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/. Embryo!, exp. Morph. Vol. 58,pp. 119-130, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Developmental fate of a distinct class of
chick myoblasts after transplantation of cloned cells
into quail embryos
By MARK D. WOMBLE 1 AND PHILIP H. BONNER 1
From the Thomas Hunt Morgan School of Biological Sciences,
University of Kentucky
SUMMARY
A technique appropriate to investigation of the developmental fates of distinct embryonic
cell types is described and the fate of a particular type of chick myoblast (CMR-I) examined.
CMR-I myoblast clones are morphologically different from other chick myoblast clone
types and can readily be identified in living cultures.
After two weeks of culture CMR-I myoblast clones were collected, aggregated, and
transplanted into the prospective dorsal thigh region of young quail embryos. After four
days of growth, cells of the transplant-containing quail legs were grown as clones. Chick
clones were located by Feulgen staining and identified as muscle or non-muscle and, if
muscle, as CMR-I or not; 91 % of the chick clones recovered from transplants were muscle
clones, and of these 97 % were CMR-I. It was concluded that CMR-I myoblasts do not
undergo a change in differentiated state identifiable by clonal analysis.
Other transplant-containing quail legs were fixed, sectioned, and Feulgen stained. The
locations of chick nuclei were determined. The only region in which chick nuclei appeared at
significantly greater frequency than in control tissue was the dorsal thigh muscle, the region
into which the cloned chick cells were placed originally. Dorsal thigh multinucleated myotubes
exhibited the highest percentage of chick nuclei of all tissues examined. It was concluded
that the fate of CMR-I myoblasts is fusion to form myotubes.
INTRODUCTION
In order to study changes in the differentiated states of individual cells or
populations of cells during development, one must be able to identify particular
cells in some initial state and follow them to a point where the cells or their
progeny can be identified as part of a particular tissue or as having attained a
differentiated state different from the original state. If a developmental progression of differentiated states is to be fully understood, the sequence of
changes that a cell undergoes before it reaches its final differentiated state must
be determined. One method of approaching this problem is to remove undifferentiated tissue from embryos and allow it to grow and differentiate in
1
Authors' address; Thomas Hunt Morgan School of Biological Sciences, University of
Kentucky, Lexington, Kentucky 40506 U.S.A.
120
M. D. WOMBLE AND P. H. BONNER
culture as an explant or as dissociated cells. Unfortunately, the exact cellular
composition of the original tissue is often not known, so that conclusions are
complicated by ignorance of the cell types which give rise to differentiated cells.
Because of this difficulty in identifying cell populations during the development
of heterogeneous tissue, the ultimate cell source of differentiated tissue usually
cannot be determined.
Another method of ascertaining a cell's fate is transplantation of embryonic
tissues between two closely related species which carry stable, definitive histological or biochemical differences in their cells (Le Douarin, 1969, 1973 a, b).
This technique is superior to the usual tissue culture methods for identification
of the tissue of origin of particular differentiated cell types and has been applied
to a number of different problems concerning the developmental origins and
fates of cells. But, like tissue culture, tissue transplantation suffers the disadvantage of an unknown degree of cell heterogeneity in the grafted tissue.
This report describes a method in which tissue culture and transplantation
are combined and which circumvents the problem of cell heterogeneity. Muscle
clones grown in culture from single chick cells are harvested, aggregated, and
transplanted into quail embryos. After a period of in vivo growth, the heterospecific, transplant-containing quail tissue is examined in vitro by clonal analysis
and the cloned progeny of the original chick cells identified by Feulgen staining.
Mesenchymal cells derived from the limbs of chick embryos younger than
Hamburger & Hamilton stage 20 can be grown in mass culture and will
occasionally exhibit muscle differentiation (fusion) (Dienstman, Biehl,
Holtzer & Holtzer, 1974) but single cells from these ages do not grow as clones.
The first clonable cells appear in the chick leg at stage 20 and the first cells
giving rise to differentiated muscle clones are found at stage 21 (Bonner &
Hauschka, 1974). Muscle-colony-forming cells become an increasingly greater
proportion of the total clonable cell population in the chick leg during the
next few days of embryonic development until, by day 10 (stage 35), approximately 85 % of the clonable cells differentiate as muscle in conditioned medium
(White, Bonner, Nelson & Hauschka, 1975). The muscle-colony-forming cell
population is composed of a number of distinct myoblast classes. Chick
myoblast classes are distinguished on the bases of clone morphology (Bonner &
Hauschka, 1974), culture medium requirements for differentiation (White et al.
