/ . Embryol exp. Morph. Vol. 54, pp. 155-170, 1979
Printed in Great Britain © Company of Biologists Limited 1979
\ 55
Development of the tibiotarsus
in the chick embryo: biosynthetic activities of
histologically distinct regions
By DAVID L. STOCUM, 1 RICHARD M.DAVIS, 2
MARILYN LEGER, 2 AND H. EDWARD CONRAD 2
From the Department of Genetics and Development and the
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
SUMMARY
The dynamics of the histological changes which occur in the distal half of the tibial portion
of the embryonic chick tibiotarsus from day 8 to day 18 of incubation are correlated with the
capacities of histologically distinct zones to incorporate isotopic precursors into mucopolysaccharides and collagen. At the distal end of the tibia, which abuts the suture line formed by
the fusion of the two tarsals with the tibia, there persists throughout embryonic development
a narrow band of small, round or oval, rapidly dividing chondrocytes which synthesize
chondroitin SO4 and collagen at low rates. Just proximal to this zone is a broader zone of
flattened, disc-shaped chondrocytes which divide more slowly and are extremely active in
chondroitin SO4 and collagen synthesis. Proximal to the zone of flattened chondrocytes is a
zone of non-dividing, hypertrophied chondrocytes which are large and round and increase
continually in size going from the distal to the proximal end of the zone. The biosynthetic
activities of the cells in this zone fall sharply with their distance from the zone of flattened
chondrocytes. Finally, there is a fourth zone, the marrow cavity, formed by a proximal to
distal disintegration of the hypertrophied chondrocytes, starting at mid-diaphysis. The marrow
cavity is surrounded by a shell of periosteal and intra-membranous bone which extends to
the distal end of the zone of hypertrophied chondrocytes. Our data suggest that as the tibiotarsus grows in length the small, round rapidly dividing cells of the tibia differentiate first
to flattened chondrocytes which synthesize matrix at a high rate and ultimately to low
activity, hypertrophying chondrocytes. This sequence proceeds in a linear fashion.
INTRODUCTION
Understanding the complex network of interactions among cells and tissues
which controls the emergence of spatial patterns is the central problem in
developmental biology (Wolpert, 1969, 1971). Extensive studies of pattern
formation in the differentiation of the embryonic chick limb bud have generated
a great deal of morphological, histological and ultrastructural information
concerning pattern organization.
1
Author's address; Department of Genetics and Development, University of Illinois,
Urbana, Illinois 61801, U.S.A.
2
Authors' address; Department of Biochemistry, University of Illinois, Urbana, Illinois
61801, U.S.A.
II
EMB 54
156
D. L. STOCUM AND OTHERS
Initial differentiation of the limb begins at stage 22 (Hamburger & Hamilton,
1951) with the successive formation of the different skeletal segments in a
proximo-distal direction as condensations of mesoderm within the bud. This
pattern sequence is highly dependent on the interaction between the mesoderm
and the apical ectodermal ridge (AER) of the limb bud (Amprino, 1965;
Saunders, 1977; Summerbell, 1974) which, according to several proposed models,
keeps the distal mesoderm in a labile and dividing condition. If the AER is
removed, the limb pattern is more or less distally truncated, depending on the
developmental stage of the bud. The ridge appears to release a diffusible
substance which maintains the 100-200 /.im zone of mesodermal cells subjacent
to it (a 'progress zone', Summerbell, Lewis & Wolpert, 1973) in a 'distal'
state that is distinctly different from that of the more proximal mesoderm (Cairns,
1975). Once having left the progress zone, cells are capable of differentiation, and
the proximal-distal sequence of segment condensation is seen as a consequence
of progressive exclusion of cells from the progress zone as it moves distally due
to cell division. The specification of the spatial pattern of this differentiation
within each segment has been proposed to occur in a variety of ways (see Stocum,
1975, for a review).
The next phase of skeletal development, the differentiation of cartilage and
bone within each limb segment, now begins. The spatial pattern of the intrasegmental differentiation of cartilage and bone is distinctly different from that
observed during the initial mesenchymal condensations, and is independent of
the apical ectodermal ridge. Several stages of chondrogenesis and then osteogenesis begin in the middle of each condensation and spread both proximally
and distally at the same time (Fell, 1925). Ultimately, the long bones are composed of bonyjdiaphyses and epiphyses with a band of growth cartilage between
them.
