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T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema The EMBO Journal Vol. 22 No. 14 pp. 3546-3556, 2003 T1a./podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema abnormal development of cutaneous lymphatic vessels Vivien Schacht, Maria I.Ramirez 1, results in lymphedema, which is associated with defects in Young-Kwon Hong, Satoshi Hirakawa, 2 tissue repair and the immune response (Mallon and Ryan, Dian Feng , Natasha Harvey3, 1994). Although the mechanisms that control the devel- 1 4 Mary Williams  , Ann M.Dvorak2, opment of the blood vascular system have been well Harold F.Dvorak , Guillermo Oliver3 and studied (Carmeliet, 2000), those of the lymphatic vessels Michael Detmar are poorly understood. Cutaneous Biology Research Center, Massachusetts General Hospital Recent analyses of Proxl -deficient mice have shown and Harvard Medical School, Charlestown, MA 02129, Pulmonary that the lymphatic vascular system, as predicted by Sabin Center, Department of Medicine and Department of Anatomy, Boston (1902), originates from the embryonic veins (Wigle and University School of Medicine, Boston, MA 02118, Departments of Oliver, 1999; Oliver and Detmar, 2002). Beginning at Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215 and Department of Genetics, embryonic day (E) 9.5 of mouse development, the St Jude Children's Hospital, Memphis, TN 38105, USA homeobox gene Proxl is specifically expressed by a 5 subpopulation of endothelial cells that are located on one Corresponding author e-mail: [email protected] side of the anterior cardinal vein. This is followed by polarized budding and migration of these Proxl-positive V.Schacht and M.I.Rarnirez contributed equally to this work lymphatic progenitor cells, which eventually form lymph- atic sacs and then the entire lymphatic vasculature. In Within the vascular system, the mucin-type trans- Proxl -null mice, the budding and sprouting of lymphatic membrane glycoprotein Tla/podoplanin is pre- endothelial cells from the veins is arrested at ~El 1.5- dominantly expressed by lymphatic endothelium, and E12.0, and these mice completely lack a lymphatic recent studies have shown that it is regulated by the vascular system (Wigle and Oliver, 1999). We and others lymphatic-specific homeobox gene Proxl. In this have shown recently that ectopic expression of Proxl in study, we examined the role of Tla/podoplanin in primary human blood vessel endothelial cells represses the vascular development and the effects of gene disrup- expression of several genes that are associated with the tion in mice. Tla/podoplanin is first expressed at blood vascular phenotype (Hong et al., 2002; Petrova et al., around Ell.O in Proxl-positive lymphatic progenitor 2002). Proxl expression was also found to upregulate the cells, with predominant localization in the luminal expression of lymphatic-specific genes (Hong et al., 2002; plasma membrane of lymphatic endothelial cells Petrova et al., 2002), indicating its function as a master during later development. TlaJpodoplanin-1- mice die control gene that determines lymphatic endothelial cell at birth due to respiratory failure and have defects in fate (Oliver and Detmar, 2002). These studies identified lymphatic, but not blood vessel pattern formation. the mucin-type transmembrane glycoprotein Tla;/podo- These defects are associated with diminished lymph- planin as one of the primary Proxl-induced genes (Hong atic transport, congenital lymphedema and dilation of et al., 2002). lymphatic vessels. Tla/podoplanin is also expressed in TlaJpodoplanin is expressed by cultured human lymph- the basal epidermis of newborn wild-type mice, but atic endothelial cells, and is one of the most highly gene disruption did not alter epidermal differenti- expressed lymphatic-specific genes (Petrova et al., 2002; ation. Studies in cultured endothelial cells indicate Hirakawa et al., 2003). In vivo expression of Tla;/ that Tla/podoplanin promotes cell adhesion, migra- podoplanin in lymphatic endothelium was first reported tion and tube formation, whereas small interfering by Wetterwald et al. (1996), who named it 'El 1 antigen'. RNA-mediated inhibition of Tla/podoplanin expres- It was characterized further under the name 'podoplanin', sion decreased lymphatic endothelial cell adhesion. because of its low level expression in kidney podocytes These data identify Tla/podoplanin as a novel critical (Breiteneder-Geleff et al., 1997). Podoplanin is homo- player that regulates different key aspects of lymph- logous to Tia, which was originally found to encode an atic vasculature formation. antigen that is selectively expressed at the apical surface of Keywords: angiogenesis/lymphangiogenesis/podoplanin/ alveolar type I cells in rat lung (Dobbs et al., 1988; Rishi Proxlffla; et al., 1995). Expression of Tla; has also been detected in the choroid plexus, ciliary epithelium of the eye, intestine, kidney, thyroid and esophagus of the fetal rat (Williams Introduction et al., 1996), and it has been shown to be homologous to The lymphatic vascular system maintains tissue fluid the OTS-8 gene, a phorbol ester-induced gene in MC3T3- homeostasis and mediates the afferent immune response, El mouse osteoblast cells (Nose et al., 1990). Other but can also aid in the metastatic spread of malignant homologs include RT/40 (Gonzalez and Dobbs, 1998), tumors (Detmar and Hirakawa, 2002). Dysfunction or murine gp38 (Farr et al., 1992), canine gp40 (Zimmer 3546 © European Molecular Biology Organization Podoplanin is required for lymphatic patterning E10.5 E12.5 E14.5 Fig. 1. Tla/podoplanin is expressed by Proxl-positive lymphatic progenitor cells during embryogenesis. (A and B) At day 10.5 of embryonic mouse development, Proxl (green) is already expressed by endothelial cells predominantly located on one side of the anterior cardinal vein (CV), whereas the expression of Tla/podoplanin (red) is still restricted to the neural tube (NT). (C and D) At El2.5, Tla/podoplanin-positive endothelial cells are present throughout the anterior cardinal vein. Budding Proxl-positive lymphatic progenitor cells also express Tla/podoplanin. (E and F) At E14.5, the expression of Tla/podoplanin becomes restricted to Proxl-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 (N). Bars = 50 µm. et al., 1997), human gp36 (Zimmer et al., 1999) and found that Tla/podoplanin-1- mice have defects in lymphatic vessel, but not blood vessel, pattern formation. murine PA2.26 (Gandarillas et al., 1997). There have been many studies of Tla/podoplanin These defects lead to diminished lymphatic transport, expression in the lymphatic vascular system (Kriehuber congenital lymphedema and dilation of cutaneous and et al., 2001; Maekinen et al., 2001; Hong et al., 2002; intestinal lymphatic vessels. Overexpression of Tla/ podoplanin in cultured vascular endothelial cells promoted Petrova et al., 2002; Hirakawa et al., 2003). In spite of the the formation of elongated cell extensions and large number of descriptive studies, little is understood significantly increased endothelial cell adhesion, migra- about Tla/podoplanin's biological function. We exam- tion and tube formation. Together, these findings suggest ined its role in lymphatic and blood vessel development by that the transmembrane glycoprotein Tla/podoplanin is examining mice that have targeted deletions in the Tla/ required to regulate key aspects of lymphatic vascular podoplanin gene (Ramirez et al., 2003). formation. Here, we show that within the vascular system, Tla/ podoplanin is first expressed between EI0.5 and El 1.5 in endothelial cells of the cardinal vein and in budding, Results Proxl-positive lymphatic progenitor cells. Tla/podopla- nin expression becomes specifically restricted to lymph- T1a/podoplanin is expressed by budding Prox1- atic endothelium during later development. Ultrastructural positive lymphatic progenitor cells analysis revealed its predominant localization to the In agreement with previous observations showing Tl al luminal plasma membrane of lymphatic vessels. We podoplanin expression in the central nervous system and 3547 V.Schacht et al. Impaired lymphatic transport and formation of lymphedema in neonatal T1a/podoplanin-deficient mice Mice with heterozygous and homozygous disruptions of the Tla/podoplanin gene (Ramirez et al., 2003) were compared with their wild-type littermates for all investi- gations. Whereas Tla/podoplanin+ - mice were healthy and fertile, and were macroscopically indistinguishable from their wild-type littermates, Tla/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). To investigate lymphatic transport, we intradermally injected Evans blue dye into the dorsum of the footpads of newborn mice. In both wild-type and Tla/podoplanin+t- mice, the dye was immediately transported through a dense network of interconnected dermal lymphatic capil- laries and larger collecting lymphatic vessels towards the popliteal lymph nodes (Figure 2B and C). In Tl al podoplanin- - mice, in contrast, only dilated lymphatic vessels were visible, and small dermal capillaries were not detectable (Figure 2D). Immediately after injection of Fig. 2. Congenital skin lymphedema and impaired lymphatic transport in homozygous Tl cr/podoplanin knockout mice. (A) Newborn wild- Evans blue into all four extremities, retroperitoneal para- type (+/+) and heterozygous mice (+/-) showed no phenotypic abnor- aortic lymph nodes and lymphatic ducts were clearly malities, whereas Tlalpodoplanin-null mice (-/-) died immediately stained blue in wild-type and in Tla/podoplanin+t- mice, after birth due to respiratory failure. Examination of Tl alpodoplanin-t- indicating efficient centripetal lymphatic transport mice revealed smoothened skin texture, thickened wrinkles, particularly (Figure 2E and F). Although immunofluorescence stain- in the neck area, and swelling of the lower extremities (inset). (B-D) Intradermal lymphatic capillaries and larger collecting lymphatic ings revealed the presence of retroperitoneal lymphatic vessels of wild-type (B) and Tlalpodoplanin+ - (C) mice were filled ducts in all of the investigated mice (data not shown), no with dye after injection of Evan's blue dye into the dorsum of the staining of para-aortic lymphatic structures was detected paws. In contrast, only enlarged subcutaneous lymphatic collectors (D, in Tla/podoplanin- - mice (Figure 2G), demonstrating that arrowheads) were detected in Tlalpodoplanin-1- mice. After intrader- mal injection of Evan's blue dye into the paws, retroperitoneal lymph- lymphatic transport was impaired. atic vessels (arrowheads) and lymph nodes (arrows) were stained blue in wild-type (E) and Tlalpodoplanin+ - (F) mice, but no dye was de- tected in the retroperitoneal lymphatics of Tlalpodoplanin-1- mice (G). Dilation of intestinal and cutaneous lymphatic IVC = inferior vena cava; A = aorta. vessels, but not of blood vessels, in T1a/ podoplanin-nu/1 mice Both the skin and intestine are characterized by their rich the foregut at around E9 (Rishi et al., 1995; Williams, lymphatic vascularization, and these tissues are highly 2003), we detected expression of Tla/podoplanin in the sensitive to impairment of lymphatic network formation. neural tube of wild-type mice at El0.5 (Figure lA). Using differential immunostains for the lymphatic-specific However, no vascular expression of Tla/podoplanin was hyaluronan receptor LYVE-1 (Prevo et al., 2001) and the detected yet at this time point, whereas the homeobox endothelial plasma membrane molecule CD31, we found gene Proxl was already expressed by a subset of several slightly enlarged submucosal lymphatic vessels in endothelial cells of the anterior cardinal vein (Figure lA the small intestine of Tl a/podoplanin+t- mice as compared and B). By Ell.5 (data not shown) and El2.5, Tla/ with wild-type mice (Figure 3A and E). In Tla/ podoplanin was expressed by all endothelial cells of the podoplanin- - mice, an increased number of severely cardinal vein and by the Proxl-positive lymphatic pro- dilated submucosal lymphatic vessels was found genitor cells that had already budded off from the (Figure 31), whereas no major alterations of subserosal embryonic veins (Figure lC and D). Two days later, lymphatic capillaries were detected. L YVE-1-positive Tla/podoplanin expression was restricted to the budded 1 lacteals were completely absent in Tla/podoplanin- - Proxl-positive lymphatic endothelial progenitor cells and mice (Figure 31), whereas these formed normally in to the Proxl-positive lymphatic endothelial cells that lined 1 wild-type and Tla/podoplanin+ - mice (Figure 3A and E, the developing primitive lymph sacs (Figure lC and F). asterisks). The number and size of CD31-positive/ After birth, vascular Tla/podoplanin expression was L YVE-1-negative intestinal blood vessels, in contrast, almost exclusively detected in lymphatic vessels (see were comparable in all genotypes (Figure 3A, B, E, F, I below). and J). 3548 Podoplanin is required for lymphatic patterning Intestine Skin LYVE:-1 (red) CD31 (green) Podoplanin (red) CD31 (greeol LYVE-1 (red) CD31 (g.reon) Podoplanin (red) CD31 {green) B C +!+ +/- . , . ' . .. ' "' -/- _. -i J , r , .._  . - . ,,  t I    .,. ' ,,: . .. 