RECONSTRUCTION OF SINGLE COMB
FROM DISSOCIATED CELLS
ON THE CHORIOALLANTOIC MEMBRANE
A Thesis
Presented to
the Faculty of the Department of Biology
East Carolina College
In Partial Fulfillment
of the Reguirements for the Degree
Master of Arts in Biology
by
Rosalie Marie Vogel
May 1967
V’Sélr
c. 1
RECONSTRUCTION OF SINGLE COMB
FROM DISSOCIATED CELLS
ON THE CHORIOALLANTOIC MEMBRANE
by
Rosalie Marie Vogel
APPROVED BY:
SUPERVISOR OF THESIS
CHAIRMAN OF DEPARTMENT
DEAN OF THE GRADUATE SCHOOL
./
243409
ABSTRACT
Reaggregation of Ii/hite Leghorn single comb cells was studied in
culture. Entire comb organs from 8 and 10 day incubated chicks were
excised and dissociated in a calcium-magnesium-free trypsin solution.
The dissociated cells were washed in Tyrode's solution, centrifuged,
transferred by pipette to the chorioallantois of host chicks, and
developed for approximately 10 days.
Some comb structure was reconstructed in the grafts. Two organ
patterns formed; elongate ridge and irregular points. Several tissues
developed. Typical comb epidermis formed in many grafts. Dense regular
dermis and a comb-like vascular pattern formed in all grafts. Loose
irregular tissue was frequent. Several grafts formed non-comb tissues
of cartilage and bone.
Tissue interactions between epidermis and dermis and between
cartilage and dermis were apparent. Where thin epidermis formed on the
free margin of the comb ridge, underlying dermis was usually organized
in typical comb pattern. Cartilage formed an elongate rod, and when
present, comb ridge was prominent and the vascular and dermal patterns
were most typical. There was evidence that mass of tissue was significant:
cartilage formed in all whole comb cellular suspensions, but was missing
in most of the partial combs.
Ccmb mesenchymal cells possess some pleuripotency; a capacity
expressed in formation of cartilage and bone. Pleuripotency did not
include feather papillae. Since comb is an integumentary derivative,
the lack of feather formation in any of the grafts is considered
significant.
/
ACKNOWLEDGEMENTS
The writer would like to express her sincere appreciation to the
following people for their unselfish assistance in this study:
To Dr. Graham J. Davis, who made it possible for the writer to
attend graduate school and who served on my committee;
To Dr. Irvin E. Lawrence, Jr., who served as advisor for this
study and offered guidance and much patience;
To Dr. Joseph G. Boyette for his encouragement and confidence as
my friend;
To Mr. David G. Cargo, of the Chesapeake Biological Laboratory
at Solomons, Maryland, and Dr. Leonard Schultz, of the Smith-
sonian Institution, for their encouragement and assistance;
To the entire staff of the Biology Department for their guidance
and ideas;
To all the graduate students that have offered their encouragement;
To Eunice Dorsey, my typist, and to Mark Stern, Dave Hardy, and
Frank Goodwyn, my photographers; and
To my parents, Mr. and Mrs. William J. Vogel.
TABLE OF CONTENTS
Page
INTRODUCTION 1
REVIEW OF LITERATURE 4
METHOD AND MATERIALS 7
RESULTS 10
DISCUSSION 15
SUMMARY 20
REFERENCES CITED 21
LIST OF ILLUSTRATIONS
Page
PlatIII eII sI 22Figure1 A-C Graphic Reconstructions of grafts. Lateral view 222 A-C Graphic Reconstructions of grafts. Vertical view 2223Figure3 Graft Number 38. Section through middle of graft. 244 Graft Number 38. Basal area near end of graft. 245 Graft Number 38. Entire section near end of graft. 246 Graft Number 38. Free margin of Figure 4. 247 Graft Number 38. Entire section opposite end ofgraft. 2425
Figure
8 Graft Number 93. Section through one end of graft. 26
9 Graft Number 93. Section through other level of
graft. 26
10 Graft Number 85. Section near center of graft. 26
11 Graft Number 85. Over all section of graft. 26
12 Graft Number 192. Section near center of graft. 26
13 Graft Number 73. Section of cartilage rod. 26
14 Graft Number 143. Ridge formed in graft. 26
INTRODUCTION
The purpose of this study was to find out if the phenomenon of
aggregation of embryonic chick comb would occur and to what extent the
geometric pattern of comb organogenesis could be reconstructed.
