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Fig. 1. Developmental anatomy of the thymus. A: The thymus primordia are recognizable at around stage 47 (arrows). C: Transverse section of a stage-47 tadpole, at the level of the pharynx (ph); dorsal towards the top. The thymus primordia (arrows) are located lateral and ventral to the hindbrain (hb), between the eye and the developing inner ear (ie), as seen on a longitudinal section (D). F: Higher magnification view of the right thymic primordium shown in E. Longitudinal section of a stage-47 tadpole, anterior towards the top. G: At stage 50, the thymus primordia have increased in size but retained their dorsolateral position (arrows). G, I: Phase contrast. H: Hematoxylin and eosin staining of the same section. K,L: Expression of p63, an epithelial stem cell factor. I,J are a higher magnification view of G,H. L is a higher magnification view of K. Transverse sections, dorsal towards the top. M,N: At stage 55, the thymus has been extensively colonized by the NC-derived pigment cells (arrows). N is a higher magnification view of the thymus shown in M. Dorsal views, anterior toward the top. O: On a transverse section, the cortex (cx) and me- dulla (md) of the thymus are now clearly distinguishable. The dashed circle outlines the medulla in Q. P and Q are higher magnification views of O. Scale bars (A, D) 1â„4 500 mm; (B, C, E, G, H, N, P) 1â„4 200 mm; (F, I, J, K, Q) 1â„4 100 mm; (M) 1â„4 2 mm; (O) 1â„4 1 mm.
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Fig. 2. Early development of the thymus primordia. Histological sections illustrating the formation of the thymus primordium (arrows) from the pharyngeal pouch epithelium (arrowheads) in Xenopus tadpoles at stages 415. For each stage, both sides of the pharyngeal region are shown (lateral panels). The central panels are lower magnification views, composite pictures of both sides. Dorsal bilateral outpocketings of the pharyn- geal epithelium are first visible at stage 41. The thymus primordium is completely separated from the pharyngeal pouch by stage 45. Transverse sections through the hindbrain region at a level posterior to the eye and anterior to the developing inner ear, dorsal to top. The scale bars in the lateral and middle panels 1⁄4 100 and 200 mm, respectively.
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Fig. 3. Developmental expression of gcm2. A,B: gcm2 was first detected at stage 31 by whole mount in situ hybridization in two longitudinal stripes in the pharyngeal region. At stage 32 (C,D) and stage 34 (E,F), gcm2 was now expressed in three distinct stripes. At stage 40 (G,H) and by stage 41 (I), gcm2 expression appeared to be confined to the developing gills (arrows). A: Lateral views, dorsal towards the top, anterior to the left. B, D, F, H are higher magnification views of the embryos shown in A, C, E, G. In situ hybridization on longitudinal sections of stage-32 (J,K) and stage-34 (L,M) embryos confirmed the expression of gcm2 in the endoderm and ectoderm components of the second, third, and fourth pha- ryngeal arches, with stronger expression in the third arch. For J,K and L,M, the level of the section is indicated by a white line in D and F, respec- tively. In J, anterior towards the top. N,O: Hematoxylin and eosin staining of a section through the thoracic region of a 3-month-old (90 days) froglet. The parathyroid (arrow) was located in the vicinity of the aortic arch arteries. O is a higher magnification view of the parathyroid shown in N. aa, aortic arch artery; ca, carotid artery; pa, pulmonary artery; ph, pharynx; pp, pharyngeal pouches; IIV, pharyngeal arches. In each panel, the stage/age is indicated in the lower left corner. The scale bars (A, C, E and G) 1â„4 500 mm; (B, D, F, H, and I) 1â„4 250 mm; (J, L, and N) 1â„4 200 mm; (K, M, and O) 1â„4 100 mm. P: In 3-month-old froglets, Gcm2 was specifically detected in the parathyroid (Pa) by real time RT-PCR, while in other tis- sues, including brain (Br), heart (He), and thymus (Th), gcm2 was essentially undetectable.
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Fig. 4. Developmental expression of hoxa3. A: hoxa3 expression at stage 22 (A,B) and stage 24 (C,D) was restricted to the spinal cord with an anterior boundary of expression corresponding to rhombomere 5. hoxa3 was also expressed in the migrating NC cells. At stage 32 (E) and stage 34 (I), NC cells had colonized the third and fourth pharyngeal arches (IIIV) where they still expressed hoxa3. At this stage, hoxa3 was also detected in the pharyngeal endoderm epithelium of the arches, but was excluded from the mesoderm core. The level of the sections is indi- cated by a black line in F, J, N. At stage 41 (M), the primordium of the thymus emerging from the second pharyngeal pouch was surrounded by hoxa3-negative NC cells (arrows). For whole mount in situ hybridization, anterior to left, dorsal to top except for A, C, which are dorsal view. G, H, K, L are longitudinal sections, anterior towards the top. O, P are transverse sections, dorsal to top. ph, pharynx; pp, pharyngeal pouches; IIV, pharyngeal arches. In each panel, the stage is indicated in the lower left corner. The scale bars in (A, I, and M) 1â„4 500 mm; (F, J, and N) 1â„4 250 mm; (G, K, and O) 1â„4 200 mm; (H, L, and P) 1â„4 100 mm.