'L K14 K10 Loricrin a, J:j .,, +/+ .; ., .,, !£ -0 iii ' ~' "'· - . "'.-r . .... r\~ . -. +! - N 'i;' .': - :!l _.:, 'ill ., "' - "' ~ -,.,, ~-:-~-~ !l! . -' v'"'" ,<#'' t 1 .._,_,,,,..rt,·' .._ .. \ . J <ii -/- .c '  .; . ·' 0. .: "'  , 0, [ ' ,,  ~-... . \.,\ - ...J Fig. 3. Tla/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 CD3 l (green) and L YVE-1 (red, C, G and K) or Tla/podoplanin (red, B, F, J, D, Hand L) revealed slightly enlarged lymphatic vessels of the submucosal plexus of TJCJ/podoplanin+ - mice (E), whereas TJCJ/podoplanin-1- mice have greatly enlarged lymphatics (I). Lacteals of wild-type and Tla/podoplanin+ - mice were LYVE-1 positive (asterisks, A and E), whereas no LYVE-1-positive lacteals were detected in TJCJ/podoplanin-1- mice (I). Lymphatic expression of TJCJ/podoplanin was confirmed in the ileum and the skin of wild-type (B and D) and TJCJ/podoplanin+ - (F and H) mice (asterisks= lacteals), but was not detected in TJCJI podoplanin-1- 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 TJCJI podoplanin-1- mice. Because some Tla/podoplanin-expressing basal keratinocytes were found in the epidermis of wild-type and TJCJ/podoplanin+ - mice (D and H; arrowheads), the comparative expression of the epidermal differentiation markers K14, KIO and loricrin was investigated (O-W). No differences in the expression of these markers were seen in the three genotypes. Bars = 50 µm. The Tlalpodoplanin-1- mice also had enlarged lympha- et al., 1992), for Tla/podoplanin (Figure 3B, D, F, H, J and L). tics in the skin, as compared with wild-type and Tlal podoplanin+ - mice (Figure 3C, G and K). Computer- assisted morphometric image analyses confirmed that the T1a/podoplanin deficiency results in impaired average area of dermal L YVE-1-positive lymphatic ves- patterning of lymphatic capillary networks sels was significantly increased in Tlalpodoplanin-1- mice We investigated whether Tla/podoplanin deficiency, in (Figure 3N), whereas no differences in the size of blood addition to causing the enlargement of intestinal and vessels were found (Figure 3M). lmrnunofluorescence cutaneous lymphatic vessels, might also affect the pat- analysis of the intestine and the skin confirmed that terning of the lymphatic network in the same anatomical lymphatic vessels in both wild-type and Tlalpodopla- regions. To this end, we performed imrnunostain analysis nin+t- mice strongly expressed Tla/podoplanin. In con- of L YVE-1 expression on tissue samples from the intestine trast, no imrnunoreactivity was detected in Tlal (ileum) and from the ear skin of all newborn genotypes. podoplanin- - mice, confirming the complete disruption We observed a dense and well-organized network of this gene and the specificity of the 8.1.1 antibody, of intestinal lymphatic capillaries in wild-type and that was originally raised against the gp38 antigen (Farr Tl alpodoplanin+t- newborn mice (Figure 4A and E). 3549 V.Schacht et al. Newborn mice Adult mice Intestine Ear Intestine Ear +/+ +/- -!- Fig. 4. Abnormal patterning of lymphatic capillaries in Tlcx/podoplanin-deficient mice. (A-H) L YVE-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 Tla/podoplanin+ - mice. The network patterning was completely irregular and the diameter of lymphatics was strikingly increased in Tla/podoplanin-1- mice (I). The lymphatic vessels in the ear of Tla/podoplanin-1- mice also devel- oped 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 Tla/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 Tla/podoplanin+ - mice. Bars for (A), (B), (E), (F) and (J) = 100 µm, (C) and (G) = 200 µm, (D) and (H) = 300 µm. Fig. 5. Ultrastructural localization of Tlcx/podoplanin in murine intestinal lymphatic vessels, but not in blood vessels, by immuno-nanogold staining. (A) In newborn wild-type mice, membrane-bound Tlcx/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 Tlcx/podoplanin was detected within lymphatic endothelial cells. (B) Tlcx/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-Tlcx/podoplanin antibody. (D) Lymphatic endothelial cells of a newborn Tla/podoplanin-1- mouse do not react with the anti-Tlcx/podoplanin antibody. Bars for (A) = 0.4 µm, (B) = 0.2 µm, (C) = 0.3 µm, (D) = 0.5 µm. 3550 Podoplanin is required for lymphatic patterning Fig. 6. Comparable ultrastructural localization of L YVE-1 in intestinal lymphatic vessels of wild-type and T 1 a/podoplanin--1- mice. (A) High levels of immuno-nanogold labeling for L YVE-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 L YVE-1 was absent from the cyto- plasm. (B) L YVE-1 expression was absent from blood vascular endothelium. (C and D) Lymphatic endothelial cells in the intestine of Tia/ podoplanin--1- mice also had high levels of L YVE-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 L YVE-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 Tla/podoplanin-null mice (F). Bars for (A), (C), (D) and (E) = 0.5 µm, (B) and (F) = 0.4 µm . Tl a/podoplanin- - newborn mice, in contrast, developed areas of dilated lymphatic vessels in the ear skin (Figure 4H) and in the intestine of Tla/podoplanin+t- areas of extremely enlarged lymphatic vessels, and the mice, in addition to incomplete network formation pattern of these vessels was completely disorganized (Figure 4G) that was not observed in wild-type littermates (Figure 41). Analysis of the ear skin of wild-type and Tl al (Figure 4C and D). podoplanin+t- mice revealed well-organized networks of L YVE-1-positive lymphatic capillaries (Figure 4B and F). Most of these capillaries were interconnected, and only a Ultrastructural localization of T1a/podoplanin few blind beginning lymphatic capillaries were detected in intestinal lymphatic vessels, but not in (Figure 4B and F). In contrast, the formation of lymphatic blood vessels capillary networks was impaired in the ear skin of Tla/ To investigate the ultrastructural localization of Tl a/ podoplanin- - mice. These mice possessed an increased podoplanin in the vascular system, we performed immuno- number of non-anastomozing, blind beginning cutaneous nanogold electron microscopy, using intestinal tissue lymphatic capillaries (Figure 4J, arrowheads), indicative samples obtained from newborn mice. In wild-type mice, of impaired lymphatic network patterning. high levels of membrane-bound Tla/podoplanin were Because there were no clear-cut differences in detected at the luminal side of intestinal lymphatic vessels lymphatic network patterns between newborn Tl a/podo- (Figure SA). Fewer nanogold particles were observed on planin+I- and wild-type mice, we performed immuno- the abluminal plasma membrane, and no labeling was histochemical analyses on intestine (ileum) and ear skin detected within the cytoplasm (Figure SA). Occasionally, tissue obtained from adult (4-month-old) Tla/podo- the lateral plasma membranes between adjacent cells were planin+t- mice and their littermates. Although the defects also labeled. Tla/podoplanin expression was completely in lymphatic network patterning were not as striking as absent from blood vessel endothelial cells in the intestine those seen in newborn Tla/podoplanin- - mice, there were of newborn wild-type mice (Figure SB). Lymphatic 3551 V.Schacht et al. Rat podoplanln F-actln Merge Q c 3-,------------, 20 ,g e2:.5 -~ E 2 e GI 1.6 ]l 10 G) :I !! 1 :E 0.5 LL o~~C_o_n-tr_o_l ~-Podoplanln Control Podoplanfn Fig. 7. Overexpression of Tla/podoplanin in HMEC-1 cells enhances endothelial cell migration and adhesion in vitro. (A-C) Stable transfection of HMEC-1 cells with rat Tla/podoplanin cDNA resulted in the production of the rat Tla/podoplanin protein (green; A and C) and in the formation of long filopodia, which were not seen in cells that did not overexpress Tl a/podoplanin. Some of the rat Tl a/podoplanin-expressing cells showed a marginal accumulation of F-actin bundles (red, B and C). Bar= 150 µm. (D and E) Overexpression of Tla/podoplanin significantly stimulated cell migration (D) and adhesion (E) of HMEC-1 cells. **P < 0.01; ***P < 0.001. endothelial cells in the small intestine of Tl al expression of several markers of epidermal differentiation. podoplanin- - mice were not immunolabeled by the anti- A comparable expression pattern of keratin 14 (K14), Tla/podoplanin antibody (Figure 5D), confirming the which is expressed by proliferative basal keratinocytes disruption of the Tla/podoplanin gene in these mice. (Fuchs and Byrne, 1994), was found in all three groups (Figure 30, R and U). Expression patterns of the early and late epidermal terminal differentiation markers KIO T1a/podoplanin deficiency does not alter the (Figure 3P, S and V) and loricrin (Figure 3Q, T and W) ultrastructural architecture of intestinal lymphatic were also similar in the skin in all three genotypes. vessels or the distribution of LYVE-1 expression Computer-assisted morphometric image analyses demon- We next investigated whether loss of Tla/podoplanin strated comparable thickness of the epidermis in all might result in abnormal ultrastructural architecture of genotypes (data not shown), and no major histological lymphatic vessels or in altered distribution of the L YVE-1. differences of epidermal structure were detected, indicat- We detected high levels of immuno-nanogold labeling for ing that Tla/podoplanin deficiency does not affect the L YVE-1 at both the luminal and the abluminal plasma formation or structure of the epidermis. 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 T1a/podoplanin promotes endothelial cell the exception of contact areas between adjacent cells. migration, adhesion and tube formation L YVE-1 expression was completely absent from To characterize further the biological function of Tl a/ blood vessel endothelium (Figure 6B), confirming the podoplanin, we isolated lymphatic endothelial cells from specificity of L YVE-1 for lymphatic endothelium. the skin of newborn mice of all three genotypes. However, Lymphatic endothelial cells of Tla/podoplanin- - mice we were unable to expand lymphatic endothelial cell also showed strong L YVE-1 immunogold labeling at cultures obtained from all genotypes, even after addition both the luminal and the abluminal plasma membranes at of recombinant vascular endothelial growth factor levels similar to those observed in wild-type mice (VEGF)-C to the growth medium (data not shown). We (Figure 6C and D). No ultrastructural differences in therefore decided to test the effects of Tla/podoplanin lymphatic vessel structure were observed between the overexpression. Immortalized human microvascular different genotypes. endothelial cells (HMEC-1) were stably transfected with a plRES2-rat Tla/podoplanin expression vector and T1a/podoplanin deficiency does not impair pooled cells were used for subsequent in vitro studies. epidermal differentiation Immunostains revealed high levels of rat Tla/podoplanin We occasionally detected Tla/podoplanin expression in protein in the stably transfected cells (Figure 7 A) that basal keratinocytes of the epidermis in wild-type and Tla/ projected extremely long and thin cell extensions, which were podoplanin+ - mice (Figure 3D and H). To determine not seen in control cells that did not express rat Tl a/ whether Tla/podoplanin deficiency might also affect podoplanin (Figure 7B and C). Some of the stably Tla/ epidermal structure or differentiation, we studied the podoplanin-transfected cells also formed phalloidin-positive 3552 Podoplanin is required for lymphatic patterning 10~--------, A B Podoplanin .. 8 ~30 II> g 25 E a -70 kD ]i II) 'aj 4 f 15 (.) .: 10 I&. 2 C1 C2 R1 R2 lnnn 50--------~ o C1 C2 CJ P1 P2 PJ B Controls Podoplanin E30 U) a, 20 .L..Lc~1 -----'-----'~ c~2 .