An Analysis of the Postgastrula Differentiation of the Hypomere

An Analysis of the Postgastrula Differentiation of
the Hypomere
III. The Histogenetic Competence of Salamander Hypomere in
Heterotopic Transplantation
by CYRIL V. FINNEGAN 1
From the Department of Zoology, University of British Columbia
WITH TWO PLATES
INTRODUCTION
F O L L O W I N G its involution around the lateral lips of the blastopore, the
potential hypomere (lateral plate mesoderm) of Urodeles undergoes a dorsal
convergence, along with other portions of the mesoderm mantle and then, during
neurulation, initiates an independent ventrad migration (Nieuwkoop, 1947).
As the hypomere migrates ventrally, it becomes further removed from the influences shown by Yamada (1950) to emanate from the mid-dorsal axial system
and its deep layer is in contact with a tissue (endoderm) which seems to be
influential in the differentiation of this mesoderm (Bacon, 1945; Jacobson,
1960; Nieuwkoop, 1947, 1950; Finnegan, 1953, 1955, 1961a, 1961ft).
A further understanding of the postgastrula development of the trunk hypomere seemed to require information of the possible restriction, in time, of the
competence of this mesoderm or, alternatively, its degree of self-differentiation
within an embryonic system for in vitro observations may indicate histogenetic
potencies unrelated to normal development (Finnegan, 1961a, 1961ft).
The experimental analysis was attempted by heteroplastic and homoioplastic
transplantation of portions of neurula and tail-bud trunk hypomere to a dorsal
position overlying the host's somitic mesoderm. In making heterotopic mesodermal transplantations, Hoadley (1931) noted that three kinds of results may
be obtained: (1) the self-differentiation of the transplanted material; (2) the
incorporation of the transplanted material into host structures; or (3) some
combination of the above responses. In this work, transplanted hypomere produced results of the third type and interesting observations concerning the host
response to the transplanted material were obtained.
1
Author's address: Department of Zoology, University of British Columbia, Vancouver 8, Canada.
[J. Embryol. exp. Morph. Vol. 10, Part 3, pp. 293-314, September 1962]
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C. V. FINNEGAN—POSTGASTRULA
DIFFERENTIATION
EXPERIMENTAL PROCEDURES
Neurula (stages 14-22) and tail-bud (stages 23-30) embryos of Ambystoma
punctatum and Taricha torosa were used as donors and late tail-bud stages (2832) were used as hosts. The ectoderm overlying the anterior trunk somites
(typically, somites 7-11) of the host animals was removed or lifted free of the
somites. An incision was made in the ectoderm superficial to the ventral somite
and this was continued along the anterior-posterior axis. The anterior and posterior boundaries of the ectodermal piece were delineated by cuts perpendicular
to the above. If the hypomere was transplanted without its overlying ectoderm,
the piece of host ectoderm was simply lifted away from the somites, the transplant inserted, and the host ectoderm returned to position. If the transplant
included its own ectoderm, the host dorsal ectoderm was removed by a fourth
cut made some distance lateral to the neural tube-neural crest region in order not
to disturb the host's axial tissues.
From the donor embryos at the time of operation the most ventral portion of
the trunk hypomere, constituting the material from a point anterior to the blastopore or anus cephalad to the level of the liver (but exclusive of the more ventral
angioblastema), was excised and placed on the exposed host somites. In a few
experiments the more dorsal hypomere was removed from the trunk area
immediately ventral to the nephrogenic mesoderm in a manner that avoided the
anterior limb-bud area by initiating the incision posterior to that region. In
some of these experiments involving T. torosa hosts the superficial portion of the
host's somites were scraped prior to the placing of the transplant in an attempt
to remove the possibly more undifferentiated somite cells shown previously
(Finnegan, 1961c) to be present in this species during development stages
28-40.
Aseptic techniques were used throughout and the host animals reared in
Urodele growing medium at 18° C. (Rugh, 1948). The majority were cultured
for 10-40 days following the operation while a few animals in each group were
maintained for longer periods (up to 3 months). In all, over 200 viable cases were
observed macroscopically and approximately one-fifth (45) were prepared for
histological examination by being fixed in 70 per cent, alcohol, Bouin's and
Michaelis' solutions (Rugh, 1948), sectioned at 10/z and stained with haematoxylin and eosin.
RESULTS
Three types of host-donor response were revealed by the macroscopic observations of the host animals. One, the response of the host chromatophores to
the hypomeric mesoderm, had been the subject of previous reports (Finnegan,
1955, 1958) and will not be considered herein. A second response was the production on the host flank of supernumerary projections or limbs. The third
response, frequently made more easily visible in older A. punctatum hosts by
OF THE HYPOMERE
295
a dorsal convexity of the trunk, was a lack of ventro-lateral musculature in the
trunk region ventral to the graft locus. Histological examination of the host
animals has provided further information on these responses and on the differentiation of the hypomere. It has also revealed a fourth type of response involving nephric differentiation. Control experiments, in which donor ventral
ectoderm alone was transplanted to the dorsal graft site or in which the host
dorsal ectoderm was lifted free and then allowed to heal, produced none of the
results to be discussed.
For purposes of clarity and continuity in the following, each of these responses
will be considered and discussed separately.
A. Limb-projection formation
Ventral hypomere ( ± ectoderm) as transplant
The results pertaining to limb-projection production following the transplantation of the ventral hypomere are presented in Table 1. In the composition
of the various groups from the experimental data the following criteria were
used. The structure was considered to be a limb if the outgrowth could definitely
be seen to contain cartilage. Its appearance as an elongated, digitiform, mesenchyme-containing element served to mark it as a projection. In addition, there
were many cases in which a bump, not unlike an early limb-bud, was developed
and maintained for some time on the flank in the graft area. These were classified as negative for limb-projection formation, although Newth (1958), in
a report on regeneration of induced supernumerary limbs, had considered a
similar response as positive limb induction.