I used a modification of Moscona's experimental technique to study
aggregation of single comb cell suspension on the chorioallantoic mem-
brane (Moscona, 1952). A specific objective was to determine whether
dissociated and isolated comb cells from 9 and 10 day embryos would ag-
gregate to form comb.
The form of the single comb is basically a dorsoventrally symmet-
rical ridge. The highest part of the ridge is found in the posterior
blade. From this peak, the ridge decreases in size in a series of
points anteriorly. The lowest section of the ridge is an anterior un-
serrated division. Histologically, typical comb epidermis is thin and
without feathers and can be distinguished from the surrounding epider-
mis. The dermis is chiefly fibrous and consists of three strata. The
fibers are most compact in the peripheral layer and are loose and more
random in the center (Lawrence, 1963). These morphological patterns
are easily recognized, and results from cellular aggregation experi-
ments should lend themselves to reasonable interpretation.
Organogenesis is brought about by a series of genetically deter-
mined interactions which control characteristic tissue arrangements of
a given organ. Organ-specific regional environments develop which
determine, in part, the nature of the developing cells. The cells be-
come determined as specific types at certain stages in development;
2
and, following this, environmental influences may become less pronounced.
If these cells are transplanted to another area on the embryo at an
older stage, they will develop into a "determined" type and assume organ
specific tissue patterns. Whole organs may self-differentiate in un-
characteristic areas.
Several related experiments have been performed to determine wheth-
er a group of cells transplanted as an intact tissue contained an extra-
cellular determinant which may cause characteristic organ formation, or
whether each individual cell contained an intracellular determinant
which may cause characteristic organ formation. For example, H. V.
Wilson (1908) dissociated adult sponge cells by passing them through silk
bolting. In culture, these sponge cells clump together and aggregate as
small sponges with epidermis, collar cell chambers, and archeocytes, in
the same relative positions as in the original sponge.
Later dissociation studies have included vertebrate embryos. Dis-
sociation of tissue cells can be brought about in various ways: 1) me-
chanical, by straining through silk or nylon or by forcing the cells a-
part with a mortar and pestle; 2) chemical, by raising the pH of the
surrounding medium which results in the breaking of the connections
between the cells; and 3) enzymatical, by means of a digestive enzyme
which destroys intercellular bonds (Weiss and Taylor, 1960).
If the cells of an amphibian embryo in the late blástula or early
gastrula stage are dissociated and isolated in culture, the presumptive
ectoderm, mesoderm, and endoderm, will assume characteristic positions.
A. A. Moscona (1952) was a pioneer in enzymatical dissociation of
embryonic cells. He successfully used trypsin. He found that briefly
3
exposed cells will not be damaged even in relatively large concentrations
of the enzyme. At first he placed the cells in in vitro culture where
aggregation occurred without obvious outward stimulation, and found that
the cell numbers must be of a critical mass before they would aggregate.
This was also observed by Weiss and Taylor (1960). Rotating flasks were
used to speed clumping of the cells to the critical mass for aggregation.
This method improves reproducibility and consistency of results (Moscona,
1961a). The cellular mass in in vitro cultures would reach a certain
size and then cease to grow. Cessation of growth was attributed to a
lack of vascularity in the tissues (Weiss and Taylor, 1960; and Moscona,
1961b) so in vivo culture was tried. The chorioallantoic membrane of
host chick embryos can be used to provide a vascular system sufficient
for further growth on a relatively indifferent medium (Hamburger, 1966).
In this case dissociated cells are centrifuged to form a pellet of cells
before they are grafted to the membrane. This technique has been used
in studying several vertebrate organs.
REVIEW OF LITERATURE
The capacity of cells to aggregate after partial or complete dis-
sociation has been investigated extensively. It is well established
that the organs of certain invertebrates can be broken down and the sep-
arated cells will associate into characteristic structures (Weiss and
Taylor, 1960). Wilson (1908) pioneered such work with his studies of
sponges. The capacity to aggregate is found in vertebrates also. Holt-
fréter (1948) observed that early amphibian embryonic cells would aggre-
gate in the same relative positions as in whole embryos. Furthermore,
it was found that cellular suspensions of chick and mannal tissue would
form characteristic patterns (Moscona, 1952; and Weiss and Andres, 1952).