__ R 1 R2 Fig. 9. Inhibition of Tla/podoplanin expression by siRNA transfection ,_ 100 E reduces human lymphatic endothelial cell adhesion to type I collagen. E BOO (A) Four days after siRNA transfection of human primary lymphatic -E 560 endothelial cells, endogenous Tla/podoplanin protein levels, but not L YVE-1 levels, were decreased by two different siRNA oligonucleo- [400 tides (RI and R2), as compared with control cells (Cl, control vector cDNA; C2, sham-transfected cells). (B) Human dermal lymphatic en- dothelial cells showed significantly reduced adhesion to type I collagen 4 days after siRNA transfection (Rl and R2), as compared with control cells (Cl and C2). *P < 0.05; **P < 0.01. o C1 Controls Podoplanln Fig. 8. Overexpression of Tla/podoplanin in EOMA cells enhances podoplanin significantly enhanced EOMA cell migration cell migration, adhesion and tube formation in vitro. (A and B) Stable transfection of EOMA cells with rat Tla/podoplanin cDNA resulted in towards type I collagen and also promoted cell adhesion in significantly enhanced haptotactic cell migration and adhesion to type I all three clones tested, as compared with the control clones collagen in all three clones tested (Pl-P3), as compared with control (Figure 8A and B). Tla/podoplanin-overexpressing clones (Cl-C3). (C and D) Enhanced formation of tube-like structures EOMA cell clones also showed a significantly increased by Tla/podoplanin-overexpressing EOMA cells after seeding onto ability to form tube-like structures after plating onto Matrigel (24 h; D), as compared with control vector-transfected cells (C). Bar= 50 µm . (E) Overexpression of Tla/podoplanin significantly Matrigel (Figure 8C-E). enhanced the formation of tube-like structures in all three Tla/podopla- nin-overexpressing clones (Pl-P3) as compared with control clones (Cl-C3). Inhibition of T1a/podoplanin expression by siRNA transfection reduces cell adhesion of human lymphatic endothelial cells F-actin bundles at the periphery (Figure 7B and C), indicating To investigate further the role of Tla/podoplanin in cell that Tla/podoplanin expression might control endothelial adhesion, we studied the effects of small interfering RNA cell cytoskeletal organization. We next studied whether (siRNA)-mediated inhibition of endogenous Tla/podo- overexpression of Tla/podoplanin could affect endothelial planin expression in cultured human dermal lymphatic cell migration or adhesion. We found a >2-fold (P < 0.001) endothelial cells that are characterized by high expression increase in haptotactic cell migration towards fibronectin in levels ofTla/podoplanin (Hirakawa et al., 2003). Two out stably transfected cells, compared with controls (Figure 7D). of three siRNAs tested efficiently reduced endogenous Tl a/podoplanin-overexpressing HMEC-1 cells also adhered Tla/podoplanin protein expression by 49 and 34%, more tightly to immobilized fibronectin (P < 0.01 ; Figure 7E). respectively, at 4 days after transfection, whereas the To confirm these results further in a second, independ- expression of another 1 ymphatic marker, L YVE-1, re- ent cell line, murine hemangioendothelioma-derived mained unchanged (Figure 9A). Accordingly, lymphatic EOMA cells were stably transfected with the same endothelial cell adhesion to type I collagen, which is pIRES2-rat Tla/podoplanin-enhanced green fluorescent closely associated with lymphatic vessels in vivo (Skobe protein (EGFP) construct or with the control vector. Three and Detmar, 2000), was significantly inhibited by both of clones with high expression of the transfected Tl al these siRNAs, as compared with control or sharn- podoplanin, along with three control clones, were used transfected control cells, at 4 days after transfection for subsequent experiments. Overexpression of Tla/ (Figure 9B). 3553 V.Schacht et al. podoplanin-1- mice is even more pronounced, we expect Discussion that lipid uptake in the intestine would also be defective. Previous studies have identified Tla/podoplanin as a The observed enlargement of both dermal and submucosal Proxl-induced gene (Hong et al., 2002) that is predom- intestinal lymphatics of Tla/podoplanin-1- mice is most inantly expressed, within the vascular system, in lymph- likely to be related to the lack of connecting lymphatics atic endothelium (W etterwald et al., 1996; Kriehuber et al., between the superficial and deep networks, indicating that 2001; Maekinen et al., 2001; Hirakawa et al., 2003). We Tla/podoplanin is required for the formation of these show that similarly to VEGF receptor-3 (Wigle et al., specific lymphatic vessels. 2002), another Proxl target gene (Hong et al., 2002; We were unable to propagate lymphatic endothelial Petrova et al., 2002), Tla/podoplanin is expressed cells that were isolated from both wild-type and Tl al throughout the endothelium of the anterior cardinal vein podoplanin- - neonatal mice by using a modification of our at E12.5 of embryonic mouse development and later recently established purification method for human dermal becomes restricted to the budding Proxl-positive lymph- lymphatic endothelial cells (Hirakawa et al., 2003). At atic progenitor endothelial cells and to lymphatic present, there are no published reports on the successful endothelial cells of the embryonic lymph sacs and of propagation of primary murine lymphatic endothelial lymphatic vessels. In contrast to Proxl -null mice, which cells, and suitable culture techniques still remain to be fail to develop any lymphatic vasculature (Wigle and established. Therefore, we investigated the cellular effects Oliver, 1999), Tla/podoplanin-1- mice develop a periph- of Tla/podoplanin overexpression in two types of eral lymphatic vascular system; however, it exhibits immortalized human and murine vascular endothelial pronounced defects in lymphatic vascular organization cells that express only moderate levels ofTla/podoplanin and function. Our findings indicate that (Hong et al., 2002). The induction of elongated endothelial Tl a/podoplanin is cell extensions and cytoskeletal reorganization, together important to regulate different aspects related to the later stages of lymphatic development and patterning. In with the enhanced adhesion and migration of stably Tla/ contrast to other recently described gene targeting models, podoplanin-transfected endothelial cells are in agreement including mice deficient for angiopoietin-2 (Gale et al., with its effects on the motility of immortalized keratino- 2002) or VEGF receptor-3 (Dumont et al., 1998), Tla/ cytes (Scholl et al., 1999) and indicate that this protein podoplanin deficiency selectively affects the lymphatic controls endothelial cell functions that are required for vascular system without any detectable effect on the normal lymphatic patterning during development. These development of the blood vascular system. Similarly to findings were confirmed further by the reduced endothelial neuropilin-2 mutant mice which show only reduction of cell adhesion observed after inhibition of endogenous small lymphatic vessels but no alteration of the blood Tla/podoplanin expression by siRNA transfection. vascular system (Yuan et al., 2002), our findings in Tia/ Because efficient cell migration is dependent upon the podoplanin-deficient mice are in agreement with the polarity of the migrating cells, and since Tla/podoplanin relatively late onset of Tl alpodoplanin expression during is also expressed on the apical surface of polarized vascular development, and with its predominant expres- alveolar epithelial cells (Dobbs et al., 1988; Rishi et al., sion on lymphatic vascular endothelium. 1995), it may exert an important role for the polarization of Importantly, Tla/podoplanin-1- mice were character- cells and for the stabilization of cellular protrusions at the ized by congenital lymphedema, as manifested by the leading edge of migrating cells. This proposed function is pronounced swelling of the limbs at birth. lntradermal dye supported further by our findings that overexpression of injection into the foot pads of Tla/podoplanin-1- mice Tla/podoplanin in endothelial cells promoted the forma- revealed several enlarged, plump lymphatic vessels, but tion of tube-like structures on Matrigel which provide a failed to visualize the characteristic dermal capillary link to the in vivo findings of incomplete lymphatic networks seen in wild-type and in Tl a/podoplanin+t- network formation in Tla/podoplanin-1- mice. mice. These networks were most probably filled from Tla/podoplanin is a mucin-type glycoprotein with deeper lymphatic vessels through anastomozing vessels extensive O-glycosylation and a high content of sialic acid (Gonzalez and Dobbs, 1998). This negatively charged that still lack valves at the early stages of pre- and post- natal lymphatic development (Polonskaja, 1935). Because structure probably is resistant to proteases and provides a histological examination revealed the presence of dermal physical barrier that protects cells from environmental capillaries in Tla/podoplanin-1- mice, these findings agents (Zimmer et al., 1999). Immuno-nanogold electron indicate an insufficient formation of anastomozing lymph- microscopy showed that Tla/podoplanin is predominantly localized to the apical, luminal plasma membrane of atic vessels between the superficial and subcutaneous lymphatic networks. intestinal lymphatic endothelial cells. This localization is In the intestine of Tl a/podoplanin-1- mice, lacteals were similar to that reported for other cell types (Dobbs et al., not detectable and many of the lymphatics of the 1988; Rishi et al., 1995; Williams et al., 1996; submucosal plexus were enlarged. The physiological Breiteneder-Geleff et al., 1997; Zimmer et al., 1997) and consequences of these developmental defects on the is compatible with a protective function towards the uptake of lipids from the intestine are not known, since proteinase-containing lymph (Bartos et al., 1979). It is of these mice died immediately after birth (before the first interest that Tla/podoplanin is also expressed by alveolar feeding) and chyle transport could not be investigated. epithelial cells, cells of the choroid plexus, ependymal However, as previously described (Gale et al., 2002), epithelia and mesothelia that are also exposed to an angiopoietin-2-null mice develop chylous ascites shortly external or internal fluid compartment. after birth, due to insufficient formation of lacteals. In conclusion, we propose that Tla/podoplanin is Because the observed morphological defect in Tl al required to control different aspects of normal lymphatic 3554 Podoplanin is required for lymphatic patterning controls included replacement of the primary antibody with irrelevant vasculature formation. Lack of Tla/podoplanin leads to rabbit lgG and omission of the specific primary antibody. alterations in the final patterning of the lymphatic vasculature as well as in lymph transport. The future Cell culture and transfection generation of a conditional inactivation of Tl a/podoplanin hnmortalized HMEC-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) that contained 10% fetal bovine serum (PBS), will be a valuable tool to understand further its role in the 2 mM L-glutarnine and antibiotics (Life Science, Grand Island, NY). The normal function and in pathological alterations of the murine hemangioendothelioma-derived cell line EOMA (Obeso et al., lymphatic vasculature. 1990) was maintained in DMEM containing 20% PBS and 4 mM L-glutamine. Primary human dermal lymphatic endothelial cells were isolated and propagated as recently described (Hirakawa et al., 2003). The rat Tla/podoplanin coding sequence (Rishi et al., 1995; GenBank Materials and methods accession No. U07797) was cloned into a plRES2-EGFP vector (Clontech, Palo Alto, CA) which contains a cytomegalovirus (CMV) Generation of T1a/podoplanin knockout mice enhancer promoter and a neomycin selection cassette. The nucleotide Tl a/podoplanin mutant mice were generated as described (Ramirez et al., sequence was verified using the Applied Biosystems Big Dye Terminator 2003). Fragments were designed to replace ~1.5 kb of the promoter kits. After linerarization with Bsal, HMEC-1 and EOMA cells were sequence, 210 bp of the untranslated region (UTR), the first exon (67 bp) transfected either with the full-length rat Tla/podoplanin cDNA or with and 181 bp of the first intron. The efficiency of the disruption of the T 1 al plRES2-EGFP vector alone using the SuperFect transfection reagent podoplanin gene was confirmed by northern and western blot analyses of (Qiagen, Chatsworth, CA). Stably transfected cells were selected in newborn lung RNA and protein extracts that demonstrated the absence of growth medium containing 200 µg/ml neomycin (Gibco, Carlsbad, CA). Tla/podoplanin mRNA or protein in Tla/podoplanin--1- mice and For all experiments, pooled transfected HMEC-1 cells and three clones of reduced Tla/podoplanin mRNA levels in Tla/podoplanin+ - mice stably transfected EOMA cells were used for Tla/podoplanin over- (Ramirez et al., 2003). expression and for vector controls. To determine Tla/podoplanin expression levels, we performed SYBR Green-based real-time RT- Tissue processing and immunostaining PCRs as described (Hong et al., 2002), using the ABI Prism 7000 Tissue samples were obtained from newborn Tl a/podoplanin ( +/+ ), ( +/-) Sequence Detection System. The following forward and reverse and (-/-) littermates that had been killed by intraperitoneal injection of primers were used: 5'-GACATGGTGAACCCAGGTCT-3 and 5'- barbital sodium within 10 min after birth and from wild-type embryos at AATGGGAGGCTGTGTTGGTA-3. Total RNAs were isolated using days El0.5, E12.5 and E14.5, and were fixed in 4% paraformaldehyde Tri-reagent (Sigma, St Louis, MO) and were treated with RNase-free RQ- (Fluka, Buchs, Switzerland). hnmunofluorescence stains were performed DNase (Promega, Madison, WI). A 100 ng aliquot of RNA was used for on 6 µm cryostat sections of dorsal skin and of ileum, as well as on 10 µm each reaction. SYBR Green PCR Master Mix (Applied Biosystems, sections of wild-type mouse embryos as described (Wigle et al., 2002), Foster City, CA) was used for reactions with the addition of MultiScribe using polyclonal rabbit antibodies against Proxl (Wigle et al., 2002) and reverse transcriptase (Applied Biosystems). Protein expression was LYVE-1 (Prevo et al., 2001; kindly provided by Dr D.Jackson, Oxford, confirmed by immunofluorescence staining with a specific monoclonal UK), a monoclonal rat antibody against CD31 (BD Pharmingen, San anti-rat Tla/podoplanin antibody (Wetterwald et al., 1996). For F-actin Diego, CA), a hamster antibody against murine gp38 (Farr et al., 1992; staining, phalloidin-tetramethylrhodamine B isothiocyanate conjugate clone 8.1.1, Developmental Studies Hybridoma Bank, University of (Sigma) was used (Scholl et