DEVELOPMENTAL BIOLOGY 28, 142-161 (1972)

Early Regionalization of the Semitic Mesoderm as Studied by the

Development of the Axial Skeleton of the Chick Embryo

M. KIENY, A. MAUGER, AND P. SENGEL

Lubomtoire de Zoologie, Uniuersitt! Scientifique et Mkdicale de Grenoble,
38-Saint-Martin-d’Hkres, France

Accepted January 19, 1972

The excision of a portion of unsegmented somitic mesoderm from the thoracic region has no
or little effect on the development of vertebrae and ribs. Its orthotopic replacement by thoracic
somitic mesoderm obtained from another embryo does not cause important deficiencies of the
rib basket. But its replacement by nonsomitic tissue (neural tube, midgut, somatic mesoderm)
not only causes the formation of a costal gap within the thoracic region, but also leads to the pro-
duction of a defective spine on the operated side.
Segmented or unsegmented somitic mesoderm from the cervical region, implanted in place
of unsegmented thoracic somitic mesoderm, differentiates according to its origin: no ribs de-
velop in the host embryo at the operation level. Conversely, segmented or unsegmented somitic
mesoderm from the thoracic region, implanted in place of unsegmented cervical somitic meso-
derm, also differentiates according to its origin, giving rise to supernumerary ribs in front of the
host’s rib basket. These extra ribs, however, always lack a sternal component.
It is concluded that the level-specific morphogenetic capacity of the cervical and thoracic
somitic mesoderm is determined before metamerization occurs. Despite this early determination,
the unsegmented somitic mesoderm is endowed with regulative properties.

INTRODUCTION ments between thoracic and cervical so-

mitic mesoderm.
The vertebral column of amniotes is The chick possesses 14 cervical and 7
classically regarded as being subdivided thoracic vertebrae. The latter are char-
in five specialized regions, namely cer- acteristically equipped with a pair of
vital, thoracic, lumbar, sacral, and caudal. ribs. The posterior five pairs consist of
The vertebrae within each of these re- distinct vertebral and sternal com-
gions are characterized by particular ponents separated by a costal suture.
morphological features, among which pres- The anterior two pairs lack the sternal
ence or absence of ribs is most conspic- component and do not reach the sternum.
uous and most easily detected in embryos. It is widely accepted, although not
As it is well established that the verte- quite conclusively demonstrated, that
brae arise from the chondrogenic differ- at least the vertebral components of the
entiation of the sclerotomal part of the ribs originate from somites (Seno,
somites, the development of the axial 1961; Murillo-Ferrol, 1963; Sweeney and
skeleton appears to be an adequate sys- Watterson, 1969a). The strongest evi-
tern for the study of the regionalization dence for the somitic origin of the verte-
of the segmental plates. In other words, bra1 component of the rib was provided
at what stage are the different regions of by Pinot (1969), who showed that ver-
the vertebral column determined? Is there tebral ribs not only could develop within
a relationship between this regional de- isolated somites, but also apparently did
termination and the segmentation of the not need a somatopleural matrix to dif-
somitic mesoderm? We have chosen to ferentiate (cf. Sweeney and Watterson,
approach this problem in the chick em- 1969a).
bryo by means of translocation experi- As concerns the origin of the sternal
142
Copyright 0 1972 by Academic Press, Inc
 KIENY, MALJGER, AND SENCEL Regionalization of Semitic Mesoderm 143
component no convincing evidence is as excised; (b) the excised thoracic somitic
yet available in the literature. For some mesoderm was replaced by homologous
(Straus and Rawles, 1953) it derives (thoracic) somitic mesoderm from another
entirely from somatopleural mesoderm; embryo; (c) the excised thoracic somitic
for others (Seno, 1961; Pinot, 1969; mesoderm was replaced by nonsomitic
Sweeney and Watterson, 1969a,b) it tissue.
originates from the somites. Donors. The donor blastoderm was
The results reported below show con- transferred to a calcium- and magne-
clusively that the regional determination sium-free Earle’s solution. Incubation of
of the somitic mesoderm takes place be- the blastoderm for 15-30 min at room
fore metamerization. They also shed temperature sufficed to loosen the en-
some light on the origin and mechanism doderm and the ectoderm so that they
of differentiation of the sternal com- could be peeled from the embryo with
ponents of ribs. glass needles. A portion of endoderm-
and ectoderm-free segmented and/or
MATERIALS AND METHODS unsegmented somitic mesoderm was then
The experiments were performed on excised and placed in Tyrode’s solution.
White Leghorn chick embryos during Carbon particles were routinely placed
day 2 or 3 of incubation. The exact stage at the anterior end of the dorsal side of the
attained at the time of operation is speci- graft.
fied by the number of pairs of somites. Hosts. The host embryo was stained
The cervical vertebrae arise from so- in ouo with neutral red-impregnated agar
mites 5-18, the thoracic rib-bearing ones strips moistened with Tyrode’s solution.
from somites 19-26 (cf. Seno, 1961; Pinot, After the vitelline membrane had been
1969). torn apart with watchmaker’s forceps,
In the breed of fowl we used, the pres- the implantation site was prepared in
ence of the sternal component of the third the following way. In all but 11 embryos
pair of ribs was inconstant. It was missing of the third series of experiments the
in 10% of the unoperated control embryos. excised portion of the somitic mesoderm
Consequently its absence in the experi- was entirely comprised within the yet
mental embryos was not counted as a de- unsegmented plate, in which case its an-
ficiency, unless the corresponding verte- terior edge was made to coincide with
bral component was shortened or itself the posterior edge of the last-formed
lacking. Moreover, in about 5% of the somite. In the 11 exceptions of the third
White Leghorn embryos (cf. Herrick and series of experiments, the excised piece
Herrick, 1956) used in this study, an of cervical somitic mesoderm comprised
eighth rib, more or less completely de- an anterior part of three to five somites
veloped, with or without sternal com- and a posterior part of variable length
ponent, was present on one or both sides of yet unsegmented somitic plate. The
of the first lumbar vertebra. latter is easily removed completely with-
The experimental design consisted in out damaging neural tube, endoderm or
implanting cervical somitic mesoderm in lateral plates. On the contrary, somites
place of thoracic somitic mesoderm and are almost impossible to excise entirely
vice versa. These translocation experi- without puncturing major vessels, be-
ments make up the second and third cause once it is segmented the somitic
series of experiments. In a first series of mesoderm sits right on top of the paired
experiments, three types of control ex- dorsal aortae. Consequently, whenever
periments were performed in which (a) somitic mesoderm was excised in its seg-
the thoracic somitic mesoderm was simply mented state, ventral and medial portions
144 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972

