde, 25% saturated
picric acid and 1% glacialacetic acid) for lh,washed three times in
0.4% saponin-PBSfor 30min each, and taken through an ethanol
dehydrationseries at 4°C. Fixed specimens were incubated in
collagenase( lmgmr1; Sigma, type XI) for lh, followed by extinction
ofendogenous peroxidase activity with 0.75 % hydrogen per-oxide in
methanol. Specimens were then blocked with goatserum (5 nig ml"1;
Sigma lot no. 28F-9401) and bovine serumalbumin (1% w/v) in 0.4%
saponin-PBS for 2h.
A three-tier antibody system using the
peroxidase-anti-peroxidase (PAP) method was employed to identify
cells withCAP-like immunoreactivity. Primary antibody (6C5;
dilution1:1000), secondary antibody (whole molecule, goat
anti-mouse IgG; dilution 1:100), and tertiary antibody
(mouseperoxidase anti-peroxidase (PAP); dilution 1:100) were
sus-pended with 1 % BSA in 0.4% saponin-PBS. Each
antibodysuspension was incubated for 24 h at 4°C. Specimens
werewashed five times in five hours with 0.4% saponin-PBScontaining
1 % BSA between successive incubations. Follow-ing equilibration in
3,3'- diaminobenzidine (DAB), immuno-reactivity was visualized by
incubation in a solution of 0.8 %saponin-PBS containing DAB
(O.Smgml"1), hydrogen per-oxide (0.003 % v/v) and NiCl2 (0.001 %
w/v) until complete,usually 10-15 min. Visualized embryos were
taken through anethanol dehydration series, equilibrated in xylene,
andmounted in Permount for observation with Nomarski optics.
CAP extractionDevelopmentally staged embryos (Dorn et al. 1987;
Broadie,1989e) were heat-treated for 5 min at 80°C, ice-cooled,
andhomogenized in double-distilled H2O (ddH2O; 10^1/embryo)in a
ground glass homogenizer. The homogenate was centri-fuged (15 min,
12000g, at 4°C) and the supernatant collected.The pellet was
re-suspended in ddH2O (20 A«l/embryo), re-ground, centrifuged, and
the supernatants from both extrac-tions pooled. The combined
supernatant fraction was loadedonto a MeOH-activated, water-rinsed
Waters C-18 Sep-Pakcartridge and washed in five times its volume
with ddH2O.This was followed by step-wise applications of 20 % and
80 %acetonitrile (HPLC grade; J.T. Baker no. 9017-2) in ddH2O.From
earlier studies (Tublitz and Truman, 1985a; Tublitz andEvans,
1986), it was known that both CAP] and CAP2 elute inthe 80%
acetonitrile fraction. Accordingly, this fraction wascollected,
frozen in dry ice, and lyophilized to powder.Lyophilized samples
were stored at —20°C for up to onemonth before use. Immediately
prior to bioassay, sampleswere brought to room temperature and
re-hydrated in normalManduca saline.
CAP bioassayAn in vitro heart bioassay was used to quantify
relative CAPlevels in each fraction as described earlier (Tublitz
andTruman, 1985a). In short, a portion of the abdominal heartwas
dissected from a pharate adult male immediately prior toeclosion.
One end of the heart tissue was pinned in a smallsuperfusion
chamber; the other end was attached with finesuture thread
(Ethicon, 6-0) to a Bionix F-200 isotonic-displacement transducer
powered by a Bionix ED1-1A Pow-erpack. The signal was amplified and
displayed on a HitachiVC-6026 oscilloscope. Concurrently, the
signal from the forcetransducer was passed through a frequency
converter with awindow discriminator to determine instantaneous
heart rate.
Heart amplitude and contraction frequency were
recordedcontinuously on a Gould 2200 chart recorder for later
analysis.
Manduca saline of the following composition was used in
allexperiments: Pipes biological buffer (dipotassium salt;Sigma),
5mM; CaCl2, 5.6mM; NaCl, 6.5mM; KC1, 28.5mM;MgCl2, 16 mM; dextrose,
173 min. The final pH was adjustedto 6.7±0.1 using a concentrated
solution of HC1. During eachbioassay. saline flow rate was
maintained at approximately80mlh through the open superfusion
chamber containingthe isolated heart. 100//I test samples were
directly injectedinto the saline flow with a gas-tight Hamilton
syringe.
Each injection of embryonic extract was bracketed withseveral
graduated injections of known adult CAP activity. Theresultant
dose-response curves of adult CAP activity enableda precise
determination of an adult CAP activity equivalentfor the embryonic
extract. Thus, the CAP level in eachembryo fraction was expressed
as a percentage of the standardCAP levels in the abdominal nerve
cord of the adult moth.Using these adult activity equivalents,
measurements of theamount of CAP in embryo development stages could
bequantitatively compared within the same bioassay, or be-tween
different heart preparations.
Immunoprecipitation75 % DT Manduca embryos were extracted using
the CAPextraction procedure as described above. The
lyophilizedsample was resuspended in Manduca saline and half of
thesample incubated with the 6C5 antibody at a dilution of 1
partantibody: 10 parts saline. The other remaining aliquot
wasincubated with a non-reactive protein (1.0% BSA) as acontrol.
Each aliquot was incubated at 4°C for 30min. Thesupernatant from
each fraction was collected and bioassayedfor cardioacceleratory
activity on the isolated pharate adultManduca heart as described
above. Other controls included6C5 alone, BSA alone, 6C5+serotonin
(Sigma) and6C5+peptide F (a crustacean cardioactive
neuropeptide;Trimmer et al. 1987). Bioactivity of each treatment
wascompared to that of an untreated control.
HPLCSep-Paked embryo extracts were chromatographed on aBrownlee
Alltech C-18, reverse-phase HPLC column(4.6x250mm, 300jan particle
size). An acetonitrile-watersolvent gradient with 0.1%
trifluoroacetic acid (TFA)counter-ion was used in all trials. A
linear acetonitrile-watergradient was used with the acetonitrile
concentration increas-ing at 1.5% per min (Tublitz and Evans,
1986). For eachchromatography run, 30 separate 1 ml fractions were
collectedat lmin intervals. Each fraction was lyophilized, stored
at—20°C, and later re-suspended in Manduca saline for bioassayon
the isolated heart.