al., 1999). Iowa), antibodies against murine K14, KlO and loricrin (Babco, Richmond, CA) and corresponding secondary antibodies labeled with Cell transfection with human T1a/podoplanin siRNAs AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, OR). Cell The following siRNA oligonucleotides were synthesized by Dharmacon nuclei were counterstained with 20 µg/ml Hoechst bisbenzimide (Lafayette, CO): (Rl) 5'-GCGAAGACCGCUAUAAGUCdTdT-3', (R2) (Molecular Probes). Whole-mount samples of mouse ears and of ileum 5'-AAGAUGGUUUGUCAACAGUdTdT-3' and (R3) 5'-AGAUGA- were stained as described elsewhere (Gale et al., 2002). The tissues were CACUGAGACUACAdTdT-3'. Primary human lymphatic endothelial examined by using a Nikon E-600 microscope (Nikon, Melville, NY) and cells (Hirakawa et al., 2003) were transfected or not with siRNA images were captured with a SPOT digital camera (Diagnostic oligonucleotides (500 nmol) or with equimolar concentrations of control Instruments, Sterling Heights, Ml). Computer-assisted morphometric plasmid vector by using the Nucleofector kit (Amaxa, Cologne, analysis of lymphatics and blood vessels within the dermis of eight Germany) according to the manufacturer's instructions. Cells were animals per group (three fields per sample at 20X magnification) were harvested at 2 and 4 days after transfection. For western analyses, cell performed as described previously (Streit et al., 1999), using the IP-LAB lysates were obtained as described (Hong et al., 2002) and 30 ng of software (Scanalytics, Fairfax, VA). The differences in vessel size were protein per sample were immunoblotted with an antibody against human analyzed by the two-sided unpaired Student's t-test. Tla/podoplanin (Zimmer et al., 1997; kindly provided by Dr G.Herrler, Hanover, Germany) and with an antibody against human L YVE-1 (kindly lmmuno-nanogold electron microscopy provided by Dr D.Jackson, Oxford, UK). Small intestine samples were collected from genetically modified mice and their wild-type littermates immediately after birth. Tissue samples Cell migration, adhesion and tube formation assays For cell migration assays, 24-well FluoroBlok inserts (Falcon, Franklin were fixed in 4% paraformaldehyde and transferred to 30% sucrose/ phosphate-buffered saline (PBS). After embedding the tissues in OCT Lakes, NJ; 8 µm pore size) were coated on the underside with 10 µg/ml compound (Miles, Elkhart, IN), cryostat sections (5 µm) were immersed fihronectin (BD Bioscience, Bedford, MA) or with 50 µg/ml type I in 50 mM glycine. After washing, sections were immersed in normal goat collagen (Vitrogen, Palo Alto, CA) for 1 h, followed by addition of serum (Vector Laboratories, Burlingame, CA) and incubated with anti- 100 µg/ml bovine serum albumin (BSA; Sigma) to block the remaining LYVE-1 or 8.1.1 antibody, followed by incubation with goat anti-rabbit protein-binding sites. Cells (2 X 10 cells/ml) were seeded in serum-free Fab', conjugated with 1.4 nm nanogold particles (Nanoprobes, Stony EBM-2 medium (Clonetics, Watersville, MD) containing 0.2% de- Brook, NY), or with goat anti-hamster IgG, conjugated with 0.8 nm lipidized BSA into each well and cells were incubated for 3 h at 37°C. nanogold particles (Aurion, Wageningen, The Netherlands). Sections Cells on the underside of inserts were stained with Calcein AM were post-fixed in 1 % PBS-buffered glutaraldehyde, developed with HQ (Molecular Probes), and the fluorescence intensity was measured using silver enhancement solution (Nanoprobes), fixed in 5% sodium the Victor2 Fluorometer (PerkinElmer, Boston, MA). Cell adhesion thiosulfate, post-fixed in 1 % osmium tetroxide in sym-collidine buffer, assays were performed as described (Streit et al., 1999). Twelve-well washed in 0.05 M sodium maleate buffer, and stained with 2% uranyl plates were coated with 10 µg/ml fibronectin or with 50 µg/ml type I acetate in 0.05 M sodium maleate buffer. Tissues were dehydrated in collagen for 1 hat 4°C, followed by blocking with 100 µg/ml BSA. Cells graded ethanols, infiltrated with propylene oxide-eponate (Ted Pella, (1 X 105 cells in 200 µI of serum-free DMEM) were added to each well Redding, CA) and embedded by inverting eponate-filled plastic capsules and were incubated at 37°C for 20 min. Unattached cells were removed over the slide-attached tissue sections. After polymerization, eponate by three gentle washes with PBS; attached cells were fixed with 4% blocks were separated from the glass slides and thin sections were cut on paraformaldehyde, stained with Hoechst bisbenzimide and the number of an ultratome (Reichert illtracuts, Austria), collected on uncoated 200 attached cells/mm was determined. Tube formation assays were mesh copper grids (Ted Pella) and examined with a CM 10 transmission performed on Matrigel-coated 24-well plates (BD Bioscience) as electron microscope (Philips, Eindhoven, The Netherlands). Specificity described previously (Obeso et al., 1990). EOMA cells were seeded at 3555 V.Schacht et al. a density of 2 X 105 cells/ml in growth medium and were incubated for Mallon,E.C. and Ryan,T.J. (1994) Lymphedema and wound healing. 24 h at 37°C. After fixation with 4% paraformaldehyde, images were Clin. Dermatol., 12, 89-93. captured and the total length of tube-like structures per area was measured Nose,K., Saito,H. and Kuroki,T. (1990) Isolation of a gene sequence using the IP-LAB software. All studies were performed in triplicate. induced later by tumor-promoting 12-O-tetradecanoylphorbol-13- Statistical analyses were performed using the unpaired Student's t-test. acetate in mouse osteoblastic cells (MC3T3-El) and expressed constitutively in ras-transformed cells. Cell Growth Differ., 1, 511- Obeso,J., Weber,J. and Auerbach,R. (1990) A hemangioendothelioma- Acknowledgements derived cell line: its use as a model for the study of endothelial cell The authors thank J.Bertoncini, L.Janes, M.Constant and G.Millien for biology. Lab. Invest., 63, 259-269. technical assistance, Dr D.Jackson for the gift of the L YVE-1 antibody, Oliver,G. and Detmar,M. (2002) The rediscovery of the lymphatic Sam W.Lee for valuable advice regarding siRNA experiments, and system. Old and new insights into the development and biological Kathryn Pyne for photographic assistance with the electron micrographs. function of lymphatic vascular system. Genes Dev., 16, 773-783. This work was supported by NIH grants CA69184, CA86410, CA92644 Petrova,T.V. et al. (2002) Lymphatic endothelial reprogramming of (M.D.), HIA7049 (M.C.W.), Al33372 and Al44066 (A.M.D.), by vascular endothelial cells by the Prox-1 homeobox transcription American Cancer Society Program Project Grant 99-23901 (M.D.), by factor. EMBO J., 21, 4593-4599. the Deutsche Forschungsgemeinschaft (V.S.), by the Francis Family Polonskaja,R. (1935) The connections between the superficial and deep Foundation (M.I.R.) and by the Cutaneous Biology Research Center lymphatic vessels of the lower limb. Anat. Anz., 81, 247-256. through the Massachusetts General Hospital/Shiseido Co. Ltd Agreement Prevo,R., Banerji,S., Ferguson,D.J., Clasper,S. and Jackson,D.G. (2001) (M.D.). Mouse L YVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium. J. Biol. Chem, 276, 19420-19430. Ramirez,M.I., Millien,G., Hinds,A., Cao,Y., Seldin,D.C. and References Tla, a lung type I cell differentiation gene, is Williams,M.C. (2003) required for normal lung cell proliferation and alveolus formation at Bartos,V., Kolc,J., Vanecek,R. and Malek,P. (1979) Lymph and blood Dev. Biol., 256, 61-72. birth. enzymes and pathologic alterations in canine experimental pancreatitis Rishi,A.K., Joyce-Brady,M., Fisher,J., Dobbs,L.G., Floros,J., after administration of benzo-pyrones. Scand. J. Gastroenterol., 14, VanderSpek,J., Brody,J.S. and Williams,M.C. (1995) Cloning, 343-347. characterization and development expression of a rat lung alveolar Breiteneder-Geleff,S., Matsui,K., Soleiman,A., Meraner,P., type I cell gene in embryonic endodermal and neural derivatives. Dev. Poczewski,H., Kalt,R., Schaffner,G. and Kerjaschki,D. (1997) Biol., 167, 294-306. Podoplanin, novel 43-kd membrane protein of glomerular epithelial Sabin,F.R. (1902) On the origin of the lymphatic system from the veins cells, is down-regulated in puromycin nephrosis. Am J. Pathol., 151, and the development of the lymph hearts and thoracic duct in the pig. 1141-1152. Am. J. Anat., 1, 367-389. Carmeliet,P. (2000) Mechanisms of angiogenesis and arteriogenesis. Scholl,F.G., Gamallo,C., Vilaro,S. and Quintanilla,M. (1999) Nat. Med., 6, 389-395. Identification of PA2.26 antigen as a novel cell-surface mucin-type Detmar,M. and Hirakawa,S. (2002) The formation of lymphatic vessels glycoprotein that induces plasma membrane extensions and increased and its importance in the setting of malignancy. J. Exp. Med., 196, motility in keratinocytes. J. Cell Sci., 112, 4601-4613. 713-718. Skobe,M. and Detmar,M. (2000) Structure, function and molecular Dobbs,L.G., Williams,M.C. and Gonzalez,R. (1988) Monoclonal control of the skin lymphatic system. J. Investig. Dermatol. Symp. antibodies specific to apical surfaces of rat alveolar type I cells bind Proc., 5, 14-19. to surfaces of cultured, but not freshly isolated, type II cells. Biochim Streit,M., Velasco,P., Brown,L.F., Skobe,M., Richard,L., Riccardi,L., Biophys Acta, 970, 146-156. Lawler,J. and Detmar,M. (1999) Overexpression of thrombospondin-1 Dumont,D.J., Jussila,L., Taipale,J., Lymboussaki,A., Mustonen,T., decreases angiogenesis and inhibits the growth of human cutaneous Pajusola,K., Breitman,M. and Alitalo,K. (1998) Cardiovascular squamous cell carcinomas. Am J. Pathol., 155, 441-452. failure in mouse embryos deficient in VEGF receptor-3. Science, Wetterwald,A., Hoffstetter,W., Cecchini,M.G., Lanske,B., Wagner,C., 282, 946-949. Fleisch,H. and Atkinson,M. (1996) Characterization and cloning of the Farr,A.G., Berry,M.L., Kim,A., Nelson,A.J., Welch,M.P. and Aruffo,A. Ell antigen, a marker expressed by rat osteoblasts and osteocytes. (1992) Characterization and cloning of a novel glycoprotein expressed Bone, 18, 125-132. by stromal cells in T-dependent areas of peripheral lymphoid tissues. Wigle,J.T. and Oliver,G. (1999) Proxl function is required for the J. Exp. Med., 176, 1477-1482. development of the murine lymphatic system. Cell, 98, 769-778. Fuchs,E. and Byme,C. (1994) The epidermis: rising to the surface. Curr. Wigle,J.T., Harvey,N., Detmar,M., Lagutina,I., Grosveld,G., Gunn,M.D., Opin. Genet. Dev., 4, 725-736. Jackson,D.G. and Oliver,G. (2002) An essential role for Proxl in the Gale,N. et al. (2002) Angiopoietin-2 is required for postnatal induction of the lymphatic endothelial cell phenotype. EMBO J., 21, angiogenesis and lymphatic patterning and only the latter role is 1505-1513. rescued by angiopoietin-1. Dev. Cell, 3, 411-423. Williams,M.C. (2003) Alveolar type I cells: molecular phenotype and Gandarillas,A., Scholl,F.G., Benito,N., Gamallo,C. and Quintanilla,M. development. Annu. Rev. Physiol., 65, 669--695. (1997) Induction of PA2.26, a cell-surface antigen expressed by active Williams,M.C., Cao,Y., Hinds,A., Rishi,A.K. and Wetterwald,A. (1996) fibroblasts, in mouse epidermal keratinocytes during carcinogenesis. Tl a protein is developmentally regulated and expressed by alveolar Mal. Carcinog., 20, 10-18. type I cells, choroid plexus and ciliary epithelia of adult rats. Am. J. Gonzalez,R.F. and Dobbs,L.G. (1998) Purification and analysis of Respir. Cell Mal. Biol., 14, 577-585. RTI40, a type I alveolar epithelial cell apical membrane protein. Yuan,L., Moyon,D., Pardanaud,L., Breant,C., Karkkainen,M.J., Biochim Biophys Acta, 1429, 208-216. Alitalo,K. and Eichmann,A. (2002) Abnormal lymphatic vessel Hirakawa,S., Hong,Y.K., Harvey,N., Schacht,V., Matsuda,K., development in neuropilin 2 mutant mice. Development, 129, 4797- Libermann,T. and Detmar,M. (2003) Transcriptional profiling of isolated human lymphatic versus blood vascular endothelial cells. Am Zimmer,G., Lottspeich,F., Maisner,A., Klenk,H.D. and Herrler,G. (1997) J. Pathol., 162, 575-586. Molecular characterization of gp40, a mucin-type glycoprotein from Hong,Y.K., Harvey,N., Noh,Y.H., Schacht,V., Hirakawa,S., Detmar,M. the apical plasma membrane of Madin-Darby canine kidney cells and Oliver,G. (2002) Proxl is a master control gene in the program (type I). Biochem. J., 326, 99-108. specifying lymphatic endothelial cell fate. Dev. Dyn., 225, 351-357. Zimmer,G., Oeffner,F., Von Messling,V., Tschernig,T., Groness,H.J., Kriehuber,E., Breiteneder,G.S., Groeger,M., Soleiman,A., Klenk,H.D. and Herrler,G. (1999) Cloning and characterization of Schoppmann,S.F., Stingl,G., Kerjaschki,D. and Maurer,D. (2001) gp36, a human mucin-type glycoprotein preferentially expressed in Isolation and characterization of dermal lymphatic and blood vascular endothelium. Biochem J., 341, 277-284. endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med., 194, 797-808. Received January 20, 2003; revised May 13, 2003; Maekinen,T. et al. (2001) Isolated lymphatic endothelial cells transduce accepted May 19, 2003 growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J., 20, 4762-4773. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

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Copyright © European Molecular Biology Organization 2003