1975), and differentiative interactions with the nervous system (Bonner, 1978,
1980). While eventually we hope to examine the developmental fates of all
chick myoblast types, the present work is concerned solely with the fate of that
particular myoblast type (CMR-I) which is the first to appear during development and which gives rise to morphologically distinctive clones.
Interspecific transplantation of cloned muscle cells
121
MATERIALS AND METHODS
White leghorn or White-Rock chicken and Japanese quail eggs were incubated
at 38 ± 1 °C. Embryos were staged according to the criteria of Hamburger &
Hamilton (1951). All operations were performed using sterile technique.
Cell suspension
Techniques used for the production of single-cell suspensions from embryonic
skeletal muscle and the conditions of clonal culture have been described
previously (Bonner & Hauschka, 1974; White et ah 1975). Briefly, embryonic
chick hind limb buds (stages 26-28; 5-6 days of egg incubation) are removed,
the tissue is minced with fine forceps and incubated at 37 °C in the presence of
0-05 % crude collagenase (Worthington CLS) for 10 min. The enzymic reaction
is stopped by adding an equal volume of cold, serum-containing fresh medium
(FM). Filtration through Nitex (20 /im pore size) removes the remaining tissue
pieces and cell aggregates. The cells are pelleted, resuspended in FM and,
after dilution, are placed in gelatin-coated 60 mm tissue-culture plates (Falcon
3002) containing approximately 2-5 ml of conditioned medium. The plates are
incubated for 13-14 days at 37 °C in a water-saturated atmosphere of 95 % air
and 5 % CO2.
Culture medium
Fresh medium is 79 % Ham's F-10 nutrient solution, 1 % penicillinstreptomycin (Stock solution contains 10000 units/ml penicillin G and
0-5mg/ml of streptomycin sulfate), 15% preselected horse serum, and 5%
day-12 chick embryo extract (Konigsberg, 1968). Conditioned medium is fresh
medium that has been exposed, in 20 ml aliquots, for 24 h to confluent secondary
cultures of chick leg muscle cells grown in 100 mm petri dishes (Falcon 3003)
(White & Hauschka, 1971; Hauschka, 1972).
Aggregation
At the end of the culture period, living cultures were examined by phase
microscopy and the locations of selected CMR-I muscle clones were marked on
the culture plate bottom surface. Individual clones were isolated with the aid
of ceramic cylinders (Penicylinder, Fisher). The cylinders were ringed with
sterile silicon grease at one end and placed over the clone, silicon side down, to
effect a tight seal. Collagenase was added to the cylinder for 10 min at 37 °C
to dissociate clone cells from the plate. The collagenase and cells were removed,
individual clones of the same type pooled when necessary, and mixed with an
equal volume of FM. The cells were washed with FM and pelleted in FMcontaining microfuge tubes. The microfuge tube containing the pellet and 1 ml
FM was left in the incubator overnight, at 37 °C under 95 % air and 5 % CO2,
to allow the cells to adhere and form a tight aggregate.
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M. D. WOMBLE AND P. H. BONNER
Transplantation
Quail eggs at 3 days of incubation (equivalent to Hamburger & Hamilton
stages 19-21) were prepared for transplantation according to the procedure of
Zwilling (1959). Eggs were prepared on the day before transplantation by
drilling a small hole in the pointed end of the egg, removing approximately
0-5 ml of albumin with a sterile syringe, and cutting a window in the top of
the egg. Cellophane tape covered the window until the eggs were used for
transplantation.
Cell aggregates were removed from microfuge tubes and placed in cold,
sterile, Puck's Saline G solution, containing a small amount of Nile Blue, for
3-5 min. This lightly stains the outer layer of cells and enables the aggregates
to be seen during transplantation. After staining, the aggregates were transferred
to a dish containing cold Saline G and cut into smaller pieces for transplantation.