Invertebrate and amphibian cellular aggregates are able to maintain
themselves in isolation for some length of time as self-contained units
(invertebrates) or with stored yolk providing nutrients (amphibians)
(A. Moscona and H. Moscona, 1952). The Mosconas found that work that is
done with chick and mammal embryos requires more exacting technique as
the nutritional and thermal conditions have to be supplied by the experi-
menter. Weiss and Andres (1952) met the requirements for dissociated
chick embryos by injecting mechanically separated cells into the vascular
route of host embryos. These cells settled in various places along the
route and continued to differentiate. Some of the aggregates formed were
located by the color difference in feathers. In these early studies,
whole embryos were used for their cellular suspensions and various types
of tissues were obtained in the aggregates. Enzymatical dissociation of
cells from homiothermic animals was employed by Moscona (1952). He
5
tested hyaluronidase for dissociation of the cells, but it brought about
irreversible abnormalities in a large number of the cells. Trypsin did
not attack the cells even in relatively large concentrations so this
enzyme was used in his later studies. With enzyme dissociation, the num-
ber of viable cells in a cellular suspension was increased. He used a
calcium-and magnesium-free Tyrode's solution with 1% salt-free trypsin
and found that the absence of calcium and magnesium salts decreases the
stability of the intercellular materials and the mutual adherence of
cells. The digestive enzyme, trypsin, in a calcium-and magnesium-free
saline solution, attacks selectively the peptide linkage adjacent to
lysil and arginyl residues (Moscona, 1963). The procedure of disso-
ciation is effective in most cases for chick embryos up to about 10 days.
After this time, the basement membrane becomes resistant to the trypsin
and another enzyme is needed (Grover, 1960). Collagenase may be used for
basement membrane breakdown.
In his initial studies. Moscona cultured the tissues in a hanging
drop (Moscona, 1952; and A. Moscona and H. Moscona, 1952) and the cells
formed self-aggregates. The technique was later improved by the use of
rotating flasks so that the cells could be concentrated (Moscona, 1961a).
Still later the membrane of host chick embryos was used. The chorio-
allantois provided a vascular supply which supported grafts for longer
periods on a relatively indifferent medium (Hamburger, 1966; Weiss and
Taylor, 1960; and Garber and Moscona, 1964).
The first studies Moscona performed were on limb bud rudiments in
four-day chicks. He later worked with mesonephros and skin. From the
studies, he found that the dissociated cells would aggregate to form
6
characteristic tissues (Moscona, 1952; and A. Moscona and H. Moscona,
1952).
The principle of reassociation was carried over to older chick em-
bryos. It is now apparent that dissociated tissue from 7-14 day chicks
will reassociate by cell migration and selective adhesion to form char-
acteristic patterns (Weiss and James, 1955; and Steinberg, 1963).
Steinberg states the cells regroup each with others allied to it and re-
construct the various original tissues.
In an effort to find out whether a particular group of cells will
combine with cells from other tissues. Moscona used tissues from Swiss
albino mice as markers. The mouse cells have a larger nucleus and stain
differently from chick cells (Moscona, 1957). If cellular suspensions
of like organ rudiments of chick and mouse embryos are intermixed, the
cultures will aggregate and combine to form chimeric tissues. Moscona
finds that aggregates skin from chick and mouse embryos will form
feathers and hair respectively. In co-aggregates of mouse and chick
cells, however, the feathers are suppressed but hair is not (Garber and
Moscona, 1964; and H. Moscona and A. Moscona, 1965). Recent studies by
Moscona (1967) indicate that age is critical for feather formation in
the chimeric aggregates. The structure developing from cellular suspen-
sions form recognizable morphology rather than a chaotic assemblage of
cells (Balinsky, 1964). Furthermore, Weiss and Taylor (1960) find evi-
dence of aggregated liver and kidney tissues being functional. Such
studies indicate controlled aggregation takes place which results in a
characteristic pattern.
METHOD AND MATERIALS
The White Leghorn breed was used in this study. Eggs were obtained
directly from the Castlebury Poultry Farm, Apex, North Carolina. Many
of the eggs were stored for short intervals at before incubating.