of the somites presumably remained in side were characterized as follows. (A)

situ. Rib baskets: (1) complete (7 ribs which
The excised somitic mesoderm was then may be normal or subnormal, duplicated
replaced by the graft, which was made to or deficient); (2) incomplete (fewer than 7
fit exactly within the prepared hole. The ribs, whatever their morphology). (B) In-
level of excised tissue and of graft origin dividual ribs: (1) normal or subnormal
will be given as appropriate in subse- (normal, or slightly thinner than normal,
quent sections of the paper. or tortuous, but complete with sternal
Embryos were sacrificed at 11 days of components for ribs 3-7); (2) duplicated
incubation and fixed in Bouin-Hollande’s (branched within their vertebral com-
solution. The embryos served two pur- ponents); (3) deficient (shorter than nor-
poses. They were used to study the de- mal, without sternal components in ribs
velopment of the feather pattern (Mauger, 3-7).
1972a, b) and in the present investigation
RESULTS
to study the regionalization of the somitic
mesoderm. Consequently, the spinal The results of the first and second
pteryla of each embryo was photographed series of experiments are summarized in
before Lundvall’s staining method was two different ways. In Tables 1 and 2 are
applied for cartilage visualization. In recorded the absolute numbers of em-
quite a few instances, the conformation bryos with characteristic types of rib
of the dorsal feather pattern helped in the baskets and individual ribs on the
interpretation of skeletal modifications. operated side, regardless of the exact
Characterization of abnormalities. The level of the operation. In Figs. 1 and 14,
abnormalities observed in each host rib the percentages of normal, deficient and
basket and individual rib on the operated duplicated ribs are diagrammatically
TABLE 1
CONTROL EXPERIMENTS PERFORMED AT THE THORACIC (RIB-FORMING) LEVEL
Morphology of the rib basket

vum- Complete rib basket T Incomplete rib bask@ by the absence of:
ber
Operation Nature of of
type graft em- t
bryos 1 2 3 4 5 6 1
Dt?. rib ribs ribs ?bs ,ibs I ,ibs ribs
Du$;;ted Kent
ribs
- - -
Excision -
Orthotopic re- Segmented 2
placement somitic
mesoderm

Unsegmented 23 4(2+, 1”) 1

somitic
mesoderm

Replacement Neural tube 1

by nonsomitic Midgut 3
tissues Somatic
mesoderm
RRibs present at normal rib-forming
1 4

levels may be normal, duplicated or deficient. The nur

6

hers in each
column correspond to specimens lacking the indicated quantity (not the number) of ribs.
b The number of rib baskets with one (+) or two (++) duplicated ribs is given in parentheses.
c One of the five rib baskets is overdeveloped, as illustrated by Fig. 4.
 KIENY, MAUGER, AND SENCEL Regionalization of Semitic Mesoderm 145

represented according to the various First Series: Control Experiments

levels of the operation, which, for the
sake of simplicity, have been grouped as
follows: 16-17, 18-19, 20-21, 22-23, and
24. These numbers refer to the posterior
somite left in place in front of the excised
fragment of somitic mesoderm. (For exam-
ple, host embryos grouped under “18-19”
have had their somitic mesoderm excised
unilaterally immediately behind the 18th
or 19th somite).
1. Excision of unsegmented somitic
mesoderm. In 21 embryos, a portion of
somitic plate corresponding to approxi-
mately six future somites was completely
removed together with the overlying ec-
toderm, leaving the endoderm in place.
Minor effects on rib development are
summarized in Table 1 and Fig. 1A. Nine
embryos were normal (4 cases) (Fig. 2) or