Embryonic heart and gut bioassaysManduca embryos were dissected
free of their chorion andyolk, and development stage was determined
(Dorn et al.1987; Broadie et al. 1989a). Specific target organs,
either theembryonic hindgut or heart, were exposed by making
in-cisions through the body wall and pinning back the epidermiswith
minute steel pins. When necessary, minimal surgery wasperformed to
clearly expose the target organ. This semi-intactpreparation was
placed in a small superfusion chamber andperfused with Manduca
saline at 60 ml h"1.
HPLC-purified CAP2, and a range of other known neuro-hormones
and transmitters, were applied to the preparationand the effect on
myogenic contraction frequency in the heartand gut quantified. The
applied substances included: smallcardioactive peptideB (SCPB;
Lloyd, 1978), FMRFamide
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62 K. S. Broadie and others
(Greenberg and Price, 1979), peptide F (Trimmer et al.
1987),proctolin (Sigma), serotonin (5-HT; Sigma),
octopamine(Sigma), and acetylcholine. All substances were dissolved
in100 fi\ of Manduca saline and injected directly into the
salineflow with a gas-tight Hamilton syringe.
The target tissue was observed under high magnification(x800)
with a Wild binocular dissecting microscope, andmyogenic
contractions counted during 30 s intervals for up to8min following
an injection. From these observations, con-traction frequency over
30 s intervals was computed. A secondinjection was applied if and
only if the organ returned rapidlyto a basal contraction
frequency.
Results
Spatial distribution of CAP-like immunoreactivityDistinctive
patterns of CAP-like immunoreactivitywere observed repeatedly in
the posterior abdominalganglia during the late stages (70-100 % DT)
of Man-duca embryonic development. Cells showing
CAP-likeimmunoreactivity fit into two spatially distinct groups.The
first group consisted of a pair of large neurons thatlie along the
posterior midline of the caudal abdominalganglia (A4-A8; Figs 1 and
2). The axons from thesecells bifurcate and exit the ganglion via
each ventralnerve, ultimately projecting posteriorly to both
ipsilat-eral and contralateral transverse nerves (data notshown).
The second group consisted of lateral neuro-secretory cell clusters
that lie at the base of each ventralroot in the abdominal ganglia
(A2-A8; Figs 1 and 2).These lateral cells also have processes in
the ventralnerve leading to the transverse nerve. Midline
stainingwas observed in an average of two medial neurons(range: 1-3
cells) in all four posterior abdominalganglia. The mean number of
CAP-like immuno-reactive cell bodies in each lateral cluster varied
greatlydepending on position in the VNC (range: 2-8 cells/cluster).
In general, more caudal ganglia contained ahigher number of
immunoreactive somata in the lateralclusters as compared to the
rostral ganglia (Fig. 2). Thecellular morphology of both types of
CAP-like immuno-reactive cells was commensurate with observations
ofother insect neurosecretory cells (Rowell, 1976): largeand
clearly evident cell bodies displaying the TyndallBlue effect
characteristic of neurosecretory function.
CAP-like immunoreactivity appeared earliest in de-velopment and
at the highest intensity in the fusedterminal abdominal ganglion
(A7/A8; Figs 3A and4A). Both immunoreactive midline and lateral
cellswere observed in the terminal ganglion. The midlinecells in A8
appeared anterior and dorsal, just posteriorof the transverse nerve
branching, whereas the midlinecells in A7 were found very
posteriorly, just anterior tothe juncture between A7 and A8. These
midline cellswere very large relative to other neurosecretory cells
inthe ganglion, with cell body diameter ranging from15 fim to 20
/im. The cell bodies of these cells displayedstrong CAP-like
immunoreactivity throughout late em-bryonic development (Figs 1, 3A
and 4A).
The lateral cell clusters in the fused terminal ganglionalso
stained intensely throughout the last quarter of
embryonic development. The lateral cells in A8 showeda
distribution different from that of A7 and the anteriorunfused
ganglia, in that the more numerous cell bodiesin A8 were less
clustered and tended to be isolated intoone or two cells lying at
the bases of the major posteriorventral root branches (Figs 1 and
2C). In this ganglion(A8), an average of six cell bodies containing
CAP-likeimmunoreactivity were observed, ranging from 2-10cells per
preparation. In contrast, the lateral cellclusters in A7 formed
close-knit, bilaterally symmetri-cal clusters at the bases of the
posterior ventral root(Figs 1 and 2C). Both groups of lateral cells
in theterminal ganglion (A7/A8) had relatively small somata,with
diameters in the range of 6^m to 11 fun each. In allpreparations,
only the cell bodies of the lateral cellswere intensely
immunoreactive.
In general, more CAP-like immunoreactive cellbodies were located
in ganglion A8 than in the moreanterior ganglion. We surmise that
this is due to the factthat ganglion 'A8' is actually a
condensation of multipleneuromeres found during the initial
formation of theCNS (Jacobs and Murphey, 1987). This unique
develop-ment explains the different distribution of
CAP-likeimmunoreactive cells in A8 relative to A7 and the
moreanterior, unfused abdominal ganglia (Fig. 2).
Unfused abdominal ganglia showed immunoreactivepatterns similar
to A7 (Figs 1 and 2). An average of twomidline cells per ganglion
stained in ganglia A4 to A6(range: 1-3 immunoreactive cells per
ganglion). Thecell bodies lay along the posterior, dorsal midline
andwere often positioned asymmetrically across theganglion midline,
with one cell body lying slightlyanterior relative to the other.
The morphology and sizeof these cells was comparable to the midline
cells in thefused terminal ganglion (Figs 1 and 2). The
midlinecells in the unfused ganglia were less immunoreactivethan in
the fused terminal ganglia, with intensity of theDAB reaction
product progressively decreasing fromA6 to A4 (Fig. 4A). No midline
cells showed CAP-likeimmunoreactivity in the first three abdominal
gangliaduring any stage of embryo development.
The lateral cell bodies in the unfused abdominalganglia remained
clustered at the base of the posteriorventral root in a pattern
similar to A7 (Figs 1 and 2).The number of cells in the lateral
clusters decreased inthe more anterior unfused ganglia: an average
of fourcells per cluster in A6 (range: 4-5), three cells in
A5(range: 2-4), and two lateral cells, one cell at the baseof each
ventral root, in A2-A4 (range: 1-2). As withthe midline cells,
immunoreactive staining intensityprogressively decreased in the
lateral cells of the moreanterior abdominal ganglia: A6 lateral
cells were themost intensely stained, and A2 lateral cells were
theleast immunoreactive (Fig. 4B). Lateral cell body mor-phology
and size were similar in all the abdominalganglia. CAP-like
immunoreactivity was not detectablein the lateral cells of ganglion
Al.