Following the healing of the operational wound, the graft ectoderm could
be seen, particularly in the xenoplastic transplantations (e.g. A. punctatum on T.
torosa), to spread dorso-posteriorly, frequently sufficiently far to become incorporated into the developing dorsal fin. Histological examination of host
animals showed the occasional case, classified as negative in Table 1, in which
the donor T. torosa tissue had developed an elongated cartilage rod similar
to the cartilage units found in the positive limb cases. The rod continued from
the vicinity of the ventral somite dorso-posteriorly superficial to the lateral face
of the somite without appearing to interrupt the normal flank contour of the
host (see Plate 1, fig. F). A somewhat similar situation appears with some of the
A. punctatum hosts to which T. torosa hypomere plus ectoderm had been grafted
(Text-fig. 1). In such cases it was frequently observed that the donor ectoderm
was sloughed off and apparently replaced by host ectoderm prior to 20 days of
culture (host stage 40~). In the sectioned material it could be seen that a cartilage rod developed with the dorso-posterior orientation as in the above but
with an ectodermal-covered mesenchyme-filled projection rising from the more
dorsal termination of the cartilage piece. (Such cases were classified as positive
for projection formation in Table 1 since the cartilage development was visible
5584.10
U
296
C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
only on histological examination.) In another similar case it was found that the
cartilage rod was continuous with a cartilage-like cell orientation of the mesoderm within the dorsal projection.
TEXT-FIG. 1. The graft flank of a section, at 30 days after transplantation, through
an outgrowth developed from an A. punctatum St. 15 hypomere graft on a T.
torosa host. The host axial structures are to the left and the donor tissue is to the
right in the illustration. The donor cartilage unit extends dorsally from its origin
in the vicinity of the host's ventral somite tip and an area of degenerating donor
tissue (light colour) is present dorsal to the cartilage. Further dorsally and superficially a group of deeply staining donor cells (dark colour) continue into the
projection. (Traced from a photograph, approx. x 300.)
These results leave one with the impression that the mesodermal development
does not appear externally on the flank because of the failure of the overlying
ectoderm to respond properly, and this impression is strengthened by noting the
unusually thick basement membrane on the inner surface of the overlying
ectoderm. Balinsky (1957) has called attention to the possible interfering role of
this extracellular structure in the induction of supernumerary limbs. Furthermore it would appear that in A. punctatum hosts the more dorsal ectoderm did
respond whereas the more ventral host ectoderm (or the donor ventral ectoderm
if it remained) did not, a not unlikely situation in the light of Bodenstein's (1952)
demonstration of the variation along the dorso-ventral axis of the ectodermal
response to neural crest induction of dorsal fin.
The data in Table 1 indicate that when positive results were obtained the older
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OF THE HYPOMERE
donors (stages 25-30) developed projections (see Plate 2,fig.G) while the younger
ones formed both limbs and projections. Although the ectodermal factor, that is,
the dorsal recession in time of the ectodermal ability to respond, undoubtedly
plays a role in these results there is the additional factor of the reduction of the
competence of the hypomere to respond to the host's limb influence. In grafts
from young donors a small amount of donor-developed striated muscle was
often found superficial to the long cartilage element, the latter of no discernable
TABLE 1
Outgrowth production by ventral hypomere transplants
Donor-host
1. T. torosa-T. torosa
Donor age
St. 15-20 group
St. 25-30 group
Totals
2. T. torosa-A. punctatwn
st. 15-20 group
st. 25-30 group
Totals
3. A. punctatum-A. punctatum
st. 20 group
st. 25-30 group
Totals
4. A. punctatum-T. torosa
st. 15-20 group
st. 25-30 group
Totals
No. of cases Projection
Limb
% positive
16
21
3
2
2
0
31
10
37
5
2
18
9
20
0
3
0
0
15
29
3
0
10
3
34
0
3
0
0
9
37
3
0
7
30
12
7
4
1
0
27
33
42
11
1
28
specific morphology (see Plate 1, fig. F). The major portion of a limb or a projection contained mesenchyme tissue with a large amount of ground substance
(see Plate 2, fig. G). In grafts from older donors there was no evidence of striated
muscle, the cartilage element was often reduced or absent and the major portion of the projection contained a fibrous connective tissue. It was also noticed
that regardless of the host species, the A. punctatum hypomere developed the
cartilage element more rapidly than did the T. torosa grafted tissue, and the former produced fibrous tissue at an earlier developmental age than did the latter.
The experimental results for T. torosa hypomere grafted on both host species
were used to test the hypothesis that the ventral hypomeres from the stages used
(15, 20, 25, 30) are equally competent to produce positive results. The probability
of this being the case is small (p = < 0-05 > 0-025; Chi-square = 8-89). Thus
it may be inferred that the ventral hypomere of T. torosa, during the period of its
ventrad migration, does not remain equally competent to respond to the limbprojection influence of the host, a conclusion substantiated by the histological
evidence.
Another consideration is the variability between the two species as to the host
influence on the grafted tissue. It can be seen from Table 1 that a larger number
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C. V. FINNEGAN—POSTGASTRULA
DIFFERENTIATION
of positive cases are recorded with T. torosa as the host animal (19 out of 79;
24 per cent.) than with A. punctatum (6 out of 66; 9 per cent.). The experimental
results for A. punctatum hypomere grafted on the two host species were utilized
in testing the assumption that the hypomere limb-projection response of A.
punctatum is similar in both host species. The probability of there being no
difference between the two hosts in this respect is small (p = < 005 > 0-025;
Chi-square = 4-7). From this it may be inferred that the two host species are
not similar in their limb-projection influence on the grafted A. punctatum
hypomere. The data suggest that the T. torosa hosts retain to a later stage the
ability to influence the grafted material than do the A. punctatum hosts. The
data also suggest that the ventral hypomere of A. punctatum is competent to
respond for longer than it evidences on its own species since the older hypomere grafts gave a larger positive response on T. torosa (33 per cent.) than on
A. punctatum (9 per cent.). On the other hand, the older ventral hypomere
of T. torosa shows a reduced capacity to respond on both its own species and
on A. punctatum hosts, indicative of a loss of competence in the tissue itself.
While it is evident that, during the ventral migration of the hypomere, changes
have occurred in the capacity of the tissue to respond histogenetically to host
influences, it is also true that the transplanted material of A. punctatum is a
larger mass, both absolutely and relative to host size, at all the developmental
stages studied. Balinsky (1957) has emphasized the role of the accumulation of
mesenchyme into a mass in supernumerary limb induction, and previous reports
from this laboratory (Finnegan, 1961a, 19616) have noted the histogenetic
effects in these two species of varying the mass of hypomere available for differentiation.
