|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 14 November 2007
doi: 10.1242/dev.004697
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 School of Biological Sciences, University of Reading, Reading RG6 6AJ,
UK.
2 Royal Veterinary College, London NW1 0TU, UK.
3 Institute of Anatomy I, University Erlangen-Nuremberg, Krankenhausstrasse 9,
91054, Germany.
4 Pediatrics I, Children's Hospital, Robert-Koch-Strasse 40, 37075 Goettingen,
Germany.
5 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK.
* Author for correspondence (e-mail: valasekpetr{at}hotmail.com)
Accepted 20 September 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Lymph, Heart, Avian, Embryo, Oedema, Disease, Skeletal muscle, ACh receptor, Immobility, crooked neck, Tailless, Rumpless, Araucana
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). In the
chick, the lymph hearts are functional only in ovo, returning the lymph from
the extraembryonic membranes (Wilting et
al., 1999), and after hatching they partially degenerate
(Bischof and Budras, 1993). In
some birds, e.g. duck and emu, the lymph assists copulatory organ erection of
male adults and lymph hearts function postnatally to return lymph from the
lymphatic erectile phallus to the venous system
(Budras and Berens von Rautenfeld,
1984). Unlike birds, amphibians have several pairs of lymph hearts
along the vertebral column which remain functional into adult life. Mammals do
not have lymph hearts as their extra-embryonic membranes and placenta are
drained by uterine circulation. Propulsion of lymph in adult mammals is
achieved by smooth muscle in lymph collectors, contraction of adjacent
skeletal muscles and through the action of respiratory pressure changes
(Jeltsch et al., 2003).
The avian lymph heart contains endothelial, smooth and striated muscle
layers. Its striated musculature has ultra-structural features of both cardiac
and smooth muscle so a separate histological category has been proposed (e.g.
Budras et al., 1987).
Controversy remains regarding its origin with some researchers suggesting it
develops either from local mesenchyme or from myotomes
(Waldeyer, 1864;
Schipp and Flindt, 1968), or
is derived from the embryonic venous system
(Fedorowicz, 1913;
Jolly and Lieure, 1934). A
general agreement is that the lymph heart is mesodermal in origin like all
other musculature.
Striated skeletal musculature of the body originates from the somites
(Christ and Ordahl, 1995)
which give rise to the epaxial muscles that remain locally in the back and to
the hypaxial muscles which shift to a more ventral position and form the
ventral trunk and limb musculature. Cardiac muscle develops from specialised
splanchnic mesoderm (Brand,
2003). Smooth muscle develops locally from mesodermal and
mesectodermal cells (Le Douarin,
1982; Wilting et al.,
1995).
We re-investigated the composition and development of the lymph heart and for the first time experimentally evaluated its function. We found that it is initially made exclusively of striated skeletal muscle with no smooth musculature. Using the chick-quail lineage tracing, we have shown that cells of the lymph heart originate from somites 34-41. Furthermore we show that the lymph heart has unique properties regarding its innervation profile and its response to neuromuscular blockers. Finally, we demonstrate that simple mechanical obstruction of the lymph heart or complete surgical ablation of the tissue or its genetic absence all resulted in gross oedema formation.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) with
the overlying ectoderm (to facilitate graft orientation) was removed from
quail donors (stage HH17-18) (Hamburger
and Hamilton, 1951) and used for non-orthotopic
transplantations.
Chick recipient embryos were manipulated in ovo at HH17-21. After windowing
the shell the embryo was floated with PBS and antibiotics
(Hara, 1971) and reflective
aluminium foil was temporarily placed under the tail for better accessibility.
Somite I and its surface ectoderm were removed with tungsten needles and
microcapillary suction. Viral marking of interlimb somites was carried out at
this stage with retrovirus expressing heat-resistant placental alkaline
phosphatase, facilitating future exact localisation of operation site
(Valasek et al., 2005). The
quail somite was transferred with a thin glass pipette and positioned by glass
needles. Albumin (2 ml) was removed, the window sealed using surgical tape and
embryos re-incubated for 6-7 days until HH35-36.
In the case of dermomyotomal transplants, only dorsomedial or ventrolateral thirds of the dermomyotome with ectoderm (somites V-X) were substituted at the position of somite 37.
Tissue fixation, processing and histology
Embryos at embryonic day (E) 10 and E18 were decapitated and cut
transversally below the thorax. The caudal part of the embryo was fixed in
Serra's fixative (Serra,
1946), dehydrated in ethanol, CNP30 and embedded in Paraplast.
Serial 10 µm transverse sections were deparaffinised and processed for
immunohistochemistry. Pre-incubation in 10% heat-inactivated goat serum in PBS
was followed by primary antibody incubation (overnight at 4°C). PBS washes
included a 10 minute wash with H2O2 (0.3% v/v in PBS)
for endogenous peroxidase inactivation. Secondary rabbit anti-mouse
biotinylated antibody (DAKO E0354; 1:200) was applied for 1 hour followed by
ABC signal amplification and DAB colour substrates (Vector). Alternatively,
goat anti-mouse-AP (DAKO D0486; 1:1000) with alkaline phosphatase colour
substrates (Roche) were used. Sections were counterstained with Eosin or
Alcian Blue (0.5% w/v in 0.5% v/v glacial acetic acid in water), before
dehydration and mounting with DPX (VWR, BDH 360292F, UK).
The following primary antibody dilutions were used: MyHC (anti-myosin heavy
chain A4.1025; DSHB; supernatant 1:4, biotinylated 1:1000; required citrate
antigen retrieval), SMA (anti-smooth muscle alpha actin; Sigma, clone 1A4,
1:5000), QCPN (anti-quail nuclei; DSHB; supernatant 1:1), QH1 [anti-quail
endothelia (Pardanaud et al.,
1987); DSHB; supernatant 1:10], 3A10 (anti-neurofilament; DSHB;
supernatant 1:50).
