1 Veterinary Basic Sciences, Royal Veterinary College, London NW1 0TU, UK
2 Anatomy Institute, First Medical Faculty, Charles University, Prague, Czech
Republic
3 Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton BN1
9PX, UK
4 INSERM UMR 623, Developmental Biology Institute of Marseille, Campus de
Luminy, Case 907, 13288 Marseille Cedex 09, France
* Authors for correspondence (e-mail: valasekpetr{at}hotmail.com; kpatel{at}rvc.ac.uk)
Accepted 26 October 2004
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SUMMARY |
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Key words: Striated sphincter muscles, Cloaca, Perineum, Chick/quail chimera, Somite, Segmental origin, limbless, MyoD, Pax3, Pax7, Met, Lbx1, Meox2
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Introduction |
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The degree of evolutionary homology of muscles in birds and mammals has
facilitated the use of the avian model to decipher the origin and formation of
distinct muscles in mammals. In particular, the chick/quail chimeras
(Le Douarin, 1969) have been
used to determine the origin of a plethora of tissues. Furthermore this
technique has been used to determine the axial origin of numerous muscles.
These studies have shown that skeletal muscles of the head originate from the
non-segmented head mesoderm (Noden,
1983
; Wachtler et al.,
1984
), whereas all skeletal muscles of the body originate from the
somites (Christ and Ordahl,
1995
).
Epaxial musculature of the back develops locally from the dorsomedial part
of the somites (Brand-Saberi et al.,
1996a) whereas the lateral aspect of the somites form the hypaxial
musculature that comprises the ventral body wall muscle and those found in the
limbs. Hypaxial muscle forms by two distinct mechanisms: (1) extension of the
myotomal sheet for the body wall; or (2) migration of individual muscle
precursors for limb muscles (Dietrich et
al., 1998
). The migratory mechanism is also deployed for the
formation of the tongue and the diaphragm muscles of mammals
(Dietrich, 1999
). Migration of
muscle precursors is dependent upon Scatter Factor/Hepatocyte Growth Factor
(Heymann et al., 1996
;
Brand-Saberi et al., 1996b
),
which acts on its receptor, Met, which is expressed by migrating myogenic
cells. Genetic deletion of either molecule results in complete absence of
migratory muscles (Bladt et al.,
1995
; Dietrich et al.,
1999
). During migration, the muscle precursors express
transcription factors Pax3 and Pax7, and only initiate the
expression of MyoD having once reached their final location
(Amthor et al., 1998
). By
contrast, myotomal extension for the body wall musculature involves
MyoD-expressing cells from the very beginning.
In this study, we have determined the axial origin of the cloacal skeletal muscles and investigated the mechanism of how these muscles develop both in birds and mammals. Using two independent labelling techniques (chick/quail chimeras and retroviral labelling of somitic cells), we have established the somitic origin of the cloacal striated muscles in chick. Surprisingly, the cloacal muscles derive in large part from the same somites as those that give rise to leg muscles. A study of the early muscle development in chick using a panel of markers, including MyoD, showed that cloacal muscles were derived from the ventral muscle mass of the leg. Homologous perineal muscles in mouse were also found to develop from the ventral muscle mass of the hindlimb. We provide evidence that the perineal muscles are migratory, in the same way as the muscles of the limbs, as they all are absent in the metd/d mutant embryo. Furthermore, we show through the use of surgical procedures and genetic mutants that the development of the cloacal muscles is dependent on the development of the limbs.
The results from this study show that the cloacal muscles develop via novel
mechanism that involves an initial phase of myogenic precursors migration from
the somites into the limb. These precursors then extend out from the limbs
towards the ventral midline and the process is subsequently followed by
differentiation into individual muscles. The knowledge of the early
development of these muscles will allow us to understand the aetiology, or at
least the consequences, of a variety of human conditions that affect the
caudal part of the body. These include imperforate anus
(Tichy et al., 1998),
persistent cloaca, caudal dysgenesis
(Bohring et al., 1999
;
Inomata et al., 1989
) and body
wall closure anomalies (Hartwig et al.,
1991
).
