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• W2099366913 abstract "Article15 July 2003free access T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema Vivien Schacht Vivien Schacht Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Maria I. Ramirez Maria I. Ramirez Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Young-Kwon Hong Young-Kwon Hong Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Satoshi Hirakawa Satoshi Hirakawa Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Dian Feng Dian Feng Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Natasha Harvey Natasha Harvey Department of Genetics, St Jude Children's Hospital, Memphis, TN, 38105 USA Search for more papers by this author Mary Williams Mary Williams Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Department of Anatomy, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Ann M. Dvorak Ann M. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Harold F. Dvorak Harold F. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Guillermo Oliver Guillermo Oliver Department of Genetics, St Jude Children's Hospital, Memphis, TN, 38105 USA Search for more papers by this author Michael Detmar Corresponding Author Michael Detmar Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Vivien Schacht Vivien Schacht Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Maria I. Ramirez Maria I. Ramirez Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Young-Kwon Hong Young-Kwon Hong Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Satoshi Hirakawa Satoshi Hirakawa Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Dian Feng Dian Feng Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Natasha Harvey Natasha Harvey Department of Genetics, St Jude Children's Hospital, Memphis, TN, 38105 USA Search for more papers by this author Mary Williams Mary Williams Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Department of Anatomy, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Ann M. Dvorak Ann M. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Harold F. Dvorak Harold F. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Guillermo Oliver Guillermo Oliver Department of Genetics, St Jude Children's Hospital, Memphis, TN, 38105 USA Search for more papers by this author Michael Detmar Corresponding Author Michael Detmar Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA Search for more papers by this author Author Information Vivien Schacht1, Maria I. Ramirez2, Young-Kwon Hong1, Satoshi Hirakawa1, Dian Feng3, Natasha Harvey4, Mary Williams2,5, Ann M. Dvorak3, Harold F. Dvorak3, Guillermo Oliver4 and Michael Detmar 1 1Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129 USA 2Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA 3Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215 USA 4Department of Genetics, St Jude Children's Hospital, Memphis, TN, 38105 USA 5Department of Anatomy, Boston University School of Medicine, Boston, MA, 02118 USA ‡V.Schacht and M.I.Ramirez contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3546-3556https://doi.org/10.1093/emboj/cdg342 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Within the vascular system, the mucin-type transmembrane glycoprotein T1α/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-specific homeobox gene Prox1. In this study, we examined the role of T1α/podoplanin in vascular development and the effects of gene disruption in mice. T1α/podoplanin is first expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1α/podoplanin−/− mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1α/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1α/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1α/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1α/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation. Introduction The lymphatic vascular system maintains tissue fluid homeostasis and mediates the afferent immune response, but can also aid in the metastatic spread of malignant tumors (Detmar and Hirakawa, 2002). Dysfunction or abnormal development of cutaneous lymphatic vessels results in lymphedema, which is associated with defects in tissue repair and the immune response (Mallon and Ryan, 1994). Although the mechanisms that control the development of the blood vascular system have been well studied (Carmeliet, 2000), those of the lymphatic vessels are poorly understood. Recent analyses of Prox1-deficient mice have shown that the lymphatic vascular system, as predicted by Sabin (1902), originates from