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Abstract

The EMBO Journal Vol. 22 No. 14 pp. 3546-3556, 2003 T1a./podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema abnormal development of cutaneous lymphatic vessels Vivien Schacht, Maria I.Ramirez 1, results in lymphedema, which is associated with defects in Young-Kwon Hong, Satoshi Hirakawa, 2 tissue repair and the immune response (Mallon and Ryan, Dian Feng , Natasha Harvey3, 1994). Although the mechanisms that control the devel- 1 4 Mary Williams  , Ann M.Dvorak2, opment of the blood vascular system have been well Harold F.Dvorak , Guillermo Oliver3 and studied (Carmeliet, 2000), those of the lymphatic vessels Michael Detmar are poorly understood. Cutaneous Biology Research Center, Massachusetts General Hospital Recent analyses of Proxl -deficient mice have shown and Harvard Medical School, Charlestown, MA 02129, Pulmonary that the lymphatic vascular system, as predicted by Sabin Center, Department of Medicine and Department of Anatomy, Boston (1902), originates from the embryonic veins (Wigle and University School of Medicine, Boston, MA 02118, Departments of Oliver, 1999; Oliver and Detmar, 2002). Beginning at Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215 and Department of Genetics, embryonic day (E) 9.5 of mouse development, the St Jude Children's Hospital, Memphis, TN 38105, USA homeobox gene Proxl is specifically expressed by a 5 subpopulation of endothelial cells that are located on one Corresponding author e-mail: [email protected] side of the anterior cardinal vein. This is followed by polarized budding and migration of these Proxl-positive V.Schacht and M.I.Rarnirez contributed equally to this work lymphatic progenitor cells, which eventually form lymph- atic sacs and then the entire lymphatic vasculature. In Within the vascular system, the mucin-type trans- Proxl -null mice, the budding and sprouting of lymphatic membrane glycoprotein Tla/podoplanin is pre- endothelial cells from the veins is arrested at ~El 1.5- dominantly expressed by lymphatic endothelium, and E12.0, and these mice completely lack a lymphatic recent studies have shown that it is regulated by the vascular system (Wigle and Oliver, 1999). We and others lymphatic-specific homeobox gene Proxl. In this have shown recently that ectopic expression of Proxl in study, we examined the role of Tla/podoplanin in primary human blood vessel endothelial cells represses the vascular development and the effects of gene disrup- expression of several genes that are associated with the tion in mice. Tla/podoplanin is first expressed at blood vascular phenotype (Hong et al., 2002; Petrova et al., around Ell.O in Proxl-positive lymphatic progenitor 2002). Proxl expression was also found to upregulate the cells, with predominant localization in the luminal expression of lymphatic-specific genes (Hong et al., 2002; plasma membrane of lymphatic endothelial cells Petrova et al., 2002), indicating its function as a master during later development. TlaJpodoplanin-1- mice die control gene that determines lymphatic endothelial cell at birth due to respiratory failure and have defects in fate (Oliver and Detmar, 2002). These studies identified lymphatic, but not blood vessel pattern formation. the mucin-type transmembrane glycoprotein Tla;/podo- These defects are associated with diminished lymph- planin as one of the primary Proxl-induced genes (Hong atic transport, congenital lymphedema and dilation of et al., 2002). lymphatic vessels. Tla/podoplanin is also expressed in TlaJpodoplanin is expressed by cultured human lymph- the basal epidermis of newborn wild-type mice, but atic endothelial cells, and is one of the most highly gene disruption did not alter epidermal differenti- expressed lymphatic-specific genes (Petrova et al., 2002; ation. Studies in cultured endothelial cells indicate Hirakawa et al., 2003). In vivo expression of Tla;/ that Tla/podoplanin promotes cell adhesion, migra- podoplanin in lymphatic endothelium was first reported tion and tube formation, whereas small interfering by Wetterwald et al. (1996), who named it 'El 1 antigen'. RNA-mediated inhibition of Tla/podoplanin expres- It was characterized further under the name 'podoplanin', sion decreased lymphatic endothelial cell adhesion. because of its low level expression in kidney podocytes These data identify Tla/podoplanin as a novel critical (Breiteneder-Geleff et al., 1997). Podoplanin is homo- player that regulates different key aspects of lymph- logous to Tia, which was originally found to encode an atic vasculature formation. antigen that is selectively expressed at the apical surface of Keywords: angiogenesis/lymphangiogenesis/podoplanin/ alveolar type I cells in rat lung (Dobbs et al., 1988; Rishi Proxlffla; et al., 1995). Expression of Tla; has also been detected in the choroid plexus, ciliary epithelium of the eye, intestine, kidney, thyroid and esophagus of the fetal rat (Williams Introduction et al., 1996), and it has been shown to be homologous to The lymphatic vascular system maintains tissue fluid the OTS-8 gene, a phorbol ester-induced gene in MC3T3- homeostasis and mediates the afferent immune response, El mouse osteoblast cells (Nose et al., 1990). Other but can also aid in the metastatic spread of malignant homologs include RT/40 (Gonzalez and Dobbs, 1998), tumors (Detmar and Hirakawa, 2002). Dysfunction or murine gp38 (Farr et al., 1992), canine gp40 (Zimmer 3546 © European Molecular Biology Organization Podoplanin is required for lymphatic patterning E10.5 E12.5 E14.5 Fig. 1. Tla/podoplanin is expressed by Proxl-positive lymphatic progenitor cells during embryogenesis. (A and B) At day 10.5 of embryonic mouse development, Proxl (green) is already expressed by endothelial cells predominantly located on one side of the anterior cardinal vein (CV), whereas the expression of Tla/podoplanin (red) is still restricted to the neural tube (NT). (C and D) At El2.5, Tla/podoplanin-positive endothelial cells are present throughout the anterior cardinal vein. Budding Proxl-positive lymphatic progenitor cells also express Tla/podoplanin. (E and F) At E14.5, the expression of Tla/podoplanin becomes restricted to Proxl-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 (N). Bars = 50 µm. et al., 1997), human gp36 (Zimmer et al., 1999) and found that Tla/podoplanin-1- mice have defects in lymphatic vessel, but not blood vessel, pattern formation. murine PA2.26 (Gandarillas et al., 1997). There have been many studies of Tla/podoplanin These defects lead to diminished lymphatic transport, expression in the lymphatic vascular system (Kriehuber congenital lymphedema and dilation of cutaneous and et al., 2001; Maekinen et al., 2001; Hong et al., 2002; intestinal lymphatic vessels. Overexpression of Tla/ podoplanin in cultured vascular endothelial cells promoted Petrova et al., 2002; Hirakawa et al., 2003). In spite of the the formation of elongated cell extensions and large number of descriptive studies, little is understood significantly increased endothelial cell adhesion, migra- about Tla/podoplanin's biological function. We exam- tion and tube formation. Together, these findings suggest ined its role in lymphatic and blood vessel development by that the transmembrane glycoprotein Tla/podoplanin is examining mice that have targeted deletions in the Tla/ required to regulate key aspects of lymphatic vascular podoplanin gene (Ramirez et al., 2003). formation. Here, we show that within the vascular system, Tla/ podoplanin is first expressed between EI0.5 and El 1.5 in endothelial cells of the cardinal vein and in budding, Results Proxl-positive lymphatic progenitor cells. Tla/podopla- nin expression becomes specifically restricted to lymph- T1a/podoplanin is expressed by budding Prox1- atic endothelium during later development. Ultrastructural positive lymphatic progenitor cells analysis revealed its predominant localization to the In agreement with previous observations showing Tl al luminal plasma membrane of lymphatic vessels. We podoplanin expression in the central nervous system and 3547 V.Schacht et al. Impaired lymphatic transport and formation of lymphedema in neonatal T1a/podoplanin-deficient mice Mice with heterozygous and homozygous disruptions of the Tla/podoplanin gene (Ramirez et al., 2003) were compared with their wild-type littermates for all investi- gations. Whereas Tla/podoplanin+ - mice were healthy and fertile, and were macroscopically indistinguishable from their wild-type littermates, Tla/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). To investigate lymphatic transport, we intradermally injected Evans blue dye into the dorsum of the footpads of newborn mice. In both wild-type and Tla/podoplanin+t- mice, the dye was immediately transported through a dense network of interconnected dermal lymphatic capil- laries and larger collecting lymphatic vessels towards the popliteal lymph nodes (Figure 2B and C). In Tl al podoplanin- - mice, in contrast, only dilated lymphatic vessels were visible, and small dermal capillaries were not detectable (Figure 2D). Immediately after injection of Fig. 2. Congenital skin lymphedema and impaired lymphatic transport in homozygous Tl cr/podoplanin knockout mice. (A) Newborn wild- Evans blue into all four extremities, retroperitoneal para- type (+/+) and heterozygous mice (+/-) showed no phenotypic abnor- aortic lymph nodes and lymphatic ducts were clearly malities, whereas Tlalpodoplanin-null mice (-/-) died immediately stained blue in wild-type and in Tla/podoplanin+t- mice, after birth due to respiratory failure. Examination of Tl alpodoplanin-t- indicating efficient centripetal lymphatic transport mice revealed smoothened skin texture, thickened wrinkles, particularly (Figure 2E and F). Although immunofluorescence stain- in the neck area, and swelling of the lower extremities (inset). (B-D) Intradermal lymphatic capillaries and larger collecting lymphatic ings revealed the presence of retroperitoneal lymphatic vessels of wild-type (B) and Tlalpodoplanin+ - (C) mice were filled ducts in all of the investigated mice (data not shown), no with dye after injection of Evan's blue dye into the dorsum of the staining of para-aortic lymphatic structures was detected paws. In contrast, only enlarged subcutaneous lymphatic collectors (D, in Tla/podoplanin- - mice (Figure 2G), demonstrating that arrowheads) were detected in Tlalpodoplanin-1- mice. After intrader- mal injection of Evan's blue dye into the paws, retroperitoneal lymph- lymphatic transport was impaired. atic vessels (arrowheads) and lymph nodes (arrows) were stained blue in wild-type (E) and Tlalpodoplanin+ - (F) mice, but no dye was de- tected in the retroperitoneal lymphatics of Tlalpodoplanin-1- mice (G). Dilation of intestinal and cutaneous lymphatic IVC = inferior vena cava; A = aorta. vessels, but not of blood vessels, in T1a/ podoplanin-nu/1 mice Both the skin and intestine are characterized by their rich the foregut at around E9 (Rishi et al., 1995; Williams, lymphatic vascularization, and these tissues are highly 2003), we detected expression of Tla/podoplanin in the sensitive to impairment of lymphatic network formation. neural tube of wild-type mice at El0.5 (Figure lA). Using differential immunostains for the lymphatic-specific However, no vascular expression of Tla/podoplanin was hyaluronan receptor LYVE-1 (Prevo et al., 2001) and the detected yet at this time point, whereas the homeobox endothelial plasma membrane molecule CD31, we found gene Proxl was already expressed by a subset of several slightly enlarged submucosal lymphatic vessels in endothelial cells of the anterior cardinal vein (Figure lA the small intestine of Tl a/podoplanin+t- mice as compared and B). By Ell.5 (data not shown) and El2.5, Tla/ with wild-type mice (Figure 3A and E). In Tla/ podoplanin was expressed by all endothelial cells of the podoplanin- - mice, an increased number of severely cardinal vein and by the Proxl-positive lymphatic pro- dilated submucosal lymphatic vessels was found genitor cells that had already budded off from the (Figure 31), whereas no major alterations of subserosal embryonic veins (Figure lC and D). Two days later, lymphatic capillaries were detected. L YVE-1-positive Tla/podoplanin expression was restricted to the budded 1 lacteals were completely absent in Tla/podoplanin- - Proxl-positive lymphatic endothelial progenitor cells and mice (Figure 31), whereas these formed normally in to the Proxl-positive lymphatic endothelial cells that lined 1 wild-type and Tla/podoplanin+ - mice (Figure 3A and E, the developing primitive lymph sacs (Figure lC and F). asterisks). The number and size of CD31-positive/ After birth, vascular Tla/podoplanin expression was L YVE-1-negative intestinal blood vessels, in contrast, almost exclusively detected in lymphatic vessels (see were comparable in all genotypes (Figure 3A, B, E, F, I below). and J). 3548 Podoplanin is required for lymphatic patterning Intestine Skin LYVE:-1 (red) CD31 (green) Podoplanin (red) CD31 (greeol LYVE-1 (red) CD31 (g.reon) Podoplanin (red) CD31 {green) B C +!+ +/- . , . ' . .. ' "' -/- _. -i J , r , .._  . - . ,,  t I    .,. ' ,,: . .. 'L K14 K10 Loricrin a, J:j .,, +/+ .; ., .,, !