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Materials and methods |
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Each chick recipient embryo was manipulated in ovo at somite stages 29-36
(HH17-19). After windowing the eggshell and the subjacent shell membrane, the
embryo was floated with sterile PBS/antibiotics to facilitate the operation
procedure (Hara, 1971). The
most newly formed somite (I) was removed along with its surface ectoderm using
a tungsten needle. Remaining cells were removed with a microcapillary. Viral
marking of a more cranial interlimb somite was carried out at this stage (see
below). The donor quail somite was transferred to the chick surgery site with
a thin glass Pasteur pipette and positioned using drawn glass needles. The
surface ectoderm allowed orientation of the graft. Albumin (1-2 ml) was
removed, the egg was closed with a surgical tape and re-incubated for 6-7 days
until HH35-36.
Orthotopical transplantations of quail leg bud was carried out at stage HH23 after ablation of the chick leg. The proximal leg field of the chick was left intact. Retention of the transplanted limb was achieved by closure of the amnion. Embryos were reincubated until HH35-36.
Tissue fixation and processing
Embryos at stage HH35-36 were decapitated and cut transversally below the
thorax. The caudal part of the embryo was fixed in Serra's fixative
(Serra, 1946), then dehydrated
in ethanol and processed for paraffin embedding using CNP30 and Fibrowax.
Serial 10 µm transverse sections were mounted on albumen-glycerin coated
slides. After de-paraffinisation, sections were treated in PBS with 0.1%
Tween, rinsed in PBS and pre-incubated in 1% dried skimmed milk powder in PBS.
QCPN anti-quail monoclonal antibody (Developmental Studies Hybridoma Bank,
University of Iowa) was applied in the spin-cleared milk solution over night
at 4°C. Secondary Goat anti-mouse antibody conjugated to alkaline
phosphatase (DAKO) was used 1:1000 in PBS for 1 hour. Blue colour AP
substrates (Roche) in NTMT buffer revealed the presence of quail nuclei in the
host tissue. After eosin counterstaining, sections were dehydrated and mounted
in DPX.
Somite counting and segmental level determination
One or more interlimb somite (20-26) was marked at the time of surgery at a
known distance from the operated somite
(Fig. 1A). This allowed us to
locate exactly at the end of the experimental period which caudal somite was
operated with respect to the reference point. Somites were marked by injecting
a small volume of avian retrovirus coding for heat resistant alkaline
phosphatase RCAS(A)-AP (titre ca. 108IU/ml with 0.1% Fast green)
using a hand held microcapillary. At time of fixation the thoracic region was
fixed in 4%PFA washed in PBS, followed by heat inactivation of endogenous
alkaline phosphatase. The enzymatic colour reaction in the infected
intercostal muscle (Fig. 1B)
was carried out as for in situ whole mounts with AP substrates. Only embryos
with identifiable normal seven ribs were included in the analysis.
|
Whole-mount in situ hybridisation
In situ hybridisation was performed according to Nieto et al.
(Nieto et al., 1996). Briefly,
chick embryos HH17-36 and mouse embryos (C57BL6) E11.5-E15.5 were fixed in
4%PFA/PBS/0.1%Triton, dehydrated in methanol, re-hydrated, treated with
proteinase K and re-fixed. For good quality in situ hybridisation, chicken
HH32 and mouse E14.5 and older embryos were skinned while in methanol at this
stage. Antisense RNA probes of chicken MyoD (a 1518 bp probe, Dr
Bruce Paterson), mouse MyoD (Myod1 - Mouse Genome
Informatics), Meox2 (1833 bp, Dr Baljinder Mankoo), chicken
Pax3 (645 bp, Dr Martin Goulding) and Pax7 (582 bp, Dr
Susanne Dietrich) were labelled with digoxigenin. Fab fragments of sheep
antibody to digoxigenin conjugated to alkaline phosphatase mediated the
visualisation (1:5000, Roche).
Chick legless models
Unilateral legless chicks were produced by ablating the limb anlage
(ranging from lateral somatopleura to a limb bud, HH16-23) either with
tungsten needles or with ophthalmology scissors. On HH34, the embryos were
processed for MyoD whole-mount in situ hybridisation. Spontaneously
amelic chicken embryos limbless were kindly provided by Professor
John Fallon (University of Wisconsin, USA)
(Prahlad et al., 1979;
Fallon et al., 1983
).