One or more masses of cells appeared in the graft area associated with the
ventral somite tips from which the projection and the limbs appeared to develop,
although it must be noted that many of the cases did not proceed beyond the
limb-bud stage. Examination of this limb-bud area in the sectioned material,
particularly grafts of A. punctatum hypomere on T. torosa at 10 days, revealed
that a group of basiphilic donor cells was present in the vicinity of the host's
ventral somite tip, and that mitotic activity was high in both the donor tissue
and the juxtaposed host somite tip (see Plate 1, fig. C). At a later period (20-30
days) the donor cells had developed the single long cartilage element typical of
these cases. A similar mass of donor cells was found, though much less frequently, in the region of the dorsal somite tip when the transplant was placed
dorsally or when its size was such as to extend it into that area. A recent study
of in situ differentiation of somites in T. torosa (Finnegan, 1961c) indicated that
the superficial cells in the ventral somite tip and in the lateral face of the trunk
somites represent a population of relatively undifferentiated cells for 20 days
after transplantation (stages 28-40) (see Plate 1, fig. B). It is also known that
a somite growth-centre is present in the dorsal tip area (Holtzer & Detwiler,
1953, for A. punctatum] Finnegan, 1961c, for T. torosa) in which relatively
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OF THE HYPOMERE
undifferentiated somite cells are present during this developmental period.
Balinsky (1957) has called attention to an increased mitotic activity in the lateral
plate cells responding to nasal placode induction of supernumerary limbs and
he has demonstrated increased basiphilia in these cells, the latter attributed to a
high concentration of RNA in the cytoplasm. It would appear that, in these
hypomere transplants, active basiphilic donor cells have accumulated in the
vicinity of the less differentiated somite tip and that this cell group has then
developed, to the best of its histogenetic competence, a limb outgrowth. In this
latter respect it is of interest to note that Newth (1958) remarks on the paucity of
muscle tissue in nasal placode induced supernumerary limbs and the frequency
with which cartilage is present in the outgrowth as a single morphologically
unidentifiable element.
Dorsal hypomere ( i ectoderm) as transplant
It seemed pertinent to investigate whether the non-limb dorsal hypomere of
neurula and tail-bud stages would demonstrate a greater competence for limbprojection production under the experimental conditions than had the ventral
hypomere. Thus the hypomere lying immediately ventral to the intermediate
mesoderm (mesomere) was excised from neurula and tail-bud donors and transplanted to the dorsal somite site as in the preceding experiments. While an
attempt was made to exclude the donor anterior limb area, it is possible, particularly with T. torosa hypomere, that the more posterior portion of the potential limb area may have been included in the graft. (In A. punctatum this area
forms shoulder and body-wall; Nicholas, 1955.) The results are presented in
Table 2 and the same criteria for the designation of the outgrowths were used
in assembling these data as had been used in Table 1.
TABLE 2
Outgrowth production by dorsal hypomere transplants
Donor-host
1. T. torosa-T. torosa
Donor age
St. 15-20 group
st. 25-30 group
Totals
2. A. punctatum-A. punctatum
st. 15-20 group
st. 25-30 group
Totals
3. A. punctatum-T. torosa
st. 15-20 group
st. 25-30 group
Totals
No. of cases Projection
Limb
% positive
21
13
4
1
12
5
76
46
34
5
17
64
4
4
1
0
1
0
50
8
1
1
25
11
4
3
0
1
0
36
15
3
1
26
The results obtained when T. torosa dorsal hypomere was transplanted to T.
torosa hosts are made more significant because of the various experimental
300
C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
manoeuvres performed to test the possible role of the host environment. In one
case, neurula stage hypomere was placed on the host somites with its ectoderm
side against the host somite tissue and the whole covered with host dorsal ectoderm so that the donor hypomere lay between two layers of ectoderm and without direct contact with the host somites. No local cell mass (limb-bud) was
observed and no outgrowth was recorded in this case. Amano (1960) has
reported negative results when neural tissue was interposed between lateral plate
mesoderm and epibrachial somites, a situation which otherwise resulted in limb
development. Negative results were also obtained in four other cases when a
small amount of hypomere from late neurula donors was placed dorsally in
association with the dorsal tip region of the host somites.
In a number of cases (13) the T. torosa host animals were prepared as
previously described (see Experimental Procedures), and then the superficial
somite area, involving the ventral tip and the lateral face, was scraped with a
sharp needle and hair-loop in an attempt to remove the less-differentiated cells
of those areas (Finnegan, 1961c). Late neurula (stage 19) donor hypomere was
observed, in three of these cases, to develop an aggregation of cells within the
scraped area near the region of the missing ventral tip. This cell group remained
but a short time while, simultaneously, two other such aggregations of donor
cells appeared; one at the anterior edge of the scraped area where the graft tissue
overlapped the ventral tip region of unscraped somites, and a second group at
the posterior edge of the scraped area where a similar situation existed. Outgrowths were developed from both of these buds in all three cases. Thus, since
the tissue was transplanted as an undisturbed sheet, it is possible that the initial,
or even one of the secondary, cell aggregations may represent an inadvertently
grafted portion of the donor limb field, but it does not seem reasonable that all
three of these physically separated responses by the grafted hypomere should
be so. And while the anterior outgrowth may result from a close association with
the host's anterior limb region, the posterior outgrowth would have had no
such origin. This production of outgrowths from the anterior, or posterior, or
both edges of the scraped somite region was observed in all cases where donor
tissue from neurula and early tail-bud stages exceeded the region of scraped host
somites. Five additional cases were prepared in which a smaller hypomere
transplant was excised and placed entirely within the scraped somite area of the
host. No outgrowths developed from any of these transplants. The removal of
the somites and their replacement with dorsal hypomere does not appear to
produce these outgrowths (Detwiler, 1937), nor does the simple scraping of the
somites.
These results demonstrate, as did those with the ventral hypomere, that an
association between the donor tissue and the superficial ventral somite tissue
is required to produce an outgrowth. It should be emphasized that in describing
the results in this report the term iimb', as previously defined, covers an array
of morphological entities that might perhaps be better acknowledged with some
OF THE HYPOMERE
301
other identifying label (outgrowths?). Undoubtedly this phenomenon is a
reflection of the competence of the hypomere to respond to the host's limb
influence and, in this sense, it was noticeable that the dorsal hypomere developed
more typical limbs than did the ventral hypomere. The histological examination
of the dorsal hypomere outgrowths gave further evidence on this point, for
striated muscle was more abundant than it had been in the ventral hypomere
grafts, the cartilage elements more frequently resembled recognizable limb units,
and fibrous connective tissue was not the dominant element it had been in the
older ventral hypomere outgrowths. Application of the Chi-square test to the
tabulated data for both dorsal and ventral T. torosa hypomere transplanted to
T. torosa hosts was made on the assumption that there is no difference in the
outgrowth response of ventral and dorsal hypomere in the experimental situation. It was found that the probability of these two regions of the hypomere
being equally competent in this respect is very small (p = < 0-01; Chi-square
= 16).