£ -0 iii ' ~' "'· - . "'.-r . .... r\~ . -. +! - N 'i;' .': - :!l _.:, 'ill ., "' - "' ~ -,.,, ~-:-~-~ !l! . -' v'"'" ,<#'' t 1 .._,_,,,,..rt,·' .._ .. \ . J <ii -/- .c '  .; . ·' 0. .: "'  , 0, [ ' ,,  ~-... . \.,\ - ...J Fig. 3. Tla/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 CD3 l (green) and L YVE-1 (red, C, G and K) or Tla/podoplanin (red, B, F, J, D, Hand L) revealed slightly enlarged lymphatic vessels of the submucosal plexus of TJCJ/podoplanin+ - mice (E), whereas TJCJ/podoplanin-1- mice have greatly enlarged lymphatics (I). Lacteals of wild-type and Tla/podoplanin+ - mice were LYVE-1 positive (asterisks, A and E), whereas no LYVE-1-positive lacteals were detected in TJCJ/podoplanin-1- mice (I). Lymphatic expression of TJCJ/podoplanin was confirmed in the ileum and the skin of wild-type (B and D) and TJCJ/podoplanin+ - (F and H) mice (asterisks= lacteals), but was not detected in TJCJI podoplanin-1- 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 TJCJI podoplanin-1- mice. Because some Tla/podoplanin-expressing basal keratinocytes were found in the epidermis of wild-type and TJCJ/podoplanin+ - mice (D and H; arrowheads), the comparative expression of the epidermal differentiation markers K14, KIO and loricrin was investigated (O-W). No differences in the expression of these markers were seen in the three genotypes. Bars = 50 µm. The Tlalpodoplanin-1- mice also had enlarged lympha- et al., 1992), for Tla/podoplanin (Figure 3B, D, F, H, J and L). tics in the skin, as compared with wild-type and Tlal podoplanin+ - mice (Figure 3C, G and K). Computer- assisted morphometric image analyses confirmed that the T1a/podoplanin deficiency results in impaired average area of dermal L YVE-1-positive lymphatic ves- patterning of lymphatic capillary networks sels was significantly increased in Tlalpodoplanin-1- mice We investigated whether Tla/podoplanin deficiency, in (Figure 3N), whereas no differences in the size of blood addition to causing the enlargement of intestinal and vessels were found (Figure 3M). lmrnunofluorescence cutaneous lymphatic vessels, might also affect the pat- analysis of the intestine and the skin confirmed that terning of the lymphatic network in the same anatomical lymphatic vessels in both wild-type and Tlalpodopla- regions. To this end, we performed imrnunostain analysis nin+t- mice strongly expressed Tla/podoplanin. In con- of L YVE-1 expression on tissue samples from the intestine trast, no imrnunoreactivity was detected in Tlal (ileum) and from the ear skin of all newborn genotypes. podoplanin- - mice, confirming the complete disruption We observed a dense and well-organized network of this gene and the specificity of the 8.1.1 antibody, of intestinal lymphatic capillaries in wild-type and that was originally raised against the gp38 antigen (Farr Tl alpodoplanin+t- newborn mice (Figure 4A and E). 3549 V.Schacht et al. Newborn mice Adult mice Intestine Ear Intestine Ear +/+ +/- -!- Fig. 4. Abnormal patterning of lymphatic capillaries in Tlcx/podoplanin-deficient mice. (A-H) L YVE-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 Tla/podoplanin+ - mice. The network patterning was completely irregular and the diameter of lymphatics was strikingly increased in Tla/podoplanin-1- mice (I). The lymphatic vessels in the ear of Tla/podoplanin-1- mice also devel- oped 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 Tla/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 Tla/podoplanin+ - mice. Bars for (A), (B), (E), (F) and (J) = 100 µm, (C) and (G) = 200 µm, (D) and (H) = 300 µm. Fig. 5. Ultrastructural localization of Tlcx/podoplanin in murine intestinal lymphatic vessels, but not in blood vessels, by immuno-nanogold staining. (A) In newborn wild-type mice, membrane-bound Tlcx/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 Tlcx/podoplanin was detected within lymphatic endothelial cells. (B) Tlcx/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-Tlcx/podoplanin antibody. (D) Lymphatic endothelial cells of a newborn Tla/podoplanin-1- mouse do not react with the anti-Tlcx/podoplanin antibody. Bars for (A) = 0.4 µm, (B) = 0.2 µm, (C) = 0.3 µm, (D) = 0.5 µm. 3550 Podoplanin is required for lymphatic patterning Fig. 6. Comparable ultrastructural localization of L YVE-1 in intestinal lymphatic vessels of wild-type and T 1 a/podoplanin--1- mice. (A) High levels of immuno-nanogold labeling for L YVE-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 L YVE-1 was absent from the cyto- plasm. (B) L YVE-1 expression was absent from blood vascular endothelium. (C and D) Lymphatic endothelial cells in the intestine of Tia/ podoplanin--1- mice also had high levels of L YVE-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 L YVE-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 Tla/podoplanin-null mice (F). Bars for (A), (C), (D) and (E) = 0.5 µm, (B) and (F) = 0.4 µm . Tl a/podoplanin- - newborn mice, in contrast, developed areas of dilated lymphatic vessels in the ear skin (Figure 4H) and in the intestine of Tla/podoplanin+t- areas of extremely enlarged lymphatic vessels, and the mice, in addition to incomplete network formation pattern of these vessels was completely disorganized (Figure 4G) that was not observed in wild-type littermates (Figure 41). Analysis of the ear skin of wild-type and Tl al (Figure 4C and D). podoplanin+t- mice revealed well-organized networks of L YVE-1-positive lymphatic capillaries (Figure 4B and F). Most of these capillaries were interconnected, and only a Ultrastructural localization of T1a/podoplanin few blind beginning lymphatic capillaries were detected in intestinal lymphatic vessels, but not in (Figure 4B and F). In contrast, the formation of lymphatic blood vessels capillary networks was impaired in the ear skin of Tla/ To investigate the ultrastructural localization of Tl a/ podoplanin- - mice. These mice possessed an increased podoplanin in the vascular system, we performed immuno- number of non-anastomozing, blind beginning cutaneous nanogold electron microscopy, using intestinal tissue lymphatic capillaries (Figure 4J, arrowheads), indicative samples obtained from newborn mice. In wild-type mice, of impaired lymphatic network patterning. high levels of membrane-bound Tla/podoplanin were Because there were no clear-cut differences in detected at the luminal side of intestinal lymphatic vessels lymphatic network patterns between newborn Tl a/podo- (Figure SA). Fewer nanogold particles were observed on planin+I- and wild-type mice, we performed immuno- the abluminal plasma membrane, and no labeling was histochemical analyses on intestine (ileum) and ear skin detected within the cytoplasm (Figure SA). Occasionally, tissue obtained from adult (4-month-old) Tla/podo- the lateral plasma membranes between adjacent cells were planin+t- mice and their littermates. Although the defects also labeled. Tla/podoplanin expression was completely in lymphatic network patterning were not as striking as absent from blood vessel endothelial cells in the intestine those seen in newborn Tla/podoplanin- - mice, there were of newborn wild-type mice (Figure SB). Lymphatic 3551 V.Schacht et al. Rat podoplanln F-actln Merge Q c 3-,------------, 20 ,g e2:.5 -~ E 2 e GI 1.6 ]l 10 G) :I !! 1 :E 0.5 LL o~~C_o_n-tr_o_l ~-Podoplanln Control Podoplanfn Fig. 7. Overexpression of Tla/podoplanin in HMEC-1 cells enhances endothelial cell migration and adhesion in vitro. (A-C) Stable transfection of HMEC-1 cells with rat Tla/podoplanin cDNA resulted in the production of the rat Tla/podoplanin protein (green; A and C) and in the formation of long filopodia, which were not seen in cells that did not overexpress Tl a/podoplanin. Some of the rat Tl a/podoplanin-expressing cells showed a marginal accumulation of F-actin bundles (red, B and C). Bar= 150 µm. (D and E) Overexpression of Tla/podoplanin significantly stimulated cell migration (D) and adhesion (E) of HMEC-1 cells. **P < 0.01; ***P < 0.001. endothelial cells in the small intestine of Tl al expression of several markers of epidermal differentiation. podoplanin- - mice were not immunolabeled by the anti- A comparable expression pattern of keratin 14 (K14), Tla/podoplanin antibody (Figure 5D), confirming the which is expressed by proliferative basal keratinocytes disruption of the Tla/podoplanin gene in these mice. (Fuchs and Byrne, 1994), was found in all three groups (Figure 30, R and U). Expression patterns of the early and late epidermal terminal differentiation markers KIO T1a/podoplanin deficiency does not alter the (Figure 3P, S and V) and loricrin (Figure 3Q, T and W) ultrastructural architecture of intestinal lymphatic were also similar in the skin in all three genotypes. vessels or the distribution of LYVE-1 expression Computer-assisted morphometric image analyses demon- We next investigated whether loss of Tla/podoplanin strated comparable thickness of the epidermis in all might result in abnormal ultrastructural architecture of genotypes (data not shown), and no major histological lymphatic vessels or in altered distribution of the L YVE-1. differences of epidermal structure were detected, indicat- We detected high levels of immuno-nanogold labeling for ing that Tla/podoplanin deficiency does not affect the L YVE-1 at both the luminal and the abluminal plasma formation or structure of the epidermis. 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 T1a/podoplanin promotes endothelial cell the exception of contact areas between adjacent cells. migration, adhesion and tube formation L YVE-1 expression was completely absent from To characterize further the biological function of Tl a/ blood vessel endothelium (Figure 6B), confirming the podoplanin, we isolated lymphatic endothelial cells from specificity of L YVE-1 for lymphatic endothelium. the skin of newborn mice of all three genotypes. However, Lymphatic endothelial cells of Tla/podoplanin- - mice we were unable to expand lymphatic endothelial cell also showed strong L YVE-1 immunogold labeling at cultures obtained from all genotypes, even after addition both the luminal and the abluminal plasma membranes at of recombinant vascular endothelial growth factor levels similar to those observed in wild-type mice (VEGF)-C to the growth medium (data not shown). We (Figure 6C and D). No ultrastructural differences in therefore decided to test the effects of Tla/podoplanin lymphatic vessel structure were observed between the overexpression. Immortalized human microvascular different genotypes. endothelial cells (HMEC-1) were stably transfected with a plRES2-rat Tla/podoplanin expression vector and T1a/podoplanin deficiency does not impair pooled cells were used for subsequent in vitro studies. epidermal differentiation Immunostains revealed high levels of rat Tla/podoplanin We occasionally detected Tla/podoplanin expression in protein in the stably transfected cells (Figure 7 A) that basal keratinocytes of the epidermis in wild-type and Tla/ projected extremely long and thin cell extensions, which were podoplanin+ - mice (Figure 3D and H). To determine not seen in control cells that did not express rat Tl a/ whether Tla/podoplanin deficiency might also affect podoplanin (Figure 7B and C). Some of the stably Tla/ epidermal structure or differentiation, we studied the podoplanin-transfected cells also formed phalloidin-positive 3552 Podoplanin is required for lymphatic patterning 10~--------, A B Podoplanin .. 8 ~30 II> g 25 E a -70 kD ]i II) 'aj 4 f 15 (.) .: 10 I&. 2 C1 C2 R1 R2 lnnn 50--------~ o C1 C2 CJ P1 P2 PJ B Controls Podoplanin E30 U) a, 20 .L..Lc~1 -----'-----'~ c~2 .