Compared to the histological, cytological, and experimental morphological
information that has been accumulated on avian limb development, there is
little biochemical data to enable an understanding of the process at the molecular
level of organization. Biochemical studies of chick limb mesoderm during the
establishment of the initial skeletal pattern (Searls, 1965; Medoff, 1967;
Toole, 1972; Toole, Jackson & Gross, 1972; Linsenmayer, Toole & Trelstad,
1973) have demonstrated significant increases in the levels of chondroitin SO4,
hyaluronidase, and type-II collagen during progressive determination of the
skeletal tissue. In addition, several studies (Matukas, Panner & Orbison, 1967;
Oohira et at. 1974; Kimata, Okayama, Oohira & Suzuki, 1974) have correlated
histological, ultrastructural, and biochemical properties of long bones of 12day chick embryos, a time at which chondrogenesis and osteogenesis are well
under way. However, the sequential biochemical changes in the chondrogenic
and osteogenic differentiation patterns of such bones throughout their embryonic
development have not been reported. Such data would be of interest because
they may give some insight into the factors which control the sequence of
Development of tibiotarsus in chick embryo
157
chondrogenic differentiations observed histologically. This paper describes the
changes in the capacities of histologically distinct regions along the proximaldistal axis of the embryonic chick tibiotarsus to synthesize collagen and chondroitin SO4 from day 8 to 18 of incubation. These activities are correlated with
the pattern of chondrogenic differentiation as the bone develops and lengthens.
Experimental procedures
Histological studies. White Leghorn eggs were incubated at 37° and embryos
were staged according to the Hamburger-Hamilton system (1951). All limb
tissues were preserved in Bouin's solution, sectioned at 10 /mi, and stained with
iron hematoxylin and eosin. Whole hindlimb buds of 4-6 days of incubation
(HH stages 23-28) were sectioned for the study of early tibiotarsus development. To study development from days 8 to 18 of incubation, the tibiotarus
was separated from the rest of the limb and sectioned after removal of skin and
muscle and decalcincation in Jenkin's fluid.
Lengths of the different histological zones within two to four tibiotarsi
were measured at each developmental stage from median longitudinal or frontal
sections with an ocular micrometer mounted in one eyepiece of the microscope
and calibrated in mm. Since the zones grade into one another, their boundaries
were arbitrarily denned as the regions where the cell type of the adjacent zone
began to predominate.
Mitotic indices of the histological zones were determined on the same sections
with the aid of a 1 mm2 grid divided into 0-1 mm2 subunits, mounted in the eyepiece of the microscope. The number of cells contained in a random selection of
five of the subunits was counted at a magnification of 400 x and multiplied by
20 to give the total number of cells contained within the grid. All of the anaphase and metaphase mitotic figures lying within the 1 mm2 boundaries of the
grid were then counted and the mitotic index expressed as the percent of the
total cells that were in mitosis. Counts were performed on three sections within
each histological zone for two to four specimens at each stage of development.
These sections were located 30 /im apart within a 60 /im thick block of tissue in
the geometrical center of each zone. Only about one-half of the grid was utilized
to make counts in Zone 1, due to the narrowness of this zone. Within the two
differentiation centers of the tarsal portion of the bone, similar sets of histological zones develop concentrically (see Results), but it was not feasible to count
each zone of these regions separately. For this reason the mitotic index was
calculated only for one of the centers and includes counts from more than one
histological zone (especially in the younger embryos).
Culture of tibiotarsus sections. Tibiotarsi were removed from White Leghorn
chick embryos and placed in Dulbecco's pH 7-4 Tris-saline solution containing
CaCl2 (0-26 g per 1.) and MgSO 4 .7H 2 O (0-2 g per 1.) and were freed of all
adhering tissues. The bones were cut into histologically distinct segments,
the lengths of which were determined for each age embryo as described above
158
D. L. STOCUM AND OTHERS
(see Results). Zones 2 and 3 were cut in half to give distal (Zones 2a and 3a)
and proximal (Zones 2b and 3b) halves, except in the early embryos when these
zones were not sufficiently elongated. For each incubation the number of segments sufficient to give 0-2-0-3 mg protein were placed in a 30 mm Falcon
plastic tissue culture dish and cultured in 2 ml Dulbecco's Modified Eagle's
Medium (GIBCO) containing 2 g D-glucose per 1., 5 mg streptomycin per 1.,
100000 units penicillin per 1., and 10% fetal calf serum. For cultures used to
measure mucopolysaccharide synthesis 20 fid 35SO42~ (New England Nuclear)
was added per ml of medium to give a ^SCV" specific activity of 19-4 mCi/
m-mole (Kim & Conrad, 1976). Under these incubation conditions 35SO42~
was incorporated linearly into mucopolysaccharides for more than 24 h. When
tissues were labeled in this manner and then transferred to fresh medium, no
degradation was observed; consequently, the rate of isotope incorporation
measured is a good approximation of the biosynthetic activity of these tissues.