the embryonic veins (Wigle and Oliver, 1999; Oliver and Detmar, 2002). Beginning at embryonic day (E) 9.5 of mouse development, the homeobox gene Prox1 is specifically expressed by a subpopulation of endothelial cells that are located on one side of the anterior cardinal vein. This is followed by polarized budding and migration of these Prox1-positive lymphatic progenitor cells, which eventually form lymphatic sacs and then the entire lymphatic vasculature. In Prox1-null mice, the budding and sprouting of lymphatic endothelial cells from the veins is arrested at ∼E11.5–E12.0, and these mice completely lack a lymphatic vascular system (Wigle and Oliver, 1999). We and others have shown recently that ectopic expression of Prox1 in primary human blood vessel endothelial cells represses the expression of several genes that are associated with the blood vascular phenotype (Hong et al., 2002; Petrova et al., 2002). Prox1 expression was also found to upregulate the expression of lymphatic-specific genes (Hong et al., 2002; Petrova et al., 2002), indicating its function as a master control gene that determines lymphatic endothelial cell fate (Oliver and Detmar, 2002). These studies identified the mucin-type transmembrane glycoprotein T1α/podoplanin as one of the primary Prox1-induced genes (Hong et al., 2002). T1α/podoplanin is expressed by cultured human lymphatic endothelial cells, and is one of the most highly expressed lymphatic-specific genes (Petrova et al., 2002; Hirakawa et al., 2003). In vivo expression of T1α/podoplanin in lymphatic endothelium was first reported by Wetterwald et al. (1996), who named it ‘E11 antigen’. It was characterized further under the name ‘podoplanin’, because of its low level expression in kidney podocytes (Breiteneder-Geleff et al., 1997). Podoplanin is homologous to T1α, which was originally found to encode an antigen that is selectively expressed at the apical surface of alveolar type I cells in rat lung (Dobbs et al., 1988; Rishi et al., 1995). Expression of T1α has also been detected in the choroid plexus, ciliary epithelium of the eye, intestine, kidney, thyroid and esophagus of the fetal rat (Williams et al., 1996), and it has been shown to be homologous to the OTS-8 gene, a phorbol ester-induced gene in MC3T3-E1 mouse osteoblast cells (Nose et al., 1990). Other homologs include RTI40 (Gonzalez and Dobbs, 1998), murine gp38 (Farr et al., 1992), canine gp40 (Zimmer et al., 1997), human gp36 (Zimmer et al., 1999) and murine PA2.26 (Gandarillas et al., 1997). There have been many studies of T1α/podoplanin expression in the lymphatic vascular system (Kriehuber et al., 2001; Maekinen et al., 2001; Hong et al., 2002; Petrova et al., 2002; Hirakawa et al., 2003). In spite of the large number of descriptive studies, little is understood about T1α/podoplanin's biological function. We examined its role in lymphatic and blood vessel development by examining mice that have targeted deletions in the T1α/podoplanin gene (Ramirez et al., 2003). Here, we show that within the vascular system, T1α/podoplanin is first expressed between E10.5 and E11.5 in endothelial cells of the cardinal vein and in budding, Prox1-positive lymphatic progenitor cells. T1α/podoplanin expression becomes specifically restricted to lymphatic endothelium during later development. Ultrastructural analysis revealed its predominant localization to the luminal plasma membrane of lymphatic vessels. We found that T1α/podoplanin−/− mice have defects in lymphatic vessel, but not blood vessel, pattern formation. These defects lead to diminished lymphatic transport, congenital lymphedema and dilation of cutaneous and intestinal lymphatic vessels. Overexpression of T1α/podoplanin in cultured vascular endothelial cells promoted the formation of elongated cell extensions and significantly increased endothelial cell adhesion, migration and tube formation. Together, these findings suggest that the transmembrane glycoprotein T1α/podoplanin is required to regulate key aspects of lymphatic vascular formation. Results T1α/podoplanin is expressed by budding Prox1-positive lymphatic progenitor cells In agreement with previous observations showing T1α/podoplanin expression in the central nervous system and the foregut at around E9 (Rishi et al., 1995; Williams, 2003), we detected expression of T1α/podoplanin in the neural tube of wild-type mice at E10.5 (Figure 1A). However, no vascular expression of T1α/podoplanin was detected yet at this time point, whereas the homeobox gene Prox1 