In summary, CAP-like immunoreactivity during em-bryogenesis was
restricted to midline and lateral neuro-secretory cells in the
abdominal ganglia, predominantlythe posterior five ganglia (A4-A8).
Immunoreactivity
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§
Fig. 1. Immunocytochemical labeling of neurosecretory cells in
the embryonic abdominal ganglia of Manduca sexta.(A) Terminal,
fused ganglion (A7/A8) in a 75% DT embryo. Notice intensely
immunoreactive medial and lateral cellbodies. (B) Terminal ganglion
(A7/A8) at 80% DT. Immunoreactivity in lateral and medial cells is
maintained only in theterminal ganglion. (C) Unfused (A5) ganglia
in a 75% DT embryo showing immunoreactive midline and lateral cell
bodies.(D) Ganglion A5 at 80 % DT with selectively decreased
immunoreactivity in lateral cells. Note that medial cells still
displaystrong CAP-like immunoreactivity. (E) Terminal ganglion
(A7/A8) in the 100 % DT embryo prior to hatching,
intenselyimmunoreactive lateral and midline cells. (F) Terminal
ganglion (A7/A8) 1 h after hatching of first instar larvae. Notice
theselective decline in medial cell immunoreactivity relative to
staining in lateral cells. (G) Unfused ganglion (A5) at 100 %
DTprior to hatching. (H) Unfused (A3) ganglion 1-2 h after
hatching. Observe selective decrease in medial cell
labelingcompared to (G). (I) Pre-incubation of the anti-CAP
antibody with HPLC-purified CAP2 abolished observedimmunoreactivity
patterns. Pre-incubation with four other invertebrate
cardioexcitatory peptides (see Materials andmethods) had no effect
on observed immunoreactivity. Scale bar, 50/on.
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Peptidergic modulation of the insect embryonic gut 63
A . Abdominal VentralNerve Cord (VNC)
A1
A2
Midline Cells Lateral Cells
VN
Midline'Cell
Lateral'Cell
Unfused AbdominalGanglion (A4)
A3
A4
A5
A6
A7
A8
Fused, TerminalGanglia (A7/A8)
Fig. 2. Spatial location of CAP-like immunoreactive cells inthe
abdominal VNC of Manduca sexta embryo.(A) Schematic representation
of the abdominal VNCshowing all cells with CAP-like
immunoreactivity presentduring embryogenesis. Mean number and
relative positionof medial (midline) and lateral cells
demonstrating CAP-like immunoreactivity are indicated. (B) Camera
lucidadrawing of lateral and medial immunoreactive cell bodies inan
unfused abdominal ganglion (A4) of a 85 % DT embryo,tn, transverse
nerve; dn, dorsal nerve; vn, ventral nerve.(C) Camera lucida
drawing of immunoreactive cell bodiesin the fused, terminal
abdominal ganglion (A7/A8) of a85 % DT embryo.
was limited to the cellular cytoplasm with no observednuclear
staining. In most preparations, CAP-like immu-noreactivity was
limited to the cell body; however, thebase of the transverse nerve
also demonstrated CAP-like immunoreactivity in approximately 10% of
ourtrials. No CAP-like reactivity was observed in thethoracic
ganglia or in the brain during embryonicdevelopment.
Temporal pattern of CAP-like immunoreactivityCAP-like
immunoreactivity first appeared in the 60%developed embryo. The
first cells to demonstrate im-munoreactivity were the two pairs of
large, dorsalmidline cells in the terminal abdominal ganglion
(A7/
A1
v A2Vn
i wco3 A4cooo AS
1 «A7 : i
JO K «U
iii\r
••••R S 8 8 S S R 8 3 8
Percent Embryonic Development
Fig. 3. Percentage of trials possessing CAP-likeimmunoreactive
cells in the abdominal ganglia during lateembryonic development
(60-100% DT). (A) Percent ofembryo preparations with immunoreactive
midline cells.Notice the gradual increase in the number of ganglia
withimmunoreactive cells and the percent increase in positivetests
throughout embryogenesis, followed by a drop athatching. (B)
Percent of embryo specimens withimmunoreactive lateral cells.
Notice the drop in positivestaining trials between 75 % and 85 %
development.
A8; Figs 3A and 4A). Immunoreactive intensity wasvery weak in
these cells in the 60 % developed embryos,and positively stained
cells were only observed in asmall fraction of our animals (12%;
Figs 3A and 4A).
The number of immunoreactive midline cells and theimmunoreactive
intensity of these cells increased gradu-ally during the later
stages of embryonic development(70-100 % DT; Figs 3A and 4A). After
the first appear-ance of CAP-like immunoreactivity in the midline
cellsof the fused, terminal ganglion, the homologous cells inan
unfused abdominal ganglion (A6) first appeared inthe 70% developed
embryo. However, only a smallfraction (10%) of the preparations
exhibited positivestaining. By 75 % DT, the percentage of
preparationswith immunoreactive midline cells in A6 had increasedto
25 %, and the immunoreactive intensity of these cellshad become
more pronounced (Figs 3A and 4A).Midline cells in A5 first
demonstrated immunoreactivityat 80% development. From 80 to 95% DT,
no newmidline cells with CAP-like immunoreactivity appeared(Fig.
3A). Immunoreactive intensity also remainedfairly constant from 80
to 90% DT, but began toincrease in the midline cells of the last
four abdominalganglia by 95% DT (Fig. 4A). Immunoreactive A4midline
cells, the most anterior midline cells to haveCAP-like
immunoreactivity during embryogenesis,were first detected in the
100%, fully developed em-bryo, just prior to hatching. In the fully
developedembryo, a high percentage of preparations had
immu-noreactive midline cells in the last five abdominal
-
64 K. S. Broadie and others
A Midline Cells B Lateral Cells
F 1 " J13
i!i40
Iir
J
!
n
100 (»
3
2 K R 8 2
Percent Embryonic Development
Fig. 4. Intensity of immunocytochemical reaction inembryonic
abdominal VNC cells. (A) Midline cells.Relative intensity of
immunochemical reaction scaled withthe most intense reaction
arbitarily assigned maximumintensity (1=1) and immunoreactive
intensity in othermedial cells scaled relative this standard.