Mouse models
To partially rescue placental phenotype of metd/d
mutant embryos (Maina et al.,
1996), heterozygous males on a mixed C57BL/6x129/sv background
were inter-crossed with heterozygous females on outbred strain CD1
(Maina et al., 2001
). Mice
null for p63, Meox2 and Lbx1 were produced by simple
heterozygous breeding programmes. Mouse mutants were embedded in tissue
freezing medium (Leica) by slow freezing in isopentane on liquid nitrogen.
Embryos were sectioned parallel to the axis of rectum. Serial cryosections (16
µm) were re-hydrated in PBS, fixed in 4%PFA/PBS for 3 minutes, rinsed in
PBS and preblocked in 10% heat-inactivated goat serum in PBS. Sections were
incubated for 2 hours with biotinylated mouse monoclonal antibody against
myosin heavy chain (DSHB A41025, 1:1000), which was developed with ABC
streptavidin/peroxidase kit and DAB staining (Vectorlabs). Sections were
counterstained with Haematoxylin and Alcian Blue.
Anatomy and terminology
We use the descriptive terms cranial/caudal rather than anterior/posterior
in order to avoid confusion with their usage in human anatomy
(Baumel et al., 1993). The
normal anatomy of the pelvic region was determined by studying serial 10 µm
paraffin wax embedded sections of ED10 chick and 16 µm serial cryosections
of mouse embryos. Sections were stained by a myosin heavy chain monoclonal
antibody. Photographs of serial sections were imported into Microsoft
Powerpoint, which enabled movie-like scanning and identification of single
muscles. MyoD in situ hybridisation wholemounts were used for gaining
a three-dimensional concept. Photographs were taken on a stereomicroscope with
a digital camera (Nikon). Images for Fig.
2D and Fig. 4B were
taken on Olympus SZX-12. Photos of sections were taken on Nikon Eclipse 400.
Image processing was carried out using Adobe Photoshop 5.0LE.
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Results |
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We next identified the homologous perineal muscles in mouse using sectioning and whole-mount in situ hybridisation with MyoD probe. The single muscles are recognisable from E15.5 (Fig. 1E,F). Similar to the chick, we could discern three groups of muscles in the pelvic outlet.
Cloacal muscles develop predominantly from hindlimb somites
To identify the somites from which the cloacal muscles arise, we first used
chick/quail somitic chimera approach. Among the 116 chimeras, 76 survived
until stage HH36, and 71 had unequivocal reference intercostal AP marking.
Fifty-four chimeras had quail tissue, and 30 had hypaxial musculature derived
from the quail tissue. The heterotopic transplants of quail somites I-V were
adequate for fate mapping, as we found no differences between their muscle
derivatives. Using this approach, we found that somites 30-34 were the source
of myogenic cells of the cloacal sphincter muscle complex (asterisks in
Table 1). Remarkably, this
contribution domain was extremely precise and restricted, as both adjacent
somites 29 and 35 did not contribute to the formation of these muscles. Each
of the four cloacal muscles had a contribution from at least three somites.
However, the four cloacal muscles were not supplied in the same proportion by
the five somites. Fig. 2A,B
show an example of contribution of quail-derived cells from somite 31 to the
formation of cloacal and leg muscles. In addition, as previously described
(Lance-Jones, 1988;
Rees et al., 2003
), we found
that leg muscles originate from somites 26-33
(Fig. 2B; Table 1). Interestingly, these
transplantation experiments revealed that whenever a cloacal muscle developed
that contained quail cells from somites 30-33, there were also quail cells
integrated within the leg muscles (see Fig.
2B). This was not the case for somite 34, which gave rise to
cloacal muscle cells but not to cells within leg muscles.
|
Taken together with the leg muscles somitic origin from somites 26-33 and the fact that somites 30-33 gave rise to both groups of muscles, we suggest that cloacal muscles develop together with leg muscles.