A 600 egg capacity cabinet incubator was used. Average dry bulb temper-
ature was while wet bulb readings ranged from 30.0 - 30.5°C.
These are optimal value and were recommended by the manufacturer of the
incubator.
The technique for dissociation followed the standard procedure des-
cri bed by Moscona (1952) with some modifications. A one percent trypsin
solution was made, using crystalline 2x salt-free trypsin (obtained from
Nutritional Biochemicals Corporation, Cleveland, Ohio) in calcium-and
magnesium-free (CMF) Tyrode's solution. This solution was warmed to in-
cubation temperature before using.
Eggs were incubated for nine or ten days, which allows the embryos
to develop to stage 36 or 37 (Hamburger and Hamilton, 1951). At these
stages, the comb ridge is conspicuous and the points are visible. Be-
fore operation, the eggs were candled and were divided into donor and
host groups. Initially the grouping was arranged to insure a host em-
bryo for every donor. Later one donor was used for every four or six
hosts. The donor eggs were prepared by opening them into a medium size
finger bowl and removing the extraembryonic membranes from around the
embryo. The exposed embryos were than placed in a watch glass contain-
ing warm, sterile 0.86% NaCL solution and staged. Watchmaker's forceps
were used to hold the embryo in position during the operation. The comb
8
organ was removed with iridectomy scissors by cutting through the skin
posteriorly and anteriorly to the ridge, and then laterally along each
side. The entire organ was severed from adjacent tissues by making a
fifth and last incision at right angles to and underneath the comb
ridge. The excised organ was removed to a dish, minced with the seis-
sors and placed in a small sterilized test tube which contained approx-
imately 2 ml of 1% trypsin in CMF Tyrode's solution. The cells were
then incubated at VJ°Z in the trypsin for 30-45 minutes or until the
cells were dissociated. The tubes were shaken at intervals and the
medium agitated by rapidly pipetting it a number of times. Occasionally
a portion of the cellular mass was examined under the microscope to con-
firm cellular dissociation.
The tubes containing dissociated comb cells were lightly centrifuged
using an International Clinical Centrifuge to concentrate the cells. The
trypsin solution was decanted and the cells washed with complete Tyrode's
solution (Davenport, 1960) to remove any remaining enzyme. The cells
were washed in repeated changes of Tyrode's ten times, centrifuging and
decanting the solution after each wash. Following the washing, the tubes
containing the dissociated cells were placed in the incubator no longer
than 15-30 minutes while the hosts were prepared.
The host chicks were exposed by cutting a circular opening in the
shell above the embryo using a drill provided with a circular abrasive
dental disc. The shell membranes over the air sac were punctured to
cause the embryo to settle to a lower place inside the shell. This al-
lowed more room beneath the opening for the donor cells to develop
(Wenger, 1951). The dissociated clump of donor cells was then transferred
9
from the tube to the chorioallantoic membrane by means of the standard
pipette. The opening through the host membranes was then covered with
a sterile, circular cover glass and sealed with melted paraffin. The
host egg was identified by a number and returned to the incubator for
eight or nine more days. Chorioallantoic grafts were removed at around
nineteen days incubation before the host membranes started to dry prior
to hatching. Some of the comb grafts were transferred to a second ten
day host for another nine day period of development. These cellular
masses were permitted to grow for a more extended length of time in
order to develop larger grafts.
The grafts were excised at the termination of development and
placed in Bouin's fixative. Following fixation, the grafts were stained
with iron hematoxylin in toto (Wenger, 1951), embedded in paraffin, sec-
tioned at 10 microns, and mounted with Permount for histological obser-
vation. Graphic reconstructions were made by projecting the histological
sections with a bioscope and marking the vertical and horizontal dimen-
sions for every fifth section. Finally, photomicrographs were taken of
representative tissues.
RESULTS
Table 1 presents a summary of the experimental operations performed
during the investigation. Grafts were made of whole comb and partial
comb cellular aggregations. Host and graft survival was greater in the
cases where only a portion of the tissue was used. A whole comb was ap-
parently too large for some host embryos to support. Table 1 shows that
of 39 sectioned grafts, 25 developed comb-like tissue or comb organ.
Some comb organ structure was recognizable in all grafts and usually con-
sisted of an elongate ridge with some irregular surface projections.
Five other grafts were not sectioned. The remaining 29 survivors did not
yield successful grafts.