To measure collagen synthesis, 5 /*Ci of L- [2, 3-3H]proline (New England
Nuclear) and 0-6 /*mole of unlabeled L-proline were added to each dish to give
a final L-proline concentration of 0-3 mM and a specific activity of 8-33 mCi/
m-mole. These cultures also received 100 /ig ascorbic acid per ml. In these
incubations 3 H was incorporated into collagen and converted to L [3H]hydroxyproline at linear rates for more than 24 h. The labeled collagen in prelabeled
tissues was not degraded on further incubation.
Determination of the rates of chondroitin SO± and collagen synthesis. The
above cultures were incubated for 20 h at 37° in a humidified incubator gassed
with 7 % CO 2 ,93 % air. At the end of each incubation period the culture medium
was removed with a Hamilton syringe and saved for analysis, and the tissue
was washed twice with pH 7-5 phosphate-buffered saline to remove excess
labeled medium. The tissue from each culture was homogenized in 200/d
H 2 O in a glass homogenizer and aliquots were withdrawn for assay of protein
by the method of Lowry, Rosebrough, Farr & Randall (1951) and DNA by
the 4', 6-diamidino-phenylindole (Polysciences) fluorescence method of Kapuscinski & Skoczylas (1977). The 35SO4-labeled cultures were analyzed for labeled
chondroitin-4-SO4 and chondroitin-6-SO4 by the chondroitinase digestion
methodology described previously (Kim & Conrad, 1974, 1976). The number
of cpm in each product was converted to n-mole using the known specific
activity of the 35SO42~ used in the incubation medium.
The rate of collagen synthesis was expressed as the rate of appearance of
labeled hydroxyproline in protein. The amount of labeled hydroxyproline in
protein was determined by mixing an aliquot of the [3H]proline-labeled homogenate with an equal volume of concentrated HC1 and heating the solution at
100° in the sealed tube for 18 h to release free hydroxyproline. The hydrolysate
was evaporated to dryness, redissolved in water, and spotted on a one-inch
wide strip of Whatman No. 3 chromatography paper for separation of labeled
proline from labeled hydroxyproline by descending chromatographic develop-
Development of tibiotarsus in chick embryo
159
ment for 44 h in «-butanol, methyl ethyl ketone, water (2:1:1). The dried strip
was cut into \ in. segments, each of which was counted in a scintillation counter
using a scintillation fluid containing 4 g diphenyloxazole per 1. of toluene. The
total cpm in the [3H]hydroxyproline peak was converted to n-mole using the
known specific activity of the [3H]proline used in the incubation medium.
RESULTS
General histological features of the tibiotarsus. Figure 1A shows a longitudinal
section of the distal half of the tibiotarsus of a 12-day chick embryo. The
tibiotarsus is formed by fusion of three distinct mesenchymal condensations the tibia, and two tarsals, the peroneal and the tibiale (Johnson, 1883). The
region originating from the two tarsals, referred to here as the tarsus ('T'),
in Fig. 1A, forms the distal epiphysis. As reported previously (Fell, 1925;
Oohira et at. 1974), the tibia forms all of the diaphysis and exhibits several
regions of unique chondrocyte morphology referred to as Zones 1, 2, 3 and 4
and shown at higher magnification in Fig. 1B. Zone 1 is the least differentiated
and consists of a narrow band of round, oval and polygonal chondrocytes
surrounded by small amounts of metachromatic extracellular matrix. Zone 2 is
a wide band of disc-shaped chondrocytes whose long dimensions are perpendicular to the longitudinal axis of the bone and which are surrounded by more
abundant matrix. Zone 3 consists of a wide band containing the most differentiated (hypertrophied) chondrocytes with large, swollen nuclei. Zone 4, the
marrow cavity, results from destruction of the hypertrophied cartilage and its
replacement by marrow elements.
