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Impaired development of the thymic primordium after neural crest ablation.

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THE ANATOMICAL RECORD 228:185-190 (1990)
Impaired Development of the Thymic Primordium
After Neural Crest Ablation
SHIGERU KURATANI AND DALE E. BOCKMAN
Department of Anatomy, Medical College of Georgia, Augusta, Georgia
ABSTRACT
Impaired thymic development as a result of ablation of neural
crest has been observed in embryos late in development. The present study was
initiated to determine what changes are effected early in thymic development by
neural crest ablation. The epithelial primordia of the thymus were studied in chick
embryos on the sixth day of incubation. Embryos with neural crest ablations were
compared with sham-operated and untreated controls. Neural crest ablation inhibited formation of epithelial thymic primordia. Primordia in experimental embryos were fewer in number and were smaller than in shams and untreated controls. When primordia from shams and controls were transplanted to the
chorioallantoic membrane of chick hosts, they were able to develop into organs
with the typical features of embryonic thymus. Similar transplantation from neural crest-ablated animals, on the other hand, led to small, predominantly epithelial
structures with meager lymphoid development. These findings are consistent with
the hypothesis that mesenchyme derived from cranial neural crest is critical in
initiating and sustaining the development from pharyngeal pouches of epithelial
structures competent to attract and support the proliferation and differentiation of
lymphoid stem cells.
The definitive thymus is derived from three major
sources. The epithelial primordium comes from the endoderm of pharyngeal pouches. As the epithelial primordium develops, it attracts lymphoid stem cells
which are brought into the area from yolk sac andlor
fetal liver by blood vessels. The lymphoid stem cells
proliferate and differentiate within the specialized microenvironment provided by the epithelial reticulum.
The connective tissue of the thymus is derived from
cranial neural crest, which migrates through the pharyngeal arches and differentiates into mesenchyme
early in embryonic development (Le Lievre and Le
Douarin, 1975).
We have shown previously that surgical ablation of
cranial neural crest early in development leads to inhibited or impaired development of the thymus when
that development is assessed late in embryonic development (Bockman and Kirby, 1984). This led to the
hypothesis that thymic development was inhibited because neural crest-derived mesenchyme failed to initiate and sustain the proper development of the epithelial component of the thymus.
The present study was designed to study directly the
epithelial primordia of the thymus. The primordia
were studied in situ after neural crest ablation, and
were transplanted to the chorioallantoic membrane of
host chicks to assess their competence to attract and
support the proliferation and differentiation of lymphoid cells. The results are consistent with defective
development being the result of improper development
of the epithelial primordium.
c
1990 WILEY-LISS. INC
MATERIALS AND METHODS
Fertilized Arbor Acre eggs were incubated at 38°C
and constant humidity approximately 30 hours until
stage 8-11 of Hamburger and Hamilton (1951). Experimental animals were stained in situ through a window
in the shell prior to carefully tearing the vitelline
membrane in order to expose the neural folds. Portions
of neural folds from mid-otocyst level through somite 3
were ablated bilaterally by microcautery as has been
described previously (Kirby et al., 1983). This area corresponds to the neural crest which will seed arches 3 , 4 ,
and 6. Shams were windowed and stained, and the vitelline membrane was torn, but the embryos were not
altered. The windows were sealed and incubation was
continued. Controls were incubated without windowing. Experimental, sham, and control embryos were
harvested on the sixth incubation day, fixed in Bouin’s
solution, and prepared routinely for paraffin embedding. Embryos were oriented for longitudinal sectioning. Serial sections 10 pm thick were stained with hematoxylin and eosin (H&E), or occasionally with
Mallory’s triple stain.
The presence or absence of each epithelial thymic
primordium was determined from the serial sections.
Received November 29, 1989; accepted January 12, 1990.
Address reprint requests to Dale E. Bockman, Department of Anatomy, Medical College of Georgia, Augusta, GA 30912-2000.
186
S. KURATANI AND D.E. BOCKMAN
Fig. 1. Sagittal section of control chick embryo a t day 6 of incubation. These two epithelial thymic primordia (T3 and T4) are derived
from the third and fourth pharyngeal pouches, respectively, on one
side of the embryo. x 280.
Ftg. 2. Similar preparation of a sham embryo. The dense outer epithelium and loose middle are evident. The surrounding mesenchyme
is beginning to form a capsule. x 470.
The size of each thymic primordium was determined by
drawing the outline of the primordium on paper with
the aid of a drawing tube attached to the microscope,
then determining the area using a digitizing pad and
computer. The total for each animal was then calculated and this size estimate was compared among the
three treatment groups. The statistical significance
was determined by Student’s t test.
In order to transplant thymic primordia to the chorioallantoic membrane of host chicks, the region containing pharyngeal pouches 3, 4, and 6 was excised
from embryos on the sixth day of incubation. To encourage vascularization, the transplant was secured in
a small hole which was made in the chorioallantoic
membrane of chicks at the 10-12th day of incubation.
After the transplants were allowed to develop for 6
days on the chorioallantoic membrane, they were fixed
in Bouin’s solution and serial sections stained with
H&E were studied. A total of 29 transplants were used
for the present evaluation-9 after neural crest ablation, 13 shams, and 7 non-operated controls.