Quail embryos were prepared for transplantation by cutting a slit through the
membranes covering the right leg bud. A piece of the clone aggregate was then
placed in the tip of a drawn-out Pasteur pipette, the pipette tip inserted into the
prospective thigh region of the leg bud, and the aggregate gently blown into
the leg bud as the pipette was slowly withdrawn.
Four days after transplantation the quail right and left legs were separately
prepared for clonal analysis. The untreated left leg served as a control for the
chick-cell-containing right leg. No developmental abnormalities due to the
transplantation procedure of the quail legs were seen. The experimental right
leg of each treated embryo was of similar size and developmental stage as the
control left leg. After two weeks of clonal culture the dishes were fixed with
3:1 ethanol: glacial acetic acid for 5 min and rinsed with tap water before
staining.
Histology
Transplant-containing and control quail embryo legs were removed at various
times between 7 and 10 days of incubation (4-7 days post-transplantation),
washed in Puck's Saline G and fixed in a 3:1 (v/v.) mixture of 100 % ethanol
and glacial acetic acid. Fixed tissues were embedded in Paraplast (Fisher),
sectioned at 6 [im and stained with either hematoxylin and eosin, or the Feulgen
procedure with a fast green counterstain.
Stained culture dishes were flooded with distilled water to retard fading and
individual clones were examined to determine whether they contained quail or
chick cells. The locations of all chick clones were marked and the dishes
counter stained with Harris' hematoxylin. The hematoxylin-stained quail and
chick clones were then scored as muscle (myotubes present) or as non-fusing
(no myotubes); all chick muscle clones were then scored as CMR-I or CMR-II,
CMR-III (see Results for definitions of chick muscle clone types). Using these
Interspecific transplantation of cloned muscle cells
123
methods one can determine the proportion of all chick clones which are
differentiated, and the types of chick muscle clones recovered from the transplanted quail legs.
Histological analysis of sectioned tissues
Sections which had been stained by the Feulgen procedure with a fast green
counterstain were examined at 400 x magnification. Quail nuclei characteristically exhibit one or more darkly Feulgen-positive spots while chick nuclei
generally stain relatively homogeneously with a few small, lightly Feulgenpositive spots. With experience, the two nucleus types can be distinguished
without much difficulty. For quantitative scoring of nuclei a grid reticule was
inserted in the microscope ocular and all nuclei appearing within the grid
limits were noted as either quail or chick. At 400 x magnification, 200 to 400
nuclei were contained within the grid. In some cases, only those nuclei seen to
be in myotubes were scored. Since it is difficult in many instances to determine
by light microscopy whether or not nuclei are in myotubes, only the best
examples of myotube nuclei were scored. Here, both sides of the myotube
were visible and the nuclei were clearly in the center of and in the same focal
plane as the myotube. In sections cut from older tissue (10- to 12-day-old host
quail embryos) cross-striations often aided identification of myotubes. Since
the myotubes were generally sectioned at various oblique angles the number of
nuclei seen per myotube was small, usually from 2 to 8.
The difference in proportion of nuclei scored as chick between transplantcontaining and pure quail tissue is fairly small. To assure ourselves that bias in
scoring nuclei did not affect the data, three persons unfamiliar with the project
scored representative fields of transplant-containing and control quail sections.
The ratios of percentage chick nuclei in transplant regions to percentage chick
nuclei in non-transplant or control regions in these blind scorings were nearly
identical to those determined by us.
RESULTS
Developing chick embryo leg skeletal muscle contains a number of different
clonable myoblast classes. Some of these, termed CMR, require the presence
of conditioned medium for in vitro differentiation. The first class of CMR
clonable myoblasts to appear during development is the 'early embryo'
(Bonner & Hauschka, 1974), or CMR-I class. CMR-I is the only type of
clonable myoblast derived from chick legs until about stage 25; after stage 25,
the proportion of CMR-I declines and two other conditioned-mediumrequiring myoblast types appear - CMR-II and CMR-III. CMR-II and
CMR-III clones are morphologically identical to each other but different from
CMR-I (White et al. 1975). CMR-I clones are characterized by the presence of
short, stubby myotubes containing relatively few (10-20) nuclei, and the
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124
M. D. WOMBLE AND P. H. BONNER
myotubes are arranged in an unpatterned, irregular manner. CMR-II and
CMR-III muscle clones exhibit myotubes which contain up to hundreds of
nuclei each. These myotubes are much longer than those of CMR-I clones and
are arranged in swirling patterns.