Table 2 presents a tissue analysis of the 25 sectioned grafts of ag-
gregated comb. Vascularity and dense regular dermis are characteristic
of comb and were used as criteria in establishing the presence or absence
of comb. In all cases, dermis with dense regular organization and a
vascular pattern were seen. Thin comb-like epidermis developed in 17 of
the grafts. Generally, where characteristic epidermis formed, the dermal
pattern was more organized and was similar to the dense regular tissue of
the peripheral layer in the normal comb (Plate III, Figure 10). Some
non-comb tissues differentiated; these were two grafts with bone, six
with cartilage, twelve with reticular, and ten with loose irregular con-
nective tissue. However, the loose irregular tissue resembled, in pat-
tern, that tissue forming the central layer of normal comb. No feather
papillae were noted in any of the comb skin grafts.
Table 1. Summary of Experimental Operations on Dissociated Comb
Amount Number Host survival Number of Sectioned
of Comb of graftsgrafts number % grafts number number
recovered sectioned with comb
Whole comb 88 16 18.2 10 5 2
Portions
of comb 103 57 55.3 34 34 23
Totals 191 73 38.2 44 39 25
12
Table 2. Tissue Analysis of Sectioned Grafts of Aggregated Comb
Differentiated Tissue
Code Bone Carti- Dermis Dermal organization Epidermis
Number lage comb-like dense irr. loose
Complete
comb
27 - + + + + +
38 + + + + + +
Partial
comb
73 + + +
76
79 +
80 +
81 +
84 + +
85 +
90 + + +
93 + +
94 + +
96 +
100 + +
104 + +
no
114 +
132
133 +
140 +
142 +
143 + +
191 + +
192 + +
196 + + +
Totals 2 6 25 25 12 10 17
13
Plate I presents graphic reconstructions of three of the grafts.
Figures 1-a, b, and c show the lateral views and Figures 2-a, b, and c
are diagrams of partial comb grafts. A comb ridge was seen in all three
cases. The ridge is irregular and contains projections which may be
comparable to points found on normal comb.
Plate II contains photomicrographs of graft Number 38. The levels
of the photographs on the tissue are indicated in Figures la and 2a. Un-
like normal comb, the dense dermis is central in position with loose ma-
terial on the periphery. Figures 4 and 6 show different levels taken of
the same tissue section from graft Number 38. A cartilage rod is under
the middle portion of the graft but does not extend the entire length of
the graft. Bone is near the center of the section (note Figure 5 also).
Bone and cartilage were found in close proximity in the two cases where
bone occurred. Figures 3, 5, 6, and 7 show the projecting free surface
of the graft and clearly show dermis as a core in the center of the
graft. A very distinct epidermis is present. In spite of the fact that
the dermal layers are reversed, there is no lack of organization even in
these layers. Figure 5 is of a complete tissue section from the middle
of the graft and the presence of the cartilaginous rod is conspicuous.
On the other hand. Figure 7 is from a section near one end of the graft
and the cartilage rod has almost disappeared. There was no cartilage
beyond this point.
Plate III and Figures 8 and 9 are photomicrographs of graft Number
93. The levels of these photographs are given in Figures Ic and 2c.
There is a distinct comb-like pattern of the tissues in this case with
the dense regular dermis being found outside the loose irregular and
14
vascular tissue in the center. Though typical epidermis is not present,
the aggregate is similar to normal comb in the pattern of dermal tissues.
Plate III and Figure 12 was taken of graft Number 92. This specimen also
shows organization of tissue that is characteristic of comb. In graft
Number 92, typical comb epidermis is present. Organization of dermis is
more regular in the comb-like process of graft Number 85 (Plate III,
Figures 10 and 11). In this specimen, typical comb-like epidermis covers
the free surface.
Plate III and Figure 13 shows the cartilage rod that formed in graft
Number 73. As noted above, this rod does not extend the entire length
of the graft. Graft Number 143 is shown in Figure 14 (Plate III). A
definite ridge with a dermal pattern typical of comb formed. Cartilage
was present in a basal position throughout most of the graft.
DISCUSSION
The material presented in this study indicates that dissociated
single chick comb cells cultured in vivo will aggregate to form comb-
like tissue and some comb organ structure.