Notice theincrease in relative reaction intensity during
embryodevelopment and the drop in intensity at hatching.(B) Lateral
cells. Relative intensity scaled to most intenselateral cell
immunoreactivity (1=1). A large drop in CAP-like immunoreactivity
occurs between 75 % and 80 %development.
ganglia, ranging from 75 % positive staining in A8 downto 10%
staining in A4 (Fig. 3A). In addition, theintensity of CAP-like
immunoreactivity peaked in themidline cells within the last two
hours of embryonicdevelopment.
A marked decrease in midline cell CAP-like immuno-reactivity was
observed at hatching. During larvalemergence, the number of
immunoreactive midlinecells and the intensity of their staining
decreased(Figs 1E-H, 3A, and 4A). Specifically, midline
cellCAP-like immunoreactivity in ganglia A4 and A5disappeared
completely and the midline cell immuno-reactive intensity decreased
markedly in A6 through A8(Figs 3A and 4A).
By way of contrast, the lateral neurosecretory cellsacquired
CAP-like immunoreactivity in a developmen-tally unique pattern,
distinct from the midline cells.First appearing at 70% DT, lateral
cell immunoreac-tivity peaked in the 75 % developed embryo (Figs
1A,Cand 4B). At this stage we saw the largest number ofCAP-like
immunoreactive lateral cells (Fig. 3B), themost intense CAP-like
reactivity in these cells(Fig. 4B), and the greatest number of
abdominalganglia (A2-A8) possessing immunoreactive lateralcells
(Fig. 3B). A sharp decline in lateral cell immuno-reactivity was
observed between 75 % and 80 % DT:lateral cell immunoreactivity in
the more anterior twoabdominal ganglia (A2-A3) disappeared
completely,while the percentage of CAP-like reactive cells and
the
immunoreactive intensity of these cells declined mark-edly in
all the remaining posterior abdominal ganglia(A4-A8; Figs 3B, 4B
and 1A-D). By 85% DT, bothimmunoreactive intensity and the total
percentage ofCAP-like immunoreactive lateral cells began to
recoverin A5 through A8, whereas no recovery was detected inthe
lateral cells of ganglion A4 at this stage of develop-ment (85 %
DT).
CAP-like immunoreactivity of the lateral neuro-secretory cells
continued to recover during the last 15 %of development. By 95 %
DT, a large number (Fig. 3B)of intensely immunoreactive lateral
cells were onceagain observed in A4-A8. Less intense staining
wasseen in ganglia A3. The immunoreactivity of the lateralcells
plateaued at this level for the remainder ofembryonic development
(Figs 3B and 4B). Unlike themidline cells, lateral cell
immunoreactivity remainedconstant during hatching behavior (Fig.
1E-H).
In summary, we found that levels of the CAP-likeantigen were
modulated independently in the twoabdominal ganglia cell types.
Expression of the CAP-like antigen in the midline cells was
characterized byearly development (60% DT), a gradual
continuousincrease in immunoreactivity during embryogenesis,and a
marked drop in the levels of the immunoreactiveantigen during
hatching. In contrast, lateral cells ex-pressed the CAP-like
antigen later at 70 % DT, rapidlyaccumulated levels of the antigen
during the next 5 % ofdevelopment, and demonstrated a dramatic drop
inCAP-like immunoreactivity between 75 % and 80 % ofembryo
development.
Antibody specificityTo reaffirm its specificity, our primary
antibody (6C5)was pre-absorbed with a variety of antigens to
helpverify the nature of molecule responsible for the ob-served
immunoreactivity patterns. All CAP-like immu-noreactivity was
completely abolished after pre-incu-bation of the primary antibody
in CAP2 (Fig. li). Incontrast, pre-incubation of 6C5 with other
invertebratecardioactive peptides (peptide F, SCPB and FMRF-amide)
in no way altered the immunoreactivity patternsobserved with the
free antibody (data not shown).
Levels of cardioacceleratory activity during
embryonicdevelopment in ManducaThe immunohistochemical observations
describedabove clearly demonstrated the presence of a
CAP-likeantigen in a well defined subset of neurosecretory cellsin
the embryonic caterpillar. We next carried out aseries of
biochemical tests to establish the relationshipbetween this antigen
and the CAPs. Embryos at variousdevelopmental stages were extracted
for cardioacceler-atory activity as described in Materials and
methods,with the crude homogenate partially purified through aC-18
Sep-Pak. The resultant fractions then were as-sayed for biological
activity on the isolated Manducaheart. Using this procedure, we
determined that allembryonic development stages possessed some
degreeof cardioacceleratory bioactivity. Even samples of
-
Peptidergic modulation of the insect embryonic gut 65
o
i
Io
a.
i"c««
Q-
20-•
10--
un 0 10 20 30 40 50 60 70 75 80 85 90 95 100pro/post
Percentage Embryo Development Time
Fig. 5. Changes in cardioacceleratory levels duringManduca
embryogenesis. CAP2 activity was assayed on thein vitro pharate
adult Manduca heart as described inMaterials and methods. An ANC
unit refers to the CAPactivity level found in the standardized
pharate adult mothabdominal nerve cord. Activity was first detected
at 40% ofembryo development. Two drops in cardioacceleratorylevels
occurred during development: between 75% and80% development, and at
hatching. Cardioacceleratoryactivity in embryo extracts of all
developmental stages wascorrected for the minor yet detectable
activity found in theunfertilized eggs (UN), as described in the
Results. Pre/Post 100% DT refers to 1-2 h before hatching and 1-2
hfollowing hatching, respectively. Each bar represents
themean±s.E.M. of at least ten independently
pooleddeterminations.
unfertilized Manduca eggs, purified with the
identicalpurification scheme, contained very low yet
measurablelevels of cardioacceleratory activity (0.9% of the
ac-tivity in an adult abdominal nerve cord (ANC); «=10).Thus, all
measurements of the embryonic cardioexcit-atory levels found at
later development stages werecorrected for this background
cardioacceleratory ac-tivity by subtracting the level of
unfertilized egg bioac-tivity from the observed response of the
pharate adultheart to a given sample.