was already expressed by a subset of endothelial cells of the anterior cardinal vein (Figure 1A and B). By E11.5 (data not shown) and E12.5, T1α/podoplanin was expressed by all endothelial cells of the cardinal vein and by the Prox1-positive lymphatic progenitor cells that had already budded off from the embryonic veins (Figure 1C and D). Two days later, T1α/podoplanin expression was restricted to the budded Prox1-positive lymphatic endothelial progenitor cells and to the Prox1-positive lymphatic endothelial cells that lined the developing primitive lymph sacs (Figure 1C and F). After birth, vascular T1α/podoplanin expression was almost exclusively detected in lymphatic vessels (see below). Figure 1.T1α/podoplanin is expressed by Prox1-positive lymphatic progenitor cells during embryogenesis. (A and B) At day 10.5 of embryonic mouse development, Prox1 (green) is already expressed by endothelial cells predominantly located on one side of the anterior cardinal vein (CV), whereas the expression of T1α/podoplanin (red) is still restricted to the neural tube (NT). (C and D) At E12.5, T1α/podoplanin-positive endothelial cells are present throughout the anterior cardinal vein. Budding Prox1-positive lymphatic progenitor cells also express T1α/podoplanin. (E and F) At E14.5, the expression of T1α/podoplanin becomes restricted to Prox1-positive lymphatic endothelial cells of the lymph sac (LS), whereas no or only low-level expression is detected on endothelial cells of the jugular vein (JV). Bars = 50 μm. Download figure Download PowerPoint Impaired lymphatic transport and formation of lymphedema in neonatal T1α/podoplanin-deficient mice Mice with heterozygous and homozygous disruptions of the T1α/podoplanin gene (Ramirez et al., 2003) were compared with their wild-type littermates for all investigations. Whereas T1α/podoplanin+/− mice were healthy and fertile, and were macroscopically indistinguishable from their wild-type littermates, T1α/podoplanin−/− mice died immediately after birth, due to respiratory failure caused by impaired formation of alveolar airspace, associated with reduced numbers of differentiated type I alveolar epithelial cells in the lung (Ramirez et al., 2003). The skin of these mice was cyanotic and its texture was smoothened (Figure 2A). The lower limbs were markedly swollen, and thickened skin folds were clearly detectable in the neck area, indicative of cutaneous lymphedema (Figure 2A). Figure 2.Congenital skin lymphedema and impaired lymphatic transport in homozygous T1α/podoplanin knockout mice. (A) Newborn wild-type (+/+) and heterozygous mice (+/−) showed no phenotypic abnormalities, whereas T1α/podoplanin-null mice (−/−) died immediately after birth due to respiratory failure. Examination of T1α/podoplanin−/− mice revealed smoothened skin texture, thickened wrinkles, particularly in the neck area, and swelling of the lower extremities (inset). (B–D) Intradermal lymphatic capillaries and larger collecting lymphatic vessels of wild-type (B) and T1α/podoplanin+/− (C) mice were filled with dye after injection of Evan's blue dye into the dorsum of the paws. In contrast, only enlarged subcutaneous lymphatic collectors (D, arrowheads) were detected in T1α/podoplanin−/− mice. After intradermal injection of Evan's blue dye into the paws, retroperitoneal lymphatic vessels (arrowheads) and lymph nodes (arrows) were stained blue in wild-type (E) and T1α/podoplanin+/− (F) mice, but no dye was detected in the retroperitoneal lymphatics of T1α/podoplanin−/− mice (G). IVC = inferior vena cava; A = aorta. Download figure Download PowerPoint To investigate lymphatic transport, we intradermally injected Evans blue dye into the dorsum of the footpads of newborn mice. In both wild-type and T1α/podoplanin+/− mice, the dye was immediately transported through a dense network of interconnected dermal lymphatic capillaries and larger collecting lymphatic vessels towards the popliteal lymph nodes (Figure 2B and C). In T1α/podoplanin−/− mice, in contrast, only dilated lymphatic vessels were visible, and small dermal capillaries were not detectable (Figure 2D). Immediately after injection of Evans blue into all four extremities, retroperitoneal para-aortic lymph nodes and lymphatic ducts were clearly stained blue in wild-type and in T1α/podoplanin+/− mice, indicating efficient centripetal lymphatic transport (Figure 2E and F). Although immunofluorescence stainings revealed the presence of retroperitoneal lymphatic ducts in all of the investigated mice (data not