RESULTS
Thymic primordia were present in only 24% of the 29
embryos which were subjected to neural crest ablation.
All control and sham embryos, on the other hand, had
thymic primordia. In controls (N = 51, all animals had
four thymic primordia, except for one which was missing one primordium on one side. In shams (n = l o ) , two
animals were missing thymic primordia on one side,
while four primordia were present in each of the remaining animals.
Of the seven experimental (neural crest-ablated) animals with thymic primordia, four had primordia only
on one side, one had three primordia, and two animals
had four primordia.
The thymic primordia in control (Fig. 1 ) and sham
(Fig. 2) animals were well-formed, elongate epithelial
outgrowths of pharyngeal pouches 3 and 4. By following the serial sections, it was possible to determine that
each was connected with its corresponding pharyngeal
pouch, frequently by a cord of epithelial cells. The ep-
IMPAIRED DEVELOPMENT OF THYMIC PKIMORDIUM
187
Fig. 3. Sagittal section through pharyngeal pouches 3 and 4 (P3and
P4) in a chick embryo on the sixth day of incubation, after neural crest
ablation. An abortive thymic primordium (T3) arises from pouch
three, and the only indication of primordium formation in pouch four
is a thickening of the epithelium of the pouch. X 290.
Fig. 4. Similar preparation of another experimental embryo. No
epithelial thymic primordium is present in association with pharyngeal pouch four (P4).The cluster of cells a t left center is part of the
nodose ganglion. x 320.
ithelial cells of the primordium were closely packed
around the periphery and more loosely arranged toward the center (Figs. 1,2). The primordia were surrounded by abundant mesenchyme. The mesenchymal
cells adjacent to the thymic epithelium formed the beginning of a capsule.
The morphology of the epithelial thymic primor-
dium, when present, in animals subjected to neural
crest ablation was heterogeneous. In some animals the
morphology was similar to that of controls and shams,
although the mean size of individual primordia was
significantly (P<0.05) smaller (0.0084 mm2 t 0.0012)
than that of controls (0.0127 mm’ t 0.0015). Shams
were intermediate in size (0.0095 mm2 ? 0.0007). In
188
S. KURATANI AND D.E. BOCKMAN
5
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0.05
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0.04
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0.03
a
0.02
0.01
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SHAM
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Fig. 5. Comparative mean content of epithelial thymic primordia in control, sham, and experimental
embryos. The area (in mm2) for each embryo was calculated by summing the maximum areas of each
primordium. Total primordial area in experimental animals is significantly less than controls and
shams. Controls and shams are not significantly different.
other animals, the only indication of thymus formation epithelial primordia failed to develop, or were smaller,
was small extensions of epithelium from the dorsum of in experimental animals. As shown by transplantation
the pouch (Fig. 3). The epithelium of the pouch com- to the chorioallantoic membrane of host chicks that
monly showed no elaboration which could be inter- were not subjected to neural crest ablation, the defecpreted a s indicative of thymic epithelial proliferation tive epithelial primordia were not competent to attract
circulating lymphoid precursor cells and support their
(Fig. 4).
The mean quantity of total thymic primordium in proliferation and differentiation.
experimental animals (0.006 ? 0.0027 mm2) was also
These deficiencies due to the lack of mesenchymal
significantly (P<O.OOl) less than that in control (0.048 interaction with epithelium are consistent with the
0.0089) or sham (0.034 ~t0.0049) animals (Fig. 5). studies of Auerbach (1960). He showed that when the
Shams were not significantly different from controls.
epithelial component of mouse thymus was cultured
After residence on the chorioallantoic membrane of with thymic mesenchyme, there was the expected, vighost embryos, the epithelial thymic primordia from orous growth. When mesenchymes from various other
control (Fig. 6) and sham embryos (Fig. 7) were able to sources were substituted, growth was delayed, or was
develop into obvious lymphoid organs with the lobula- supported poorly or not at all.
tion and zonation characteristic of thymus. TransAblation of cardiac neural crest causes a quantitaplants from control, sham, and experimental animals tive decrease in the mesenchyme of the pharyngeal
were vascularized. However, in marked distinction to arches (Bockman et al., 1989).Thus, it appears that the
controls and shams, lymphoid development within the failure of neural crest-derived mesenchyme to arrive a t
developing thymus of experimental animals was mark- the proper region of pharyngeal endoderm, in the apedly reduced (Figs. 8, 9); the structure of most re- propriate quantities and a t the appropriate time,
mained that of a predominantly epithelial primordium. causes in t u r n a failure of the endodermal epithelium
The mean area of the thymus in transplants from ex- to produce epithelial thymic primordia, or a failure of
perimental animals was 0.027 mm2
0.008 S.E.M., the primordia to achieve complete competence.
while it was 0.049
0.009 mm2 in shams and 0.050
Absent or poorly developed thymic primordia in exmm2 t 0.013 in unoperated controls.
perimental animals a t day 6 of incubation could be due
to a simple delay in development. The results of the
DISCUSSION
present and other studies show that this is not the case.