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Fig. 5. Sequence and developmental expression of foxn1. A: The deduced amino acid sequences of Foxn1 from human, mouse, chicken, and zebrafish were aligned using ClustalW2 and compared to the partial amino acid sequence of Xenopus laevis Foxn1. The highly conserved fork- head domain is underlined
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Fig. 5. Sequence and developmental expression of foxn1. B: By whole mount in situ hybridization, foxn1 was first detected at stage 47 (arrows) lateral to the hindbrain (hb). In situ hybridization on transverse sections of stage-47 (C,D) and stage-48 (E,F) tadpoles. foxn1 was completely restricted to the thymus primordium (arrows). foxn1 expression in the thymus persisted at least up to stage 57 (G), at which stage foxn1 was weakly detected throughout the entire organ. D, F are higher magnification views of the thymus primordium shown in C, E, respectively. In each panel the stage is indicated in the lower left corner. The scale bar (B) 1â„4 500 mm; (C, E, and G) 1â„4 200 mm; (D and F) 1â„4 200 mm.
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Fig. 6. RT-PCR analysis of the developmental expression of hoxa3, gcm2, and foxn1. The stages are according to Nieuwkoop and Faber (1967). Th, thymus from stage-55 tadpoles. Orni- thine decarboxylase (odc) is shown as a loading control.
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Fig. 7. NC contribution to the thymus. A: Experimental design to analyze the NC contribution to the thymus. Two-cell-stage embryos were injected in the animal pole with mRNA encoding Red Fluorescent Protein (RFP). At stage 17, the RFP-labeled cardiac NC was transplanted onto the same region of an unlabeled host embryo (NC graft). B: RFP-labeled NC graft at stage 26. Lateral views of the same embryo. Dorsal towards the top, anterior to the right (B,C) and left (D,E). Cells derived from the RFP-labeled NC graft were confined to the three most posterior streams of cranial NC, as previously described (Lee and Saint-Jeannet, 2011). At stage 41 (F), cells derived from the RFP-labeled NC graft (JO) formed most of the head mesenchyme, surrounding the thymus primordial (arrows). A fraction of the RFP-labeled NC cells also express hoxa3; however, these cells remained confined to a region posterior to the thymus primordia (G, I, M, J, N). H, L are higher magnification of F. I, M are higher magnification of G. Longitudinal sections, anterior towards the top. K and O are overlay of H and L, respectively. Insets in K, O are higher magnification views of the thymus primordia (arrows). P: At stage 47, the thymus primordia can be identified by Foxn1 expression (Q, S, W). A small number of RFP-labeled NC cells have colonized the thymus (T, U, X, Y). R are higher magnification views of the thymus primordium (arrows) in P, Q. R and V show two representative tadpoles form two independent experiments. U and Y are overlay of R and V, respec- tively. The scale bars (B, P, and Q) 1â„4 200 mm; (H and R) 1â„4 50 mm.
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Fig. 8. NC cells are dispensable for thymus development. A: Diagram illustrating cardiac NC ablation at stage 17. B: sox10 expression in con- trol and NC-ablated embryos at stage 17 (B and E), and stage 25 (C,D and F,G). The red brackets in control (B) and NC-ablated (E) embryos indi- cate the position of cardiac NC territory ablated at stage 17. NC ablation resulted in the bilateral loss of the three most posterior NC streams at stage 25 (F,G) as compared to control (red brackets in C and D). In NC-ablated embryos, sox10 expression in the otic vesicle is unaffected (arrows). B and E are dorsal views, anterior to top. C and D (control) and F and G (NC-ablated) are showing both sides of the same embryo. Dor- sal to top, anterior to right (C and F) or to left (D and G). H: Transverse sections (dorsal to top) through two stage-47 tadpoles (H and K) following NC ablation at stage 17. foxn1 expression was detected in the thymus primordia of both tadpoles. H, J and K, M are higher magnifica- tion views of the thymi shown in I and L, respectively. I, L: The arrows point to the thymus primordia expressing foxn1. The scale bars in (B) 1â„4 500 mm; (H, J, K, and M) 1â„4 50 mm; (I and L) 1â„4 200 mm. N,O: The thymi of control (n1â„49) and NC-ablated (n1â„49) tadpoles were dissected at stage 48 and analyzed by real time RT-PCR for foxn1 (N) and rag1 (O) expression. foxn1 and rag1 expression was normalized to the expression of the TEC-specific marker, keratin 8. Statistical significance was determined using the t-test. The values are presented as mean SEM.
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gcm2 (glial cells missing homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32 ,dorsal view, anterior left, dorsal up, and longitudinal section, anterior top.
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gcm2 (glial cells missing homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40 ,dorsal view, anterior left, dorsal up.
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hoxa3 (homeobox A3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22,dorsal view, anterior left, dorsal up.
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hoxa3 (homeobox A3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32,dorsal view, anterior left, dorsal up, and longitudinal section, anterior up.
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Thymus primordium, in X. laevis NF stage 47 tadpole, indicated by arrows, via gene expression of foxn1 (forkhead box N1), visualised via in situ hybridization.( hb is hindbrain ).
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