Whole-mount in situ hybridisation
For good penetration of probes, embryos E7 and older were skinned during
methanol dehydration. In situ hybridisation
(Nieto et al., 1996) was
performed with the following digoxigenin-labelled probes: Myf5 (1.1
kb; a gift from Dr Anthony Graham, King's College London, UK) MyoD
(1518 bp probe; Dr Bruce M. Paterson, NIH, Bethesda, MD), Pax3
(645bp; Dr Martyn D. Goulding, Salk Institute, San Diego, CA) and
Pax7 (582bp; Dr Susanne Dietrich, King's College London, UK).
Innervation studies
E10, 15 and 18 chick embryonic pelves were fixed in 4% PFA-PBS for 2-4
hours, followed by washes in PBS and cryoembedding. 20-µm-thick cryostat
sections were mounted on poly-L-lysine-coated slides and air-dried (1
hour).
For immunocytochemistry, sections were preincubated with 1% BSA, 5% normal donkey serum, and 0.5% Triton X-100 (for 1 hour). After rinsing in Tris-buffered saline (TBS; pH 7.4) mouse anti-neurofilament (Chemicon; mAb1592, 1:4000), mouse anti-tyrosin hydroxylase (Chemicon; mAb318, 1:2000) or rabbit anti-CGRP (Peninsula; T-4032, 1:5000) antibodies were applied overnight in a humid chamber at room temperature. Following three washes with TBS, donkey anti-rabbit Alexa Fluor 488 and/or donkey anti-mouse Alexa Fluor 555 (Molecular Probes, A21206 and A31570, 1:1000) were applied for 1 hour.
Some sections were additionally incubated with Alexa-Fluor-594-labelled alpha-bungarotoxin (Molecular Probes B-13423, 1:1000) diluted in TBS. The sections were rinsed, mounted in TBS-glycerol (1:1; pH 8.6) and a coverslip added.
For NADPH-diaphorase activity, sections were washed in 0.1 M phosphate buffer pH 7.4 and then incubated with 0.3% Triton X-100, 0.2 mg/ml nitrobluetetrazoliumchloride (NBT) and 1.0 mg/ml beta-NADPH (1-2 hours in a humid chamber at 37°C).
For the demonstration of AChE in motor endplates we used
acetylthiocholiniodide solutions for 30-105 minutes as described by Karnovsky
and Roots (Karnovsky and Roots,
1964) with modifications by Gruber and Zenker
(Gruber and Zenker, 1978).
Iso-OMPA (tetraisopropylpyrophosphoramide, 0.1 mM; Sigma T-1505) was used as
an inhibitor of non-specific cholinesterases.
Whole-mount immunofluorescence detection
Whole embryos were processed as for in situ hybridisation, followed by
pre-incubation with 10% goat serum in PBS-0.5% Triton X-100 (for 24 hours) and
incubation with primary antibody (MyHC-biotin or 3A10) for 24 hours. Several
washes in PBS-0.5% Triton X-100 were followed by overnight incubation with
Avidin-Cy3 (Amersham, 1:200) or secondary goat-anti-mouse-Alexa Fluor 488
(Invitrogen Molecular Probes, 1:200). Extensive washes were followed by
confocal imaging.
Electron microscopy
Chick embryonic pelves at E10, 15, 19 were skinned, fixed [4%
glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 hours], and washed in
0.2 M cacodylate buffer (30 minutes). Lymph hearts were dissected with
adjacent tissue and fixed for an additional 1 hour in 1% osmium tetroxide in
0.1 M cacodylate buffer. Dehydration in graded ethanol and infiltration with
propylene oxide was followed by embedding in Epon resin. Ultrathin (70 nm)
sections were cut on a Leica Ultracut, stained with uranyl acetate, contrasted
with lead citrate and viewed on a JEOL JEM-1011 microscope.
Mechanical obstruction of lymph hearts
Chick E9 embryos were exposed in ovo, the amnion cavity opened and lymph
hearts were injected, using a microcapillary, with freshly prepared Mercox
blue resin (Norwald, Hamburg, Germany) before polymerisation. Eggs were
re-sealed and incubated for 24-28 hours.
Chick tailless/rumpless models
Tail ablations were performed by removing the tailbud at HH20-22.
Unilateral tail ablations were carried out at HH22-25. Natural rumpless
embryos were from Araucana club
(www.araucana.org.uk).
E10-HH36 embryos were photographed for presence of oedema and processed for
MyoD in situ hybridisation.
Pharmacological immobilisation
Decamethonium bromide (Sigma D-1260) 0.2% (w/v) in PBS with antibiotics was
applied to the chorioallantoic membrane in 200 µl doses on E6 and repeated
on E8 through a windowed eggshell. Pancuronium bromide (Sigma P-1918, 100
µl of 8 mg/ml in PBS) was applied on E8 or E9. Embryos were examined on
E10.
Photography
Whole embryos were photographed on a Nikon SMZ1500 stereomicroscope with a
Nikon Coolpix digital camera, and sections were photographed on a Nikon
Eclipse 400. A Leica SP2 confocal microscope was used for
Fig. 1E and
Fig. 3A. Image processing was
performed with Adobe Photoshop 5.0LE.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Musculature of the lymph heart is striated skeletal type
To determine the composition of the developing lymph heart we examined its
make-up from E10 to E18. We found that at E10 it is composed almost
exclusively of striated skeletal muscle cells (myosin heavy chain antibody
positive in Fig. 1E,F,H).
Smooth muscle protein (smooth muscle actin antibody positive) was distinctly
detected only at E18 (Fig. 1I).