A 6 C
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%

FIG. 1. Percentages of normal, deficient, and duplicated ribs (orthotopic ribs numbered l-7, heterotopic
lumbar ribs numbered 8 and 9) according to operation levels (indicated at the right-hand side of diagram by
the underlined number(s) representing the last-formed somite left in place in front of the operation site; the
underlined number also indicates the developmental stage of the embryo expressed in pairs of somites).
(A) Excision of unsegmented somitic mesoderm. (B) “Pseudo-orthotopic” replacement of unsegmented
somitic mesoderm by somites. (C) Orthotopic replacement of unsegmented somitic mesoderm by unseg-
mented somitic mesoderm. Circled figures specify number of cases.
146 DEVELOPMENTAL BIOLOGY
subnormal (5 cases). Nine other em-
bryos had also reconstituted a complete
dorsal rib pattern; in one of them the
sixth rib was bifurcated at the middle
of its vertebral component, the tip of
each branch articulating with a complete
sternal component reaching the sternum;
in the 8 remaining embryos the vertebral
component of one to four ribs was shorter
than normal and sternal components were
absent; in one case, the vertebral com-
ponent was fused to the neighboring
vertebral rib and sharing a common
sternal rib (Fig. 3). Only 3 embryos had
an incomplete rib basket: in two cases,
one rib was missing (the first or the
seventh) and, in one case, in addition to
the absence of the seventh rib, the sternal
components of ribs 4-6 were also lacking.
The rib basket of the contralateral non-
operated side of all embryos was normal.
The vertebrae of the 10 embryos with a
complete rib basket and nondeficient
ribs were considered perfectly normal
(Fig. 2). In 7 of the 11 other embryos,
fusions extending over 2-4 vertebrae af-
fected the neural arches on the operated
side. The level of the fused arches cor-
responded to the rib deficiencies al-
ready mentioned. The remaining 4 em-
bryos had normal vertebrae. These were
embryos in which only one rib was de-
ficient (Fig. 3).
These results show that the unseg-
mented somitic mesoderm is capable of
extensive skeletal regulation. Conse-
quently, it was necessary to test the abil-
ity of the embryos to build a normal rib
basket when the excised somitic meso-
derm was replaced either by orthotopic
somitic mesoderm or by nonsomitic tis-
sue. Experiments of these types consti-
tute the two following control series.
2. Orthotopic replacement of unseg-
mented somitic mesoderm by seg-
mented or unsegmented somitic meso-
derm. These control experiments were
carried out in order to test the sound-
ness of the transplantation technique
VOLUME 28, 1972
used in the experimental series, namely,
the implantation of a row of somites or
unsegmented somitic mesoderm. Since
it was expected that translocation of
cervical somitic material to the thoracic
level and vice versa would produce
anomalies in the axial skeleton, it was in-
dispensable to ascertain beforehand to
what extent orthotopic transplantations
would produce malformations. In ad-
dition, it must be remembered that it is
mechanically impossible to realize per-
fect orthotopic grafts when a row of so-
mites replaces unsegmented somitic plate,
because a chain of, say, 5 somites is
longer than the somitic plate correspond-
ing to 5 prospective somites. Such grafts
should be called “pseudo-orthotopic”
grafts. Perfect orthotopic fit can, how-
ever, be secured when host and donor
have exactly the same number of pairs of
somites if unsegmented donor somitic
plate replaces unsegmented host somitic
plate of the same level.
a. Implantation of somites. The graft
was composed of a chain of 5 or 6 somites,
the length of which fitted exactly within
the hole produced by the corresponding
excision in the host embryo. The donors
possessed from 21 to 26 pairs of somites.
The number of the anterior somite of the
graft was chosen to be the same as the
number of the next-to-be-formed somite
of the host. This procedure resulted in
the juxtaposition of the host’s last formed
small somite with the graft’s bigger anter-
ior somite but in a fairly good adjustment
of host and graft somites at this anterior
level. At the posterior level of the graft,
however, there was always a virtual gap
of 2 to 3 somites in the normal cephalo-
caudal succession.
b. Implantation of unsegmented so-
mitic plate. The donors possessed 16-22
pairs of somites. The length of the graft
varied from 0.7 to 1.0 mm and correspon-
ded to approximately 6 to 8 prospective
somites. In most cases the anterior edge
of the graft was made to correspond in
 KIENY, MAUGER, AND SENCEL Regionnlization of Somitic Mesoderm 147

FIG. 2. Excision of right prospective somites 21-26 at stage of 20 pairs of somites. Complete regulation.
FIG. 3. Excision of right prospective somites 22-26 at stage of 21 pairs of somites. Note fusion of vertebral
components of ribs 5 and 6, resulting in the development of a single sternal component (arrow).
FIG. 4. “Pseudo-orthotopic” implantation of somites. Donor: 26 pairs of somites. Graft: left somites
22-26. Host: 21 pairs of somites. Implantation site: behind somite 21, on the left side. Note duplication of rib
6 and presence of 2 supernumerary ribs on lumbar vertebrae 1 and 2 (LI, L2).
FIG. 5. “Pseudo-orthotopic” implantation of somites. Donor: 23 pairs of somites. Graft: left somites
19-23. Host: 19 pairs of somites. Implantation site: behind somite 19, on the left side. Note marked distortion
of rib pattern and spine.
FIG. 6. Orthotopic implantation of unsegmented somitic mesoderm. Donor and host: 21 pairs of somites.
Graft and implantation site: behind somite 21. Graft length: 1 mm, right side. Note duplication of vertebral
components of ribs 5 and 6, the 4 tips of which are prolonged by complete sternal components.
FIG. 7. Orthotopic implantation of unsegmented somitic mesoderm. Donor and host: 16 pairs of somites.
Graft and implantation site: behind somite 16. Graft length 0.8 mm, left side. Note absence of rib 1 (arrow)
and duplication of rib 3, with 2 correspondent sternal components.
148 DEVELOPMENTAL BIOLOGY
level with the posterior edge of the host’s
last-formed somite. In a few cases, how-
ever, there was a difference of plus or
minus one somite between host and do-
nor age.
c. Results. The results of both series
may be described together and compara-
tively. The malformations were always
more pronounced in the “somite” series
than in the “unsegmented plate” series
(Table 1, Figs. lB, C, 4-7). In the for-
mer, the vertebral column of all embryos
(except three which were normal) was
malformed, displaying various fusions
and distortions of its cartilaginous ele-
ments (Fig. 4). The most severe axial mal-
formations were often accompanied by an
abnormal rib pattern (Fig. 5) character-
ized by fusions, anastomoses, branching
and various other defects of the vertebral
rib components. Some ribs were lacking.
In all embryos where the graft had been
isolated behind the 16th or 17th somite
and therefore contained one or more cer-
vical somites at its anterior tip, the rib
basket lacked at least the first rib and of-
ten also the second to fourth ribs.
The embryos that had received an un-
segmented graft were comparatively less
affected by the operation (Figs. 6 and 7).
The vertebral column was almost always
straight and quite normally developed,
with the exception of some slight for-
ward or backward shifting of the trans-
verse processes. The rib basket was
complete in half of the cases. The ribs
themselves were frequently branched
within their vertebral component at
any level between the capitulum and the
junction between vertebral and sternal
portions. Again, whenever the vertebral
component was shorter than normal, the
sternal part was missing. Conversely,
whenever the vertebral component of
ribs 3-7 was branched and the branches
attained a normal length, each branch
was prolonged by a complete sternal ele-
ment. The girdles were normal or sub-
normal (Figs. 4-7).
VOLUME 28. 1972
To summarize the results of both
series, it may be said that in general the
“pseudo-orthotopic” implantation of
somites and the orthotopic implantation
of unsegmented somitic mesoderm, al-
though causing more severe modifica-
tions than excision experiments, did not
produce defects that were too serious to
use the transplantation technique to test
the developmental capacities of the
somitic mesoderm from various levels.
3. Replacement of unsegmented so-
mitic cord by nonsomitic tissues. Three
types of tissues were used as graft: neural
tube from 2-day embryos, midgut from
8- to g-day embryos and somatic meso-
derm from the lateral plates of 2- and
4-day embryos. The grafts were cleaned
from all undesired attached tissue frag-
ments (such as ectoderm, endoderm, so-
mitic mesoderm, and notocord) either by
short trypsin digestion or by prolonged
incubation in Ca- and Mg-free Earle’s
solution, after which they were cut to
appropriate size (0.7-1.3 mm in length)
so that they would fit tightly within the
implantation site. The midgut was cut
longitudinally into quarters before strips
were grafted with their intestinal epi-
thelium facing the host’s endoderm.
Nine of the 40 embryos operated in
that manner developed a complete rib
basket (Table 1, Fig. 14A). Three of them
were perfectly normal, the other 6 had one
or several deficient ribs. Thirty-one
embryos developed an incomplete rib
basket, lacking one to seven ribs on the
operated side (Figs. 8-13). The remain-
ing ribs, when adjacent to the costal
gap, were often shorter than normal;
those that were further away from the
gap were normally developed. The
position of the costal gap, i.e., the serial
number of the missing ribs, was in good
correspondence with the level of the
excised somitic mesoderm (Fig. 1). In
embryos operated behind the 16-17th
somite, ribs numbers 1, 2, and 3 were
always missing (Figs. 8, 14A). Most em-
 KIENY, MAUGER, AND SENGEL Regionulization of Semitic Mesoderm 149