__ R 1 R2 Fig. 9. Inhibition of Tla/podoplanin expression by siRNA transfection ,_ 100 E reduces human lymphatic endothelial cell adhesion to type I collagen. E BOO (A) Four days after siRNA transfection of human primary lymphatic -E 560 endothelial cells, endogenous Tla/podoplanin protein levels, but not L YVE-1 levels, were decreased by two different siRNA oligonucleo- [400 tides (RI and R2), as compared with control cells (Cl, control vector cDNA; C2, sham-transfected cells). (B) Human dermal lymphatic en- dothelial cells showed significantly reduced adhesion to type I collagen 4 days after siRNA transfection (Rl and R2), as compared with control cells (Cl and C2). *P < 0.05; **P < 0.01. o C1 Controls Podoplanln Fig. 8. Overexpression of Tla/podoplanin in EOMA cells enhances podoplanin significantly enhanced EOMA cell migration cell migration, adhesion and tube formation in vitro. (A and B) Stable transfection of EOMA cells with rat Tla/podoplanin cDNA resulted in towards type I collagen and also promoted cell adhesion in significantly enhanced haptotactic cell migration and adhesion to type I all three clones tested, as compared with the control clones collagen in all three clones tested (Pl-P3), as compared with control (Figure 8A and B). Tla/podoplanin-overexpressing clones (Cl-C3). (C and D) Enhanced formation of tube-like structures EOMA cell clones also showed a significantly increased by Tla/podoplanin-overexpressing EOMA cells after seeding onto ability to form tube-like structures after plating onto Matrigel (24 h; D), as compared with control vector-transfected cells (C). Bar= 50 µm . (E) Overexpression of Tla/podoplanin significantly Matrigel (Figure 8C-E). enhanced the formation of tube-like structures in all three Tla/podopla- nin-overexpressing clones (Pl-P3) as compared with control clones (Cl-C3). Inhibition of T1a/podoplanin expression by siRNA transfection reduces cell adhesion of human lymphatic endothelial cells F-actin bundles at the periphery (Figure 7B and C), indicating To investigate further the role of Tla/podoplanin in cell that Tla/podoplanin expression might control endothelial adhesion, we studied the effects of small interfering RNA cell cytoskeletal organization. We next studied whether (siRNA)-mediated inhibition of endogenous Tla/podo- overexpression of Tla/podoplanin could affect endothelial planin expression in cultured human dermal lymphatic cell migration or adhesion. We found a >2-fold (P < 0.001) endothelial cells that are characterized by high expression increase in haptotactic cell migration towards fibronectin in levels ofTla/podoplanin (Hirakawa et al., 2003). Two out stably transfected cells, compared with controls (Figure 7D). of three siRNAs tested efficiently reduced endogenous Tl a/podoplanin-overexpressing HMEC-1 cells also adhered Tla/podoplanin protein expression by 49 and 34%, more tightly to immobilized fibronectin (P < 0.01 ; Figure 7E). respectively, at 4 days after transfection, whereas the To confirm these results further in a second, independ- expression of another 1 ymphatic marker, L YVE-1, re- ent cell line, murine hemangioendothelioma-derived mained unchanged (Figure 9A). Accordingly, lymphatic EOMA cells were stably transfected with the same endothelial cell adhesion to type I collagen, which is pIRES2-rat Tla/podoplanin-enhanced green fluorescent closely associated with lymphatic vessels in vivo (Skobe protein (EGFP) construct or with the control vector. Three and Detmar, 2000), was significantly inhibited by both of clones with high expression of the transfected Tl al these siRNAs, as compared with control or sharn- podoplanin, along with three control clones, were used transfected control cells, at 4 days after transfection for subsequent experiments. Overexpression of Tla/ (Figure 9B). 3553 V.Schacht et al. podoplanin-1- mice is even more pronounced, we expect Discussion that lipid uptake in the intestine would also be defective. Previous studies have identified Tla/podoplanin as a The observed enlargement of both dermal and submucosal Proxl-induced gene (Hong et al., 2002) that is predom- intestinal lymphatics of Tla/podoplanin-1- mice is most inantly expressed, within the vascular system, in lymph- likely to be related to the lack of connecting lymphatics atic endothelium (W etterwald et al., 1996; Kriehuber et al., between the superficial and deep networks, indicating that 2001; Maekinen et al., 2001; Hirakawa et al., 2003). We Tla/podoplanin is required for the formation of these show that similarly to VEGF receptor-3 (Wigle et al., specific lymphatic vessels. 2002), another Proxl target gene (Hong et al., 2002; We were unable to propagate lymphatic endothelial Petrova et al., 2002), Tla/podoplanin is expressed cells that were isolated from both wild-type and Tl al throughout the endothelium of the anterior cardinal vein podoplanin- - neonatal mice by using a modification of our at E12.5 of embryonic mouse development and later recently established purification method for human dermal becomes restricted to the budding Proxl-positive lymph- lymphatic endothelial cells (Hirakawa et al., 2003). At atic progenitor endothelial cells and to lymphatic present, there are no published reports on the successful endothelial cells of the embryonic lymph sacs and of propagation of primary murine lymphatic endothelial lymphatic vessels. In contrast to Proxl -null mice, which cells, and suitable culture techniques still remain to be fail to develop any lymphatic vasculature (Wigle and established. Therefore, we investigated the cellular effects Oliver, 1999), Tla/podoplanin-1- mice develop a periph- of Tla/podoplanin overexpression in two types of eral lymphatic vascular system; however, it exhibits immortalized human and murine vascular endothelial pronounced defects in lymphatic vascular organization cells that express only moderate levels ofTla/podoplanin and function. Our findings indicate that (Hong et al., 2002). The induction of elongated endothelial Tl a/podoplanin is cell extensions and cytoskeletal reorganization, together important to regulate different aspects related to the later stages of lymphatic development and patterning. In with the enhanced adhesion and migration of stably Tla/ contrast to other recently described gene targeting models, podoplanin-transfected endothelial cells are in agreement including mice deficient for angiopoietin-2 (Gale et al., with its effects on the motility of immortalized keratino- 2002) or VEGF receptor-3 (Dumont et al., 1998), Tla/ cytes (Scholl et al., 1999) and indicate that this protein podoplanin deficiency selectively affects the lymphatic controls endothelial cell functions that are required for vascular system without any detectable effect on the normal lymphatic patterning during development. These development of the blood vascular system. Similarly to findings were confirmed further by the reduced endothelial neuropilin-2 mutant mice which show only reduction of cell adhesion observed after inhibition of endogenous small lymphatic vessels but no alteration of the blood Tla/podoplanin expression by siRNA transfection. vascular system (Yuan et al., 2002), our findings in Tia/ Because efficient cell migration is dependent upon the podoplanin-deficient mice are in agreement with the polarity of the migrating cells, and since Tla/podoplanin relatively late onset of Tl alpodoplanin expression during is also expressed on the apical surface of polarized vascular development, and with its predominant expres- alveolar epithelial cells (Dobbs et al., 1988; Rishi et al., sion on lymphatic vascular endothelium. 1995), it may exert an important role for the polarization of Importantly, Tla/podoplanin-1- mice were character- cells and for the stabilization of cellular protrusions at the ized by congenital lymphedema, as manifested by the leading edge of migrating cells. This proposed function is pronounced swelling of the limbs at birth. lntradermal dye supported further by our findings that overexpression of injection into the foot pads of Tla/podoplanin-1- mice Tla/podoplanin in endothelial cells promoted the forma- revealed several enlarged, plump lymphatic vessels, but tion of tube-like structures on Matrigel which provide a failed to visualize the characteristic dermal capillary link to the in vivo findings of incomplete lymphatic networks seen in wild-type and in Tl a/podoplanin+t- network formation in Tla/podoplanin-1- mice. mice. These networks were most probably filled from Tla/podoplanin is a mucin-type glycoprotein with deeper lymphatic vessels through anastomozing vessels extensive O-glycosylation and a high content of sialic acid (Gonzalez and Dobbs, 1998). This negatively charged that still lack valves at the early stages of pre- and post- natal lymphatic development (Polonskaja, 1935). Because structure probably is resistant to proteases and provides a histological examination revealed the presence of dermal physical barrier that protects cells from environmental capillaries in Tla/podoplanin-1- mice, these findings agents (Zimmer et al., 1999). Immuno-nanogold electron indicate an insufficient formation of anastomozing lymph- microscopy showed that Tla/podoplanin is predominantly localized to the apical, luminal plasma membrane of atic vessels between the superficial and subcutaneous lymphatic networks. intestinal lymphatic endothelial cells. This localization is In the intestine of Tl a/podoplanin-1- mice, lacteals were similar to that reported for other cell types (Dobbs et al., not detectable and many of the lymphatics of the 1988; Rishi et al., 1995; Williams et al., 1996; submucosal plexus were enlarged. The physiological Breiteneder-Geleff et al., 1997; Zimmer et al., 1997) and consequences of these developmental defects on the is compatible with a protective function towards the uptake of lipids from the intestine are not known, since proteinase-containing lymph (Bartos et al., 1979). It is of these mice died immediately after birth (before the first interest that Tla/podoplanin is also expressed by alveolar feeding) and chyle transport could not be investigated. epithelial cells, cells of the choroid plexus, ependymal However, as previously described (Gale et al., 2002), epithelia and mesothelia that are also exposed to an angiopoietin-2-null mice develop chylous ascites shortly external or internal fluid compartment. after birth, due to insufficient formation of lacteals. In conclusion, we propose that Tla/podoplanin is Because the observed morphological defect in Tl al required to control different aspects of normal lymphatic 3554 Podoplanin is required for lymphatic patterning controls included replacement of the primary antibody with irrelevant vasculature formation. Lack of Tla/podoplanin leads to rabbit lgG and omission of the specific primary antibody. alterations in the final patterning of the lymphatic vasculature as well as in lymph transport. The future Cell culture and transfection generation of a conditional inactivation of Tl a/podoplanin hnmortalized HMEC-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) that contained 10% fetal bovine serum (PBS), will be a valuable tool to understand further its role in the 2 mM L-glutarnine and antibiotics (Life Science, Grand Island, NY). The normal function and in pathological alterations of the murine hemangioendothelioma-derived cell line EOMA (Obeso et al., lymphatic vasculature. 1990) was maintained in DMEM containing 20% PBS and 4 mM L-glutamine. Primary human dermal lymphatic endothelial cells were isolated and propagated as recently described (Hirakawa et al., 2003). The rat Tla/podoplanin coding sequence (Rishi et al., 1995; GenBank Materials and methods accession No. U07797) was cloned into a plRES2-EGFP vector (Clontech, Palo Alto, CA) which contains a cytomegalovirus (CMV) Generation of T1a/podoplanin knockout mice enhancer promoter and a neomycin selection cassette. The nucleotide Tl a/podoplanin mutant mice were generated as described (Ramirez et al., sequence was verified using the Applied Biosystems Big Dye Terminator 2003). Fragments were designed to replace ~1.5 kb of the promoter kits. After linerarization with Bsal, HMEC-1 and EOMA cells were sequence, 210 bp of the untranslated region (UTR), the first exon (67 bp) transfected either with the full-length rat Tla/podoplanin cDNA or with and 181 bp of the first intron. The efficiency of the disruption of the T 1 al plRES2-EGFP vector alone using the SuperFect transfection reagent podoplanin gene was confirmed by northern and western blot analyses of (Qiagen, Chatsworth, CA). Stably transfected cells were selected in newborn lung RNA and protein extracts that demonstrated the absence of growth medium containing 200 µg/ml neomycin (Gibco, Carlsbad, CA). Tla/podoplanin mRNA or protein in Tla/podoplanin--1- mice and For all experiments, pooled transfected HMEC-1 cells and three clones of reduced Tla/podoplanin mRNA levels in Tla/podoplanin+ - mice stably transfected EOMA cells were used for Tla/podoplanin over- (Ramirez et al., 2003). expression and for vector controls. To determine Tla/podoplanin expression levels, we performed SYBR Green-based real-time RT- Tissue processing and immunostaining PCRs as described (Hong et al., 2002), using the ABI Prism 7000 Tissue samples were obtained from newborn Tl a/podoplanin ( +/+ ), ( +/-) Sequence Detection System. The following forward and reverse and (-/-) littermates that had been killed by intraperitoneal injection of primers were used: 5'-GACATGGTGAACCCAGGTCT-3 and 5'- barbital sodium within 10 min after birth and from wild-type embryos at AATGGGAGGCTGTGTTGGTA-3. Total RNAs were isolated using days El0.5, E12.5 and E14.5, and were fixed in 4% paraformaldehyde Tri-reagent (Sigma, St Louis, MO) and were treated with RNase-free RQ- (Fluka, Buchs, Switzerland). hnmunofluorescence stains were performed DNase (Promega, Madison, WI). A 100 ng aliquot of RNA was used for on 6 µm cryostat sections of dorsal skin and of ileum, as well as on 10 µm each reaction. SYBR Green PCR Master Mix (Applied Biosystems, sections of wild-type mouse embryos as described (Wigle et al., 2002), Foster City, CA) was used for reactions with the addition of MultiScribe using polyclonal rabbit antibodies against Proxl (Wigle et al., 2002) and reverse transcriptase (Applied Biosystems). Protein expression was LYVE-1 (Prevo et al., 2001; kindly provided by Dr D.Jackson, Oxford, confirmed by immunofluorescence staining with a specific monoclonal UK), a monoclonal rat antibody against CD31 (BD Pharmingen, San anti-rat Tla/podoplanin antibody (Wetterwald et al., 1996). For F-actin Diego, CA), a hamster antibody against murine gp38 (Farr et al., 1992; staining, phalloidin-tetramethylrhodamine B isothiocyanate conjugate clone 8.1.1, Developmental Studies Hybridoma Bank, University of (Sigma) was used (Scholl et al., 1999). Iowa), antibodies against