Skeletal muscle fibres were thin and relatively sparse with frequent branching
creating a mesh appearance (Fig.
1E). The over-bridging trabeculae also contained striated
musculature (not shown). Similarly, when we examined the expression of muscle
determining factors MyoD (Fig.
1B-D) and Pax3, Pax7, Myf5 (not shown) during earlier
development (HH18-36) we observed a clear distinction from both smooth and
cardiac musculature (Table 1).
However, at E18, a distinct layer of smooth muscle had developed in the
subendothelial layer, well separated from the striated muscle
(Fig. 1I,L) by loose connective
tissue.
|
|
) were also detected (data not shown).
We failed to find any neuromuscular junctions at this stage. These observations suggest that the musculature of the chick lymph heart has striated skeletal muscle characteristics alone during the first half of in ovo development. Smooth muscle subsequently develops during the second half of in ovo development.
The lymph heart originates from somites 34-41
We next determined the origin of the lymph heart. The developing lymph
hearts are composed of skeletal muscle and as all skeletal muscle of the
vertebrate body develops from somites
(Christ and Ordahl, 1995), we
postulated somitic origin of this musculature. Therefore, we determined its
segmental origin using the chick-quail chimeras.
Grafting experiments showed that the striated muscle of the lymph heart originated from somites 34-41 (Table 2). Neighbouring somites (33 and 42) never gave rise to this musculature despite forming adjacent local tissues. From somite 38 caudally, the proportionate contribution gradually decreased, with somite 41 contributing only a few myogenic cells to the caudal end of the lymph heart. This was consistent with the pattern observed during the early differentiation of the myogenic cells directly from the somites/dermomyotomes (Fig. 1B,C). Furthermore the transplanted quail somitic cells formed all the other cell types of the lymph heart, i.e. the connective tissue (Fig. 2B) and the endothelial cells (Fig. 2C).
|
The lymph heart is a hypaxial structure
Having established that the lymph heart is somitic in origin we next
determined which specific part of the somite contributes to the lymph
heart.
We substituted the dorsal portion of the newly formed somite I (at the
position of somite 37) of the chick host with corresponding tissue of quail
origin (Fig. 2A). These
experiments showed that quail cells contributed to the lymph heart, same as
following whole somite grafts (n=10; data not shown). The dorsal
portion of somite I forms the complete dermomyotome
(Christ and Ordahl, 1995).
In order to define which portion of the dermomyotome contributes to the
development of the lymph heart we next substituted the dorsomedial or
ventrolateral thirds (Huang and Christ,
2000) of the dermomyotome of somite 37 at older somite stages
(V-X) with quail dermomyotomal tissue (Fig.
2D,F). These experiments showed that the ventrolateral third of
the dermomyotome gives rise to the lymph heart and hypaxial tissues, whereas
epaxial m. levator caudae remained unpopulated by quail cells
(n=4, Fig. 2G). We did
not observe any decrease in the quantity of quail cells in the lymph heart
(not shown) compared to whole somite substitutions. The dorsomedial third
(n=4) gave rise only to the epaxial structures and there were no
quail cells in the lymph heart (Fig.
2E). These experiments show that the ventrolateral lip is the
source of somitic cells that form the lymph heart and thus lymph heart belongs
to the hypaxial territory.
Innervation of the lymph heart
To complement the developmental and morphological data concerning the lymph
heart musculature with more functional aspects, we investigated the
innervation of this organ.
Whole-mount neurofilament immunostaining revealed five to six segmental
branches in the vicinity of the lymph heart at E10
(Fig. 3A). These fibres
originate from a plexus which innervates local skin, dermis and adjacent
muscles. It is supplied by ventral rami of the spinal nerves 29-33 (LS9-Co1,
corresponding to somites 35-39), confirming that the innervation usually
reflects the segmental origin (Kida,
1997).
As the lymph heart musculature is initially only of striated skeletal muscle type, we first investigated the distribution of classical neuromuscular junctions. Fluorescently labelled alpha-bungarotoxin detected nicotinic acetylcholine receptors of the endplates in both lymph heart and body muscle fibres at E10 in a scattered pattern (Fig. 3B,C). At this stage intimate contacts with nerve fibres were scarce in both tissues (Fig. 3B,C). However, by E18 the endplates of the body skeletal muscle became more organised and formed close contacts with nerve fibres (Fig. 3E). By contrast, the lymph musculature became devoid of nerve fibres and bungarotoxin labelling remained scattered (Fig. 3D). These immunohistological findings are in agreement with our electron microscopy data that detected the presence of neuromuscular junctions in the lymph heart only at E10 (Fig. 1K).
Specific acetylcholinesterase (AChE) is classically restricted to the neuromuscular junctions, whereas non-specific activity can be detected throughout the muscle fibres. The lymph heart musculature exhibited only diffuse activity with no reaction product on the alpha-bungarotoxin positive endplates (Fig. 3F). Furthermore this activity was non-specific as it was abolished by the use of iso-OMPA - an inhibitor of pseudocholinesterases (data not shown).
The lymph heart at later stages also develops a layer containing smooth
muscle protein, so we examined autonomic nervous system innervation. In the
stages examined (E10-19) we could not detect any significant signal for either
NADPH-diaphorase - a marker for parasympathetic efferents
(Grozdanovic et al., 1992) or
tyrosine hydroxylase - sympathetic efferents
(Yurkewicz et al., 1981) in
the wall of the lymph heart (Fig.
3H,J). These markers were robustly expressed in other parts
(Fig. 3I,K) of the embryo at
all stages.