shown), no staining of para-aortic lymphatic structures was detected in T1α/podoplanin−/− mice (Figure 2G), demonstrating that lymphatic transport was impaired. Dilation of intestinal and cutaneous lymphatic vessels, but not of blood vessels, in T1α/podoplanin-null mice Both the skin and intestine are characterized by their rich lymphatic vascularization, and these tissues are highly sensitive to impairment of lymphatic network formation. Using differential immunostains for the lymphatic-specific hyaluronan receptor LYVE-1 (Prevo et al., 2001) and the endothelial plasma membrane molecule CD31, we found several slightly enlarged submucosal lymphatic vessels in the small intestine of T1α/podoplanin+/− mice as compared with wild-type mice (Figure 3A and E). In T1α/podoplanin−/− mice, an increased number of severely dilated submucosal lymphatic vessels was found (Figure 3I), whereas no major alterations of subserosal lymphatic capillaries were detected. LYVE-1-positive lacteals were completely absent in T1α/podoplanin−/− mice (Figure 3I), whereas these formed normally in wild-type and T1α/podoplanin+/− mice (Figure 3A and E, asterisks). The number and size of CD31-positive/LYVE-1-negative intestinal blood vessels, in contrast, were comparable in all genotypes (Figure 3A, B, E, F, I and J). Figure 3.T1α/podoplanin deficiency leads to dilation of lymphatics, but not of blood vessels, in the skin and intestine. (A–L) Immunofluorescence stains of the ileum (A, B, E, F, I and J; L = lumen; AC = abdominal cavity) and the skin (C, D, G, H, K and L) for CD31 (green) and LYVE-1 (red, C, G and K) or T1α/podoplanin (red, B, F, J, D, H and L) revealed slightly enlarged lymphatic vessels of the submucosal plexus of T1α/podoplanin+/− mice (E), whereas T1α/podoplanin−/− mice have greatly enlarged lymphatics (I). Lacteals of wild-type and T1α/podoplanin+/− mice were LYVE-1 positive (asterisks, A and E), whereas no LYVE-1-positive lacteals were detected in T1α/podoplanin−/− mice (I). Lymphatic expression of T1α/podoplanin was confirmed in the ileum and the skin of wild-type (B and D) and T1α/podoplanin+/− (F and H) mice (asterisks = lacteals), but was not detected in T1α/podoplanin−/− mice (J and L). Staining for CD31 revealed no differences of the size of blood vessels in all of the mice. These findings were confirmed by computer-assisted image analysis which revealed a significant increase in the size of lymphatics (N), but not of blood vessels (M), in T1α/podoplanin−/− mice. Because some T1α/podoplanin-expressing basal keratinocytes were found in the epidermis of wild-type and T1α/podoplanin+/− mice (D and H; arrowheads), the comparative expression of the epidermal differentiation markers K14, K10 and loricrin was investigated (O–W). No differences in the expression of these markers were seen in the three genotypes. Bars = 50 μm. Download figure Download PowerPoint The T1α/podoplanin−/− mice also had enlarged lymphatics in the skin, as compared with wild-type and T1α/podoplanin+/− mice (Figure 3C, G and K). Computer-assisted morphometric image analyses confirmed that the average area of dermal LYVE-1-positive lymphatic vessels was significantly increased in T1α/podoplanin−/− mice (Figure 3N), whereas no differences in the size of blood vessels were found (Figure 3M). Immunofluorescence analysis of the intestine and the skin confirmed that lymphatic vessels in both wild-type and T1α/podoplanin+/− mice strongly expressed T1α/podoplanin. In contrast, no immunoreactivity was detected in T1α/podoplanin−/− mice, confirming the complete disruption of this gene and the specificity of the 8.1.1 antibody, that was originally raised against the gp38 antigen (Farr et al., 1992), for T1α/podoplanin (Figure 3B, D, F, H, J and L). T1α/podoplanin deficiency results in impaired patterning of lymphatic capillary networks We investigated whether T1α/podoplanin deficiency, in addition to causing the enlargement of intestinal and cutaneous lymphatic vessels, might also affect the patterning of the lymphatic network in the same anatomical regions. To this end, we performed immunostain analysis of LYVE-1 expression on tissue samples from the intestine (ileum) and from the ear skin of all newborn genotypes. We observed a dense and well-organized network of intestinal lymphatic capillaries in wild-type and T1α/podoplanin+/− newborn mice (Figure 4A and E). T1α/podoplanin−/− newborn mice, in contrast, developed areas of extremely enlarged lymphatic vessels, and the pattern of these vessels was completely