Histological changes during tibiotarsus development. In the present study
the origins of the zones of the tibiotarsus and the histological changes which
they undergo from day 4 to day 18 are consistent with those described earlier
(Johnson, 1883; Fell, 1925; Hampe, 1959; Jurand, 1965). Briefly, the mesenchymal condensation for the tibia appears at day 4 (HH stage 24) and those for
the peroneal and tibiale appear just distal to the tibia at 4-5-5 days (HH stage
25-26). Between days 6 and 8 the two tarsals fuse and attach to the tibia by a
'suture line' ('S' in Fig. 1A) of connective tissue cells which are continuous
with the perichondrium. Beginning at 4-5 days the tibial mesenchyme undergoes
a series of differentiations, resulting in the establishment of the several histologically distinct zones along its length (Fig. IB). These differentiations are
initiated in the mid-diaphysis and spread proximally and distally so that at any
stage of development, the least differentiated cells (Zone 1) are located at the
distal ends of the tibia and the most differentiated cells (Zone 3) at the midregion. By day 8 the first three zones are established and at day 10 the marrow
cavity makes its appearance. Beginning at day 6 a thin shell of intramembranous
bone is laid down in the mid-diaphyseal region by osteoprogenitor cells of the
periosteum. At later stages of development, this bone shell surrounds zones
160
1a
D. L. STOCUM AND OTHERS
Development of tibiotarsus in chick embryo
161
3 and 4, and becomes thicker and trabecular in nature (Fig. 1 A). The histological appearance of the four zones of differentiation does not change appreciably
from the 10th to the 18th day of incubation, except that, by day 18, the flattened
chondrocytes of Zone 2 have become arranged in discrete stacks, the suture line
disappears, and the marrow cavity extends as a tunnel through Zone 3 to its
distal end.
Separate differentiation centers arise in the peroneal and in the tibiale at
about day 6-5. The same sequence of cellular differentiations is observed in each
of these centers as in the tibia, but differentiation proceeds radially instead of
linearly, so that, by day 18, Zone 1-like chondrocytes are on the periphery
of each center and hypertrophied chondrocytes are in the middle. However, no
bone or marrow differentiation has occurred in the epiphysis at this time. An
interesting observation is that a region of polygonal chondrocytes, identical in
appearance and amount of surrounding matrix to that in the tarsus, comes to
lie between Zone 1 and the suture line, forming part of the epiphysis. The origin
of these cells is not certain, but they are most likely derived from tibial Zone-1
cells; thus, the distal end of the tibia may contribute to the proximal portion of
the distal epiphysis.
Growth of tibial zones. Table 1 shows the lengths of the zones in the distal
half of the tibia and the percentages of the total length of the tibia for the several
embryonic ages. For these measurements the boundaries between the histologically distinct zones are defined as the point in the overlapping regions where
the numbers of cells of the two distinct shapes are equal. The length of Zone 1
does not change between day 8 and day 14, while the tarsus lengthens slowly
during this same period. The tarsus elongation continues through day 18 where
Zone 1 and the tarsus are treated together. Zone 2 shows a modest increase in
length until day 12 and then maintains a constant length for the remaining
period of embryonic development. The percentage length of the tibia occupied
by both Zones 1 and 2 decreases substantially throughout the developmental
period. The mean length of Zone 3 increases very rapidly throughout develop-
Fig. 1. (A) Low magnification view of a longitudinal section through the anterior
side of the distal half of a 12-day tibiotarsus. T, tarsus, which makes up the bulk
of the epiphysis; S, suture line between the tarsus and the tibia; G, space representing the groove on the anterior side of the tibia, into which fits a cartilage projection,
P, of the tarsus. Arrows point to the differentiation centers of the tarsus, which develop into the condyles. The histologically distinct zones of the tibia are indicated,
by the numbers at the right-hand side of the figure. PB, periosteal bone; IB, intramembranous bone; PC, perichondrium. Bright field, x40. (B) Series of phase
contrast, high magnification (all x 630) views showing, from top to bottom, the
histological details of sections through the centers of the tarsus and Zones 1, 2, 3a,
and 3b of the tibia. Letters and numbers for each zone correspond to those in (A).
In all of these photographs it is the nuclei of the chondrocytes that stand out most
clearly. Note the progressive distal to proximal increase in nuclear size within the
tibia.