The stability of the CMR-I clonal morphology was tested by subcloning.
Cells derived from young chick embryos were grown for two weeks in primary
clonal culture and CMR-I clones identified by phase microscopy. The primary
CMR-I clones were isolated with a ceramic cylinder and the cells removed
from the plate surface with collagenase. Single cells were then added to fresh
petri plates containing conditioned medium for another 2-week period of clonal
growth and differentiation. Of the 453 clones recovered after subcloning, 91 %
were differentiated, and virtually all the muscle clones were identified as
CMR-I. These results show that clonal morphology is a stable indicator of this
muscle clone type and that CMR-I myoblasts in culture do not spontaneously
change to the more advanced CMR types.
To test the viability of cloned CMR-I cells which had been formed into
multicell aggregates two techniques were employed. First, aggregates formed
overnight were redissociated to single cells, and the ability of the cells to
exclude the vital dye Nigrosin was tested. In three experiments, 70-1 ±1-1
(S.E.M.) % of the cells remained viable and excluded the dye. Second, chick
CMR-I cell aggregates were transplanted into quail embryo leg buds for only
12 h before dissociation of the transplant-containing quail legs and inoculation
of the cells into culture dishes. During such a short period of transplantation
most cells are not expected to divide, since collagenase-dissociated cells generally
remain dormant for 6-12 h after treatment (personal observations on cultured
cells). Any cells capable of division immediately after transplantation should do
so only once in view of the 10-12 h generation time of similar cells (Janners &
Searls, 1970; Buckley & Konigsberg, 1974; Zalin, 1979). When placed into
culture, the chick cells should be essentially the same ones which were transplanted. The cultured cells were fixed at 12 and 24 h post-inoculation to,
again, avoid significant expansion of either the chick or quail cell populations
by mitotic proliferation. Some dishes were stained by the Feulgen procedure to
distinguish chick and quail cells while others were stained with hematoxylin to
determine the total number of cells on the dishes and thus the plating efficiency.
Culture for 12 or 24 h gave identical results: 38 ± 1-0 (S.E.M.) % of the cells added
to the dishes adhered to the plate surface, spread out, and were stained by
hematoxylin; of these attached cells, 26-5 ± 0-5 (S.E.M.) % were chick. Aggregates
of chick CMR-I cells thus contain significant numbers of viable cells, as is
also shown by the clonal analysis data below.
Clonal analysis of transplanted chick CMR-I myoblasts
Quail legs containing chick CMR-I myoblasts were subjected to clonal
analysis four days after transplantation (Table 1 and Fig. 1). Of the 65 chick
Interspecific transplantation of cloned muscle cells
125
Table 1. Clonal analysis of transplant-containing quail leg skeletal muscle
Experiment
Total
quail
clones
Quail
muscle
clones
Total
chick
clones
Chick
muscle
clones
Chick
CMR-I
clones*
1
2
3
4
5
6
Totals
21
18
50
726
744
322
1881
16
11
3
590
598
293
1511
3
2
2
30
18
10
65
3
1
1
29
15
10
59
3
1
1
28
14
10
57
* All chick muscle clones were examined for the distinctive characteristics of CMR-I as
described above. Of all chick muscle clones seen (59), 97% were CMR-I.
The contralateral, non-transplanted quail leg was also cloned in each experiment. The
percentage of muscle clones from the control legs was 79-7 (1416/1776) while that of quail
clones from transplanted legs was 80-3 (columns 2 and 3).
Most cells of chick clones from transplant quail legs had no Feulgen-positive spots. In
some clones up to 10-20% of the cells did show spots, but these were generally more lightly
stained and fewer per nucleus than those of cells in quail clones on the same plates. No
clones from control quail tissue were ever seen in which more than 5-10% of the cells lacked
the distinctive quail stain. Quail nuclei of cultured cells are not identical to those of sectioned
tissue after Feulgen staining. The spots of cultured nuclei tend to be larger, more diffuse
and more lightly stained than sectioned nuclei. These differences are probably due to the
flattening of nuclei during culture.
clones recovered in six experiments, 91 % differentiated as muscle. Of these
chick muscle clones, 97 % (57 of 59) were identified as CMR-I. These results
indicate that those cells which survive the transplantation and cloning procedures remain CMR-I. No difference in percentage muscle colony differentiation was noted between quail clones grown from transplant-containing or
contralateral, transplant-free, quail leg cells.