murine K14, KlO and loricrin (Babco, Richmond, CA) and corresponding secondary antibodies labeled with Cell transfection with human T1a/podoplanin siRNAs AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, OR). Cell The following siRNA oligonucleotides were synthesized by Dharmacon nuclei were counterstained with 20 µg/ml Hoechst bisbenzimide (Lafayette, CO): (Rl) 5'-GCGAAGACCGCUAUAAGUCdTdT-3', (R2) (Molecular Probes). Whole-mount samples of mouse ears and of ileum 5'-AAGAUGGUUUGUCAACAGUdTdT-3' and (R3) 5'-AGAUGA- were stained as described elsewhere (Gale et al., 2002). The tissues were CACUGAGACUACAdTdT-3'. Primary human lymphatic endothelial examined by using a Nikon E-600 microscope (Nikon, Melville, NY) and cells (Hirakawa et al., 2003) were transfected or not with siRNA images were captured with a SPOT digital camera (Diagnostic oligonucleotides (500 nmol) or with equimolar concentrations of control Instruments, Sterling Heights, Ml). Computer-assisted morphometric plasmid vector by using the Nucleofector kit (Amaxa, Cologne, analysis of lymphatics and blood vessels within the dermis of eight Germany) according to the manufacturer's instructions. Cells were animals per group (three fields per sample at 20X magnification) were harvested at 2 and 4 days after transfection. For western analyses, cell performed as described previously (Streit et al., 1999), using the IP-LAB lysates were obtained as described (Hong et al., 2002) and 30 ng of software (Scanalytics, Fairfax, VA). The differences in vessel size were protein per sample were immunoblotted with an antibody against human analyzed by the two-sided unpaired Student's t-test. Tla/podoplanin (Zimmer et al., 1997; kindly provided by Dr G.Herrler, Hanover, Germany) and with an antibody against human L YVE-1 (kindly lmmuno-nanogold electron microscopy provided by Dr D.Jackson, Oxford, UK). Small intestine samples were collected from genetically modified mice and their wild-type littermates immediately after birth. Tissue samples Cell migration, adhesion and tube formation assays For cell migration assays, 24-well FluoroBlok inserts (Falcon, Franklin were fixed in 4% paraformaldehyde and transferred to 30% sucrose/ phosphate-buffered saline (PBS). After embedding the tissues in OCT Lakes, NJ; 8 µm pore size) were coated on the underside with 10 µg/ml compound (Miles, Elkhart, IN), cryostat sections (5 µm) were immersed fihronectin (BD Bioscience, Bedford, MA) or with 50 µg/ml type I in 50 mM glycine. After washing, sections were immersed in normal goat collagen (Vitrogen, Palo Alto, CA) for 1 h, followed by addition of serum (Vector Laboratories, Burlingame, CA) and incubated with anti- 100 µg/ml bovine serum albumin (BSA; Sigma) to block the remaining LYVE-1 or 8.1.1 antibody, followed by incubation with goat anti-rabbit protein-binding sites. Cells (2 X 10 cells/ml) were seeded in serum-free Fab', conjugated with 1.4 nm nanogold particles (Nanoprobes, Stony EBM-2 medium (Clonetics, Watersville, MD) containing 0.2% de- Brook, NY), or with goat anti-hamster IgG, conjugated with 0.8 nm lipidized BSA into each well and cells were incubated for 3 h at 37°C. nanogold particles (Aurion, Wageningen, The Netherlands). Sections Cells on the underside of inserts were stained with Calcein AM were post-fixed in 1 % PBS-buffered glutaraldehyde, developed with HQ (Molecular Probes), and the fluorescence intensity was measured using silver enhancement solution (Nanoprobes), fixed in 5% sodium the Victor2 Fluorometer (PerkinElmer, Boston, MA). Cell adhesion thiosulfate, post-fixed in 1 % osmium tetroxide in sym-collidine buffer, assays were performed as described (Streit et al., 1999). Twelve-well washed in 0.05 M sodium maleate buffer, and stained with 2% uranyl plates were coated with 10 µg/ml fibronectin or with 50 µg/ml type I acetate in 0.05 M sodium maleate buffer. Tissues were dehydrated in collagen for 1 hat 4°C, followed by blocking with 100 µg/ml BSA. Cells graded ethanols, infiltrated with propylene oxide-eponate (Ted Pella, (1 X 105 cells in 200 µI of serum-free DMEM) were added to each well Redding, CA) and embedded by inverting eponate-filled plastic capsules and were incubated at 37°C for 20 min. Unattached cells were removed over the slide-attached tissue sections. After polymerization, eponate by three gentle washes with PBS; attached cells were fixed with 4% blocks were separated from the glass slides and thin sections were cut on paraformaldehyde, stained with Hoechst bisbenzimide and the number of an ultratome (Reichert illtracuts, Austria), collected on uncoated 200 attached cells/mm was determined. Tube formation assays were mesh copper grids (Ted Pella) and examined with a CM 10 transmission performed on Matrigel-coated 24-well plates (BD Bioscience) as electron microscope (Philips, Eindhoven, The Netherlands). Specificity described previously (Obeso et al., 1990). EOMA cells were seeded at 3555 V.Schacht et al. a density of 2 X 105 cells/ml in growth medium and were incubated for Mallon,E.C. and Ryan,T.J. (1994) Lymphedema and wound healing. 24 h at 37°C. After fixation with 4% paraformaldehyde, images were Clin. Dermatol., 12, 89-93. captured and the total length of tube-like structures per area was measured Nose,K., Saito,H. and Kuroki,T. (1990) Isolation of a gene sequence using the IP-LAB software. All studies were performed in triplicate. induced later by tumor-promoting 12-O-tetradecanoylphorbol-13- Statistical analyses were performed using the unpaired Student's t-test. acetate in mouse osteoblastic cells (MC3T3-El) and expressed constitutively in ras-transformed cells. Cell Growth Differ., 1, 511- Obeso,J., Weber,J. and Auerbach,R. (1990) A hemangioendothelioma- Acknowledgements derived cell line: its use as a model for the study of endothelial cell The authors thank J.Bertoncini, L.Janes, M.Constant and G.Millien for biology. Lab. Invest., 63, 259-269. technical assistance, Dr D.Jackson for the gift of the L YVE-1 antibody, Oliver,G. and Detmar,M. (2002) The rediscovery of the lymphatic Sam W.Lee for valuable advice regarding siRNA experiments, and system. Old and new insights into the development and biological Kathryn Pyne for photographic assistance with the electron micrographs. function of lymphatic vascular system. Genes Dev., 16, 773-783. This work was supported by NIH grants CA69184, CA86410, CA92644 Petrova,T.V. et al. (2002) Lymphatic endothelial reprogramming of (M.D.), HIA7049 (M.C.W.), Al33372 and Al44066 (A.M.D.), by vascular endothelial cells by the Prox-1 homeobox transcription American Cancer Society Program Project Grant 99-23901 (M.D.), by factor. EMBO J., 21, 4593-4599. the Deutsche Forschungsgemeinschaft (V.S.), by the Francis Family Polonskaja,R. (1935) The connections between the superficial and deep Foundation (M.I.R.) and by the Cutaneous Biology Research Center lymphatic vessels of the lower limb. Anat. Anz., 81, 247-256. through the Massachusetts General Hospital/Shiseido Co. Ltd Agreement Prevo,R., Banerji,S., Ferguson,D.J., Clasper,S. and Jackson,D.G. (2001) (M.D.). Mouse L YVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium. J. Biol. Chem, 276, 19420-19430. Ramirez,M.I., Millien,G., Hinds,A., Cao,Y., Seldin,D.C. and References Tla, a lung type I cell differentiation gene, is Williams,M.C. (2003) required for normal lung cell proliferation and alveolus formation at Bartos,V., Kolc,J., Vanecek,R. and Malek,P. (1979) Lymph and blood Dev. Biol., 256, 61-72. birth. enzymes and pathologic alterations in canine experimental pancreatitis Rishi,A.K., Joyce-Brady,M., Fisher,J., Dobbs,L.G., Floros,J., after administration of benzo-pyrones. Scand. J. Gastroenterol., 14, VanderSpek,J., Brody,J.S. and Williams,M.C. (1995) Cloning, 343-347. characterization and development expression of a rat lung alveolar Breiteneder-Geleff,S., Matsui,K., Soleiman,A., Meraner,P., type I cell gene in embryonic endodermal and neural derivatives. Dev. Poczewski,H., Kalt,R., Schaffner,G. and Kerjaschki,D. (1997) Biol., 167, 294-306. Podoplanin, novel 43-kd membrane protein of glomerular epithelial Sabin,F.R. (1902) On the origin of the lymphatic system from the veins cells, is down-regulated in puromycin nephrosis. Am J. Pathol., 151, and the development of the lymph hearts and thoracic duct in the pig. 1141-1152. Am. J. Anat., 1, 367-389. Carmeliet,P. (2000) Mechanisms of angiogenesis and arteriogenesis. Scholl,F.G., Gamallo,C., Vilaro,S. and Quintanilla,M. (1999) Nat. Med., 6, 389-395. Identification of PA2.26 antigen as a novel cell-surface mucin-type Detmar,M. and Hirakawa,S. (2002) The formation of lymphatic vessels glycoprotein that induces plasma membrane extensions and increased and its importance in the setting of malignancy. J. Exp. Med., 196, motility in keratinocytes. J. Cell Sci., 112, 4601-4613. 713-718. Skobe,M. and Detmar,M. (2000) Structure, function and molecular Dobbs,L.G., Williams,M.C. and Gonzalez,R. (1988) Monoclonal control of the skin lymphatic system. J. Investig. Dermatol. Symp. antibodies specific to apical surfaces of rat alveolar type I cells bind Proc., 5, 14-19. to surfaces of cultured, but not freshly isolated, type II cells. Biochim Streit,M., Velasco,P., Brown,L.F., Skobe,M., Richard,L., Riccardi,L., Biophys Acta, 970, 146-156. Lawler,J. and Detmar,M. (1999) Overexpression of thrombospondin-1 Dumont,D.J., Jussila,L., Taipale,J., Lymboussaki,A., Mustonen,T., decreases angiogenesis and inhibits the growth of human cutaneous Pajusola,K., Breitman,M. and Alitalo,K. (1998) Cardiovascular squamous cell carcinomas. Am J. Pathol., 155, 441-452. failure in mouse embryos deficient in VEGF receptor-3. Science, Wetterwald,A., Hoffstetter,W., Cecchini,M.G., Lanske,B., Wagner,C., 282, 946-949. Fleisch,H. and Atkinson,M. (1996) Characterization and cloning of the Farr,A.G., Berry,M.L., Kim,A., Nelson,A.J., Welch,M.P. and Aruffo,A. Ell antigen, a marker expressed by rat osteoblasts and osteocytes. (1992) Characterization and cloning of a novel glycoprotein expressed Bone, 18, 125-132. by stromal cells in T-dependent areas of peripheral lymphoid tissues. Wigle,J.T. and Oliver,G. (1999) Proxl function is required for the J. Exp. Med., 176, 1477-1482. development of the murine lymphatic system. Cell, 98, 769-778. Fuchs,E. and Byme,C. (1994) The epidermis: rising to the surface. Curr. Wigle,J.T., Harvey,N., Detmar,M., Lagutina,I., Grosveld,G., Gunn,M.D., Opin. Genet. Dev., 4, 725-736. Jackson,D.G. and Oliver,G. (2002) An essential role for Proxl in the Gale,N. et al. (2002) Angiopoietin-2 is required for postnatal induction of the lymphatic endothelial cell phenotype. EMBO J., 21, angiogenesis and lymphatic patterning and only the latter role is 1505-1513. rescued by angiopoietin-1. Dev. Cell, 3, 411-423. Williams,M.C. (2003) Alveolar type I cells: molecular phenotype and Gandarillas,A., Scholl,F.G., Benito,N., Gamallo,C. and Quintanilla,M. development. Annu. Rev. Physiol., 65, 669--695. (1997) Induction of PA2.26, a cell-surface antigen expressed by active Williams,M.C., Cao,Y., Hinds,A., Rishi,A.K. and Wetterwald,A. (1996) fibroblasts, in mouse epidermal keratinocytes during carcinogenesis. Tl a protein is developmentally regulated and expressed by alveolar Mal. Carcinog., 20, 10-18. type I cells, choroid plexus and ciliary epithelia of adult rats. Am. J. Gonzalez,R.F. and Dobbs,L.G. (1998) Purification and analysis of Respir. Cell Mal. Biol., 14, 577-585. RTI40, a type I alveolar epithelial cell apical membrane protein. Yuan,L., Moyon,D., Pardanaud,L., Breant,C., Karkkainen,M.J., Biochim Biophys Acta, 1429, 208-216. Alitalo,K. and Eichmann,A. (2002) Abnormal lymphatic vessel Hirakawa,S., Hong,Y.K., Harvey,N., Schacht,V., Matsuda,K., development in neuropilin 2 mutant mice. Development, 129, 4797- Libermann,T. and Detmar,M. (2003) Transcriptional profiling of isolated human lymphatic versus blood vascular endothelial cells. Am Zimmer,G., Lottspeich,F., Maisner,A., Klenk,H.D. and Herrler,G. (1997) J. Pathol., 162, 575-586. Molecular characterization of gp40, a mucin-type glycoprotein from Hong,Y.K., Harvey,N., Noh,Y.H., Schacht,V., Hirakawa,S., Detmar,M. the apical plasma membrane of Madin-Darby canine kidney cells and Oliver,G. (2002) Proxl is a master control gene in the program (type I). Biochem. J., 326, 99-108. specifying lymphatic endothelial cell fate. Dev. Dyn., 225, 351-357. Zimmer,G., Oeffner,F., Von Messling,V., Tschernig,T., Groness,H.J., Kriehuber,E., Breiteneder,G.S., Groeger,M., Soleiman,A., Klenk,H.D. and Herrler,G. (1999) Cloning and characterization of Schoppmann,S.F., Stingl,G., Kerjaschki,D. and Maurer,D. (2001) gp36, a human mucin-type glycoprotein preferentially expressed in Isolation and characterization of dermal lymphatic and blood vascular endothelium. Biochem J., 341, 277-284. endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med., 194, 797-808. Received January 20, 2003; revised May 13, 2003; Maekinen,T. et al. (2001) Isolated lymphatic endothelial cells transduce accepted May 19, 2003 growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J., 20, 4762-4773.

Journal

The EMBO Journal – Springer Journals

Published: Jul 15, 2003

Keywords: angiogenesis; lymphangiogenesis; podoplanin; Prox1; T1α

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