FIGS. 8-13. Replacement of somitic mesoderm by nonsomitic tissues.

FIGS. 8 and 9. Implantation of B-day midgut. Hosts: 16 and 19 pairs of somites. Implantation sites: be-
hind somite 16 (Fig. 8) and 19 (Fig. 9). Note absence of ribs and also of the vertebral halves on the operated
right side. Scapula is absent (Fig. 8) or shorter than normal (Fig. 9).
FIGS. 10 and 11. Implantation of 2-day presumptive ventral somatic mesoderm. Hosts: 18 and 21 pairs of
somites. Implantation sites: behind somite 18 (Fig. 10) and 21 (Fig. ll), on the right side. Note development
of cartilaginous elements along lateral edge of vertebrae and concomitant absence of ribs.
FIG. 12. Implantation of 4-day presumptive ventral somatic mesoderm. Host: 21 pairs of somites. Im-
plantation site: behind somite 21, on the right side. Note absence of right vertebral halves and of corre-
sponding ribs.
FIG. 13. Implantation of 2-day neural tube. Host: 18 pairs of somites. Implantation site: behind somite
18, on the right side. Note extensive formation of extra cartilaginous elements, curved in tunnel fashion,
above host and grafted neural tubes.
 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972

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FIG. 14. Percentages of normal and deficient ribs (orthotopic ribs numbered 1-7, heterotopic lumbar ribs
numbered 8 and 9) according to operation levels (indicated at the right-hand side of diagram by the under-
lined number(s) representing the last-formed somite which is left in place in front of operation site; the
underlined number also indicates the developmental stage of the embryo expressed in pairs of somites).
(A) Replacement of unsegmented somitic mesoderm by nonsomitic tissues; (B) by cervical somites; (C) by
cervical unsegmented somitic mesoderm. Circled figures specify number of cases.

bryos operated behind the 1%19th so-
mites lacked ribs altogether on the
operated side (Figs. 9, 10, 14A). In em-
bryos operated at more posterior levels,
behind the 20-21st (Figs. 11, 12, 14A) or
22-23rd and 24th somite, the missing
ribs were always the more posterior ones.
The vertebrae of the embryos in which
midgut or 4-day somatic mesoderm had
been implanted formed only the centrum
and neural arch moieties on the unopera-
ted side. The other half was always ab-
sent, resulting in a characteristic cur-
vature of the spine (Figs. 8, 9, and 12).
In the embryos in which neural tube (Fig.
13) or 2-day somatic mesoderm (Figs. 10
and 11) had been grafted, however, not
only were the vertebrae generally de-
veloped on both sides of the axis, but
various supernumerary cartilaginous ele-
ments had also developed along the
operated side of the vertebrae. In these
embryos the spine was generally straight.
In addition to vertebral and costal
 KIENY, MAUGER, AND SENGEL
malformations, deficiencies could also be
observed in the girdles. The scapula on
the operated side was always shorter
than normal whenever the costal gap en-
croached upon the space in front of the
fifth rib, which is the level the tip of the
scapula normally reaches. In no case, when
less than 4 ribs had developed did the
scapula extend posteriorly within the
costal gap, so that when all ribs were ab-
sent, the scapula was frequently but
not always, completely missing (Figs.
8-13).
The iliac cartilage of the pelvic girdle
was frequently reduced in size whenever
the implantation site extended caudad
into the lumbar region (posteriorly to the
26th pair of somites). The ischium and
pubis were generally not affected but, in
a few cases, were also slightly under-
developed. In the most severe cases the
pubis was missing and the ischium re-
duced to a thin cartilaginous rod.
These experiments show that a foreign
nonsomitic tissue will impede the regula-
tion processes within the somitic material
and thus preclude rib formation.
B. Second Series: Implantation of Cer-
vical Semitic Mesoderm into the
Thomcic (Rib-Forming) Region
1. Implantation of somites. The do-
nors ranged in developmental stages
from 12 to 25 pairs of somites. The graft,
composed of a row of 5 to 8 somites,
was obtained from the cervical region
between somites 4 and 18. In 45 cases
the donor was younger than the host (by
2 to 9 pairs of somites) ; in 4 cases it was
older (by 1 or 2 pairs of somites); and in
one case donor and host were of equal
age.
The results were very clear-cut (Table
2, Fig. 14B). Out of 50 embryos, 40 de-
veloped an incomplete rib basket which
was characterized by the absence of 1
to 7 ribs, according to the level of im-
plantation (Figs. 15 and 16). Whenever
Regionalization of Semitic Mesoderm 151
cervical somites occupied the level of
thoracic somites, no ribs differentiated
at that level. Thus, when the operation
was performed behind the 18-19th so-
mites, most embryos possessed no ribs
on the operated side. When the opera-
tion was performed at a more posterior
level, the intact thoracic somites in front
of the graft developed according to their
normal destiny, with some deficiencies
in lengths and sternal components in
those ribs that formed adjacent to the
anterior limit of the costal lacuna (Figs.
15 and 16). In all 40 embryos, the con-
tralateral ribs of the unoperated side de-
veloped normally. The 10 remaining em-
bryos formed a complete rib basket, of
which 9 were perfectly normal and one
had ribs shorter than normal.
The vertebrae of 46 embryos displayed
an abnormal morphology. The vertebral
halves that developed on the operated
side were obviously of a different shape
than those on the other side. In some
cases (Fig. 16), the typical cervical
morphology could be recognized on the
operated side. In others, extensive longi-
tudinal fusions had taken place and it
was difficult to identify the individual
half vertebrae. Despite the fact that the
implanted somites were not in meta-
merit register with the prospective so-
mites of the contralateral somitic plate,
particularly in the posterior half of the
graft, a fairly good correspondence was
often observed between the contralateral
thoracic half of the vertebra and the
heterotopic cervical one (Figs. 15 and 16),
especially within the anterior half of the
operation site. In the 4 remaining embryos
the vertebral column and rib basket were
normal. Their morphology was thus sim-
ilar to that of the control embryos from
which a portion of the unsegmented so-
mitic plate of the prospective thoracic
level had been removed and not replaced
by a graft. The normal development of
these 4 embryos could therefore be in-.
terpreted as the result of an early re-
152 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972