Histological analysis of transplant-containing quail legs
Since clonal analysis did not indicate a significant transition of CMR-I cells
to either CMR-II or CMR-III, a likely alternative fate of CMR-I could be to
fuse with other chick or quail muscle cells to form myotubes. To investigate
these possibilities, transplant-containing quail legs were sectioned and examined,
after Feulgen staining, to determine where chick nuclei are found. Sections of
control quail tissue exhibit significant proportions of nuclei which do not show
the darkly stained nuclear spots characteristic of quail, and, consequently,
appear indistinguishable from chick nuclei (Table 2). Therefore, simple
observation proved inadequate to locate tissues composed of more-thanbackground proportions of chick nuclei, and quantitation of chick and quail
nuclei proportions in microscope fields was necessary.
Cell aggregates were placed in the prospective dorsal thigh region. Therefore,
9-2
126
M. D. WOMBLE AND P. H. BONNER
*fc
Fig. 1. Clones grown for two weeks in conditioned medium from cells of transplantcontaining quail legs. (A) A portion of a quail cell clone. The nuclei exhibit the
darkly Feulgen-positive spots characteristic of quail cells. 200 x magnification. (B)
A portion of a chick CMR-I muscle clone. Note the absence of nuclear stain. 200 x
magnification.
transplanted and control legs were sectioned longitudinally and the following
tissues examined: dorsal thigh muscle, ventral thigh muscle, and calf muscle.
The percentages of chick and quail nuclei found in 400 x microscope fields
were determined and, in addition, nuclei which were clearly contained in
myotubes were scored as chick or quail. Figure 2 contains photographs of
representative fields.
From the data in Table 2 it can be seen that the proportion of chick nuclei
in microscope fields of transplant dorsal thigh muscle is significantly greater
than the proportion seen in any other region, greater even than the ventral
thigh muscle, suggesting that the chick cells remain near the site of transplantation and do not migrate away to any great extent. The proportion of
chick nuclei in frank myotubes of the dorsal thigh (36-7 %) is considerably
higher than it is when all nuclei of a field are considered (19-8 %). The presence
Interspecific transplantation of cloned muscle cells
127
Table 2. Location of chick nuclei in transplants
Percentage of chick nuclei (mean±s.E.M.)*
Region examined
Dorsal Thigh
Fields
Myotubes
Ventral Thigh
Fields
Myotubes
Calf
Fields
Myotubes
Transplantcontaining
quail legs
Control quail
legs
Control chick
legs
19-8±O-8
n = 24
36-7±2-6
n = 56
6-8±l-2
n= 9
10-3 ± 2 0
n = 20
98-7±0-9
n=3
100
/i = 5
6-4 ±0-7
n = 14
7-7 ±2-2
n = 20
7-2 ±0-6
n = 14
11 -5 ± 2 0
n = 30
—
—
99-9 ± 0 1
n =3
—
* Mean percentage of chick nuclei was determined by scoring all nuclei in 400 x microscope
fields (ji = number of fields) or in frank myotubes (here, n is the number of mytobulesexamined)
for the absence of characteristic Feulgen-positive spots. The total number of nuclei scored
is 26344. The number of nuclei scored in each tissue is: dorsal thigh fields of transplantcontaining quail legs, 8995; of chick control, 1053; of quail control, 3142. Dorsal thigh
myotubes of transplants, 533; chick controls, 46; quail controls, 181. Ventral thighfieldsof
transplants, 5733. Ventral thigh myotubes of transplants, 147. Calf fields of transplants,
5524; chick controls, 990. Sections from five different transplanted embryos were examined.
Significance of differences between some sets of data was tested by Student's two-tailed
t test following an arcsine transformation of the percentages. The differences between the
mean of transplant dorsal thigh fields and the means of transplant ventral thigh fields or
quail control fields were significant at P < 0001. The differences between means of the
corresponding myotubes were also significant at P < 0001. By an a priori analysis of
variance test it was determined that the dorsal thigh and myotube means were significantly
different (P < 0001) from all other means and that these were the only significant differences
among transplant and quail control data sets.
of chick nuclei in myotubes shows that the CMR-I cells were capable of fusion,
and the relatively high proportion of them in myotubes suggests further that
fusion is their fate.
