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First published online December 7, 2008
doi: 10.1242/10.1242/dev.028456

Development 136, 29-34 (2009)
Published by The Company of Biologists 2009
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Lymph sacs are not required for the initiation of lymph node formation

Mark F. Vondenhoff1,*, Serge A. van de Pavert1,*, Miriam E. Dillard2, Mascha Greuter1, Gera Goverse1, Guillermo Oliver2,{dagger} and Reina E. Mebius1,{dagger}

1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, PO box 7057, 1007 MB Amsterdam, The Netherlands.
2 Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.


Figure 1
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  		Fig. 1. Characterization of lymphatic endothelial cells in wild-type developing axillary lymph nodes. Lymph nodes (LNs) were examined at E14.5 (A,C,E,G) and E16.5 (B,D,F,H). (A,B) LNs were identified by combined staining for CD4 (green), which is expressed by LTi cells, IL7R{alpha} (red), which is expressed by LTi cells and their precursors, and CD45 (blue), which is expressed by all hematopoietic cells. (C-H) Subsequent sections were stained to detect (C,D) the lymphatic endothelial cell (LEC) marker LYVE1(blue) in combination with the stromal marker MAdCAM1 (red) and the endothelial cell marker VE-cadherin (green), (E,F) the LEC marker PROX1 (green) in combination with the LN stromal cell marker podoplanin (red) and the vascular endothelial cell marker VEGFR3 (blue), and (G,H) the vascular endothelial cell markers VEGFR2 (green), MECA32 (red) and VEGFR1 (blue). Arrows in C and E indicate the lining of the lymphatic endothelium. Arrowheads in G and H indicate blood vessels. Scale bars: 75 µm in A-H.

 

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  		Fig. 2. Lymph node anlagen are present in the absence of lymphatic vasculature in Prox1-/- mouse embryos. Combined staining for MAdCAM1 (green), CD4 (red) and CD45 (blue) indicates that most LN anlagen are present in E14.5 Prox1-/- embryos. Shown are (A-C) mesenteric LNs, (D-F) renal LNs and (G-I) axillary LNs in wild-type, Prox1+/- and Prox1-/- embryos. Arrows indicate MAdCAM1+ cells that encapsulate hematopoietic cells (G) or disorganized clusters of hematopoietic and MAdCAM1+ cells (I). Scale bars: 75 µm in A-I.

 

Figure 3
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  		Fig. 3. Lymph node anlagen develop normally in Prox1-heterozygous and Prox1-null mouse embryos. To determine whether PROX1 deficiency affects LN development in a dose-dependent manner, we compared the LN anlagen of E14.5 wild-type, Prox1+/- and Prox1-/- embryos. (A-C) Sections were stained for CD45 (blue), CD4 (green) and IL7R{alpha} (red) to identify clusters of LTi cells, which form the LN anlagen. (D-I) Adjacent sections were stained for (D-F) VEGFR1 (green), MECA32 (red) and VEGFR2 (blue) to detect endothelial cells, and for PROX1 (G,H, green), β-galactosidase (β-gal) (I, green), podoplanin (G-I, red) and MAdCAM1 (G-I, blue) to detect lymphatic endothelium and stromal cells within the LN anlagen. LN anlagen of wild-type and Prox1+/- embryos appeared indistinguishable, although the organization of the lymphatic epithelium and blood endothelium within the LN anlagen was disorganized in Prox1-/- embryos. Arrows in D and E indicate blood vessels, and arrowheads indicate small blood vessels. Arrows in G and H indicate the lining of the lymphatic endothelium. Scale bars: 75 µm in A-I.

 

Figure 4
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  		Fig. 4. Lymph node anlagen are present in E17.5 Prox1 conditional-null mouse embryos. (A,B) Staining of wild-type and conditional-null (Tie2-Cre/Prox1flox/flox) brachial LNs with antibodies against LYVE1 (green), MAdCAM1 (red) and CD4 (blue) revealed that LYVE1+ LECs that coexpress MAdCAM1 are absent from the LN anlagen of Tie2-Cre/Prox1flox/flox embryos. (C,D) Staining of adjacent sections with antibodies against MAdCAM1 (green), VCAM1 (red) and podoplanin (blue) indicated that the MAdCAM1+ VCAM1+ stromal organizer cells, which are abundant in the wild-type LN anlagen (C), are greatly reduced in the Tie2-Cre/Prox1flox/flox LN anlagen (D). Scale bars: 75 µm in A-D.

 

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