Cloacal muscle precursors migrate from the somites into the developing leg bud
Having established that individual somites provide cellular contribution
for both leg and cloacal muscles, we next focused on how these two different
muscle regions recruit their myogenic content. Migration of single muscle
precursors from the somite would be characterised by Pax3 and
Pax7 expression, without MyoD. However, myotomal extension
presents as an elongation of a process continuous with the myotome. It
expresses not only the Pax genes but also MyoD
(Amthor et al., 1998).
In the first instance, we studied the expression of Pax3 at the
levels of somites 30-34. We detected cells leaving these somites between
HH17-20, which subsequently populated the hindlimb bud as we previously
described (Amthor et al.,
1998). We were unable to detect migration of cells from the
somites after stage HH20 using in situ hybridisation. To confirm this
molecular observation, we labelled the ventrolateral region of the somites
after HH20 with the lacZ expressing reterovirus but were never able
to detect any cells the limbs (n=5, data not shown). Furthermore, we
were never able to detect a population of migrating cells moving from the
somites towards the developing cloaca using either molecular or cellular
techniques (n>50, data not shown). We then investigated the
development of myotomal extension from the somites of interest using in situ
hybridisation with MyoD (Amthor et
al., 1998
) or virally labelled cells. We were unable to find any
elongation of myotomes from the somites 30-34 towards the developing cloaca
(data not shown; see also Fig.
3A,B).
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Cloacal muscles develop from the ventral muscle mass of the hindlimb
To our surprise, we had only detected one wave of migration from somites
30-34. However these cells had all entered the developing leg bud. However,
our fate-mapping studies had shown that these somites contributed to cloacal
muscles. We therefore followed the myogenic events of the leg at later stages.
The ventral and dorsal muscle masses of the hindlimb begin to express
MyoD at stage HH23. The masses expand within the limb bud until
HH25. At this stage there were still no myogenic cells in the cloacal region
(Fig. 3A). At stage HH27, when
the cleavage of ventral and dorsal masses into muscle groups is initiated
(Schroeter and Tosney, 1991
),
we observed a ventrocaudal elongation of the ventral muscle mass towards the
cloaca (Fig. 3B). The
elongation of the muscle mass continues
(Fig. 3C) until HH31, when the
cloacal anlage reaches the vicinity of cloacal tubercle (arrow in
Fig. 3D) and separates at the
other end from the future flexor cruris lateralis (FCLP) and
caudofemoralis (CFC) muscles (arrowhead in
Fig. 3D). The cloacal anlage at
HH33 extends around the tubercle (Fig.
3E). At HH35, the single muscles of cloaca are discernible
(Fig. 3F). Thus, from a
molecular perspective, cells from the ventral muscle mass exit the developing
limb bud and form the muscles of the cloaca. To investigate this at the
cellular level, we first determined that cloacal muscle precursors do reside
at some point in the leg bud. To this end, we replaced homotopically chick leg
bud with a quail leg bud at stage HH23 when migration from somites is
completed. We replaced only part of leg bud containing muscle masses, leaving
the proximal limb field intact. The resulting HH35 chimera showed all leg and
cloacal muscles to be of quail origin, while all material of pelvic bones was
formed by the chick recipient (Fig.
4A). Having established that cloacal muscle precursors temporarily
reside in the limb, we investigated their distribution within the bud. To this
end, we virally labelled cells in one of four regions of the leg bud
(proximoventral, proximodorsal, mid-length and distal) and then determined
which site(s) contributed to cloacal musculature. Eleven labelling procedures
were performed and we were only able to detect a contribution of the ventral
proximal of the limb bud to the formation of cloacal muscles
(Fig. 4B and
Table 2).
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Cloacal muscle anlage translocation resembles myotomal extension
In order to analyse the mechanism of the cloacal anlage translocation from
the leg bud to the cloaca, we deployed again the comparison of Pax3/7
and MyoD gene expression. The presence of population of cells
expressing only Pax3/7 in front of the MyoD domain would
suggest involvement of precursor migration. On the other hand, co-expression
of the Pax genes and MyoD throughout the translocation would indicate
a mechanism akin to myotomal extension.