Cardioacceleratory activity substantially above back-ground
levels was first detected in the 40 % developedembryo (Fig. 5).
Extracts of this stage had 2.0±0.8%(mean±s.E.M.) of the
cardioacceleratory activity in anadult ANC as determined by
quantitative bioassay onthe isolated pharate adult heart (Fig. 5).
This activityincreased rapidly between 40 % and 75 % DT, where
itpeaked at 14±3.8% of the adult ANC levels. Cardioac-celeratory
bioactivity in the entire embryo decreasedmarkedly between 75 % and
80 % DT, dropping to4.5±1.2% of adult ANC cardioacceleratory
activity(Fig. 5). A gradual increase in bioactivity was
observedfrom 80 % to 100 % DT, with fully developed
embryoscontaining 8.2±1.8 % adult ANC units of CAP activity.A
second, substantial drop in cardioacceleratory ac-tivity was
recorded at hatching when bioactivity levelsdecreased by
approximately 50 %, with newly-hatchedfirst instar larvae having
4.3±0.9 % of adult ANC levels(Fig. 5).
oDoin
100-
75-
50-
2 5 - •
75X DT EmbryoExtract
7 5 * DT EmbryoExtract
+6C5 Antibody
Fig. 6. Immunoprecipitation of embryonic cardio-acceleratory
activity by the anti-CAP antibody, 6C5.Bioactivity is expressed as
a percentage of cardioexcitatoryactivity in the experimental sample
compared to that of theuntreated embryonic extract as determined on
the in vitroheart. 6C5 had no effect on the activity of biogenic
amines(5-HT) or another invertebrate cardioactive peptides(peptide
F). 6C5 alone did not effect cardiac contractionfrequency. Each bar
represents the mean±s.E.M. of at leastfive determinations.
Table 1. An anti-CAP antibody (6C5) selectivelyprecipitated the
cardioactive factor found in the
partially-purified Manduca embryo extracts. 6C5 hadlittle or no
effect on a biogenic amine (5-HT) or on
another arthropod cardioactive neuropeptide(peptide F)
Treatment % Bioactivity
CAP2 (control)CAP2+6C55-HT (control)5-HCT+6C5Peptide F
(control)Peptide F+6C5
Each value represents the mean±s.E.M.determinations.
100±4%19±5%
100±2%100±3%100±4 %96±7%
of at least five
Biochemical identification of cardioacceleratory activityin the
Manduca embryoAs a first step in identifying the cardioactive
factorfound in the partially-purified embryonic extracts,
amonoclonal anti-CAP antibody, 6C5, (Taghert et al.1983, 1984) was
tested for its ability to precipitate thebioactive factor. This
antibody has been previouslydemonstrated to specifically recognize
an epitope com-mon to CAP1 and CAP2 (Tublitz and Evans, 1986).When
extracts from 75 % developed embryos wereincubated with the 6C5
antibody for 30min prior tobioassay on the in vitro Manduca heart,
bioactivity wasreduced by 81 % when compared to untreated
controls(Fig. 6; Table 1). The 6C5 antibody alone produced
nodetectable effect when applied to the isolated pharateadult
heart. Furthermore, the 6C5 antibody had noeffect on the
bioactivity of a biogenic amine (serotonin)
-
66 K. S. Broadie and others
10
a
6-
4
2
CAPn
rh
PHARATEADULT
CAP,
60
50
12 13 14 15 16 17 18 19 20 21 22
Qution Time (min)
30
ftoo
EZ
°1
8
6'
4-
2
0
7 5 * DTEMBRYOS
rh
T 60
50
3012 13 14 15 16 17 18 19 20 21 22
Qution Time (min)
Fig. 7. Cardioacceleratory activity in Manduca embryoextracts
co-elute with CAP2 when purified with highpressure liquid
chromatography (HPLC). (A) The elutionprofile of both CAPs isolated
from the abdominal VNC ofthe pharate adult moth. An
acetonitrile/water gradient wasused starting with 20% acetonitrile
at t=0 and withacetonitrile concentration increasing at
1.5%/minute. CAP2elutes at 15 min (42.5% acetonitrile) and CAPi at
19 min(49% acetonitrile). After separation on the HPLC
column,fractions were bioassayed on the in vitro heart as
describedin Materials and methods. (B) Elution profile
ofcardioacceleratory activity from 75 % DT embryos. Allbioactivity
co-eluted with CAP2 (15 min; 42.5 %acetonitrile) in a single peak.
No other cardioactivity wasfound to co-elute with CAPi or elsewhere
on the column.Bars represent the mean±s.E.M. of at least
tendeterminations.
or on peptide F (Trimmer, 1987), another arthropodcardioactive
peptide (Table 1).
To unequivocally ascertain the relationship betweenthe CAPs and
the cardioactivity in embryos, partially-purified material from
embryos at various stages waschromatographed on a HPLC using a
reverse phaseC-18 column, and the resultant fractions bioassayed
onthe in vitro Manduca heart. Chromatographic profiles,such as the
one shown in Fig. 7, indicated the presenceof a single peak of
cardioexcitatory activity during lateembryonic development. This
activity co-eluted withpurified CAP2 from the pharate adult moth.
No detect-able cardioregulatory bioactivity eluted in the
fractionsassociated with CAPi, or elsewhere during the
chroma-tographic run (Fig. 7). This sole activity peak
accounted
Table 2. Sensitivity of the Manduca embryonic heartand hindgut
to pulse applications of CAP2 and other
selected myoactive factorsThreshold (M)
SubstanceEmbryonic
heartEmbryonic
gut
SerotoninOctopaminePeptide
FFMRFamideSCPBProctolinAcetylcholineCAP2
io-9
10"8
io-7io-5
>10"5
>io-4>10"3
0.2 ANC
io-9io-710"8
>10"4
>10~3
>10"5
>10"3
0.05 ANC
Threshold is defined as the lowest concentration of the
substancethat produces a measurable (5%) increase in contraction
frequencyin 50% of the trials. CAP2 thresholds are given in terms
of thestandard CAP2 levels in the abdominal ventral nerve cord
(ANC)of the adult moth.
for all the cardioacceleratory activity present in theembryonic
crude extracts. Cardioacceleratory activitycould not be detected in
FIPLC samples from embryosless than 50% developed, even with sample
sizes tentimes larger than those assayed with older embryos.