The question naturally arises as to why, if these transplanted hypomere areas
did not contain normal limb regions of the donor, limb-like structures should
develop along the lateral flank of the host animal since previous in vitro investigations (Finnegan, 1961a, \96\b) did not demonstrate any capacity for
limb morphogenesis by the hypomere. The initial observable event in this
development is the establishment of a group of relatively undifferentiated donor
cells in the immediate vicinity of a population of relatively undifferentiated host
cells residing in the ventral somite tip. Balinsky (1957) has remarked that, in
nasal placode induction of supernumerary limbs, the initial mesoderm mass
must be formed in the vicinity of the induction source and the present somitescraped cases would indicate that the superficial somite cells are at least
necessary to maintain the mesoderm mass. Balinsky also notes that the specific
competence to produce limbs is a factor of the lateral body-wall tissues involved
(see also Fautrez, 1951), and Holtfreter (1955), in discussing the establishment
of embryonic fields, states (p. 632) that 'the emergence of a new field . . . can be
indicated... through the aggregation of a sufficiently large mass of pluripotential
cells . . .' but 'it is the reacting cells rather than the external stimuli which make
for field organization' (see also Weiss & Moscona, 1958). The results of the
present work then would seem to show that the ventral somite tip functions to
initiate or assist in the aggregation of donor hypomere cells; the latter group
then develop, in keeping with their histogenetic competence, an outgrowth
(projection or limb). It is probable that the more slowly differentiating T. torosa
hosts retained the superficial somite population of less differentiated cells for
a longer time than did the A. punctatum hosts and thus produced a higher number
of outgrowths (see also nephric section below).
Recently Amano (1960), using Urodele hypomere from middle neurula stages
in several experimental situations, has obtained evidence which he interprets as
showing that any area of the neurula hypomere will form limb if brought into
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C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
contact with the epibrachial somite region (see Table 1 above) and he indicates
that the somitic tissue, participating in the production of the limb, differentiates
into limb tissue. The transplanted hypomere seemed to be the source of the outgrowth tissues in the present experiments but, while this is in agreement with
the observations of most workers on limb development (see Nicholas, 1955), it
must be noted that Holtfreter (1955) has remarked on the presence of histologically inconspicuous host cells in cases of tail-limb transformations and their
possible role in the development of these limbs. Such a host contribution has not
been here excluded.
In this latter respect, two other observations seemed important. The most
frequent course of events involving the ventral hypomere included the subsequent
regression of the outgrowth or the failure of the development to proceed beyond
the limb-bud stage. In the dorsal hypomere cases, limb-bud regression was
observed when the superficial cell population of the underlying somite regions
had been removed. Examination of the sectioned material disclosed that a block
of cartilage was present at the base of the retained outgrowths and the limb
cartilage element (humerus (?)) articulated with this block (girdle; scapula,
suprascapula (?)). The latter appeared to be of host ventral somite origin, as
indeed the suprascapular is considered to be in normal limb formation (Nicholas,
1955). No such structure could be found associated with the regressing outgrowths. Experiments exploring the possibility of a reciprocal relationship
between hypomere (limb) and somite ventral tip (girdle) in the development and
retention of the outgrowths will be the subject of another report.
B. Dennis formation
The major portion of the transplanted hypomere differentiated as a thick
dermis consisting of several cell layers and much ground substance lying
superficial to the somites of the host (see also Detwiler, 1937). A dermis of one
cell-layer occupied the corresponding area on the control side of the hosts. In
the dermis of the graft area there was a large cell population present immediately
superficial to the lateral face of the somite which decreased markedly toward the
overlying ectoderm, whereas the homogeneous ground substance increased in
amount toward the surface (see Plate 1, fig. E). As noted in in vitro studies
(Finnegan, 1961a, 19616), hypomere from older donor stages of both species
developed the large amount of ground substance more rapidly than did tissue
from earlier stages. When T. torosa was the host, this condition of mesenchyme
cells and homogeneous ground substance remained relatively unchanged in the
dermis for nearly 2 months, except that the donor cells in the vicinity of the
somite usually exhibited a dorso-ventral orientation by 30 days and the amount
of ground substance increased noticeably with time (40 days). When A. punctatum was the host, the increase in ground substance took place much earlier
(20 days). These fibroblasts were dorso-ventrally oriented (see Plate 2, fig. J),
OF THE HYPOMERE
303
and subsequently fibrous material (of undetermined nature) appeared in the
surrounding matrix.
It is quite possible that the host's neural crest material was contained in the
dorsal tip area in the transplant region, having been prevented from migrating
by the grafted hypomere (Finnegan, 1955), and the histological resemblance
observed between the dorsal tip region and the control side dermis (see Plate 1,
fig. F) may be due to a neural crest origin of this tissue as has been suggested by
Holtfreter (1935), Raven (1936), Detwiler (1937, 1938), Steamer (1946), and
Nieuwkoop (1947). It is also possible that some of the mesenchyme-filled dorsal
projections noted in the preceding section may have resulted from similar
collections of neural crest cells beneath responsive dorsal ectoderm, as demonstrated by Bodenstein (1952), but the cell population and the large amount of
ground substance observed in the histological examination of these projections
seems to be more in keeping with the differentiation of the hypomere under the
experimental conditions.
This evidence, in agreement with previous in vitro studies, would indicate that
the hypomere of both these species, when associated with ectoderm (see Nieuwkoop, 1947), demonstrates a developmental capacity to form a loose connectivetissue dermis; a capacity which may be modified by other host influences (see
sections A and D). The earlier production of ground substance by older donor
tissue and of this and fibroblasts in A. punctatum hosts may indicate further
basis for the fewer outgrowths (that is, lower competence) produced by the aged
transplants on T. torosa hosts and by all transplants on A. punctatum (see
Tables 1 and 2).