=
=
jc-
1
2
3
X "
1
2
3
4
x
1
2
x
f
—
—
—
—
—
018
0-23
0-21
0-27
0-32
0-39
0-33
0-59
0-52
0-45
0-52
0-62
0-61
0-62
(9)
(10)
(9)
(11)
Tarsus
1-40 (12)t
0-81
0-81
108
0-81
0-88 (9)
1-76
108
1-35
—
—
009
0-23
0-16(9)
014
009
018
0 1 4 (4)
018
014
0-20
0 1 7 (3)
014
014
0-14 (2)
—
—
—
—
Zone 1
1-04(7)
0-86
0-63
0-75 f40)
0-90
0-90
0-90
0-90 (23)
0-99
1-22
1-28
116 (21)
108
108
108(16)
0-68
108
1-22
1-35
108(11)
108
1-22
0-81
Zone 2
A
Zone length (mm)
(34)
(35)
(46)
(50)
(40)
4-50(38)
0-68
0-81
0-75
189
1-89
1-98
192
2-70
2-97
1-89
252
2-70
216
2-43
3-78
2-70
2-70
405
3-31
5-40
378
4-32
Zone 3
—
Zone 4
uoioiarsui
4-86(43)
0-68
0-45
0-59
0-57 (15)
075
1-22
1-49
115 (21)
2-43
2-84
2-64 (38)
4-86
513
4-32
378
4-52 (46)
4-86
—
ana ine nisioiogticai zones oj ine not a in ine aeveujping
11-34
1-81
1-90
1-86
3-88
3-65
404
3-86
5-21
607
5-31
5-53
6-97
6-83
6-90
1013
9-72
9-32
9-99
979
11-34
—
Total
* Values are given for measurements made on several specimens of each embryonic age and the means {x) are recorded. For days 16 and 18 values for
the tarsus plus Zone 1 of the tibia are tabulated since after day 14 of incubation the tarsus is separated from Zone 1 by the interposition of cells derived
from Zone 1 at the distal end of the tibia. Numbers in parentheses give the percentage of the total length of the tibiotarsus occupied by each zone.
t Based on specimen no. 1 only.
18
16
14
1
2
3
12
=
x=
1
2
3
x
1
2
10
8
Days of
incubation Specimen
l a o i e i. lvi eaw lengins uj ine lursus
C/5
•T
ffl
HM1
H
d
O
o
H
u
ft
d
O\
Development of tibio tarsus in chick embryo
163
Table 2. Mitotic indices in the differentiation centers of the tarsus and in
the histological zones of the tibia in the developing tibiotarsus*
Zone
Days of
incubation
Specimen
Tarsus
1
2
8
1
2
3
x=
0-50
0-96
0-39
0-47
0-39
0-97
0-45
0-51
005
0-22
0-29
018
10
1
2
3
xi"
0-96
0-32
0-55
0-60
0-88
0-39
0-54
0-60
0-59
006
016
0-25
12
1
2
3
0-24
101
0-86
x"
0-63
0-49
1-58
1-41
115
013
0-33
019
0-22
14
1
2
x=
0-26
0-74
0-50
0-37
1-38
0-93
000
015
007
16
1
2
3
x°
018
0-23
0-44
0-27
0-34
0-64
0-73
0-57
000
Oil
000
004
18
1
2
005
013
019
010
018
014
000
0-36
018
X
°
* All of the anaphase and metaphase mitotic figures lying within the boundary of the
1 mm2 grid in the microscope eyepiece were counted (see Experimental procedures). Mitotic
indices are expressed as percentages of total cells that were in mitosis. Individual counts and
means (x) are recorded. Mitotic indices in Zone 3 were 0 0 at all embryonic ages.
ment, but the percentage of the total bone that it comprises decreases slowly
after day 10 due to a more rapid destruction of its cells to form the marrow
cavity, which occupies an ever-increasing fraction of the bone as marrow
differentiation sweeps distally.
Table 2 summarizes the mitotic activity of the different regions of the tibiotarsus from day 8 to day 18. The mitotic index in Zone 3 is zero at all ages.