Lastly we examined the distribution of sensory axons in the lymph heart
using immunoreactivity against calcitonin gene-related peptide (CGRP)
(Schrodl et al., 2001). The
wall of the lymph heart was devoid of CGRP
(Fig. 3J) in contrast to the
intestinal wall (Fig. 3K).
To summarise, we detected only somatomotor innervation of the lymph heart. This was, however, atypical, as not only the axons disappeared during the later development, but the ACh receptors were devoid of AChE activity at all stages.
Embryonic oedema is caused by dysfunction of the lymph heart
The knowledge that the lymph heart is of somitic origin and that it is
composed of striated skeletal muscle with ACh receptors allowed us to examine
its role during development using surgical procedures and pharmacological
reagents.
|
A naturally occurring autosomal dominant chick mutant Araucana rumpless provided another means to examine the consequence of lymph heart absence during embryonic development. This mutant is characterised by ear-tufts of feathers and absence of the tail (parson's nose). The hatch rate of fertilised eggs is approximately 75%. Upon examination of the unhatched eggs they usually contained very oedematous dead embryos. We examined the development of Araucana rumpless and found that the complete tailless phenotype had developed in some embryos by E10 and that although they were alive they all displayed gross oedema (Fig. 4D). Their lymph hearts were either absent or only minimally developed. Remaining embryos displayed varying degrees of shortened tail with lymph hearts being partially developed, accompanied by very mild or no oedema.
These experiments show that either the surgical ablation of the tail or its absence due to a genetic mutation result in oedema.
Obstruction of the lymph heart
Absence of the whole tail does cause absence of the lymph heart, but it
also causes defects of other structures of the tail including lymph and blood
vessels. In order to examine the role of the lymph heart pump itself without
affecting the other structures, we obstructed the lumen of the lymph heart by
the injection of fast hardening Mercox resin, which solidifies in less than 2
minutes, into both lymph hearts. This resulted in a gross oedema formation
after 1 day (Fig. 4E) in all of
the embryos that survived this procedure (n=4/12).
Paralysis of the lymph heart
The fact that the lymph heart contains only skeletal muscle by E10 enabled
us to examine the effects of absence of its mechanical contractions by
neuromuscular junction blockers.
Firstly we investigated the effect of applying a depolarising competitive
blocker with a long half-life. Application of Decamethonium bromide resulted
in complete paralysis (apart from the amnionic smooth muscle contractions) and
there was no reaction of the embryonic body to mechanical stimuli at any
point. We observed mild oedema formation and death within 2-3 hours when
applied at E10, whereas earlier application at E6 allowed further development,
despite repeated doses on alternate days for sustained paralysis. These
embryos displayed gross oedema from E8 and by E10 this was severe
(Fig. 4F) and accentuated by
degenerated oedematous skeletal muscles
(Macharia et al., 2004). The
character of the oedema was otherwise similar to the models above.
|
From this surprising difference in sensitivity of the lymph heart and body musculature to the neuromuscular blockers we deduce that the oedema of the immobile embryos is caused mainly by the absence of lymph heart contractions.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
The lymph heart musculature revisited
The lymph heart musculature has peculiar properties suggesting it cannot be
ascribed to the classical groups - smooth, skeletal striated or cardiac
striated muscle (Budras et al.,
1987). Despite some species-specific differences, its histological
structure is similar to the striated skeletal muscle fibres (multinucleated,
with peripherally located nuclei) but thinner in diameter. Some fibres branch,
although they do not anastomose like cardiomyocytes
(Rumyantsev and Krylova,
1990). Also present are light-coloured cells with few filaments
that resemble conductile bundles of His, found in cardiac tissue
(Budras et al., 1987).
Furthermore, complete automaticity has been described after denervation in
amphibians (Rumyantsev and Krylova,
1990).
Our data show that the lymph heart musculature in chick is exclusively a
striated skeletal muscle in the first half of in ovo development. Later a
separate layer of myofibroblasts develops around the endothelium. This may
reflect the gradual involution of the outer striated muscle and a change of
the heart towards a lymphatic collector. Lymphatic collectors possess a wall
of smooth muscle (Baumel et al.,
1993) and contract spontaneously 8-10 times/minute
(Witte et al., 2006), thereby
taking over the function of the lymph heart. Nevertheless some striated muscle
cells have been reported in the lymph heart even in 7-month-old adult chicken
(Bischof and Budras, 1993).
|
) and thus the suggestion of parasympathetic innervation is
relevant (Budras et al.,
1987). The autonomic innervation of the smooth muscle layer of the
lymphatic collectors (Witte et al.,
2006) may perhaps develop only postnatally.
The patterning and development of the lymph heart
The early development of the lymph heart is rather intriguing. All three
cell types forming the lymph heart (endothelial, muscle and connective tissue
cells) originate from the somites. Lymphendothelial cells can be clearly
demonstrated at E5.5 by their Prox1 expression
(Wilting et al., 2006), i.e.
at the same time as the myogenic cells
(Fig. 1B). It is therefore
likely, that these cells leave the somite - or more precisely the hypaxial
dermomyotome - together. A retrovirally marked single cell
(Kardon et al., 2002) in a
differentiated somite (VII) at limb level gave rise to myogenic and
endothelial cells in the limb periphery. These cells were often in close
proximity, suggesting again a common translocation or only later local
proliferation and differentiation.
The process of release of myogenic cells from the dermomyotomes 34-41 is
unusual, as these cells appear as single cells
(Fig. 1B), yet they already
express MyoD. Thus this process cannot be the classical migration of
single myogenic cells as in the limb, where the MyoD differentiation
step occurs only after they reach their final position in the limb bud
(Amthor et al., 1998).