DISCUSSION
Three different fates for CMR-I myoblasts are possible: they may die, they
may undergo differentiation and reappear as another cell type, or they may
fuse to form myotubes. Cell death as a fate cannot be ruled out by the present
work but is unlikely in view of the observation that clonable chick myoblasts
128
M. D. WOMBLE AND P. H. BONNER
"B-
Fig. 2. Feulgen- and fast-green-stained sections of muscle tissue. (A) Control quail
thigh muscle. (B) Control chick thigh muscle. (C) and (D) Transplant-containing
quail thigh muscle exhibiting both quail and chick nuclei. Arrows denote chick
nuclei. All photographs were made at 400 x magnification.
Interspecific transplantation of cloned muscle cells
129
are recovered from transplant-containing quail legs. If programmed cell death
were their normal fate then the four days spent in vivo after transplantation
should be sufficient to allow their death, since the same time span in the chick
ends with the nearly complete disappeaiance of clonable CMR-I (White et ah
1975). It should also be noted that the transplanted cells were cultured for two
weeks before and for two weeks after transplantation.
The available evidence also suggests that transplanted CMR-I cells do not
reappear as a different cell type. Certainly they do not undergo transition to
the more advanced clonable myoblast types, CMR-II and CMR-III, as these
clone types do not appear at significant levels after transplantation. CMR-I
cells also do not reappear as clonable non-muscle cells at a significant frequency
since 91 % of the chick clones derived from transplant-containing quail legs
were differentiated muscle clones. The possibility that the CMR-I cells reappear
as a different cell type which is not clonable cannot be ruled out since such
cells would not be taken into account by clonal analysis.
To further investigate the fate of CMR-I cells, Feulgen-stained sections of
transplant-containing quail legs were examined. When nuclei within microscope
fields were scored as chick or quail the region which contained the highest ratio
of chick to quail nuclei was found to be the dorsal thigh muscle, the region into
which the transplant was originally placed. Since most of the chick nuclei were
not found to be inside obvious myotubes the identity of the mononucleated
cells containing the nuclei remains in some doubt. They could be myoblasts or
connective tissue cells, especially fibroblasts. These two cell types cannot be
distinguished at the light microscope level. Other cell types found in the thigh
which might have had chick nuclei but are distinguishable by histological
criteria, such as blood vessel endothelium and lipocytes, were ruled out by
direct observation while chondrocytes, perichondrial fibroblasts and epidermal
epithelial cells were ruled out by quantitative scoring. It is most likely that such
chick cells seen in sections are indeed myoblasts because clonal analysis of
similar tissue demonstrated that nearly all of the chick clones grown from
transplant-containing quail leg cells differentiated as muscle and were therefore
derived from myoblasts. The possibility remains, however, that such putative
chick fibroblasts may not be clonable.
In order to be more certain that particular nuclei were muscle, only those
nuclei which were obviously contained in frank myotubes were scored. Again,
the only region which contained a ratio of chick to quail nuclei significantly
greater than quail controls was the dorsal thigh muscle. From these observations
we conclude that while mononucleated cells containing chick nuclei are not
necessarily all myoblasts, at least some of the transplanted chick CMR-I
myoblasts retain their myogenic properties and enter myotubes by fusion.
Clonal analysis has shown these myoblasts to be CMR-I and, further, that the
more advanced myoblast types, CMR-II and CMR-III, do not appear. It is
suggested, then, that the developmental fate of CMR-I myoblasts is to form
130
M. P. WOMBLE AND P. H. BONNER
myotubes. The data also suggest that CMR-I myoblasts do not serve as the
precursor to CMR-II or CMR-III.
This clone transplantation system should be applicable to determination of
the fate of any cell type which will recognizably differentiate in clonal culture
and which will retain its ability to differentiate after at least one subclonal
passage. This system is advantageous primarily because the donor cells are
derived from a single proliferating cell and are phenotypically identical.
This research was supported by a grant (HD-10307) to P.H.B. from the National Institute
of Child Health and Human Development.
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