Comb-like dense regular dermis and vascular pattern were recognized
in all of the sectioned grafts. The consistency in the formation of these
tissues can be correlated with development of definitive structure. In
typical comb, the most obvious histological feature is seen in the organ-
ization of dense dermis in a superficial layer. The vascular network of
the central layer is also pronounced. Less apparent is the loose irreg-
ular tissue in the intermediate layer. In the grafts, the underlying
dermis was most organized where a thin comb-like epidermis was present.
The regular alignment of these dermal cells and fibers is so striking as
to suggest the force of some important interaction between the tissue
layers. Even without epidermis, the aggregates reveal the strength of
some regulative capacity among comb cells, especially in the establish-
ment of dense regular dermis.
The dermal pattern of normal comb with dense regular tissue in the
periphery and loose irregular in the center was seen in most of the
grafts. In graft Number 38 (Plate II, Figures 3, 5, 6, and 7), the exact
opposite arrangement was present. Even here, however, there was some
formative interaction for comb organization. Several comb-like patterns
were noted in formation of an elongate ridge, a cartilaginous rod, a
dense dermis, and in vascularity. All of these tissues formed in close
association as if in an organizing center with controlled interaction.
16
Only comb tissue was excised in these experimental proceedures, and
that a variety of tissues formed is interesting. Certainly, the presence
of bone, cartilage, and reticulum indicates that comb dermal cells are
pleuripotent and can differentiate into tissues that are different from
comb and skin. The comb dermis is derived from mesenchyme. And, in
general, mesenchyme is considered to be on a low level of differentiat-
ion and is found in adult formative areas of bone, cartilage, and retie-
ular tissue. Perhaps this explains, in part, the occurrence of some di-
verse tissues in the grafts.
The fact that aggregated comb tissues are orderly and identifiable
indicates a certain amount of selectivity among the cells. In the grafts
that contained cartilage, only a single cartilage rod developed. If some
selectivity or organizing influence had not been operating, several
patches of cartilage might have been formed throughout the grafts. Fur-
thermore, aggregation into an elongate structure that conformed to comb
ridge rather than into an oval or spherical shape shows complex control
in origin of geometric pattern. The latter control may be one of
axiation or polarity.
It is to be noted that cartilage developed only in the middle
region of the combs and not under the anterior-and posterior-most por-
tions. Furthermore, the two whole comb grafts had cartilage, but carti-
lage was missing in all of the partial combs except four. In both situa-
tions, the results may be due to the number of cells used in the grafts:
the cells may not have been sufficiently populous to initiate cartilage
differentiation. The cartilaginous rod formed in these grafts is similar
to the one noted by Lawrence (1963) in undissociated primordial grafts of
17
comb cultured on the chorioallantois. In the primordial grafts the car-
til age was thought to be derived from the frontonasal system, perhaps
ethmoidal in origin. Certainly this is not the case in these aggregate
grafts, though it may be assumed that low differentiated mesenchymal
cells along the basal surface of the comb could have been included in the
excisions.
There is some correlation between prominence of comb ridge and the
development of the underlying cartilage rod. Though this rod is not
necessary for support of the comb as seen with development in most of
the grafts, it appears to have some organizing influence in comb ridge
formation. Characteristically, the comb organ is a surface protrusion
that describes an elongate ridge and it is important that many of the
aggregates formed a recognizable ridge. In association with epidermis
and cartilage, development of ridges was most distinct. As noted above,
dermal differentiation was also more typical in these combs. The inter-
actions that apparently occur between development of cartilage and dermis
and between development of epidermis and dermis may be very important in
formation of definitive pattern under both dissociated and undissociated
cellular states.
Some irregularities are seen along the comb ridge in the graphic re-
constructions. These may be points. If so, they are not arranged in a
regional pattern of distribution. Lawrence (1963) found that comb grafts
which grew on the chorioallantois were compressed and points were atypi-
cal. In more recent studies, he found that comb would not elongate with-
out beak (Lawrence, 1964). Combs that do not elongate have thick and
fewer points. It is not surprising, then, that comb aggregates from
18
atypical points in shape and number. The interest here is that comb
cells do possess some ability to regulate point formation.
There was little apparent anterior-posterior differential in pat-
tern of components seen in the grafts. Thus, graphic reconstructions
show no clear regional divisions that are characteristic of normal comb.