jection of the graft, after which regula-

FIG. 15. Replacement of unsegmented thoracic
somitic mesoderm by cervical somites. Donor: 18
pairs of somites. Graft: left somites 5-12. Host: 20
pairs of somites. Implantation site: behind somite 20,
on the left side. Note maladjustment between left
vertebral halves of cervical type and right halves of
thoracic type, absence of ribs 4-7, and shortness of
tion processes could take place.
In addition to vertebral and costal
malformations, the embryos showed var-
ious degrees of deficiencies in the gir-
dles. The defects were quite similar to
those mentioned in the preceding series
of experiments and, for that reason,
need not be described again, but are
merely exemplified by Figs. 15 and 16.
2. Implantation of unsegmented somitic
plate. The donors ranged in developmen-
tal stages from 9 to 15 pairs of somites. In
the majority of cases the graft was excised
immediately behind the last-formed
somite (18 cases); otherwise, one (8 cases:
stages with lo-13 pairs of somites) or two
(2 cases: stage 11 pairs of somites)
somites were included at the anterior tip
of the graft. The. length of the graft ranged
from 0.7 to 1.0 mm and comprised approxi-
mately 8-20 prospective somites. In all
cases the donor was younger than the host
by 6 to 13 pairs of somites. Because of this
great difference in age between host and
donor, and in order to fill the excision site
completely with the graft, a posterior por-
tion of the latter generally comprised
prospective thoracic and sometimes
even prospective lumbar somitic ma-
terial.
Of the 28 embryos operated in this
manner 26 showed abnormalities in
their axial skeleton. The other 2 were
normal and suspected of having rejected
their graft early. Effects of the operation
on rib development are summarized in
Table 2 and Fig. 14C. As in the previous
series, the replacement of prospective
thoracic somites by prospective cervical
ones resulted in the absence of the corre-
sponding ribs. The costal gap thus pro-
duced was frequently (18 cases) occupied
by various cartilaginous elements (Figs.
17-19) in the form of plates, rods or nod-
left scapula and ilium. Cervical (Cl, thoracic (‘I),
lumbar (L), and sacral (S) vertebrae numbered for
reference.
 FIGS. 16-19. Replacement of unsegmented thoracic somitic mesoderm by cervical somitic mesoderm.
FIG. 16. Donor: 18 pairs of somites. Graft: right somites 8%-15. Host: 22 pairs of somites. Implantation
site; behind somite 22, on the right side. Note absence of ribs 6 and ‘7, deficient vertebral component of rib 5
and absence of sternal components 5-7. Right transverse processes of vertebrae along operation site
(thoracic levels 6 and 7 and lumbar levels l-4) have typical cervical morphology. Note absence of pubis
and reduction of ilium.
FIG. 17. Donor: 11 pairs of somites. Graft: left somite IO-prospective somite 25. Host: 18 pairs of somites.
Implantation site: behind somite 18, on the left side. Note cervical transverse processes along rib gap
and presence of ectopic cartilage (arrow).

153
154 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972

TABLE 2
IMPLANTATION OF CERVICAL SOMITIC MESODERM INTO THE THORACIC (RIB-FORMING) REGION
Morphology of rib basket

Nature of Complete rib

Number Incomplete rib basket” by the absence of:
grafted of basket
cervical embryos
mesoderm
NOIld With wmx 1 2 3 4 5 6 7
or sub- deficient rib ribs ribs ribs ribs ribs ribs
normal ribs

Segmented 50 9 1 11 11 6 3 5 4
Unsegmented 28 2 1 1 1 8(2f, l++)b 7 W+) 2(1+) 2(lff)
n Ribs present at normal rib-forming levels may be normal or deficient. The numbers in each column corres-
pond to specimens lacking the indicated quantity (not the number) of ribs.
b The number in parentheses indicates the quantity of rib baskets in which one (+) or two (++) normal or
deficient ribs arose in the lumbar region, the contralateral side consisting of seven ribs.

ules, and in 2 cases, by articulated long
bones that were sticking out laterally per-
pendicular to the spinal column (Fig. 18),
thus having the appearance of an under-
developed supernumerary wing.
In 18 of 26 embryos ribs developed
posteriorly to the costal gap. These ribs
could be divided into two categories
according to their location, either within
the thoracic region or within the lumbar
region. In the first category, two types
of ribs could again be distinguished. One
type was borne by normal vertebrae. That
type obviously resulted from the de-
velopment of the host’s posterior thoracic
somitic mesoderm that was left in place
behind the graft. The other type was
linked to distorted and often poorly ad-
justed vertebral halves, thus indicating
that it originated by development of the
posterior portion of the graft, which, as
stated before, comprised prospective
thoracic material. The ribs of the second
category, which arose in the lumbar
region, were also linked to maladjusted or
longitudinally fused vertebral halves,
which suggested their exogenous origin
(Figs. 17 and 19). These supernumerary
ribs of graft origin were difficult to num-
ber. In order to include them into the
diagrams (Fig. 14C), they were arbitrarily
registered in correspondence with the
number of the contralateral vertebral
half that was closest to them.
In addition to the malformations of the
vertebral column mentioned before, the
spine on the operated side frequently
(13 cases) had developed supernumerary
vertebral segmental halves (neural arches
and transverse processes), the number
of which (1 to 4) was always smaller than
the number of the implanted prospec-
tive somites (Fig. 17). On account of the
extensive malformation of the vertebral
column, it was generally impossible, ex-
cept in a few cases (Fig. 17), to certify
the cervical source of the vertebral
halves adjacent to the rib lacuna.
In contrast with the previous series of
experiments in which cervical somites
had been implanted, the scapula, fol-
lowing implantation of unsegmented
cervical somitic plate, was usually nor-
mally developed or only somewhat shorter
than normal, thus extending a long way
caudad within the rib gap (Figs. 17-19).
The pelvic girdle was only seldom and

