Article Figures & Data
Figures
- FIGURE 1.
A Foxn1 promoter fragment directs Noggin expression to epithelial cells of the developing thymic anlage. A, In situ hybridizations of the thymic anlage at dE11.5 and dE12.5 are shown for the expression of Foxn1 and transgenic Noggin. Each panel is oriented such that down is ventral and left is medial. Note that transgenic Noggin expression is restricted to thymic epithelial cells and is not found in surrounding mesenchymal cells. Scale bar, 50 μm. B, A macroscopic view of a mediastinum of a 4- to 5-wk-old WT mouse (top), and a Foxn1::Xnoggin transgenic mouse of the same age (bottom) is shown. t, thymic lobe; h, heart. The thymus is absent in transgenic mice. C, Absolute number of cells that fall into the CD4 and CD8 double-positive gate by flow cytometry found in single-cell suspensions prepared from the entire contents of the mediastinum of 4- to 5-wk-old WT, nude, and Foxn1::Xnoggin mice. Data for transgenic mice derived from three independent founders are shown. Each dot represents one mouse. Cells that fall into the CD4 and CD8 double-positive gate by flow cytometry in nude mice, that lack a functional thymus, derive from autofluorescent cells, and their levels were considered background (<0.001% of normal values). D, FACS analyses of 4- to 5-wk-old WT, nude, and Foxn1::Xnoggin peripheral lymph node cells. E, Transversal, H&E-stained sections of dE15.5 Foxn1::Xnoggin embryos at the level of the developing hyoid bone. The location of dysplastic thymi that remain separated in Foxn1::Xnoggin mice are indicated by open arrows. Scale bar, 200 μm. oe, esophagus; la, larynx; hy, cartilage giving rise to the hyoid bone; sg, salivary gland.
- FIGURE 2.
Blocking BMP signals during thymus development leads to a reduced thymus size. A, In situ hybridizations of the thymic anlage at dE15.5 are shown for the expression of Foxn1 and transgenic Noggin. The inset in the WT Foxn1 panel shows the dysplastic thymic lobe of a Foxn1::Xnoggin embryo of the same age pictured to scale to visualize the size difference. Scale bar, 50 μm. B, In situ hybridization of thymic anlagen of Foxn1::Xnoggin mice at dE13.5 and dE14.5. Small epithelial cysts are found in the dE14.5 thymic anlage that increase rapidly in size (compare with dE15.5 shown in A). Scale bar, 50 μm. C, Immunohistochemical analysis of thymic anlagen of WT and Foxn1::Xnoggin mice at dE12.5 and dE15.5 stained for cytokeratin 5 (green) and cytokeratin 8 (red). Note that the WT dE15.5 panel shows only a small fraction of the thymic anlage. Scale bar, 50 μm.
- FIGURE 3.
T cell development proceeds normally in a Noggin-expressing thymus. A, FACS profiles of WT and Foxn1::Xnoggin thymocytes isolated from 5- to 7-day-old mice. The percentage of cells found in the respective quadrants is shown. B, The total number of thymocytes isolated from 5- to 7-day-old WT and Foxn1::Xnoggin mice is shown. Each dot corresponds to one mouse. C, The fractions of the different thymocyte subsets among total thymocytes (top panel) and coreceptor DN thymocytes (bottom panel) found in 5- to 7-day-old WT and Foxn1::Xnoggin mice were determined by FACS analysis. The mean and SD of at least four determinations for each subset is shown. D, The fraction of DNA-synthesizing cells was determined by BrdU pulse labeling. The results of three independent experiments are shown for 5- to 7-day-old WT and Foxn1::Xnoggin mice. E, The number of apoptotic cells as identified by annexin V staining and annexin V in conjunction with propidium iodide staining was determined in single-cell suspensions of thymocytes isolated from 5- to 7-day-old WT and Foxn1::Xnoggin mice. The numbers indicate the mean percentage and the SD of cells found in the respective gates from three determinations. F, FACS profiles of WT and Foxn1::Xnoggin thymocytes from dE16.5 embryos are shown. The percentage of cells found in the respective quadrants is shown.
- FIGURE 4.
Thymi of Foxn1::Xnoggin mice generate a polyclonal peripheral T cell repertoire. Usage of the indicated Vβ-chains in splenic CD4 single-positive mature T lymphocytes of WT and Foxn1::Xnoggin mice of the indicated age was determined by FACS analysis using specific mAbs. Each bar represents one mouse. Results are given as percentage of total splenic CD4-positive T cells.
- FIGURE 5.
The major BMP signaling components are expressed in embryonic thymic stroma. Embryonic thymic lobes of dE15.5 were treated with deoxyguanosine for 6 days to eliminate lymphocytes, and RNA was prepared for RT-PCR using primers specific for the indicated transcripts. Culture of such deoxyguanosine-treated lobes confirmed the absence of hemopoietic cells from these lobes. Mock samples run in the absence of reverse transcriptase, and water controls remained negative for all transcripts shown.
- FIGURE 6.
Blocking BMP signals during thymus development disturbs the physiological expression of BMP4 and BMP2 in epithelial as well as mesenchymal cells of the thymic anlage. In situ hybridizations for BMP4, BMP2, Msx-1, and Hoxa3 on transversal sections of the thymic anlage at the indicated time points during mouse embryogenesis are shown. Each panel is oriented such that down is ventral and left is medial. The outline of the epithelial outgrowth of the third pharyngeal pouch endoderm at dE10.75 that will give rise to parathyroid and thymus is indicated by a black line. The prospective parathyroid epithelium does not express Foxn1 but Gcm2 (3 ). An open arrow marks the lateral aspect of the thymic anlage at dE11.5 for better comparison. Bottom panels, Immunohistochemical sections of dE12.5 thymic anlagen stained with an antiserum directed against phosphorylated Smad1/5/8 proteins are shown (open arrows, nuclear staining in mesenchymal cells forming the thymic capsule; filled arrows, presence of phosphorylated Smads1/5/8-positive nuclei of neural crest-derived mesenchymal cells located ventrally of the thymic anlage). Scale bar, 50 μm.
- FIGURE 7.
Thymic lobes of 5- to 7-day-old Foxn1::Xnoggin transgenic mice contain cystic areas of dys-differentiated epithelial cells next to phenotypically normal thymic stroma. A, Immunohistochemical staining for cytokeratin 8, UEA-1, and CD8 reveals the thymus architecture in WT and Foxn1::Xnoggin mice. Scale bar, 100 μm. B, Panels show fluorescence microscopic images of stains for cytokeratin 5 (green) and cytokeratin 8 (red). Note that the cyst, which is invariably found in Foxn1::Xnoggin thymic lobes, contains epithelial cells that stain for either cytokeratin 5 or cytokeratin 8 as well as a few double-positive cells (yellow). Scale bar, 50 μm. C, cortex; M, medulla.
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