C. Host somite responses
When a small mass of hypomere was placed in the more dorsal somite area,
the juxtaposed somite tip failed to penetrate dorsad to the normal position overlying the neural tube (see Text-fig. 2). The dorsal region normally occupied by
the graft side somite tip was usually filled with mesenchyme and the neural tube
was tilted toward that side. Later the control side dorsal somite tip appeared to
have grown over the midline to approach the graft side, and the host's dorsal
fin often was developed from this atypical 'midline'. It appeared that the graft
tissue had somehow, either directly or indirectly through its confining the local
neural crest material, excluded the somite tissue from the dorsal area.
When the graft tissue was placed in the ventral somite tip region, a similar
situation developed. The ventral tip region was prevented from ventral extension
(Detwiler, 1955; Griffiths, 1959; Lewis, 1910; Liedke, 1958; Spofford, 1945; but
see also Straus & Rawles, 1953) and the ventro-lateral muscles were absent in
the graft area of the trunk but were present both anterior and posterior to it
(see Text fig. 3). It was apparent in A. punctatum hosts that the affected somites
were enlarged in the notochord-lateral face dimension as though the continuous
myogenic activity of the developing somite was being dammed up by the graft
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C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
tissue which prevented the normal ventro-lateral penetration of the ventral tip.
This phenomenon was even more obvious when the larger A. punctatum grafts
were present since often they extended sufficiently far to involve both the dorsal
and ventral somite tips, resulting in extremely fat somites confined to the notochord-neural tube level as both the ventro-lateral penetration and the dorsoventral elongation of the somite were inhibited. In T. torosa hosts the affected
somites often seemed reduced in myogenic tissue (see Plate 1, fig. F).
TEXT-FIG. 2. Sections of a T. torosa host, at 30 days after transplantation, on
which a small T. torosa st. 20 hypomere graft was placed near the dorsal somite
region. In the section through the graft area (A) the dorsal tip of the graft side
somite can be seen to reside ventral to the normal position of that area as illustrated in (B), a section of the trunk posterior to the graft area. The control side
somite tip in (A) has grown across the midline in the absence of the graft side
dorsal somite tip. (Traced from a photograph, approx. x 40.)
In a few cases where A. punctatum tissue was placed rather anterior on either
host species the graft tissue interfered with the ventral penetration of the myotomes immediately posterior to the anterior limb and it could be seen, in the
period after 20 days in A. punctatum and after 30 days in T. torosa, that the graft
flank limb remained dorsal in its attachment to the trunk whereas the control
flank limb appeared to have shifted ventrally in this respect. Detwiler (1955)
observed that various organs (eyes, spinal cord, limbs) implanted in the ventral
tip region of the somites were subsequently shifted ventrally as the myotome
extension occurred in their vicinity; this observation, taken together with the
present results, suggests that the ventral shift in the anterior limb attachment
possibly results from the establishment of the hypo-brachial musculature (Lewis,
1910). Detwiler (1955) also noted that when the postbrachial somites were
surgically deprived of their ventral tip regions the ventro-lateral muscles were
lacking in the corresponding region of the trunk. In the present work the hypomere grafts did not migrate ventrally, and it seemed that the host somites were
OF THE HYPOMERE
305
deprived of their ventral tip regions in the sense that the graft tissue may have
influenced the subsequent differentiation of this group of cells (see sections A
and D).
In animals sectioned 10 days after transplantation the lateral face of the host
somites in the vicinity of the graft tissue showed an increased population of
basiphilic cells, being 2-4 layers in the graft area as compared to the single layer
present on the control side. Mitotic figures were more evident in the former area
than in the same region of the control flank somites. In cases where the graft
TEXT-FIG. 3. A section through the graft region of an A.punctatum host at 80 days
after the transplantation of T. torosa st. 30 hypomere. The graft side somite
appears confined to a dorsal position and no ventro-lateral musculature has
developed on that side. (Traced from a photograph, approx. x 40.)
tissue was known to have lain diagonally along the flank, the increase in the
lateral face population was seen to follow the region of somite contact with the
donor tissue. These lateral face regions demonstrated, at 20-30 days, dorsoventrally oriented cells, and at 40 days the same area showed dorso-ventrally
oriented striated muscle-cells in some cases or similarly oriented nbroblasts in
other cases (see Plate 2, figs. I, J). It would seem that the initially less differentiated superficial somite cells (Finnegan, 1961c) responded to the presence of
the hypomeric mesoderm by increasing in number and then responded to host
influences by differentiating into myoblasts, particularly in A. punctatum hosts,
but with an orientation at variance with the normal more antero-posterior
myoblast orientation.
When the smaller T. torosa grafts were placed on A. punctatum hosts or when
small A. punctatum grafts were placed on either species, the ventro-lateral
306
C. V. FINNEGAN—POSTGASTRULA
DIFFERENTIATION
myotome penetration was occasionally not completely blocked. Such cases
when examined histologically showed that the orientation of the muscle fibres
in the more superficial portion of the ventro-lateral muscle was not normal.
Typically, in cross-section, the deep layer of this muscle, lying against the somatic hypomere, has fibres directed obliquely ventrad while the more superficial
layer, adjacent to the dermis, does not (see Plate 2,fig.K). In the experimental
area the superficial and the deep portions both have obliquely oriented fibres
(see Plate 2,fig.L). While it is possible that such orientations as these reflect
general unknown operational effects, as suggested by Weiss & James (1955), it
is felt that the close physical association of the dorso-ventrally oriented host
material and the grafted hypomere indicates some influence by the transplanted
tissue in this respect.
D. Nephric differentiation
At the time of transplantation the host's pronephric duct passed along the
ventral edge of the graft area, in close contact with the ectoderm, and continued
posteriorly. As development continues in both species, the pronephric duct and
the closely associated nephrogenic tissue shift medially to a position in the
dorsal somatic mesoderm ventral to the somites. In the majority of the operations performed, an attempt was made to place the transplanted hypomere
tissue dorsal and, if necessary, superficial to the duct and to remove the host
ectoderm, if that was required, so as to leave the duct undisturbed. In several
cases, however, a portion of the duct was inadvertantly removed during the
operation. Both kinds of situation are described below.
TABLE 3
Host nephric response to ventral hypomere transplants
1.
2.
3.
4.