Data from the expression of Pax3 was difficult to interpret at these later stages as this gene became also expressed in non myogenic tissues, including the ectoderm of the cloacal cleft (data not shown). We therefore concentrated on Pax7, which remains more specific for the myogenic lineage. We found that Pax7 was expressed by the ventrocaudal elongation (Fig. 3G,H) of the ventral limb muscle mass directed towards the cloaca. When we compared the expression domains of Pax7 (Fig. 3G) with that of MyoD (Fig. 3B) at stage HH27 we found that they were identical. Therefore, we were unable to detect a population of only Pax7-positive cells in more advanced positions compared with the domain expressing MyoD.
In summary, during the initial migration period, cells express Pax7 and Pax3 on route to the limb bud. These cells start to express also MyoD only once they populate the developing limb. This co-expression of the Pax and MyoD genes then persists during the extension of the muscle mass towards the cloaca and thus resembles a myotomal extension.
Mouse perineal muscles develop from the ventral muscle mass of the hindlimb
To establish whether this mechanism of cloacal muscle anlage formation is
conserved also in mammals, we followed the development of the cloacal/perineal
muscles using MyoD expression in mouse embryos between E11.5 and
E15.5. Similar to the chick, although at E11.5 we only observed
MyoD-expressing cells in the ventral muscle mass of hindlimb
(Fig. 5A), at E12.5 the
ventrocaudal edge of the mass began to extend towards the genital tubercle
(Fig. 5B). The direction of
this extension is mainly ventral, without the pronounced caudal direction as
in chick. At E13.0, this extension already reached the base of the tubercle
(Fig. 5C), and further at E13.5
began to split into future single muscles
(Fig. 5D). At later stage
E14.5, it is easy to see how the common cloacal/perineal anlage differentiates
into separate muscle branches around the openings
(Fig. 5E) of the already
septated cloaca. Thus, there is never a common cloacal sphincter, because the
muscles arrive after the septation has occurred.
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Development of the perineal muscles in metd/d, Lbx1 and Meox2 null embryos
It has been shown that the receptor Met is expressed in all migratory
myoblast precursors and is required for myoblast migration to colonise limbs,
diaphragm and tongue (Bladt et al.,
1995). As we found the intimate relationship between the
development of cloacal/perineal and leg muscles, we expected the perineal
muscles to be absent in metd/d mutant embryos. Serial
transverse cryosections of E15.0 homozygous metd/d embryos
(Maina et al., 1996
) were
compared with the wild-type siblings. Sections were stained for striated
skeletal muscle (MyHC). Fig. 5G
shows that all perineal muscles were completely absent in all examined embryos
(three embryos from three different litters). Consistent with results obtained
with legless mutant chick embryos (see below), the only muscles
present in the pelvic region were m. pelvicaudalis lateralis
(Fig. 5G) and medialis
(not shown). The subcutaneous muscle of the panniculus carnosus was completely
absent (data not shown). As previously reported, all hindlimb muscles were
absent. By contrast, abdominal muscles were normal, as were the axial muscles
of the tail.
These results demonstrate that the development of the perineal muscles in mammals is dependent on the migration of muscle precursors and thus that these muscles belong to the group of hypaxial migratory musculature.
Our molecular analysis showed that cells migrated as single entities into the limb and then moved towards the genital tubercle as a sheet. To achieve this, we studied the development of perineal muscle in the Lbx1 null mouse. This mutant is interesting since although muscle cells migrate into the hindlimb, they are unable to migrate any further and results in a hindlimb that has no distal muscle. We found that even though there are no distal limb muscles, Lbx1 null embryos showed perineal musculature development (Fig. 5H,I).
We also examined the development of the perineal muscles in the Meox2-null mouse embryos. These embryos display an abnormal limb muscle patterning that has been proposed to reflect alterations to the instructive signals. Although we are able to detect changes in the limb muscle pattern, the muscles of the perineal region were normally formed (Fig. 5J).
Proximal leg development is required for cloacal muscles
Our molecular analysis showed that the development of cloacal muscles is
intimately linked to the development of the hindlimb muscle mass. We next
wanted to test the role of limb development and verify to what extent cloacal
muscle formation depends on the hindlimb.