FIG. 18. Donor: 10 pairs of somites. Graft: left somite lo- prospective somite 22. Host: 19 pairs of somites.
Implantation site: behind somite 19, on the left side. Note development of supernumerary limb skeleton
with girdle-like base (arrow) within rib gap.
FIG. 19. Donor: 14 pairs of somites. Graft: left prospective somites 15-32. Host: 19 pairs of somites.
Implantation site: behind somite 19, on the left side. Note widespread distribution of numerous ectopic
skeletal elements within rib gap.
 KIENY, MAUGER, AND SENCEL Regionalization of Semitic Mesoderm 155
slightly affected by the operation, the apparently had no effect on the costal
ilium then being narrower than normal. development of the implanted tissue,
These experiments lead to the con- but had quite remarkable consequences
clusion that the heterotopic replacement on the regional differentiation of the ver-
of thoracic somitic mesoderm by seg- tebral halves on the operated side.
mented or unsegmented cervical somitic The segmented or unsegmented nature
material inhibits the regulative processes of the graft had no influence on the re-
and consequently precludes the differen- sults. Consequently the two types of im-
tiation of the host’s ribs. plantation experiments will be reported
together, but are summarized separately
C. Third Series: Implantation of Thomcic in Table 3. Only 4 of the 43 embryos were
Semitic Mesoderm into the Cervical normal. They had probably lost their
Region graft soon after implantation. Most of the
This series comprises 43 embryos, of remaining 39, hosts (33 cases) had de-
which 26 had received an implant of veloped an excess number of ribs (from
unsegmented somitic plate correspond- 1 to 6) in front of the thoracic region
ing to 4-8 prospective somites and 17 (Figs. 20-23). The latter was normal when-
had received an implant of 3-7 successive ever the excision site did not extend
somites from the thoracic region. The posteriorly into it (Figs. 20 and 21).
hosts ranged in developmental stages When the excision site extended into
from 9 to 16 pairs of somites. The anterior the thoracic region (15 cases) the in situ
level of the implantation site was loca- development of the first two ribs of the
ted from the posterior edge of the 7th host was suppressed and replaced by
somite to the prospective 15th somite. Its the formation of one or two graft-derived
posterior level was approximately located ribs (Figs. 22 and 23). They consequently
between prospective somites 16 and 21. belonged to the posterior levels of the
The excised portion of somitic material extra rib basket and should for this rea-
from the host therefore sometimes ex- son have borne sternal components.
tended within the prospective thoracic This, however, was never observed.
region, thus depriving the host of one or Sternal components were also always
both of its first two prospective thoracic lacking on the extra ribs that developed in
somites. Moreover, the anterior level of the cervical region, even though many of
the implantation site was not always lo- these were quite long and even curved at
cated at the posterior edge of the last- their distal tip.
formed somite; in 11 cases it was placed Except for the absence of sternal com-
three to five somites in front of the last- ponents, the implanted thoracic somitic
formed somite. As has been previously mesoderm developed a rib basket in
stated (Materials and Methods), once the concordance with its prospective fate,
somitic mesoderm is segmented, somites although the number of ribs formed by
can hardly be removed entirely without the graft was generally smaller than
puncturing the underlying dorsal aorta. might have been expected from the num-
Consequently, whenever the excised ber of prospective segments of the
thoracic somitic tissue extended an- implanted material (compare columns 1
teriorly within the segmented region, it and 5 of Table 3).
might be expected that some cells (pre- The vertebrae of the 39 abnormal em-
sumably of sclerotomal origin) re- bryos could be classified into two dis-
mained in place ventrally and medially tinct morphological categories. 1. The
along the neural tube and would there- vertebral halves on the operated side
fore underlie the graft. This circumstance were either very distorted and more or
 FIGS. 20-23. Replacement of cervical somitic mesoderm by thoracic somitic mesoderm.
FIG. 20. Donor: 26 pairs of somites. Graft: left somites 20-26. Host: 15 pairs of somites. Implantation
site: behind somite 9, on the left side. Note cervical morphology of vertebrae and presence of 5 extra vertebral
ribs at operation site.
FIG. 21. Donor: 26 pairs of somites. Graft: left somites 22-26. Host: 15 pairs of somites. Implantation
site: behind somite 11, on the left side. Note typical cervical morphology of vertebrae and presence of 5
extra vertebral ribs (with early development of uncinate processes) at operation site.
FIG. 22. Donor: 18 pairs of somites. Graft: right prospective somites 19-25. Host: 14 pairs of somites.
Implantation site: behind somite 13, on the right side. Note development of 6 extra vertebral ribs in front of
endogenous rib basket, ribs 1 and 2 of which are missing.
FIG. 23. Donor: 20 pairs of somites. Graft: left prospective somites 21-26. Host: 13 pairs of somites.
156
 KIENY, MAUGER, AND SENCEL Regionalization of Semitic Mesoderm 157
TABLE 3
IMPLANTATION OF THORACIC (RIB-FORMING) Sormnc MESODERM INTO THE CERVICAL REGION

Mean number Mean number Mean total

Quantity of rib-forming Number of Number of of ribs in the of ribs
normal number of ribs
material implanted abnormal host’s rib formed by
hosts hosts formed on the
basket the graft operated side