Donor-host
Duct size increase
T. torosa-T. torosa
T. torosa-A. punctatum
A. punctatum-A. punctatum
A. punctatum-T. torosa
2-3 x
lf-2x
2-4 x
Duct branching
Tubule rudiments
±
+
The initial response of the nephric tissue to the graft was an enlargement of
the pronephric duct, during the first 10 days, in the anterior region of the graft
area (see Plate 2,fig.H). This enlargement, which was seen histologically as an
increase in the number of cells per cross-section of the duct, seemed to proceed
posteriorly through the graft area in time (20-30 days) and to spread, less rapidly,
anteriorly toward the host's pronephros. Immediately posterior to the graft
region the duct remained normal in size. In the comparisions of the various
donor host combinations (Table 3) it can be seen that the heteroplastic combinations produced a greater size increase in the host duct than did the homoioplastic transplantations.
OF THE HYPOMERE
307
In the cases with 7". torosa hypomere on T. torosa hosts the enlarged pronephric duct remained more lateral in the region of the graft though it had moved
mediad in the flank areas beyond it. In all other experimental combinations
several cases were found in which the pronephric duct branched anteriorly and
two ducts, one lateral and one medial, traversed the graft region of the host
flank (see Plate 1, fig. A). Both of these ducts were usually enlarged to the same
extent as compared to the host's control flank pronephric duct (see Plate 1,
fig. D). Both donor and host cells formed the lateral duct whether or not it was
the only duct present, while only host cells seemed to be present in the more
medial duct when two branches were present (see Plate 1, fig. A). In A. punctatum hosts the more typical situation was for the enlarged host pronephric duct
to reside in the normal medial location and for a similarly enlarged branch
to extend laterally into the area where ventral somite and graft tissue were
juxtaposed. This branch duct was observed, most frequently, to remain in the
ventral somite region through the length of the graft and then to move medially
in the posterior graft area to rejoin the other nephric duct. In rare cases this
lateral duct was observed to pass dorso-posteriorly along the grafted hypomere,
if that were the distribution of the latter, and to end bhndly in the posterio-dorsal
portion of the graft area. In cases of A. punctatum hypomere on T. torosa hosts
the pronephric duct either produced several short branches, particularly in the
more posterior graft area or, less frequently, branched anteriorly as in the above.
No complete regeneration of the host nephric duct was observed after a portion
had been removed (but see Fraser, 1950) and no attempt at independent nephric
duct formation by donor tissue was evident.
Since the growing tip of the pronephric duct was far posterior to the graft site
at the time of operation (see also Burns, 1955), the above results lead one to conclude that the transplanted hypomere is somehow responsible for stimulating
the host's pronephric duct itself to increase in size and to branch, that is to
regenerate a non-missing duct. The apparent failure of T. torosa nephric duct to
branch when its own tissue was added, taken with the positive response of this
species when the larger mass of A. punctatum hypomere was the graft tissue,
may indicate a threshold phenomenon. Since A. punctatum hosts demonstrated
branching of the duct in response to the smaller mass of T. torosa hypomere, it
may be that the two species differ in the threshold level required to stimulate
branching of the pronephric duct. The greater size increase of the nephric ducts
in the heteroplastic combinations is not explicable as a mass phenomenon but
may represent an additional response of the stimulated host duct to the foreign
tissue.
Nieuwkoop (1947) reported duplication of the nephric duct along a portion of
its length following endoderm removal in Triton and attributed it to fission of the
original duct. The present results, in particular the blind ending of the lateral
supernumerary ducts, suggest that branching (regeneration) has occurred, and
it should be noted that the removal of the endoderm by Nieuwkoop served to
308
C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
bring the normally more ventral hypomere into close proximity to the pronephric duct. As originally demonstrated by Holtfreter (1944), it appears that
the juxtaposition of grafted hypomere and ventral somite tip offers, in addition
to the normal host hypomere-somite region, a favourable territory over which
the pronephric duct may pass and from the tissues of which it may recruit cells
in its enlargement. Holtfreter (1944), reiterating his earlier work, noted that
nephric duct was produced by gastrula hypomere in vitro (see also Finnegan,
1953) and it would seem that, while the capacity for total duct formation may
be lost, the competence to contribute cells to the nephric duct is retained by the
hypomere for some time after neurulation. As a result of vital stain experiments,
Shin-Ike (1953, 1955) has discussed such in situ (segmental) contributions of
the hypomere to the normal development of the pronephric duct in Triturus. It
was noticeable that older hypomere (stage 30), particularly T. torosa hypomere
on A. punctatum hosts, was markedly less effective in this nephric differentiation
than was the hypomere from younger donors.
When the ventro-lateral extension of the post-graft somites is encountered by
the lateral duct in these experimental animals (and perhaps also in normal
development, Liedke, 1958), it moves mediad in the host somatic mesoderm to
the more typical position ventral to the somites. It may be that the mesenchyme
of the experimentally produced region does not block the passage of the extra
duct (Burns, 1955) and that, as suggested by Holtfreter (1944), the endothelial
elements developed in this area are influential in directing, by selective adhesion,
the course of the supernumerary duct.
Both when T. torosa was the host and when T. torosa hypomere was grafted
to A. punctatum hosts, there appeared mesonephric tubule rudiments in the host
tissue adjacent to the graft-nephric duct boundary and usually anterior to the
normal location of these rudiments on control and graft flanks of the hosts.
These rudiments were present at 20 days as small cell groups (see Plate 1, figs.
A, D) collected along this region of the nephric duct (or branch) and later they
appeared as tubule units (see Plate 1, fig. E) resembling, in kind, the more
posterior early mesonephric tubule rudiments of the host (Gray, 1932). Nieuwkoop (1948) noted a more anterior production of mesonephric tubule rudiments
in Ambystoma following removal of the pronephros, which he discussed as
indicative of a possible pronephric inhibition of such development. As far as
could be ascertained in the present results only host tissue formed these supernumerary rudiments (see Plate 1,fig.E). Fales (1935) observed that neurula
hypomere of Ambystoma placed in the area of excised pronephroi did not form
tubules though Yamada (1937) obtained tubule formation by mid-neurula
(stage 14) hypomere in Triturus. When the nephric duct was doubled in the graft
area, the supernumerary mesonephric rudiments were associated with the lateral
branch while the normal host tubule rudiments were associated with the
medial pronephric duct at a more posterior level. Nieuwkoop (1948) showed that
the induction of the mesonephric tubules by the pronephric duct in Urodeles
OF THE HYPOMERE
309
occurs some time after the duct has grown posteriorly; that is, a delayed induction takes place (see also Fraser, 1950; Burns, 1955). Thus, the tubule induction
observed in the present experiments probably would have taken place concomitantly with, or following, the size increase and branching of the host
nephric duct. However, the induced tubules, in all cases, were of a similar size
to the host's early mesonephric rudiments in spite of the enlarged duct in their
vicinity, an indication of the control of this histogenesis by the responding
tissue (see Text-fig. 4).
