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 0.06 0.05 h 0.04 cu E E Y 5K 0.03 a 0.02 0.01 0.00 SHAM CONT EXP 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.