3 Somites 0 2 7 2 9
4 0 6 5.3 3 8.3
5 1 4 6.5 4.8 11.3
6 0 1 7 5 12
7 0 3 6.3 4.3 10.6
4 Future somites 1 2 4 3 7
5 1 3 6 4 10
6 0 12 5.5 4.6 10.1
7 0 5 5.4 5.4 10.8
8 1 1 5 6 11

less fused longitudinally, or they could the removal of the cervical somitic mater-
easily be recognized as being of thoracic ial was probably incomplete.
type with a conspicuous diapophysis to The scapula was absent in 4 cases; in
which the costal tuberculum was attached the other cases it was often distorted or
(Fig. 23). Even in those cases where the shorter than normal. The malformation
malformations obscured the shape of the or absence of the scapula in this series
vertebral halves, one could find a series could not be related to any other defects
of lateral spines probably corresponding or particular experimental conditions.
to the successive diapophyses of thoracic
vertebrae. In all the 28 embryos that DISCUSSION AND CONCLUSIONS
formed this type of chimeric vertebrae Results of the two experimental series
(thoracic on one side, cervical on the demonstrate that the somitic mesoderm
other) the cervical somitic material had is regionalized prior to its segmentation.
been removed in the unsegmented state, When prospective cervical somitic meso-
at a time when it could be eliminated en- erm is implanted into the prospective
tirely. 2. The vertebrae were generally thoracic region and, conversely, when
very well formed, particularly within prospective thoracic somitic mesoderm
the anterior half of the operation site, is grafted into the prospective cervical
the halves on the operated side being in region, the translocated material de-
almost perfect register with the halves velops according to its origin. The trans-
on the control side. They were bisym- plantation experiments thus lead either
metrical and of typical cervical type with to the production of a costal gap within
oblique transverse processes pointing the thoracic region or to the differentia-
anteriorly, to which the ribs were tion of ribs in a region where they usually
jointed (Fig. 21). All 11 embryos in which do not exist. The ectopic ribs consist of
this type of vertebra was found were vertebral components only.
operated partially within an already seg- Although our data cannot prove that the
mented portion of their somitic meso- vertebral components of the ribs ori-
derm (at least 3 somites) and consequently ginate from emigrated somitic cells, they

Implantation site: behind somite 13, on the left side. Note six supernumerary vertebral ribs and thoracic
morphology of transverse processes of left halves of vertebrae in front of endogenous rib basket, ribs 1 and 2
of which are missing.
158 DEVELOPMENTAL BIOLOGY VOLUME 28, 1972