TEXT-FIG. 4. A portion of a section of the somite tip region of an A. punctatum
host made 80 days after the transplantation of T. torosa st. 30 hypomere. The
enlarged nephric duct lies in the donor tissue to the right and a solid tubule rudiment is present in the host tissue to the left. The myogenic somite area of the host
lies to the far left in the illustration. (Traced from a photograph, approx. x 320.)
These accessory mesonephric tubule rudiments were found in the ventral
somite tip region and, in rare cases, dorso-posteriorly along the lateral face of
the somite (see Plate 1, fig. E). These observations would seem to indicate that
the pronephric duct under the local conditions imposed by the addition of the
hypomere, may induce a tubule response in closely associated but normally
non-nephrogenic host mesoderm. Gruenwald (1942) noted that, in older chick
embryos, the tubule-forming capacity of the mesoderm, following nephric duct
induction, probably exceeded the actual nephrogenic material by very little.
Burns (1955), in reviewing experimental work analysing mesonephric development, concluded that the nephrogenic field or inductor will convert or induce
a variety of tissue to nephric differentiation through mid-neurula stages while
Fraser (1950) concluded that indifferent nephrogenic material becomes determined at some unknown point between neurula and tail-bud stages in Urodeles.
Perhaps it is that, in the present experiments, the transplanted T. torosa hypomere has created local conditions; that is, it has acted synergistically, so that the
group of normally somitic but, at the time of the operation (host stage 30),
310
C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
relatively undifferentiated cells (Finnegan, 1961c) has responded to the subsequent tubule induction by the pronephric duct whereas the inhibited (Nieuwkoop, 1948) or the determined (Fraser, 1950) nephrogenic material of this level
of the host trunk has not. In respect to this somite response it is interesting to
note that Yamada (1937) obtained pronephric duct and tubule formation when
somite mesoderm and hypomere were similarly closely associated following
ventral transplantation of the somites from postgastrula stages.
In those cases in which the host nephric duct had been removed and failed to
regenerate there were found, in both host species, mesonephric tubule rudiments
in the normal location on both flanks. However, the rudiments on the graft
side were always solid units, less well-organized than the tubular control-side
rudiments associated with a complete pronephric duct. Such experimental results
have been frequently reported (see Burns, 1955) and this apparent degeneration
of tubule rudiments may reflect the prior mesonephric induction by neural
tissue or the original host duct; an induction which is unsustained in the
absence of continued nephric duct influence or of function.
SUMMARY
1. An experimental analysis of the competence of the Urodele hypomere for
different kinds of mesodermal histogenesis was continued by heteroplastic and
homoioplastic transplantations of portions of neurula and tail-bud trunk hypomere to a position overlying the somitic mesoderm in Ambystoma punctatum and
Taricha torosa.
2. The major portion of the transplanted hypomere differentiated, in association with the overlying ectoderm (either host or donor), into a loose connectivetissue dermis. The specific characteristics of this connective tissue differed with
the age of the donor tissue (older hypomere more rapidly developed the associated homogeneous ground substance) and with the species of the host (all graft
tissue on A. punctatum hosts developed more rapidly the loose connective-tissue
dermis and then, often, a fibrous connective tissue was formed).
3. The association of donor tissue and host tissue in the ventral somite tip
region resulted in host-donor responses which involved both tissues and which
gave evidence of further histogenetic competence on the part of the hypomere.
4. Apparently in response to influences of the ventral somite tip, the immediately superficial donor cells increased in number (by mitotic activity and aggregation) and developed an outgrowth from this 'limb-bud' mass. Younger ventral
hypomere demonstrated greater histogenetic competence in responding to this
host influence than did the older hypomere and, in T. torosa, the dorsal hypomere was more competent in this respect than was the ventral hypomere. It was
also shown that the development of the outgrowth would not occur in the
absence of the superficial somite tissue of the ventral myotome even though an
initial mass of donor cells might be produced.
OF THE HYPOMERE
311
5. Possibly in response to stimulation by the grafted hypomere, the host's
pronephric duct in the graft region increased in size (number of cells constituting
the duct wall) and branched (regenerated) with the supernumerary nephric duct
passing posteriad along the experimentally created hypomere-somite boundary.
Both host and donor cells seemed to be incorporated into this new duct.
6. Responding to tubule induction by the supernumerary pronephric duct,
the closely associated ventral somite cells attempted development of mesonephric tubule rudiments. These supernumerary rudiments were more anterior
than the normal host rudiments and were noticeably less well differentiated.
7. Such experimentally produced developmental associations deprived the
host trunk myotomes of their ventral tips, which resulted in a lack of ventrolateral myotome extension and thus in the subsequent failure of ventro-lateral
musculature to appear in the graft region of the host trunk.
RESUME
Analyse de la differentiation post-gastruleenne de Vhypomere — 777—La competence histogenetique de Vhypomere d'Urodele en transplantation heterotopique
1. On a poursuivi Panalyse experimentale de la competence de l'hypomere
d'Urodele pour divers types d'histogenese mesodermique, a l'aide de transplantations heteroplastiques et homeoplastiques de portions d'hypomere troncal
de neurula et de bourgeon caudal, en position sus-jacente au mesoderme somitique, chez Ambystoma punctatum et Taricha torosa.
2. La plus grande partie de l'hypomere transplants s'est differenciee, en
association avec l'ectoderme sus-jacent (de l'hote ou du donneur), en un tissu
dermique conjonctif lache. Les caracteres specifiques de ce tissu conjonctif
variaient selon l'age du tissu du donneur (l'hypomere age formait plus rapidement la substance fondamentale homogene associee) et selon l'espece-hote (tous
les tissus greffes sur A. punctatum ont forme plus rapidement le tissu conjonctif
dermique lache, puis, souvent, un tissu conjonctif fibreux).
3. L'association des tissus du donneur et de l'hote dans la region ventrale de
l'extremite des somites a provoque des reactions hote-donneur impliquant les
deux tissus, et a montre l'existence d'une competence histogenetique supplementaire de la part de l'hypomere.