The results of this study clearly indicate that there is When the pharyngeal regions were transplanted to the
defective development of the epithelial component of chorioallantoic membrane, those from control and
the thymus subsequent to ablation of neural crest. The sham embryos were able to produce thymuses, while
*
*
*
IMPAIRED DEVELOPMENT OF THYMIC PRIMORDIUM
189
Fig. 6. Section through a thymus which was transplanted from a
control chick on the sixth day of incubation to the chorioallantoic
membrane of a host chick and allowed to develop there. The lobulation and formation of cortical and medullary areas is evident. A plethora of lymphoid cells are present among the epithelial cells. x 190.
Fig. 7. Section of thymus from sham chick which was transplanted
to the chorioallantoic membrane of a host chick. Lymphoid cells are
numerous among the epithelial cell reticulum. x 380.
those from experimental animals were not able to develop complete cellularity. Furthermore, when thymuses were evaluated in neural crest-ablated embryos
after leaving them in situ for up to 16 days of incubation, they failed to achieve complete cellularity
(Bockman and Kirby, 1984).
If neural crest ablation causes defective initiation
and maintenance of the epithelial primordium of the
thymus through defective interaction of neural crestderived mesenchyme, genetic and environmental factors that affect either this mesenchyme, the appropriate epithelium, or both, could lead to defective thymic
development. Some thymic defects may be induced in
this way.
There are, however, thymic deficiencies which occur
in syndromes which are characterized by multiple defects and may include the cardiovascular system and
other organs. In these cases, the primary defect would
seem to be the neural crest and its derivatives (Kirby
and Bockman, 1984).
The DiGeorge syndrome, characterized by thymic,
parathyroid, and cardiovascular deficiencies, would
seem a prime example of this situation (Amman et al.,
1982; Couly et al., 1983; Kirby and Bockman, 1984).
Similar clusters of defects are seen in the fetal alcohol
syndrome (Amman et al., 1982; Daft et al., 1986) and
after exposure to retinoic acid (Lammer e t al., 1986).
In other types of thymic deficiency, the defect may be
in the lymphoid precursor cells. Defective thymic development in the present study was not due to defective
lymphoid precursor cells. The chicks which served a s
hosts for the chorioallantoic transplants were not operated, and had competent, circulating lymphoid precursor cells.
ACKNOWLEDGMENTS
The authors thank Mr. Greg Oblak for technical assistance. This work was supported by grant 2332, The
Council for Tobacco Research-USA., Inc.
190
S. KURATANI AND D.E. BOCKMAN
Flg. 8. Similar preparation to those shown in Figures 6 and 7, but
the transplant to the chorioallantoic membrane was from a chick
subjected to neural crest ablation. The primoridum is small and
poorly developed, with a paucity of lymphoid cells. x 380.
LITERATURE CITED
Amman, A.J., D.W. Wara, M.J. Cowan, D.J. Barrett, and E.R. Stiehm
1982 The DiGeorge syndrome and the fetal alcohol syndrome.
Am. J. Dis. Child., 136:906-908.
Auerbach, R. 1960 Morphogenetic interactions in the development of
the mouse thymus gland. Dev. Biol., 2t271-284.
Bockman, D.E., and M.L. Kirby 1984 Dependence of thymus development on derivatives of the neural crest. Science, 223t498-500.
Bockman, D.E., M.E. Redmond, and M.L. Kirby 1989 Alteration of
early vascular development after ablation of cranial neural crest.
Anat. Rec., 225:209-217.
Cody, G., A. Lagrue, and C. Griscelli 1983 Le syndrome de DiGeorge,
neurocristopathie rhombencephalique exemplaire. Rev. Stomatol. Chir. Maxillofac., 84:103-108.
Daft, P.A., M.C. Johnston, and K.K. Sulik 1986 Abnormal heart and
Fig. 9. Chorioallantoic membrane transplant from another neural
crest-ablated chick. Note the similarity to the transplant in Figure 8,
and the lack of thymic development shown in Figures 6 and 7. x 380.
great vessel development following acute ethanol exposure in
mice. Teratology, 33t93-109.
Hamburger, V., and H. Hamilton 1951 A series of normal stages in
the development of the chick embryo. J. Morphol., 88:49-92.
Kirby, M.L., and D.E. Bockman 1984 Neural crest and normal development: A new perspective. Anat. Rec., 209t1-6.
Kiirby, M.L., T.F. Gale, and D.E. Stewart 1983 Neural crest cells contribute to normal aorticopulmonary septation. Science, 22t10591061.
Lammer, E.J., D.T. Chen, R.M. Hoar, N.D. Agnish, P.J. Benke, J.T.
Braun, C.J. Curry, P.M. Fernhoff, A.W. Grix, I.T. Lott, J.M. Richard, and S.C. Sun 1986 Retinoic acid embryopathy. A new human teratogen and a mechanistic hypothesis. New Eng. J. Med.,
313:837-841.
Le Lievre, C.S., and N.M. LeDouarin 1975 Mesenchymal derivatives
of the neural crest: Analysis of chimeric quail and chick embryos.
J. Embryol. Exp. Morphol., 34t125-154.
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