Furthermore, we never detected the expression of scatter factor (SF/HGF) in
this region (data not shown), which would mediate the release of myogenic
cells from the dermomyotome via single cell migration
(Bladt et al., 1995;
Dietrich, 1999). It appears
that the development of the lymph heart striated muscle represents a unique
process of release of single myogenic cells from maturing dermomyotomes.
Lymph heart and embryonic oedema
We show for the first time a causative relationship between the function of
the lymph heart and embryonic oedema. Total absence of the lymph heart or
absence of lymph heart flow, either by obstruction or by its paralysis, leads
to the accumulation of lymph in the subcutaneous tissues. The limited extent
of the oedema below the wings probably reflects the leakage of lymph near the
caudal end and perhaps also the relative looseness of the subcutaneous tissue
in the younger embryos, which allows for pooling of the lymphatic fluid. The
cranial region of the embryo is drained directly to the jugular or subclavian
veins (Baumel et al., 1993)
independently of the lymph hearts. Lymphatic connection between the cranial
and caudal territories established through the development of the thoracic
duct around E10 (Romanoff,
1960) does not seem to affect the extent of oedema.
Rumplessness, oedema and further development
Certain degrees of taillessness/rumplessness occur also spontaneously.
Approximately 1% of chick embryos from our egg supplier display varying degree
of rumplessness at E10 and a few severe cases were also oedematous. Landauer
and Baumann (Landauer and Baumann,
1943) observed similar rates of spontaneous rumplessness and noted
that this can be increased by shaking eggs prior to incubation. Variations in
incubation temperature and application of chemical compounds, including
insulin, have been reported to result in a similar phenotype. Kaplan and
Grabowski (Kaplan and Grabowski,
1967) applied Trypan Blue to E2 chick eggs and observed the
formation of caudal haematoma resulting in rumplessness in 15% of embryos.
They also noted oedema in 12% of the samples but did not comment on any
relationship with the degree of rumplessness. Fulton et al.
(Fulton et al., 1987) observed
oedema in 40% of rumpless autosomal recessive Ottawa naked
chicks.
Araucana rumpless embryos have genetically shorter tails; however, they also have superimposed inter-individual variability of further tail truncations. We speculate that embryos with severe truncations resulting in complete absence of lymph hearts become so oedematous that it prevents their further development and they die in ovo after E10.
Oedema in pharmacologically immobilised embryos
Decamethonium-induced immobility was complete for both the body and the
lymph heart musculature and resulted in gross oedema formation (our
observations) (Macharia et al.,
2004; Hosseini and Hogg,
1991; Sullivan,
1966). By contrast, pancuronium, which also paralysed the body
musculature, left the lymph hearts contracting (although more slowly) and
oedema did not form. It is probable that the lymph heart muscle also has lower
sensitivity to other paralysing agents such as botulotoxin
(Drachman and Sokoloff, 1966)
or D-tubocurare (Murray and Smiles,
1965). These studies used botulotoxin A and tubocurare,
respectively, alongside decamethonium but reported oedema only in the
decamethonium-treated embryos. Another study using curare and succinylcholine
infusion (Oppenheim et al.,
1978) did not mention oedema at all and body weights suggest there
indeed was no apparent oedema.
It is evident that the difference of lymph heart sensitivity to
neuromuscular blockers cannot be explained by an additional autonomic mode of
innervation. The lymph heart does have ACh receptors and is sensitive to
decamethonium blocking. However, the lymph heart does not have abundant
neuromuscular junctions and its ACh receptor clusters do not possess specific
AChE activity, suggesting that activation of contraction may not necessarily
involve classical ACh release from a nerve fibre. It is known that some
AChE-positive endplates do not bind bungarotoxin
(Kallmünzer et al.,
2006), but in the case of the lymph heart we have the opposite
situation, i.e. binding of bungarotoxin without AChE activity on the
endplates. To our knowledge this is a unique situation.
Decamethonium not only binds to the acetylcholine receptor, but also depolarises the membrane, rendering the membrane of a myofibre incapable of excitation by, for example, mechanical stimulation or by an action potential spreading from adjacent cells (with possible pace-maker activity). The other neuromuscular blockers used in chick did not cause oedema and importantly these blockers affect only the ACh transmission [either by release of ACh - botulotoxin A and beta-bungarotoxin, or by mechanically blocking the receptor without depolarising the membrane (pancuronium, D-tubocurare)] without hindering the membrane excitation by direct mechanical stimuli.
We hypothesise that application of decamethonium at E10 caused the quick
death by prolonged depolarisation of fairly well developed muscle fibres,
leading to a gradual release/leak of a substance (e.g. potassium) leading to
cardiac arrest (Rang, 2003).
To avoid these sudden metabolic/mineral changes we started the decamethonium
application at E6, while the muscle fibres are less differentiated.
Decamethonium itself is not known to have a direct effect on cardiac muscle
(Punnen et al., 1984) and
indeed when applied at E6 the blood heart continues to function normally.
Automaticity of the lymph heart
The difference in sensitivity to the immobilising agents is not inherent
and only develops during ontogenesis. Initially the chick lymph heart and the
local tail musculature contract simultaneously. After E8 these two muscle
groups contract independently (Romanoff,
1960). Similarly the rhythmic contractions of the chick lymph
heart cease to be influenced by chloretone anaesthetic at E8, whereas younger
hearts were paralysed like other body musculature
(Romanoff, 1960). The
differences in sensitivity to chloretone as well as pancuronium suggest that
the lymph heart musculature is to a certain extent independent of the
classical neural input. This is further supported by the fact that the lymph
heart has initially somatomotor but no autonomic innervation, and that the
somatomotor innervation diminishes during development. The absence of AChE
activity from the ACh receptor clusters is also puzzling.
However, whether the chick lymph heart possesses an intrinsic pacemaking
system is as yet unclear. Witte et al.