4. Apparemment, en reponse aux influences emanant de l'extremite ventrale
des somites, les cellules du donneur, immediatement superficielles, ont augmente leur nombre (par activite mitotique et agregation) et ont forme une
excroissance a partir de cette masse de 'bourgeon de membre'. L'hypomere
ventral le plus jeune a presente une competence histogenetique plus grande, en
reagissant a cette influence de l'hote, que ne l'a fait l'hypomere age, et chez
T. torosa l'hypomere dorsal etait plus competent a cet egard que ne l'etait l'hypomere ventral. On a montre aussi que le developpement de l'excroissance n'avait
pas lieu en l'absence de tissu somitique superficiel du myotome ventral, meme si
une masse initiale de cellules du donneur pouvait se former.
5584.10
X
312
C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION
5. Peut-etre en reponse a une stimulation par l'hypomere greffe, le canal
pronephretique de l'hote, dans la region greffee, a augmente de taille (accroissement du nombre des cellules formant la paroi du canal) et s'est bifurque (regenere), le canal surnumeraire passant en direction posterieure le long de la limite
hypomere-somite creee experimentalement. Les cellules provenant a la fois de
l'hote et du donneur, semblaient incorporees dans ce nouveau canal.
6. Repondant a l'induction de tubules par le canal pronephretique surnumeraire, les cellules somitiques ventrales etroitement associees ont manifeste une
tentative de formation d'ebauches de tubules mesonephretiques. Ces ebauches
surnumeraires etaient plus anterieures que les ebauches normales de l'hote et
notablement moins bien differenciees.
7. De telles associations embryogenetiques produites experimentalement ont
prive les myotomes troncaux de l'hote de leur extremite ventrale, ce qui a provoque une absence d'extension latero-ventrale des myotomes; ainsi, la musculature ventro-laterale n'est pas apparue ensuite dans la region troncale de l'hote
portant le greffon.
ACKNOWLEDGEMENTS
This investigation was supported in part by grants from the U.S. National
Institutes of Health (RG-6178) and the National Research Council of Canada.
Grateful acknowledgement is made of the assistance of Dr. Peter Larkin in the
statistical analysis.
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C. V. F I N N E G A N
E X P L A N A T I O N OF PLATES
PLATE 1
All the photographs are oriented with the dorsal surface of the host toward the top of the page and,
except for fig. E, with the median axis toward the centre of the page. Abbreviations: N, notochord.
NT, neural tube. All figures approximately x 240.
FIG. A. A section through a somite in the graft region of the host's trunk. The donor cells are
observed to the left beneath the ectoderm and in the lower left in association with an enlarged nephric
duct. Note the two sizes of nuclei in this accessory duct. An accessory nephric rudiment lies between
the two nephric ducts. Compare with the ventral somite tip region of fig. B. (T.t.-A.p.; st. 20-20 days.)
FIG. B. A section through a somite of the control side of the host in the identical trunk region of fig.
A. Note the group of basiphilic cells of the ventral tip of this somite and the position of the pronephric
duct some distance medial to this cell population. (T.t.-A.p.; st. 20-20 days.)
FIG. C. A section through a graft of dorsal hypomere showing a mass of donor cells situated immediately superficial to the host's ventral somite tip and with a differentiating cartilage element rising
dorso-posterior from this point. Compare with fig. F. (A.p.-T.t.; st. 20-20 days.)
FIG. D. A section through the graft region in the vicinity of the ventral somite tip. An enlarged host
nephric duct (to the left) and an enlarged accessory duct (to the right) are observed with associated
basiphilic cell groups between the two ducts and dorsal to the accessory duct. (A.p.-T.t.; st. 20-20 days.)
FIG. E. A section through the graft region lateral to the host's somite with the host axial structures
off to the upper left. The donor tissue (to the right) has differentiated as a thick dermis and the lateral
aspect of the host's somite (in the centre of the figure) is occupied by apparent mesonephric tubule
rudiments. (T.t.-T.t.; st. 20-30 days.)
FIG. F. In this section through the graft region a cartilage rod, with a small amount of striated
muscle located superficial to it, is surrounded by a fibrous connective-tissue dermis apparently of
donor origin. The control side dorsal somite area can be seen dorsal to the spinal cord while the graft
side somite appears reduced, at least in respect to its myogenic portion. (A.p.-T.t.; st. 20-60 days.)
PLATE 2
All the figures are oriented with the dorsal aspect of the host animal toward the top of the page.
FIG. G. A dorsal mesenchyme-filled projection exemplifying the type of structure classified in this
category. In the original this projection was continuous anteriorly with a mass of basiphilic cells which
ultimately terminated in the vicinity of the host's ventral somite tip, as in Plate 1, fig. C. (A.p.-T.t.;
st. 20-20 days.) (Approx. x 30.)
FIG. H. A section through the anterior graft area in which the initial branching of the enlarged host
pronephric duct is visible. The nephrogenic tissue is continuous around the ventral somite tip and
along the lateral somite face. (A.p.-T.t.; st. 20-10 days.) (Approx. x240.)
FIG. I. A section through a graft region showing the more lateral aspect of the host's somite and
with the host axial structures to the left. The lateral face cells of the somite and the superficially
located donor cells show a dorso-ventral orientation while the deeper somite cells demonstrate a more
antero-posterior orientation. (A.p.-T.t.; st. 30-30 days.) (Approx. x240.)
FIG. J. A section through a graft area which demonstrates the dorso-ventrally oriented muscle-cells
of the lateral face region of an affected somite (to the right). (A.p.-A.p.; st. 25-30 days.) (Approx. x 30.)
FIG. K. An enlargement of a section through the ventro-lateral musculature of the control side of
a host animal. Only the deep portion of this musculature demonstrates an obliquely ventral orientation
of its fibres. (A.p.-A.p.; st. 25-30 days.) (Approx. x240.)
FIG. L. An enlargement of the graft side ventro-lateral musculature in the identical section of fig. K.
Both the deep and the superficial layers demonstrate an obliquely ventral orientation to the muscle
fibres. (A.p.-A.p.; st. 25-30 days.) (Approx. X240.)
{Manuscript received 21: xi: 61)
Vol. 10, Part 3
J. Embryol. exp. Morph.
C. V. FINNEGAN
Plate 1
J. Embryol. exp. Morph.
Vol. 10, Part 3
C. V. FINNEGAN
Plate 2