disorganized (Figure 4I). Analysis of the ear skin of wild-type and T1α/podoplanin+/− mice revealed well-organized networks of LYVE-1-positive lymphatic capillaries (Figure 4B and F). Most of these capillaries were interconnected, and only a few blind beginning lymphatic capillaries were detected (Figure 4B and F). In contrast, the formation of lymphatic capillary networks was impaired in the ear skin of T1α/podoplanin−/− mice. These mice possessed an increased number of non-anastomozing, blind beginning cutaneous lymphatic capillaries (Figure 4J, arrowheads), indicative of impaired lymphatic network patterning. Figure 4.Abnormal patterning of lymphatic capillaries in T1α/podoplanin-deficient mice. (A–H) LYVE-1 whole-mount stains of lymphatic capillaries in the ileum (A, C, E, G and I) and ear (B, D, F, H and J) of newborn (A, B, E, F, I and L) and adult mice (C, D, G and H) revealed a regular network of lymphatics (A and E) in the intestine of newborn wild-type and T1α/podoplanin+/− mice. The network patterning was completely irregular and the diameter of lymphatics was strikingly increased in T1α/podoplanin−/− mice (I). The lymphatic vessels in the ear of T1α/podoplanin−/− mice also developed an irregular network (J) with a higher number of blind beginnings of lymphatics (arrowheads) compared with the lymphatic networks in the ears of newborn wild-type and T1α/podoplanin+/− mice (B and F). In the intestine and ears of adult wild-type mice, regular networks of lymphatic vessels were found (C and D), whereas areas of enlarged lymphatics (G and H) and incomplete network patterning (G) were seen in adult T1α/podoplanin+/− mice. Bars for (A), (B), (E), (F) and (J) = 100 μm, (C) and (G) = 200 μm, (D) and (H) = 300 μm. Download figure Download PowerPoint Because there were no clear-cut differences in lymphatic network patterns between newborn T1α/podoplanin+/− and wild-type mice, we performed immunohistochemical analyses on intestine (ileum) and ear skin tissue obtained from adult (4-month-old) T1α/podoplanin+/− mice and their littermates. Although the defects in lymphatic network patterning were not as striking as those seen in newborn T1α/podoplanin−/− mice, there were areas of dilated lymphatic vessels in the ear skin (Figure 4H) and in the intestine of T1α/podoplanin+/− mice, in addition to incomplete network formation (Figure 4G) that was not observed in wild-type littermates (Figure 4C and D). Ultrastructural localization of T1α/podoplanin in intestinal lymphatic vessels, but not in blood vessels To investigate the ultrastructural localization of T1α/podoplanin in the vascular system, we performed immuno-nanogold electron microscopy, using intestinal tissue samples obtained from newborn mice. In wild-type mice, high levels of membrane-bound T1α/podoplanin were detected at the luminal side of intestinal lymphatic vessels (Figure 5A). Fewer nanogold particles were observed on the abluminal plasma membrane, and no labeling was detected within the cytoplasm (Figure 5A). Occasionally, the lateral plasma membranes between adjacent cells were also labeled. T1α/podoplanin expression was completely absent from blood vessel endothelial cells in the intestine of newborn wild-type mice (Figure 5B). Lymphatic endothelial cells in the small intestine of T1α/podoplanin−/− mice were not immunolabeled by the anti-T1α/podoplanin antibody (Figure 5D), confirming the disruption of the T1α/podoplanin gene in these mice. Figure 5.Ultrastructural localization of T1α/podoplanin in murine intestinal lymphatic vessels, but not in blood vessels, by immuno-nanogold staining. (A) In newborn wild-type mice, membrane-bound T1α/podoplanin was detected at the luminal (L) side of the lymphatic endothelium in the intestine (ileum). Fewer immuno-nanogold particles were observed at the abluminal plasma membrane and no T1α/podoplanin was detected within lymphatic endothelial cells. (B) T1α/podoplanin expression was completely absent from blood vascular endothelial cells in the intestine of newborn wild-type mice. (C) Absence of specific labeling of wild-type lymphatic endothelium after omission of the primary anti-T1α/podoplanin antibody. (D) Lymphatic endothelial cells of a newborn T1α/podoplanin−/− mouse do not react with the anti-T1α/podoplanin antibody. Bars for (A) = 0.4 μm, (B) = 0.2 μm, (C) = 0.3 μm, (D) = 0.5 μm. Download figure Download PowerPoint T1α/podoplanin deficiency does not alter the ultrastructural architecture of intestinal lymphatic vessels or the distribution of LYVE-1 