It would seem, then, that dissociated comb cells have limited ability to
regulate a complete anterior-posterior gradient.
The comb is a featherless integumentary derivative. That none of
the reconstructed grafts developed feathers, while some other non-comb
tissues formed, has important implications. Furthermore, these other
non-comb tissues were non-integumentary. Lawrence (1963) found in
tissue recombination grafts of feather determined ectoderm with pre-
sumptive comb mesoderm an inhibition in the development of previously
formed feather placodes. Amprino and Camasso (1959) also observed fea-
ther suppression with comb mesoderm. In chimeric aggregates of chick
and mouse skin, feather germs are usually suppressed, though hair fol-
lides will develop (Levak-Svajger and Moscona, 1964; and Moscona, 1967).
In this case an age dependent shift is seen to operate where chimeric
feathers are formed. These data suggest dermal specificity such that
comb dermis is not compatible to feather development.
These cumulative results show that dissociated comb cells possess
some capacity to aggregate comb structure. Ridge and point are organ
patterns that are regulated. Since comb will not regenerate in later
ontogeny (Hardesty, 1931), regulative ability is apparently lost at some
point during histogenesis. Dense regular dermis and an organized vas-
cular tissue are patterns that form generally through comb cell inter-
actions. The cells respond weakly in aggregating regional distribution
19
of parts along an anterior-posterior axis. Finally, comb dermal cells
are pleuripotent, but this capacity does not extend to feather papilla
formation.
SUMMARY
Comb from nine and ten day embryos was dissociated and the cells
were cultured as chorioallantoic grafts to determine whether aggregation
will occur. Based on the experiments, these conclusions were drawn:
1. Dissociated comb cells possess some ability to reconstruct comb
structure. An aggregate of such cells can form elongate ridge and ir-
regular points.
2. Characteristic dense regular dermis and vascular pattern form
as a result of tissue interactions. Some correlation is seen between
organized tissue structure and presence of thin epidermis and cartilage.
3. Dissociated comb dermal cells have a capacity to differentiate
into a variety of non comb-like tissues, i.e., bone, cartilage, and
reticular.
REFERENCES CITED
Amprino, R. and M. Camosso. 1959. Feather formation in heterotopically
grafted terminal parts of the leg bud in chicken embryos. J. Exp.
Zool. 142: 533-551.
Balinsky, B. I. 1965. An Introduction to embryology. 2nd ed. W. B.
Saunders Co., Philadelphia. 673p.
Davenport, H. A. 1960. Histological and histochemical technics. W. B.
Saunders Co., Philadelphia. 401p.
Garber, B. and A. A. Moscona. 1964. Aggregation in vivo of dissociated
cells. I Reconstruction of skin in the chorioallantoic membrane
from suspensions of embryonic chick and mouse skin cells. J. Exp.
Zool. 155: 179-202.
Grover, John W. 1961. The enzymatic dissociation and reproduceable
reaqgreqation in vitro of 11-day embryonic chick lunq. Develop.
Biol. 3(5): 555-568.
Hamburger, V. 1966. A manual of experimental embryology. The Univ-
ersity of Chicago Press, Chicago. 221p.
Hamburger, V. and H. L. Hamilton. 1951. A series of normal stages in
the development of the chick embryo. J. Morphol. 88: 49-92.
Hardesty, M. 1931. The structural basis for the response of the comb
of brown leghorn fowl to the sex hormones. Amer. J. Anat. 47:
277-323.
Holtfreter, J. 1948. Observations on the migration, aggregation and
phagocytosis of embryonic cells. J. Morphol. 80: 25-56.
Lawrence, Irvin E. Jr. 1963. An experimental analysis of comb develop-
ment in the chicken. J. Exp. Zool. 152: 205-218.
. 1964. Some effects on comb differentiation in altered position
of the primordium on the frontonasal process. Amer. Zool. 4(4):
43.
Levak-Svajger, B. and A. A. Moscona. 1964. Differentiation in grafts
of aggregates of embryonic chick and mouse cells. Exp. Cell Res.
36(3): 692-695.
Moscona, Aron. 1952. Cell suspensions from organ rudiments of chick
embryos. Exp. Cell Res. 3(3) 535-539.
. 1957. The development in vitro of chimeric aggregates of dis-
sociated embryonic chick and mouse cells. Proc. Nat. Acad. Sci.