Pharmacology of the in vitro Manduca embryonicheart and gutOur
biochemical results, combined with our previouslydescribed
immunocytochemical observations (Figs 1, 3and 4), clearly
demonstrate that the level of CAP2fluctuates dramatically during
embryogenesis, and thatthe observed drops in peptide levels may be
related toCAP2 release from individual neurosecretory cells.Hence,
we were interested in the possible physiologicalrole(s) of this
neuropeptide in the embryonic system.As a first step in addressing
this question, we analyzedthe effect of exogenously applied CAP2,
as well asseveral other known invertebrate cardioregulatory
fac-tors, on the beat frequency of myogenically activeembryonic
tissues utilizing an in vitro preparation asdescribed in Materials
and methods. In particular, wewere interested in the effects of
CAP2 on the heart andhindgut, as both tissues are known targets of
CAPmodulation during larval (N. Tublitz, unpublished ob-servations)
and adult life (Tublitz and Truman,1985a,b,c; Tublitz and Evans,
1986; Tublitz, 1989).
The contraction frequency of both the embryonicheart and hindgut
were found to be particularly sensi-tive to three known neuroactive
substances: two bio-genic amines, serotonin and octopamine, and
peptideF, a small neuropeptide isolated from crustaceans
(thr-asn-arg-asn-phe-leu-arg-phe-amide; Trimmer et al.1987; Table
2). In both embryonic tissues, serotonin (5-HT) had the lowest
threshold (10~9M), where thresholdis defined as the lowest
concentration required to evokea measureable increase (5 %) in
contraction frequencyin 50% of the trials (Table 2). The thresholds
foroctopamine and peptide F were at least an order ofmagnitude
higher for both assays. The heart was more
-
Peptidergic modulation of the insect embryonic gut 67
sensitive to octopamine (threshold 10 8M) than topeptide F
(threshold 10~7M). In contrast, the hindgutwas more sensitive to
peptide F (threshold 10~8M) thanto octopamine (threshold 10~7M;
Table 2).
The embryonic heart and hindgut were significantlyless sensitive
to the other substances applied during thisstudy. These included
three other invertebrate bioac-tive peptides, e.g. FMRFamide, small
cardioactivepeptide (SCPB), and proctolin, as well as
acetylcholine,another putative insect cardioregulatory
substance(Miller, 1979). The thresholds for the three peptideswere
of the order of 10~5M or greater when pulse-applied onto either in
vitro preparation (Table 2).Acetylcholine had no detectable effect
in either assay atconcentrations up to 10~3M.
CAP2 was found to increase contraction frequency inboth the
embryonic heart and hindgut. Of the twoorgans, the hindgut was more
sensitive to CAP2 appli-cation, with a lower threshold (0.05 adult
ANC equival-ents of HPLC-purified CAP2 activity) and a
moreprolonged response (response to 1.0 ANC equivalentCAP2:
5.0±0.3min; Fig. 8A). The dose-responserelationship followed a
sigmoidal curve with maximalresponse occurring at a pulse
application of 0.24 ANCCAP equivalents (Fig. 8B).
In contrast, the embryonic heart had a thresholdsensitivity of
0.2 ANC equivalents (Table 2). The re-sponse latency to exogenously
applied CAP2 (1.0 ANC)was similar in both organs, but the duration
of thecardiac response was significantly reduced(3.4±0.4min)
relative to that of the hindgut (Fig. 9A).A similar sigmoidal
dose-response relationship wasobserved in both organs. However, the
lower sensitivityof the embryonic heart resulted in a displacement
of thecurve to the right, with a maximal increase in
cardiaccontraction frequency observed to 0.52 ANC CAPequivalents
(Fig. 9B).
Development of spontaneous activity and CAP2sensitivity of the
embryonic Manduca gutThe spontaneous activity of the embryonic gut
wasmeasured in vivo and in vitro during the last 50%
ofembryogenesis. In vivo observations of gut activitywere possible
prior to 80% DT through a semi-translucent embryonic cuticle.
However, the increase ofcuticle pigmentation after this stage
prevented furtherin vivo observations. Comparison of contraction
ratesbetween the in vivo gut and our semi-intact in
vitropreparation indicated that contractile activity was
notperturbed during our manipulations. Specifically, con-traction
rates of the two preparations remained inagreement during early
development and gut activitydid not change in response to direct
physical stimu-lation. Consequently, both assays were used
interchan-geably to track embryonic gut activity.
The gut was quiescent in all embryos prior to 65 %DT (Fig. 10A).
Spontaneous gut contractions began at70% of embryonic development,
increased dramati-cally during the next 10% DT and reached
peakcontraction rates by 80% of development (Fig. 10A).Gut
contraction frequency remained relatively constant
B
co
uo
Adult ANC Equivalents of CAP2
Fig. 8. (A) Response of the embryonic Manduca hindgut topulse
application of 1 ANC equivalent of HPLC-purifiedCAP2. Vertical
error bars represent S.E.M. of the meanheart response; horizontal
error bars represent the S.E.M. tothe mean heart response time. (B)
Dose-responserelationship of the Manduca embryo hindgut to
pulseapplication of HPLC-purified CAP2. In each case, thepeptide
was dissolved in 100 u\ Manduca saline and directlyapplied to a
semi-intact in vitro preparation as described inMaterials and
methods. Each point represents themean±s.E.M. of at least ten
determinations.
at this level for the remainder of embryogenesis. Aslight
increase in contraction rate was observed immedi-ately following
hatching in the first instar larvae(Fig. 10A).
HPLC-purified CAP2 was applied to each of thedevelopmentally
staged hindgut preparations, and re-sponse in contraction frequency
quantified. We foundthat 0.25 ANC units of exogenously applied CAP2
hadno discernible effect on the gut prior to 70 % develop-ment
(Fig. 10B), stages when the gut is normallyinactive (Fig. 10A). In
contrast, the spontaneouslyactive gut was found to be very
responsive to pulseapplications of CAP2. Application of 0.25 ANC
units ofCAP2 at 75 % DT increased contraction frequency by40±4%
(Fig. 10B). Sensitivity to CAP2 did not changesignificantly during
the remainder of embryonic devel-opment.