expression We next investigated whether loss of T1α/podoplanin might result in abnormal ultrastructural architecture of lymphatic vessels or in altered distribution of the LYVE-1. We detected high levels of immuno-nanogold labeling for LYVE-1 at both the luminal and the abluminal plasma membrane of lymphatic endothelium in the intestine of newborn wild-type mice (Figure 6A). Some labeling of the lateral plasma membranes was also observed, with the exception of contact areas between adjacent cells. LYVE-1 expression was completely absent from blood vessel endothelium (Figure 6B), confirming the specificity of LYVE-1 for lymphatic endothelium. Lymphatic endothelial cells of T1α/podoplanin−/− mice also showed strong LYVE-1 immunogold labeling at both the luminal and the abluminal plasma membranes at levels similar to those observed in wild-type mice (Figure 6C and D). No ultrastructural differences in lymphatic vessel structure were observed between the different genotypes. Figure 6.Comparable ultrastructural localization of LYVE-1 in intestinal lymphatic vessels of wild-type and T1α/podoplanin−/− mice. (A) High levels of immuno-nanogold labeling for LYVE-1 were detected at both the luminal (L) and the abluminal plasma membrane of lymphatic endothelium in the ileum of newborn wild-type mice. Some labeling of the lateral plasma membranes was also observed, whereas LYVE-1 was absent from the cytoplasm. (B) LYVE-1 expression was absent from blood vascular endothelium. (C and D) Lymphatic endothelial cells in the intestine of T1α/podoplanin−/− mice also had high levels of LYVE-1 immuno-nanogold labeling of the luminal and abluminal plasma membranes. The lateral plasma membranes were also labeled, with the exception of punctate contact areas between adjacent cells (A, C and D, arrows). (E and F) Replacement of the primary LYVE-1 antibody with an unrelated rabbit IgG control resulted in the absence of lymphatic endothelial cell labeling in the ileum of wild-type (E) and of T1α/podoplanin-null mice (F). Bars for (A), (C), (D) and (E) = 0.5 μm, (B) and (F) = 0.4 μm. Download figure Download PowerPoint T1α/podoplanin deficiency does not impair epidermal differentiation We occasionally detected T1α/podoplanin expression in basal keratinocytes of the epidermis in wild-type and T1α/podoplanin+/− mice (Figure 3D and H). To determine whether T1α/podoplanin deficiency might also affect epidermal structure or differentiation, we studied the expression of several markers of epidermal differentiation. A comparable expression pattern of keratin 14 (K14), which is expressed by proliferative basal keratinocytes (Fuchs and Byrne, 1994), was found in all three groups (Figure 3O, R and U). Expression patterns of the early and late epidermal terminal differentiation markers K10 (Figure 3P, S and V) and loricrin (Figure 3Q, T and W) were also similar in the skin in all three genotypes. Computer-assisted morphometric image analyses demonstrated comparable thickness of the epidermis in all genotypes (data not shown), and no major histological differences of epidermal structure were detected, indicating that T1α/podoplanin deficiency does not affect the formation or structure of the epidermis. T1α/podoplanin promotes endothelial cell migration, adhesion and tube formation To characterize further the biological function of T1α/podoplanin, we isolated lymphatic endothelial cells from the skin of newborn mice of all three genotypes. However, we were unable to expand lymphatic endothelial cell cultures obtained from all genotypes, even after addition of recombinant vascular endothelial growth factor (VEGF)-C to the growth medium (data not shown). We therefore decided to test the effects of T1α/podoplanin overexpression. Immortalized human microvascular endothelial cells (HMEC-1) were stably transfected with a pIRES2-rat T1α/podoplanin expression vector and pooled cells were used for subsequent in vitro studies. Immunostains revealed high levels of rat T1α/podoplanin protein in the stably transfected cells (Figure 7A) that projected extremely long and thin cell extensions, which were not seen in control cells that did not express rat T1α/podoplanin (Figure 7B and" @default.
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• W2099366913 title "T1 /podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema" @default.
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• W2099366913 doi "https://doi.org/10.1093/emboj/cdg342" @default.
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