(Witte et al., 2006) found
that 3% of the smooth muscle cells in the tunica media of the contractile
lymph collectors have properties of pacemaker cells. However, the observed
contractions of lymph hearts in our pancuronium model at E10 are unlikely to
be controlled by smooth muscle pacemaker cells, as they have not
differentiated at this stage. Furthermore Berens von Rautenfeld and Budras
(Berens von Rautenfeld and Budras,
1981) and Budras et al.
(Budras et al., 1987) observed
on electron micrographs of the striated lymph heart musculature in some birds
that there are contracting-filament-poor fibres which resemble the conductive
bundle of His of the blood heart. Amphibian denervated lymph hearts show
automatic contractions 4 weeks after surgery
(Rumyantsev and Krylova,
1990).
Taken together these observations suggest that the lymph heart musculature is specialised and has acquired different properties to body skeletal musculature despite its common embryonic origin.
Oedema in genetically immobile embryos - crooked neck
Severe oedema is also present in an immobile naturally occurring autosomal
recessive crooked neck dwarf (cn/cn) chick mutant
(Asmundson, 1945;
Oppenheim et al., 1997). There
is an indirect defect in the ryanodine receptor alpha, which controls the
release of calcium from the sarcoplasmic reticulum following depolarisation of
the sarcolemma (Airey et al.,
1993; Ivanenko et al.,
1995; Oppenheim et al.,
1997). Thus there is no coupling of the nerve impulse and physical
muscle contraction. It has been described that these embryos also have
progressive embryonic muscle degeneration
(Kieny et al., 1983;
Kieny et al., 1988) and suffer
from oedema of the same appearance as our decamethonium-treated embryos.
Embryonic immobility per se does not cause oedema
All the oedemas of immobility were ascribed to the absence of lymph
propulsion or decreased venous return, features dependent on skeletal muscle
contractions (Macharia et al.,
2004). The following considerations enable us to challenge this
notion and ascribe a novel role for the lymph heart.
Firstly, we showed that pancuronium-immobilised embryos, which have no body muscle movement but retain some contractility of the lymph heart, fail to develop gross oedema.
Secondly, the degree of oedema of embryos immobilised by either decamethonium or genetically is very comparable to our models with lymph heart absence or its obstruction (bearing in mind, that the muscle atrophy induced by decamethonium enhances the oedematic appearance).
Lastly, if propulsion of the body lymph was dependent on the skeletal
muscle contractions, we would expect oedema to develop also in mouse embryos
lacking muscle movement. However, the murine mutant of the ryanodine receptor
(RyR-1 null - related to the above chick crooked neck) lacks
skeletal muscle contractions and yet is not oedematous
(Takeshima et al., 1994) or
has variable small oedema in the head and rump region (H. Takeshima, personal
communication).
All these observations suggest that immobilisation on its own is not sufficient to account for an obvious oedema and point to the specific role of the lymph heart.
It is noteworthy that there are also other causes of avian embryonic
oedema, for example `chick oedema disease' caused by organophosphates
(Gilbertson et al., 1991;
Brunstrom, 1988). Their
pathogenesis appears to include activation of cytochromes and effects of
arachidonic acid metabolites (Rifkind et
al., 1990) or direct cardiotoxicity with heart failure
(Walker and Catron, 2000)
whereas a direct role of lymph heart in these scenarios is unlikely.
Conclusion
Our work has resulted in three major findings: (1) lymph heart contains
striated muscle from somites 34-41; (2) lymph heart musculature displays
unique properties including bungarotoxin-binding endplates without AChE
activity; (3) embryonic lymphoedema is prevented by the mechanical action of
the lymph heart and not by mobility of the body musculature.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4427/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Airey, J. A., Baring, M. D., Beck, C. F., Chelliah, Y.,
Deerinck, T. J., Ellisman, M. H., Houenou, L. J., McKemy, D. D., Sutko, J. L.
and Talvenheimo, J. (1993). Failure to make normal alpha
ryanodine receptor is an early event associated with the crooked neck dwarf
(cn) mutation in chicken. Dev. Dyn.
197,169
-188.[Medline]
Amthor, H., Christ, B., Weil, M. and Patel, K.
(1998). The importance of timing differentiation during limb
muscle development. Curr. Biol.
8, 642-652.[CrossRef][Medline]
Asmundson, V. S. (1945). Crooked neck dwarf in
domestic fowl. J. Hered.
36,173
-176.
Baumel, J. J., King, A. S., Breazile, J. E., Evans, H. E. and
Vanden Berge, J. C. (1993). Handbook of Avian
Anatomy: Nomina Anatomica Avium (2nd edn). Cambridge, MA: Nuttall
Ornithological Club.
Berens von Rautenfeld, D. and Budras, K. D.
(1981). TEM and SEM investigations of lymph hearts in birds.
Lymphology 14,186
-190.[Medline]
Bischof, B. and Budras, K. D. (1993). The
topography of the lymph heart in the domestic chicken (Gallus domesticus).
Lymphology 26,177
-185.[Medline]
Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A. and
Birchmeier, C. (1995). Essential role for the c-met receptor
in the migration of myogenic precursor cells into the limb bud.
Nature 376,768
-771.[CrossRef][Medline]
Brand, T. (2003). Heart development: molecular
insights into cardiac specification and early morphogenesis. Dev.
Biol. 258,1
-19.[CrossRef][Medline]
Brunstrom, B. (1988). Sensitivity of embryos
from duck, goose, herring gull, and various chicken breeds to
3,3',4,4'-tetrachlorobiphenyl. Poult. Sci.
67, 52-57.[Medline]
Budras, K. D. and Berens von Rautenfeld, D.