do not contradict the views of previous from the observation that its develop-
authors (Seno, 1961; Pinot, 1969; Sweeney ment appears to be an all-or-none phe-
and Watterson, 1969a), who presented nomenon. Indeed we never found a case
evidence of this somitic origin. Conse- of fragmentary differentiation of the
quently, it seems reasonable to admit sternal ribs, even when they formed in
that, at least, the vertebral component excess numbers at the tips of branched
of the rib is formed by somitic cells. vertebral components. Whenever they
The origin of the sternal component were present, they were fully developed
of the rib, however, remains obscure. (except rib number 7, which does not al-
Nevertheless our results point to a strict ways reach the sternum even in normal
relationship between vertebral and sternal embryos). One could reason that, if the
components. Whenever the vertebral com- sternal ribs originated from somites, one
ponent was shorter than normal or absent, would occasionally find some that, for
the corresponding sternal component had lack of material in the operated embryos,
not formed (cf. Sweeney and Watterson, would be incomplete. This was never the
1969a). This strongly suggests that the case, which is a strong indication of the
distal tip of the vertebral component must local somatopleural origin of the sternal
reach a certain point in the somatopleure ribs. The data appear to suggest that the
away from the somite before a sternal rib sternal rib differentiation is the result of
can differentiate distal to it. In addition, an inductive action that the tip of the
sternal components never formed an- vertebral component exerts on contact
teriorly to their normal location. Thus even with competent somatopleural cells
when vertebral components had devel- located in ventrolateral position. How-
oped to full length within the cervical or ever that may be, it cannot be denied that
anterior thoracic regions (facing somites the development of the sternal component
19-20), they were never prolonged by ster- is closely dependent on the earlier forma-
nal components. Sternal components, tion of the vertebral part of the rib, as
however, could develop posterior to their already emphasized by Sweeney and
normal location, but only in relation with a Watterson (1969a, b).
vertebral component that was made to The scapula also appears to be some-
grow on the first lumbar vertebra. This is how dependent on the presence of the
not surprising since an 8th pair of ribs is ribs, although a definite relationship
sometimes observed in our breed of fowl; cannot as yet be established. When a
these lumbar ribs usually bear a sternal costal gap is produced by the implanta-
component. These results indicate that tion of either nonsomitic tissue or cer-
the prospective ventrolateral part of the vical somites within the prospective
somatopleure is competent for the for- thoracic region, the scapula is always
mation of sternal ribs only at somite levels shorter than normal and seems not to be
21 through 27 and is not competent an- able to develop beyond the rib in front of
terior to level 21. They appear to exclude the gap. However, when the costal gap
the possibility that sternal components is produced by implantation of unseg-
arise from emigrated somitic cells, since mented cervical somitic plate, the scap-
it would be difficult to explain why ula usually develops normally and ex-
prospective sternal chondroblasts of so- tends caudad within the costal gap. It
mitic origin could not move into the ven- is not known whether the scapula origi-
trolateral somatopleure outside the levels nates from the somites or somatopleure.
adjacent to somites 21-27. Another ar- It can be stated, however, from our data
gument in favor of the somatopleural ori- that nonsomitic tissue or nonthoracic
gin of the sternal rib components derives somites cannot support the development
 KIENY, MAUGER, AND SENCEL
of or give rise to scapular cartilage. Yet
unsegmented cervical somitic plate does
not interfere with the developement of the
scapula even though it does not form
ribs.
Vertebral halves (centra and neural
arches) develop in conformity with the
origin of the translocated somitic material,
except when the implantation site is pre-
pared by excising somites instead of so-
mitic plate. In the latter case, it is sus-
pected that some ventromedial so-
mitic (prospective sclerotomal) cells are
left in place. It is remarkable to note that,
whenever the excision was likely to be
incomplete, the vertebral halves that
developed always conformed with the
level of the operation site, and were per-
fectly symmetrical to their contralateral
counterpart on the unoperated side. Un-
der these circumstances it seems likely
that the transplanted somites contribute
very little, if anything, to the vertebrae,
even though they give rise to ribs. These
ribs are clearly articulated to the trans-
verse processes of the cervical verte-
brae, thus providing an embryological
proof of an otherwise classical notion of
serial homology in comparative anatomy.
Vertebral halves do not develop at all
on the operated side when the excised
fragment consists of unsegmented somitic
plate only and is replaced by a piece of
midgut. This can be considered as a proof
of the complete elimination of all prospec-
tive somitic cells from the implantation
site.
Some experimental conditions have re-
sulted in unexpected skeletal differ-
entiations. When 2-day somatopleural
mesoderm is grafted in place of thoracic
somitic mesoderm, a number of super-
numerary cartilaginous elements may form
along the medial edge of the graft. This
may be interpreted as a response of the
somatopleural mesoderm to the chondro-
genie induction originating from the host’s
neural tube. This view is also supported
by the production of supernumerary skele-
Regionalization of Semitic Mesoderm 159
tal elements along the lateral edge of a
graft of neural tube, which again may have
acted inductively on the adjacent somato-
pleure of the host. Evidence of such car-
tilaginous differentiation within somato-
pleure after neural tube implantation
stems not only from the only case (Table
1, Fig. 13) reported in this paper, but also
from many similar cases that we have ob-
tained at other levels than the thoracic
one (unpublished data) and which, for
that reason, have not been included in the
present results. We felt, however, that
this chondrogenic competence of the so-
matopleure deserved mention. In addition
to their excess skeletal elements, these
embryos generally also form supernumer-
ary feathers at the operation site, within
their spinal pteryla.
The rib gaps produced by the implanta-
tion of cervical somitic plate into the
prospective thoracic region are frequently
occupied by varied pieces of cartilage,
some of which are arranged to resemble an
underdeveloped supernumerary wing. In
view of what is known of the somite-de-
pendent morphogenetic activity of the
prospective somatopleural limb meso-
derm (Kieny, 1969, 1970, 1971), the pres-
ence of these ectopic cartilaginous ele-
ments is an indication of an inductive in-
fluence that may originate from future
somites 15-20 (wing-bud level) and act on
the adjacent somatopleural mesoderm.
In conclusion, the somitic mesoderm is
regionally determined before it is seg-
mented. However, it is pertinent to note
that this early determination of the somi-
tic cells does not preclude regulative proc-
esses from taking place whenever needed.
The control experiments where no graft is
implanted into the excision site convin-
cingly show that the unsegmented somitic
mesoderm is capable of extensive skeletal
regulation, leading in most cases to the
normal development of vertebrae and ribs.
In those embryos in which regulation is
subtotal, vertebrae seem to be reconsti-
tuted predominantly at the expense of the
160 DEVELOPMENTAL BIOLOGY
rib basket. In the same manner, the
“pseudo-orthotopic” implantation of so-
mites does not produce any conspicuous
deficiencies, but may rather lead to a
slight redundancy, although a virtual
gap of several somites usually occurs at
the posterior edge of the graft. This
harmonious development may again be
attributed to normal or excessive regu-
lative capacities of the unsegmented and
perhaps of the segmented somitic meso-
derm.
This result is in line with a previous
finding by Strudel (1966), who showed
that somitic mesoderm is able to regulate
through a length of 5 excised somites,
provided the endoderm is left in place.
On the contrary, in Chelydm serpentinu
(Yntema, 1970), the segmenting somitic
plate is unable to regulate and appears
to be a strict regionally determined mo-
saic. The absence of regulation in this
case may be due to the fact that the ven-
tromedial portion of the somite (pros-
pective sclerotome) was not extirpated
and could therefore, by its very presence,
block the onset of regulative proliferation.
Thus we are led to an apparently con-
tradictory conclusion: prospective so-
mitic cells of the 2-day embryo are re-
gionally determined to form certain skele-
tal derivatives, but are still capable of
extensive regulation. How can these no-
tions-determination and regulation
capacity-be reconciled? It is proposed
that determined cells will keep their
determination, i.e., the particular func-
tional state of their genes, as long as they
are not compelled to divide intensively.
They may lose their determination and
thus return to a naive totipotent state if
they are forced to enter extra mitotic
cycles belatedly, during which the particu-
lar functional state of their genie DNA
will be wiped out. These “de-determined”
cells may then again become susceptible
to their environment and acquire a new
determination in conformity with the spe-
cific level of the morphogenetic field
VOLUME 28, 1972
wherein they are located. This phenome-
non is probably similar in nature to the
now well-documented transdetermination
in Drosophila, which leads to the differ-
entiation of allotypic structures after the
cells have been forcibly and untimely kept
in an undifferentiated and proliferative
state (Hadorn, 1966; Wildermuth, 1970).
Finally, it is important to mention that
the reported experiments also have
marked effects on the development of the
spinal feather tract, the dermal cells of
which also originate from the somites
(Mauger and Sengel, 1970). Prospective
dermal cells within the somites (derma-
tomes) are regionally determined (Mauger
and Sengel, 1971; Mauger, 1972a, b) just
as are the sclerotomes and rib-forming
cells. They also manifest an extensive
regulative capacity, even after they have
undergone segmentation. The simultan-
eous observation of the morphological ef-
fects of the operations on the axial skele-
ton and the dorsal feather tract
greatly facilitated the interpretation of
many skeletal malformations and, for that
matter, of the few embryos that had de-
veloped normal vertebrae and ribs. In the
latter specimens the perfectly normal
spinal feather pattern convinced us that
the graft had been rejected early after im-
plantation. In the former specimens, gaps
in the rib basket generally corresponded
to featherless notches in the spinal
pteryla, whereas formation of supernu-
merary ribs in the cervical region was al-
ways accompanied by a widening of the
otherwise narrow cervical portion of the
tract. Thus axial skeleton and dermis of
the spinal tract, both originating from
somites, undergo a similar course of
events during their early developmental
history.
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