-
68 K. S. Broadie and others
o.c
B
A
Adult ANC Equivalents of CAP2
Fig. 9. (A) Response of the embryonic Manduca heart topulse
application of 1 ANC equivalent of CAP2 (see Fig. 5for details).
(B) Dose-response relationship of theembryonic heart to pulse
application of HPLC-purifiedCAP2. In each case, the transmitter was
dissolved in 100/JManduca saline and directly applied to the
dissected in vitrosystem as described in Materials and methods.
Each pointrepresents the mean±s.E.M. of at least ten
determinations.
Discussion
Comparison of CAP distribution in Manducathroughout
developmentTaylor and Truman (1974) were the first to identify
twodistinct groups of neurosecretory cells in the abdominalganglia
of the adult moth. Their work demonstrated theexistence of four
pairs of large (25-30 (ion) somata lyingalong the dorsal midline
and a bilateral set of foursmaller neurons that lay at the base of
each ventral root.Later work (Taghert and Truman, 1982) showed
thatboth sets of neurons projected to the transverse nerve,the
major neurohaemal release site in the insect ventralnerve cord
(Raabe, 1982). Additional investigations,using a variety of
techniques including intracellularstimulation of single identified
peptidergic neurons(Tublitz and Truman, 1985c) and
immunocytochemistry(Taghert et al. 1984, 1985; Tublitz and
Sylwester, 1988)revealed that the four pairs of
segmentally-reiteratedmedial cells in the adult moth synthesized
and releasedhigh levels of cardioacceleratory peptide (CAP)
ac-tivity, whereas the lateral cells expressed another
Notch ling
Percent Embryonic Development
B
hatching
Percent Embryonic Development
Fig. 10. (A) Spontaneous hindgut contractions in theManduca
embryo during the last 50 % of embryogenesis.The gut was inactive
prior to 70 % DT and increasesmarkedly in spontaneous activity
between 70 % and 80 %development. (B) Response of the in vitro
embryonichindgut to pulse application of 0.25 ANC equivalents
ofCAP2 during the last 50 % of embryo development. Theinactive gut
did not respond to the exogenously appliedpeptide. Each point
represents the mean±s.E.M. of 5independent determinations.
neuropeptide, bursicon (Taghert and Truman, 1982). Incontrast,
studies in larvae indicated that both cell typescontained CAP
activity including the lone pair ofmedial cells that arises
embryonically and the fourlaterally-situated pairs lying at the
base of each ventralroot. Our results with the anti-CAP antibody in
theManduca embryo (Figs 1 and 2) closely resembled thatobserved
during the initial stages of post-embryonicdevelopment. As in early
larvae (Tublitz and Sylwester,1988, 1989), staining was limited to
a single pair ofmedial cells and the lateral clusters at the base
of theventral root. All these cells had projections out theventral
nerve, apparently extending at least to the baseof the transverse
nerve, which also expressed strongCAP immunoreactivity. Given the
highly specificnature of the anti-CAP antibody (Fig. II; Tublitz
andEvans, 1986; Taghert et al. 1983, 1984) and that thisantibody
stains a set of cells in the Manduca embryothat closely resemble
the well-characterized CAP-con-taining neurons in larvae, we
conclude that these
-
Peptidergic modulation of the insect embryonic gut 69
embryonic cells are likely to express one or both of
theCAPs.
Identification of CAP2 in the Manduca embryoThree lines of
evidence strongly suggest that CAP2 ispresent during embryonic
development in M. sexta, andthat the level of this neuropeptide
changes in dramaticand potentially physiologically important ways.
First,immunohistological work with an anti-CAP antibodydemonstrated
the presence of an immunoreactive anti-gen specifically localized
in embryonic neurosecretorycells known to contain CAP2 during
larval and/or adultlife (Tublitz and Truman, 1985c; Tublitz and
Sylwester,1988, 1989). This embryonic staining pattern was
com-pletely abolished after pre-incubation of the anti-CAPantibody
with CAP2 (Fig. II). In contrast, pre-incu-bation of the anti-CAP
antibody with a range of similarinvertebrate cardioactive peptides
resulted in no changein the observed immunoreactivity pattern.
Conse-quently, we were inclined to attribute the
observedimmunoreactivity patterns to the presence of CAP2 inthe
Manduca embryo.
It is, however, very rarely possible to unequivocallyidentify an
antigen based on immunoreactivity alone.Absolute identification of
an antigen can be achievedonly by separating it from tissue
extracts, purifying it,and subjecting the pure sample to specific
analysis usingchemical and/or physiological assays. Consequently,
asa second line of evidence, extracts of embryos wereprepared
according to the isolation procedure for theadult CAPs and
quantitatively tested for CAP-likeactivity on a highly sensitive in
vitro heart bioassay(Tublitz and Truman, 1985a,b). Our biochemical
andpharmacological results showed that Manduca embryoscontained
substantial levels of CAP-like cardioaccelera-tory activity (Fig.
5) which changed during embryonicdevelopment in a manner consistent
with the changes inimmunohistological patterns and
immunoreactivityintensities (Figs 3, 4 and 5).
In general, the fluctuations in activity from
immuno-cytochemistry and the in vitro heart bioassay
followedcomparable time courses. Although the isolated
heartbioassay indicated the appearance of CAP2-like activityat an
earlier development time (40%) than the immu-nocytochemical results
(60%), it is apparent that thebiological cardioactivity of this
factor was very low,below 60% DT (Fig. 5). This discrepancy may be
dueto the appearance of another cardioexcitatory sub-stance at
stages before 60 % DT which is not recognizedby the 6C5 antibody.
Alternatively, endogenous CAP2levels before 60% DT may be below the
level ofresolution by our immunocytochemical techniques. It
isclear, however, that the first significant increase
incardioacceleratory activity (60%) precisely correlateswith the
appearance of the first anti-CAP immuno-reactive cells (Figs 3 and
5). Between 60 and 75% ofembryo development, both CAP-like
immunoreactivityand cardioacceleratory activity levels increased
sharply(Figs 3, 4 and 5). Even more compelling, the radicaldecline
in cardioacceleratory activity between 75 % and80% development
(Fig. 5) was paralleled by a similar
drop in immunoreactivity of the lateral cell clustersduring this
developmental span (Fig. 1). Similarly,immunocytochemical (Figs 3
and 4) and biochemicalevidence (Fig. 5) both indicated a gradual
increase inCAP-like activity during the remainder of
embryonicdevelopment, culminating in a second drop in
CAP-likecardioactivity and midline cell immunoreactivity
duringhatching.