The highest mean rates of cell division are seen in Zone 1. Lower rates of cell
division are observed in Zone 2. The highest mitotic indices in Zone 2 are observed in the distal region of the zone, adjacent to Zone 1; in the proximal end
of Zone 2, the mitotic indices drop almost to zero. Thus, the most active cell
164
D. L. STOCUM AND OTHERS
Table 3. Protein content of tibiotarsus zones*
Days of
Zone
A
bation
Tarsus
1
2a
2b
3a
3b
(mg protein per mg DNA)
52+17
54±6
67 ±21
31 ±3
(35-69)
(49-58)
(49-91)
(28-33)
64 ±22
36± 13
37±17
54 ±14
50+15
lot
(35-71)
(21-58)
(30-90)
(36-69)
(22-52)
12
58 + 12
61 + 11
52±2
49±9
43±13
47 + 12
(50-55)
(45-72)
(34-60)
(41-59)
(26-56)
(48-68)
14
53 ±8
44 + 2
48 ±6
54±13
58 ±5
68±15
(41-54)
(40-74)
(42-46)
(43-58)
(51-60)
(60-88)
16
67 + 14
69±13
66±18
60±27
79±19
(48-83)
(55-83)
(45-94)
(48-99)
(66-107)
76 ±20
72 ±14
83±19
77 ±12
92 ±31
18
(65-93)
(50-127)
(58-106)
(54-94)
(65-109)
* Values are averages of protein to DNA ratios assayed in six different tissue samples.
Numbers in parentheses show the range of values obtained for each tissue. The DNA content
of Zone 3b was too low in tibiotarsi from 16- and 18-day embryos to obtain reproducible
values.
t The proximal and distal halves of Zone 2 were not separated for examination of the
tibiotarsi of 8- or 10-day embryos. The two halves of Zone 3 were not separated for the 8-day
tibiotarsi.
8t
division is occurring in Zone 1, but the cells in the proximal region of Zone 2
and in Zone 3 have withdrawn from the cell cycle. All of the regions of the
bone which contain dividing cells show a peak in mitotic activity at day 12.
Biosynthetic activities. For activity measurements in embryos older than 10
days, Zone 2 was divided in half to obtain a distal subzone (2a) consisting
exclusively of flattened chondrocytes and a proximal subzone (2b) consisting of
a mixture of flattened and hypertrophied cells. Similarly, Zone 3 was divided in
half to obtain distal and proximal subzones (3a and 3b, respectively) containing
chondrocytes in early and advanced hypertrophy. Table 3 shows the protein
content of each zone during development. The data show that in the younger
embryos the protein contents of Zones 1 and 2a are generally lower than the
content of protein in the more proximal zones, consistent with the earlier
demonstration of increased collage accumulation in the more proximal zones
(Oohira et al. 1974). An even more consistent trend is seen in the increasing
protein content of all zones in the 16- and 18-day embryos.
Figures 2 and 3 show the changes in chondroitin SO4 and collagen synthesis
as development proceeds. In these experiments the isotopic precursors were incorporated into chondroitin SO4 and collagen at linear rates throughout the
assay period, and the labeled polymers were not degraded when prelabeled
tissues were further incubated in fresh, unlabeled medium for periods up to 20 h.
Development of tibiotarus in chick embryo
165
15 -
10 5-
20 15
S 10
a,
•5
25
o
•S 20
I
.? 15
| 10
T 1 2a 2b 3a 3b T 1 2a 2b 3a 3b
Zone
Fig. 2. Changes in biosynthetic activities along the distal half of the tibiotarsus at different embryonic ages. Values are averages of three separate determinations of the
rate of chondroitin SO4 synthesis. The proximal and distal halves of Zone 2 were
not separated for measurement of the activities of the tibiotarsi of 8- or 10-day
embryos. The two halves of Zone 3 were not separated for the 8-day tibiotarsi.
Thus, the values reported appear to be good approximations of the biosynthetic
rates for chondroitin SO4 and collagen. More than 95 % of the labeled polymers
formed during these assays were recovered in the washed tissues.
The data in Fig. 2 show that the peaks of chondroitin SO4 and collagen synthesis are found in Zone 3 at 8 days but move distally to Zone 2b by 12 days
where they remain through day 14. A further distal shift into Zones 1 and 2a
is seen at 16 and 18 days. The rates of chondroitin SO4 and collagen synthesis
along the tibiotarsus, for the most part, parallel each other. However, in the
10-day tibiotarsus the chondroitin SO4 synthesis peaks sharply in Zone 2
ahead of the rise in collagen synthesis in Zone 3a. The same tendency for a rise
in the rate of chondroitin SO4 synthesis in Zone 2a ahead of the rise in collagen
synthesis is seen in 12- and 14-day embryos, but is less pronounced than at 10
166
D. L. STOCUM AND OTHERS
25
2-5 -
20
20 1
<
15
1-5
10
< 10
Q
O
5
a
0-5
a.
si
o
25
o
2-5
(U
P-
itin
oto
20
2
20
X
1-5
a>>
15
o
13
>.