To address this point, unilateral hindlimb primordium surgical ablations on chick embryos were performed between stages HH16 and HH23. These resulted in unilateral absence of the leg and ipsilateral absence of all cloacal muscles (Fig. 6A,B). Neither mm. pubocaudales nor tail muscles were affected (Fig. 6A-C), and there were no malformations of the cloacal and tail regions. The extent of the limb ablation correlated inversely to the development of cloacal muscles. Thus, whenever we observed some remnants of the proximal leg muscles reaching the vicinity of the cloacal tubercle, the sphincter complex did develop (data not shown).
|
First, we examined chicken limbless autosomal recessive mutants
(Prahlad et al., 1979). These
mutants form no visible limbs (Fig.
7A), owing to the failure of apical ectodermal ridge (AER)
formation (Fallon et al.,
1983
). Limb-bud development is, however, initiated and parts of
the pectoral and pelvic girdles form
(Prahlad et al., 1979
), as do
a few muscles of the proximal hindlimb (data not shown). However, we found
that all cloacal muscles were completely absent
(Fig. 7B) in limbless.
These results show that cloacal muscles do not develop when the limb
development programme is severely curtailed.
|
In conclusion, both surgical procedures and genetic mutants showed that the development of the cloacal muscles is dependent on the degree of growth of the proximal hindlimb.
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Discussion |
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Cloacal muscles are homologous to perineal muscles
This is the first study to confirm that the avian cloacal muscles are
homologous to the perineal muscles of mammals. Homologies between species in
comparative anatomy are based on anatomical resemblance and innervation, but
mainly on the same developmental mechanism. We show both in chick and mouse
that the early anlagen of the cloacal and perineal muscles derive from the
ventral muscle mass of the hindlimb. Subsequently, these give rise to all the
muscles around the cloaca the chick and the perineum in mouse. It follows that
the situation in humans is very likely to be similar.
It has been previously suggested that the perineal muscles develop from a
common precursor - a circular cloacal sphincter
(Popowsky, 1899). We show that
this is not the case, because the muscle arrives to the vicinity of the
orifices at embryonic day 14. The cloacal membrane perforates in mouse by
E14.0 and the gross septation of the cloaca is completed by this stage
(Hynes and Fraher, 2004
).
Thus, the connection with its contraleteral counterpart occurs after splitting
into the single muscular primordia around the orifices.
Multiple fate of somitic cells and dual fate of hindlimb muscle mass cells
Our somitic fate-mapping studies have determined that the four cloacal
muscles originate from somites 30-34. Previous work, which is confirmed in
this investigation, has shown that leg muscles originate from somites 26-33
(Lance-Jones, 1988;
Rees et al., 2003
). Therefore,
cells from somites 26-34 have a number of developmental possibilities: to give
rise to interlimb musculature (somites 26-29), to give rise to limb muscle
(somites 26-33), to form both cloacal and limb muscle (somites 30-33), or to
contribute only to cloacal muscle (somite 34). Furthermore, we know that the
pelvicaudal muscles arise from somites 33 and caudally (P.V. and K.P.,
unpublished). Therefore, a major issue is how the same somites give rise to
the musculature of two different structures.
Skeletal muscle development at hypaxial sites can occur through two
mechanisms. Myogenic precursors can move as single migrating cells following
the epithelio-mesenchymal transition from the dermomyotome or form part of an
extension of the myotome (reviewed by
Galis, 2001). The most
parsimonious means of generating two separated populations of cells (one for
limb and one for cloacal) would be either: (1) using different mechanisms of
muscle development - migration being used for limb and myotomal extension for
cloacal muscle; or (2) having two separate waves of migrating myogenic cells.
Using a panel of genes and cell-labelling techniques we have not detected the
presence of myotomal extension from somites 30-34 directed towards the cloacal
tubercle. Furthermore, we have shown that the development of the
cloacal/perineal muscles is dependent on cell migration by demonstrating that
they do not develop in metd/d mutant embryos. Therefore,
we can now state that the development of cloacal/perineal muscles is dependent
on cell migration. The second possibility can also be discounted as our somite
labelling studies have detected only one wave of cells delaminating from the
somites and entering the hindlimb bud. Therefore, myogenic cells of the
hindlimb and the cloaca migrate from the somites at the same time.