(1984). [Functional and topographic anatomy of the lymph heart
and lymphatic system in the pelvic region of water-fowl and ratite birds].
Anat. Anz. 156,231
-240.[Medline]
Budras, K. D., Hullinger, R. L. and Berens von Rautenfeld,
D. (1987). Lymph heart musculature in birds. J.
Morphol. 191,77
-87.[CrossRef][Medline]
Christ, B. and Ordahl, C. P. (1995). Early
stages of chick somite development. Anat. Embryol.
191,381
-96.[CrossRef][Medline]
Dietrich, S. (1999). Regulation of hypaxial
muscle development. Cell Tissue Res.
296,175
-182.[CrossRef][Medline]
Drachman, D. B. and Sokoloff, L. (1966). The
role of movement in embryonic joint development. Dev.
Biol. 14,401
-420.[Medline]
Fedorowicz, S. (1913). Untersuchungen ueber die
Entwicklung der Lymphgefaesse bei Anurenlarven. Bull. Int. Acad.
Sci. Cracovie 13,290
-297.
Fulton, J. E., Wenger, B. S., Wenger, E. L. and Crawford, R.
D. (1987). Anatomical defects associated with a feathering
mutant (Ottawa naked) in domestic fowl. Teratology
35,137
-145.[Medline]
Gilbertson, M., Kubiak, T., Ludwig, J. and Fox, G.
(1991). Great Lakes embryo mortality, edema, and deformities
syndrome (GLEMEDS) in colonial fish-eating birds: similarity to chick-edema
disease. J. Toxicol. Environ. Health
33,455
-520.[Medline]
Grozdanovic, Z., Baumgarten, H. G. and Bruning, G.
(1992). Histochemistry of NADPH-diaphorase, a marker for neuronal
nitric oxide synthase, in the peripheral autonomic nervous system of the
mouse. Neuroscience 48,225
-235.[CrossRef][Medline]
Gruber, H. and Zenker, W. (1978).
Acetylcholinesterase activity in motor nerve fibres in correlation to muscle
fibre types in rat. Brain Res.
141,325
-334.[CrossRef][Medline]
Hamburger, V. and Hamilton, H. L. (1951). A
series of normal stages in the development of the chick embryo. J.
Morphol. 88,49
-92.[CrossRef]
Hara, K. (1971). Micro-surgical operation on
the chick embryo in ovo without vital staining. A modification of the
intra-coelomic grafting technique. Mikroskopie
27, 267-70.[Medline]
Hosseini, A. and Hogg, D. A. (1991). The
effects of paralysis on skeletal development in the chick embryo. I. General
effects. J. Anat. 177,159
-168.[Medline]
Huang, R. and Christ, B. (2000). Origin of the
epaxial and hypaxial myotome in avian embryos. Anat.
Embryol. 202,369
-374.[CrossRef][Medline]
Ivanenko, A., McKemy, D. D., Kenyon, J. L., Airey, J. A. and
Sutko, J. L. (1995). Embryonic chicken skeletal muscle cells
fail to develop normal excitation-contraction coupling in the absence of the
alpha ryanodine receptor. Implications for a two-ryanodine receptor system.
J. Biol. Chem. 270,4220
-4223.
Jeltsch, M., Tammela, T., Alitalo, K. and Wilting, J.
(2003). Genesis and pathogenesis of lymphatic vessels.
Cell Tissue Res. 314,69
-84.[CrossRef][Medline]
Jolly, J. and Lieure, C. (1934). Formation des
myofibrilles dans les coeurs lymphatiques des larves d'Anoures. C.
R. Soc. Biol. Paris 115,124
-127.
Kallmünzer, B., Sorensen, B., Neuhuber, W. L. and Worl,
J. (2006). Heterogeneity of neuromuscular junctions in
striated muscle of human esophagus demonstrated by triple staining for the
vesicular acetylcholine transporter, alpha-bungarotoxin, and
acetylcholinesterase. Cell Tissue Res.
324,181
-188.[CrossRef][Medline]
Kaplan, S. and Grabowski, C. T. (1967).
Analysis of trypan blue-induced rumplessness in chick embryos. J.
Exp. Zool. 165,325
-335.[CrossRef][Medline]
Kardon, G., Campbell, J. K. and Tabin, C. J.
(2002). Local extrinsic signals determine muscle and endothelial
cell fate and patterning in the vertebrate limb. Dev.
Cell 3,533
-545.[CrossRef][Medline]
Karnovsky, M. J. and Roots, L. (1964). A
"direct-coloring" thiocholine method for cholinesterases.
J. Histochem. Cytochem.
12,219
-221.[Medline]
Kida, M. Y. (1997). Nerve-muscle specificity.
J. Anat. 190,309
-310.[CrossRef][Medline]
Kieny, M., Mauger, A., Hedayat, I. and Goetinck, P. F.
(1983). Ontogeny of the leg muscle tissue in the crooked neck
dwarf mutant (cn/cn) chick embryo. Arch. Anat. Microsc. Morphol.
Exp. 72,1
-17.[Medline]
Kieny, M., Boutineau, A. M., Pautou, M. P. and Goetinck, P.
F. (1988). Muscular dysgenesis in fowl: ultrastructural study
of skeletal muscles in the crooked neck dwarf (cn/cn) mutant. Biol.
Struct. Morphog. 1,15
-27.[Medline]
Landauer, W. and Baumann, L. (1943).
Rumplessness of chicken embryos produced by mechanical shaking of eggs prior
to incubation. J. Exp. Zool.
93, 51-74.[CrossRef]
Le Douarin, N. M. (1982). The Neural
Crest. London: Cambridge University Press.
Macharia, R., Patel, K., Otto, W. R., McKinnell, I. W. and
Christ, B. (2004). Decamethonium bromide-mediated inhibition
of embryonic muscle development. Anat. Embryol.