10
o 10
5
c 0-5
rol
into
o
•a
c
o
o
c
O
cn
•n
15
"o
E
c 10 -
a,
m
"o 1-5
E
c
10
- 2
0-5 -
8
12
16
8
12
Days of incubation
16
Fig. 3. Changes in biosynthetic activities of the different zones of the
tibiotarsus as a function of embryonic age.
days. In the 16- and 18-day embryos the collagen synthesis rates rise faster than
the chondroitin SO4 synthesis rates. The ratios of 6-sulfated to 4-sulfated Nacetylgalactosamine residues in the labeled chondroitin SO4 recovered at the end
of the incubation period range from 15 to 2-0 in Zones 1 and 2a but rise sharply
to values up to 5 or 6 in Zones 3a and 3b.
The same data, replotted in Fig. 3 to show changes in the activities in each
zone as a function of embryonic age, show that marked increases in the biosynthetic rates of Zones 1 and 2a occur in 16- and 18-day embryos. In contrast,
early rises in activities in Zones 3a and 3b are followed by declines in activities
in older embryos. The Zone 3b activities were very low in the 16- and 18-day
embryos but could not be expressed in terms of DNA because of the low levels
of DNA in these tissues (see Table 3). In contrast to the tibial zones, the tarsus
shows little change in activities over the developmental period. The ratios of 6to 4-sufated JV-acetylgalactosamine residues remained fairly constant in the
Development of tibiotarsus in chick embryo
167
tarsus and Zones 1, 2a, and 2b throughout development. However, in Zone 3a
these ratios rose in the older embryos, while in Zone 3b the ratios rose and then
fell with increasing embryonic age.
DISCUSSION
The changes in biosynthetic activities accompanying the emergence of spatial
patterns in the embryonic differentiation of the tibiotarsus are described here.
Our observations on the histological development of the chick tibiotarsus are in
general agreement with those of Johnson (1883), Fell (1925), Hampe (1959),
and Holder (1978). In addition, our observations suggest that Zone-1 cells of
the tibia may give rise not only to Zone-2 cells but to the proximal portion of the
distal epiphysis. The zone numbering nomenclature of Oohira et al. (1974) for
the 12-day tibiotarsus is modified here to accommodate the fact that the tarsus
elements undergo a developmental sequence that is similar to, but relatively
independent of, the sequence observed in the tibial element. The primary difference is that Zone 1 of these investigators is here divided into two zones, the
tarsus and Zone 1. Zones 3 and 4 of Oohira et al. correspond to our subzones
of early and late hypertrophy, namely Zones 3a and 3b.
The only dividing cells in the tibia are those in Zones 1 and 2a. Some of the
cells that result from the Zone-1 divisions withdraw from the cell cycle and
undergo a temporal sequence of morphological and biochemical changes in
place, so that each group of newly formed cells first takes on a flattened morphology to form Zone-2 cells and subsequently hypertrophies to form Zone-3
cells. Thus, as the tibiotarsus grows in length, several states of chondrogenic
differentiation sweep through the cells that are left behind the dividing regions,
a process that continues from day 8 to day 18 with little change in the morphology
of the cells in Zones 1, 2, and 3. The same developmental sequence has also been
described for mammalian long bones (Serafini-Fracassini & Smith, 1974).
Marked changes in biosynthetic activities accompany the changes in cell
morphology along the proximal-distal axis. The biosynthetic activities are reported here in terms of the DNA content of the zones. In Zones 2b, 3a, and 3b,
where the cells have withdrawn from the cell cycle, presumably in G1} the DNA
per cell should be identical (2n) so that the DNA content of these zones is
proportional to the cell number. In Zones 1 and 2a, however, where the cells are
still dividing, the DNA content per cell will be somewhere between one and
two-fold higher than in the more proximal zones, with the actual value depending
on the proportions of the cells in G l5 S, and G2 of the cell cycle. Thus, a comparison of the biosyntheticactivities per cell in Zones 1 and 2a with the activities
per cell in Zones 2b, 3a, and 3b may require an increase in the Zones 1 and 2a
values in Fig. 2 and 3 relative to the values for the more proximal zones by up
to 50 %. This would make the rises in activities in going from Zone 2a to Zone
2b less pronounced in the 8- to 14-day embryos (Fig. 2) but would not alter the
general pattern of activity changes.