The two mechanisms postulated above for separating cloacal and limb muscle
precursors can therefore be discounted. Segregation may instead be achieved
through tissue patterning. From the results of our study, we suggest that
myogenic cells enter the limb and coalesce in a single entity before dividing
into the dorsal and ventral muscle masses
(Amthor et al., 1998). All the
cells of somite 34 would be part of the latter group. Cells of the ventral
muscle mass are then further divided into those that will remain in the limb
and those that will form cloacal muscle. Again, all the cells from somite 34
are destined to leave the limb. Cells from somites 26-29 will be distributed
between the dorsal and ventral muscle mass but never allowed to participate in
cloacal muscle development. Recent studies have shown that muscle precursors
have intrinsic patterning properties and therefore it is possible that the
fate and behaviour of the cloacal muscle cells is established prior to
migration (Alvares et al.,
2003
; Mootoosamy and Dietrich,
2002
).
Development of cloacal muscles from the ventral limb muscle mass
The whole process of cloacal muscle formation can be described in three
steps: (1) migration of somitic myogenic cells into the ventral limb muscle
mass; (2) translocation of the cloacal subpopulation from the limb to the
cloaca; and (3) differentiation into four individual muscles.
It has been proved that myogenic cells migrate from the somites into the
leg bud (Chevallier et al.,
1977; Jacob et al.,
1979
). We have detected single spindle shaped cells that express
Pax3 but not MyoD during this initial phase of movement
(Amthor et al., 1998
). However,
the situation is different as the cloacal anlage extends from the ventral
muscle mass towards the cloacal tubercle. Pax3, Pax7 and
MyoD are expressed at the same level both at the leading and trailing
edges of the cloacal anlage and thus resemble a myotomal extension. This
situation is similar to the mode of development displayed by the pectoral
muscles, which extend to gain attachment to the ventral midline.
The deployment of an extension mechanism as opposed to migration of
individual precursors is supported by our observations of SF/HGF
expression during this process (data not shown). SF/HGF expression in
the early limb bud is well documented
(Thery et al., 1995;
Heymann et al., 1996
;
Scaal et al., 1999
) and is
responsible for the entry of met-expressing myogenic cells from the
somites into the limb. The expression of SF/HGF is subsequently
downregulated and is not present at all by the time of the movement of the
cloacal anlage from the ventral muscle mass. Therefore, we can conclude that
SF/HGF does not play a role in the cloacal muscle anlage extension to
the cloacal tubercle.
Interestingly, the perineal muscles develop relatively normally in the Lbx1 mouse mutant, whereas the migratory muscles of the distal hindlimb are absent. One interpretation of this phenotype is that the cloacal muscle progenitors use different mechanism for translocation compared with those that migrate to populate the distal leg. We propose that this occurs through mechanism resembling myotomal extension and is substantiated by the co-expression of MyoD and the Pax genes.
Formation of cloacal muscles is dependent on initiation and continued development of proximal limb field
We have demonstrated that unilateral ablation of the lateral plate mesoderm
that forms leg results in the absence of ipsilateral cloacal muscles. These
results, combined with the phenotype of the metd/d mutant
embryos, suggest that the initiation of migration is an absolute requirement
for the generation of precursors that will eventually develop into the cloacal
muscles. Lateral plate mesoderm ablation experiments also show that muscle
cells from the unoperated side do not cross over the midline to populate the
cloacal region on the operated side.
The complete absence of cloacal muscles in the limbless chick
mutant shows that not only the initiation, but also the subsequent early
formation of the proximal limb field is required for the normal development of
these muscles. Previous works have suggested that the limb development
programme in these mutants is inhibited because of defects in dorsoventral
patterning that prevent the induction of the apical ectodermal ridge (AER).
These results stem from the finding that markers of dorsal identity extend
into the ventral regions. In addition, application of a bead soaked in FGF
results in the rescued outgrowth of a limb that has double-dorsal
characteristics of the feathers (Ros et
al., 1996; Grieshammer et al.,
1996
). These suggestions have been questioned by Laufer et al.
(Laufer et al., 1997
) who have
demonstrated that the AER forms independently of dorsoventral patterning. In
addition, our observations of the normal ventral pectoral muscles show that
the dorsoventral patterning is not impaired at least in the proximal region.