43(1): 184-194.
22
. 1961a. Rotation-mediated histogenetic aggregation of
dissociated cells. A quantifiable approach to cell interactions
in vitro. Exp. Cell Res. 22: 455-475.
. 1961b. Tissue reconstruction from dissociated cells, p. 197 to
220. In M. X. Zarrow (ed.) Growth in living systems A symposium.
Basic Books, Inc., New York.
. 1963. Inhabition of trypsin inhibitors of dissociation of
embryonic tissue by trypsin. Nature 199(4891): 379-380.
. 1967. Reconstruction of skin from single cells and control
of integumental differentiation in cell aggregates. Hahmemann
Symposium, April 10, 1967. (to be published)
Moscona, A. and H. Moscona. 1952. The dissociation and aggregation
of cells from organ rudiments of the early chick embryo. J. Anat.
86(3): 287-301.
Moscona, M. H. and A. A. Moscona. 1965. Control of differentiation in
aggregates of embryonic skin cells: Supression of feather morpho-
genesis by cells from other tissues. Develop. Biol. 11(3): 402-
423.
Steinberg, Malcolm S. 1963. Reconstruction of tissues by dissociated
cells. Science 141(3579): 401-408.
Weiss, P. and G. Andres. 1952. Experiments on the fate of embryonic
cells (chick) disseminated by the vascular route. J. Exp. Zool.
121: 449-488.
Weiss, P. and Ruth James. 1955. Skin metaplasia in vitro induced by
brief exposure to Vitamin A. Exp. Cell Res. Suppl. 3: 381-394.
Weiss, P. and A. C. Taylor. 1960. Reconstitution of complete organs
from single cell suspensions of chick embryos in advanced stages
of differentiation. Proc. Natl. Acad. Sci. U. S. 46: 1177-1185.
Wenger, Byron S. 1951. Determination of structural patterns in the
spinal cord of the chick embryo studied by transplantation between
brachial and adjacent levels. J. Exp. Zool. 116(1): 123-164.
Wilson, H. V. 1908. On some phenomena of coalescence and regeneration
in sponges. J. Exp. Zool. 5: 245-258.
23
Plate I. lmm=10
Figure 1 Graphie reconstruction Figure 2. Graphie Reconstruction
Lateral view Vertical view.
Legend: ^ graft Number 38, ^ graft Number 192, £ graft Number 93,
3-9 are levels of photographs in Plates II and III.
24
Plate II.
All Figures in Plate II are of graft Number 38 and show tissue sec-
tions from different levels. These levels are indicated in Plate I, Fig-
ures la and 2a.
Figure 3. The free margin near the middle of the grafts. Dense
dermis occurs centrally and loose vascular tissue periphecally. A thin
comb-like epidermis is also present.
Figure 4. The basal area near one end of the graft. Cartilage and
bone are differentiated.
Figure 5. An entire section near one end of the graft. Cartilage,
bone, and irregular dermis appear. The basal position of cartilage in
relation to graft attachment on the chorioallantois is apparent.
Figure 6. The free margin of the same section as Figure 4.
Figure 7. An entire section near the end of the graft and opposite
to Figure 5. The cartilagenous rod terminated four sections behind this
level.
Legend, c- cartilage, b- bone, 1- loose irregular tissue, d- dense
regular dermis, and e- epidermis
25
26
Plate III.
Figure 8. A cross-section taken from one end of graft Number 93.
Dense dermal organization that is comb-like is seen peripherally. The
vascular core is apparent. See Figure Ic for level.
Figure 9. A cross-section from another level of graft Number 93.
A tissue pattern similar to that of Figure 8 occurrs.
Figure 10. A cross-section of graft Number 85 taken from near
the center. The free process projecting superiorly shows a trans-
verse arrangement of dermal tissue components. Comb-like epidermis
is clearly seen along the margin.
Figure 11. An over all cross-section of graft Number 85. Show-
ing attachment to the chorioallantoic membrane.
Figure 12. A cross-section of graft Number 192 taken from the
center. The separate structure is typical comb.
Figure 13. A cross-section of a typical condition of the car-
til age rod. Graft Number 73 is shown.
Figure 14. Prominent ridge is formed in graft Number 143 and is
shown in cross-section.
27
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