208, 75-85.[CrossRef][Medline]
Murray, P. D. F. and Smiles, M. (1965). Factors
in the evocation of adventitious (secondary) cartilage in the chick embryo.
Aust. J. Zool. 13,351
-381.[CrossRef]
Nieto, M. A., Patel, K. and Wilkinson, D. G.
(1996). In situ hybridization analysis of chick embryos in whole
mount and tissue sections. Methods Cell Biol.
51,219
-235.[Medline]
Oppenheim, R. W., Pittman, R., Gray, M. and Maderdrut, J. L.
(1978). Embryonic behavior, hatching and neuromuscular
development in the chick following a transient reduction of spontaneous
motility and sensory input by neuromuscular blocking agents. J.
Comp. Neurol. 179,619
-640.[CrossRef][Medline]
Oppenheim, R. W., Prevette, D., Houenou, L. J., Pincon-Raymond,
M., Dimitriadou, V., Donevan, A., O'Donovan, M., Wenner, P., McKemy, D. D. and
Allen, P. D. (1997). Neuromuscular development in the avian
paralytic mutant crooked neck dwarf (cn/cn): further evidence for the role of
neuromuscular activity in motoneuron survival. J. Comp.
Neurol. 381,353
-372.[CrossRef][Medline]
Pardanaud, L., Altmann, C., Kitos, P., Dieterlen-Lievre, F. and
Buck, C. A. (1987). Vasculogenesis in the early quail
blastodisc as studied with a monoclonal antibody recognizing endothelial
cells. Development 100,339
-349.
Punnen, S., Gonzalez, E. R., Krieger, A. J. and Sapru, H. N.
(1984). Cardiac vagolytic action of some neuromuscular blockers.
Pharmacol. Biochem. Behav.
20, 85-89.[CrossRef][Medline]
Rang, H. P. (2003).
Pharmacology. Edinburgh: Churchill
Livingstone.
Rifkind, A. B., Gannon, M. and Gross, S. S.
(1990). Arachidonic acid metabolism by dioxin-induced cytochrome
P-450: a new hypothesis on the role of P-450 in dioxin toxicity.
Biochem. Biophys. Res. Commun.
172,1180
-1188.[CrossRef][Medline]
Romanoff, A. L. (1960). The Avian
Embryo. New York: The Macmillan Company.
Rumyantsev, P. P. and Krylova, M. I. (1990).
Ultrastructure of myofibers and cells synthesizing DNA in the developing and
regenerating lymph-heart muscles. Int. Rev. Cytol.
120, 1-52.[Medline]
Schipp, R. and Flindt, R. (1968). [Fine
structure and innervation of the musculature of lymph hearts in amphibia (Rana
temporaria)]. Z. Anat. Entwicklungsgesch.
127,232
-253.[CrossRef][Medline]
Schrodl, F., Schweigert, M., Brehmer, A. and Neuhuber, W. L.
(2001). Intrinsic neurons in the duck choroid are contacted by
CGRP-immunoreactive nerve fibres: evidence for a local pre-central reflex arc
in the eye. Exp. Eye Res.
72,137
-146.[CrossRef][Medline]
Serra, J. A. (1946). Histochemical tests for
protein and aminoacids: the characterization of basic proteins.
Stain Technol. 21,5
-18.
Sullivan, G. E. (1966). Prolonged paralysis of
the chick embryo, with special reference to effects on the vertebrate column.
Aust. J. Zool. 14,1
-17.[Medline]
Takeshima, H., Iino, M., Takekura, H., Nishi, M., Kuno, J.,
Minowa, O., Takano, H. and Noda, T. (1994).
Excitation-contraction uncoupling and muscular degeneration in mice lacking
functional skeletal muscle ryanodine-receptor gene.
Nature 369,556
-559.[CrossRef][Medline]
Valasek, P., Evans, D. J., Maina, F., Grim, M. and Patel, K.
(2005). A dual fate of the hindlimb muscle mass: cloacal/perineal
musculature develops from leg muscle cells.
Development 132,447
-458.
Waldeyer, W. (1864). Anatomische und
physiologische Untersuchungen uber die Lymphherzen der Froesche. Z.
Ration. Med. Leipzig Heidelberg 21,105
-124.
Walker, M. K. and Catron, T. F. (2000).
Characterization of cardiotoxicity induced by 2,3,7,
8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo
development. Toxicol. Appl. Pharmacol.
167,210
-221.[CrossRef][Medline]
Wilting, J., Brand-Saberi, B., Kurz, H. and Christ, B.
(1995). Development of the embryonic vascular system.
Cell. Mol. Biol. Res.
41,219
-232.[Medline]
Wilting, J., Neeff, H. and Christ, B. (1999).
Embryonic lymphangiogenesis. Cell Tissue Res.
297, 1-11.[CrossRef][Medline]
Wilting, J., Aref, Y., Huang, R., Tomarev, S. I., Schweigerer,
L., Christ, B., Valasek, P. and Papoutsi, M. (2006). Dual
origin of avian lymphatics. Dev. Biol.
292,165
-173.[CrossRef][Medline]
Witte, M. H., Jones, K., Wilting, J., Dictor, M., Selg, M.,
McHale, N., Gershenwald, J. E. and Jackson, D. G. (2006).
Structure function relationships in the lymphatic system and implications for
cancer biology. Cancer Metastasis Rev.
25,159
-184.[CrossRef][Medline]
Yurkewicz, L., Marchi, M., Lauder, J. M. and Giacobini, E.
(1981). Development and aging of noradrenergic cell bodies and
axon terminals in the chicken. J. Neurosci. Res.
6, 621-641.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