168
D. L. STOCUM AND OTHERS
The changes in biosynthetic activities along the proximal-distal axis of the
tibiotarsus of the 12-day chick embryo reported by Oohira et al. (1974) are
essentially reproduced in the 12-day panel of Fig. 2. However, the remaining
panels in Fig. 2 show that the pattern of the biosynthetic changes along the
tibiotarsus is not the same at every embryonic age. As the cells are incorporated
into successive histological zones, they first attain peaks of chondroitin SO4 and
collagen synthesis which subsequently decline rapidly. Within each zone, however, there are also large changes in the synthesis of polymers with increasing
developmental age. This point is best illustrated in Fig. 3. Chondroitin SO4 and
collagen synthesis increase rapidly in Zones 1 and 2a after 12 days of incubation
and decrease in Zones 3a and 3b from 8 days onward, while Zone 2b exhibits
considerable fluctuation. Thus, the peaks of chondroitin SO4 and collagen
synthesis move distally with time. Since the morphological appearances of the
histological zones are quite constant throughout embryonic development, these
data suggest that modulations in the biosynthetic activities of cells in a given
zone precede histological changes. Specifically, elevated chondroitin SO4 and
collagen synthesis may precede cell flattening and hypertrophy and a subsequent
decrease in synthesis may precede cytolysis.
The mechanisms which control the continuously repeated sequence of differentiative events seen during chondrogenesis and osteogenesis of the tibiotarsus are
unknown. Holder (1978) has shown by grafting and truncation experiments that
the temporal sequence of diaphyseal ossification in chick limb long bones is
autonomous and independent of any sequential inductive interactions spreading
proximally and distally from mid-diaphysis. Osteogenesis does not begin until
the underlying cartilage has hypertrophied, and it is possible that this is an
inductive interaction (see LaCroix, 1951 and Hall, 1978). If so, the temporal
sequence of chondrogenic changes observed in long bones must also proceed
autonomously. Whatever the mechanism of this 'programming' it must result
in the establishment at the distal and proximal ends of the initial mesenchymal
condensation of conditions favorable to continued cell division. Likewise, it
must establish conditions in the region between the ends which inhibit mitosis
and favor the progressive differentiation of chondroblasts to the hypertrophied
state. If the end region in which mitosis is favored is maintained at a relatively
constant size (as is Zone 1), we have in effect a 'progress zone' 1 analogous to that
demonstrated in the early limb bud (Summerbell et al. 1973; Cairns, 1975).
The temporal (and linear) sequence of chondrogenesis observed during tibial
development then follows as a consequence of movement of this progress
zone distally by cell division, leaving daughter cells behind to go through the
differentiative sequence. This progress zone is maintained for a time after hatching as the epiphyseal growth plate (Fell, 1925), and, as we show here,
1
We emphasize that the term 'progress zone' is used here to denote only a region of
dividing cells and does not imply that cells are changing positional value within it as has
been postulated for the early chick limb bud (Summerbell et al, 1973).
Development of tibiotarsus in chick embryo
169
appears to be associated with lower rates of chondroitin SO4 and collagen
synthesis.
The differentiative sequence of a single chondroblast left behind by the progress zone may itself be controlled by an epigenetic cascade of environmental
changes, each change being the result of the previous differentiative state. It
was noted above that, from 10 to 14 days, the rate of chondroitin SO4 synthesis
is elevated prior to an increase in the rate of collagen synthesis. One could suggest, therefore, that as some cells are displaced from the mitosis-promoting
environment of Zone 1 they withdraw from the cell cycle, a change that triggers
an increase in their chondroitin SO4 synthesis and a change in cell shape. The
increased synthesis and secretion of chondroitin SO4 would raise the level
extracellular chondroitin SO4 in Zone 2, and this might, in turn, stimulate
collagen synthesis and cause hypertrophy. Finally, the appearance of high levels
of chondroitin SO4 and collagen in the extracellular matrix might ultimately
turn off the synthesis of both these polymers and lead to cytolysis of the hypertrophied chondrocytes. Such a sequence of causal relationships is speculative;
the observed changes in morphologies and biosynthetic activities both may be
secondary responses to primary changes that are not yet obvious. Clearly,
however, the composition of the extracellular matrix surrounding these cells is
changing continuously with respect to chondroitin SO4 and collagen types
(Linsenmayer et al. 1973) and/or concentration during these final states of
chondroblast differentiation. Finally, there is evidence that stabilization and
expression of the cartilage phenotype in the early chick limb bud involves
responses to sequential environmental alterations produced epigenetically by
the cartilage precursor cells themselves (Ahrens, Solursh & Reiter, 1977).
This work was supported by Public Health Service Grant HD 8057.
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