As the last line of evidence, the embryonic cardioac-celeratory
activity was demonstrated to be attributableto CAP2 using two
independent tests. In the first, it wasshown that over 80 % of the
embryonic bioactivity wasimmunoprecipitated upon incubation with
the anti-CAP antibody 6C5 (Fig. 6). Additionally, CAP2 wasshown to
specifically block the CAP-like immunocyto-chemical staining
patterns in the Manduca embryo(Fig. II). In the second, the
embryonic cardioaccelera-tory activity was shown to precisely co-
elute with CAP2purified from pharate adult moths using high
pressureliquid chromatogTaphy (Fig. 7). Moreover, all
cardioac-celeratory activity present in the late embryo
stageseluted in a single peak, with no other
cardioexcitatoryfactors detected in our tissue extracts at any
embryonicstage (Fig. 7B).
The above data, taken together, strongly support thenotion that
the observed biological activities are due toCAP2. Furthermore, the
close correlation of the embry-onic CAP2 activity profile and the
observed immuno-cytochemical staining patterns, as well as the
absence ofany other cardioexcitatory factors in the tissue
extracts,strongly argues that the CAP-like immunoreactivity inthe
identified neurosecretory cells is directly attribu-table to the
expression of CAP2.
What is the physiological role(s) of CAP2 in theManduca
embryo?Biochemical, pharmacological, and immunohisto-chemical
evidence all argue that a large drop in thestorage levels of CAP2
occurs twice during embryodevelopment: between 75% and 80%
development,and during hatching (Figs 1, 3, 4 and 5). These
findingsimply that CAP2 may play an important physiologicalrole(s)
during the course of embryogenesis. In anattempt to define the
role(s) of CAP2 in the embryo,experiments were initiated to
identify possible targettissues that CAP2 may modulate during
embryo devel-opment. Our results demonstrate that the
contractileactivity of the embryonic heart and gut musculature
ispharmacologically modulated by exogenous applicationof several
myoactive factors (Table 2). In particular,HPLC-purified CAP2, when
applied to the in vitroembryo, accelerated contractile rates of
both the heartand hindgut (Figs 8 and 9).
Defining the precise physiological role of a neuro-chemical in
vivo is always a difficult task. In thisinstance, given the scale
of the Manduca embryo,direct, unequivocal identification of a
physiological rolefor this peptide during embryogenesis may prove
unfea-sible. Nevertheless, several lines of indirect evidencelead
us to propose a possible role for CAP2 during itsapparent release
between 75 % and 80 % of embryo
-
70 K. S, Broadie and others
development. First, our work on Manduca develop-ment
demonstrated that the hindgut initially becomesmyogenically active
at 70% DT (Broadie et al. 1989a),and that the rate of gut
contraction increases radically(3-4 fold) between 70% and 80% DT
(Fig. 10A).Second, ingestion of the extra-embryonic yolk com-mences
approximately at 75 % DT (Broadie, unpub-lished observations). At
this stage, the gut is empty ofyolk and, by 85 % DT, ingestion of
the extra-embryonicyolk is complete, resulting in a yolk-filled gut
(Broadie,unpublished observations). Third, the embryonic gut isvery
sensitive to CAP2 (Fig. 8), which, at physiologicalconcentrations,
accelerates contraction frequency in afashion similar to the
endogenous modulation of the gutobserved between 70% and 80% DT in
vivo(Fig. 10A). Fourth, the pharmacological sensitivity ofthe
embryonic gut to exogenously applied CAP2 in-creases markedly
between 70% and 80% DT(Fig. 10B). It is particularly striking that
the inactive gutat stages before 70 % DT is not responsive to
exogenousapplication of the peptide and only becomes sensitive
toCAP2 immediately prior to the acceleration of gutcontractions
seen at 75% DT (Fig. 10). Fifth, ourbiochemical studies of CAP2
levels in the embryoindicated a sharp drop in stored CAP2 levels
between75 % and 80 % development (Fig. 5). Lastly, our
immu-nocytochemical studies (Figs 1, 3 and 4), showed amarked
decrease in the CAP2-like immunoreactivity ofidentified lateral
neurosecretory cells in the posteriorabdominal VNC between 75 % and
80% development,confirming our biochemical results. Interestingly,
alarge proportion of this CAP2-like immunoreactivitywas localized
in the terminal abdominal ganglia, whichother studies have
indicated is the primary control siteof the digestive hindgut
(Raabe, 1982). Taken together,these data provide strong
circumstantial evidence thatCAP2 may be released from identified
lateral neurosec-retory cells in the abdominal VNC between 75 %
and80% of embryonic development, facilitating ingestionof the
extra-embryonic yolk by directly accelerating thefrequency of
hindgut contractions.
The role of CAP2 secretion during hatching behavioris less
clear. Our biochemical studies have shown that asimilar, if
somewhat smaller, decline in CAP2 levels inthe embryo occurs at
hatching (Fig. 5). Interestingly,the drop in CAP-like
immunoreactivity at hatchingoccurs exclusively in the large medial
neurosecretorycells of the abdominal VNC as opposed to the
decreasein lateral cell immunoreactivity observed at 75 % DT(Figs
1, 3 and 4). This raises the tantalizing possibilitythat the two
cell types may be independently regulated,playing distinctive roles
at different stages in the Man-duca embryo. CAP2 secretion at
hatching can behypothesized to increase heart contraction
providingthe necessary hydrostatic force to aid hatching and/or
itmay increase gut contraction to facilitate the embryo'sability to
eat its way clear of the egg shell and ingest partof the shell
immediately after emergence. However, it isobvious that additional
work must be done to betterunderstand the exact physiological role
of CAP2 duringeither embryonic period.
We would like to thank Dr J. C. Weeks for her commentson earlier
versions of this manuscript and Dr B. Trimmer forhis gift of
peptide F used in this study. We also thank Mr T.Schilling for his
input and advice during the preliminary stagesof the
immunocytochemical work. This work was supportedby NIH grant no. NS
24613, a NIH Research DevelopmentAward no. NS 01258, and a Sloan
Fellowship to N.J.T.
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(Accepted 29 September 1989)