Therefore, although at present we do not know the mechanism behind the
development of the limbless phenotype
(McQueeney et al., 2002
), it
is beyond doubt that it does not develop an AER. The limb phenotype of
limbless is much more severe than any produced by experimental
ablation of the AER. Even the removal of AER precursors leads to the
development of the proximal aspect of the femur
(Rowe and Fallon, 1982
). We
have been able to repeat these findings and have found that even the removal
of AER precursors from the developing limb bud does not prevent the
development of the cloacal muscles (P.V. and K.P., unpublished).
The cloacal muscle phenotype of limbless could be explained by the absence of precursor migration from the somites. However, we show that this is not the case, as we show both dorsal and ventral muscle develop and are well formed. Yet there is no cloacal muscle development in limbless. As the cloacal musculature develops from the ventral muscle mass, loss of cloacal muscle could be explained by dorsalisation of all muscles. The presence of near-perfect pectoral muscle (ventral in identity) argues against this scenario. In order to understand why the cloacal muscles fail to form in limbless but are present in chicks after limb truncations, it is necessary to keep in mind the developmental kinetics of the anlagen that form these muscles. Our cell labelling studies of the leg bud have shown that cloacal muscles develop from the ventroproximal region of the muscle mass. The extension occurs after the base of the limb bud has expanded in all directions including towards the cloacal tubercle. After AER removal, the stump of the leg develops normally and therefore expands as usual. However, in the case of limbless, the expansion of the proximal hindlimb field is defective. This is not the case in the p63 null mice where the proximal limb develops to a greater extent than in limbless and leads to the development of the perineal muscles. We therefore suggest the limb growth is required to bring the ventral muscle mass into range of signals that cause it to expand into the developing cloacal region. Therefore, the initiation of limb development, together with subsequent early growth, is required for cloacal muscle formation.
Thalidomide, AER removal, hindlimb and cloacal muscles development
Limb outgrowth can also be modulated through the use of pharmacological
compounds or by tissue manipulations. Thalidomide was prescribed in the 20th
century to alleviate the symptoms of morning sickness during pregnancy.
However, it impaired limb development and babies born to mothers who took the
drug displayed proximodistal truncations. Nevertheless, in all cases there is
some degree of limb outgrowth. Furthermore, there are no reports of perineal
muscle defects in individuals exposed to Thalidomide during embryogenesis. In
concordance with these observations, we have effected leg truncation through
the removal of the AER. Leg AER or even apical ectoderm removal (precursor of
AER) is not sufficient to stop the development of all limb elements. We have
found normal cloacal muscle development in all cases of AER removal (P.V. and
K.P., unpublished).
Concluding remarks
We show that the formation of cloacal sphincter muscle is dependent on limb
development. Phylogenetically, however, cloaca appeared before limbs.
Therefore, it will be intriguing to determine the mechanism used to form the
cloacal muscles in pre-limb animals (as well as highly evolved limbless
animals). One possibility is that they developed from local myotomal
extensions, similarly to the rectus abdominis muscle of higher vertebrates. A
more radical proposition would be that migration predates limb development.
The combination of migration and differential growth has been also described
(Neyt et al., 2000). We are
currently investigating these possibilities.
Despite the derivation of cloacal/perineal muscles from the limb muscle
mass, one should be aware of some important differences between the former and
leg muscles. The motoneurons that innervate the cloacal/perineal muscles
originate from the ventral horns of the spinal cord and the cell bodies are
located in a specific region called Onuf's nucleus X. Their cells bodies show
characteristic morphology (smaller than common motoneurons) and afferent
connections. Furthermore, in pathological conditions such as in amyotrophic
lateral sclerosis (ALS) or following poliomyelitis virus infection motoneurons
of skeletal muscles are affected but the motoneurons of perineal muscles
spared. However, they are strongly affected in a group of diseases that
involve selectively the autonomic visceromotoneurons such as Shy-Drager
syndrome (Schroder, 1985;
Mannen, 2000
). This might be a
reflection of the mixed voluntary and involuntary nerve control mechanisms of
the sphincter, but the underlying developmental cues remain to be
elucidated.
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ACKNOWLEDGMENTS |
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