Lymphatic endothelial differentiation: start out with Sox - carry on with Prox
© BioMed Central Ltd 2008
Published: 29 December 2008
The transcription factor Prox1 is the master regulator of lymphatic endothelial cell differentiation and its expression initiates the morphogenesis of the lymphatic vasculature in the early embryo. Two new studies now answer some fundamental questions concerning Prox1 biology.
The bodies of higher vertebrates contain two highly branched hierarchical networks of endothelial tubules. One comprises the blood vessels, which provide the conduits for the systemic circulation and transport cells, gases, nutrients and waste products to their appropriate targets. The second endothelial network, the lymphatic vasculature, carries the lymph - draining tissues of plasma, proteins, particles and cells that have actively or passively gained access to the extracellular space [1, 2]. Although blood vessels and lymphatic vessels are often found in close proximity, direct contact is avoided, thereby preventing illegitimate shortcuts between the two networks. Two defined connections do exist. These enable lymphatic vessels to return their cargo into the venous circulation after having delivered potential antigens to the adaptive immune system en route, as the lymph percolates through the lymph nodes .
Despite being first described in the 17th century by Aselli , the lymphatic system until very recently remained the Cinderella of the vascular family. However, the discovery of positively identifying marker proteins for lymphatic endothelial cells (LECs), such as the homeobox transcription factor Prox1, and the generation of targeted gene deletions causing lymphatic defects in the mouse have led to unprecedented progress in our understanding of the biology of lymph vessel formation.
Initiation and maintenance of lymphatic differentiation
Endothelial expression of Prox1 is first detected in mice at embryonic day (E)10.5 in a dorsal subset of endothelial cells of the cardinal veins. Prox1-positive cells adopt a lymphatic identity and under the influence of vascular endothelial growth factor-C (VEGF-C) bud from the veins, migrate away and reorganize themselves in the primary lymph sacs of the jugular and mesonephric region . Prox1-deficient embryos do not accomplish specification of the emerging lymphatic subpopulation and lack the subsequent upregulation of lymphatic markers. Endothelial cells bud from the cardinal veins but keep on expressing blood vascular markers and fail to organize into lymph sacs. The result is a complete arrest of lymphatic development [6, 7]. The knockout therefore indicates that Prox1 serves as a master gene for lymphatic identity, a notion further bolstered by reports demonstrating that forced expression of Prox1 in blood endothelial cells (BECs) led to the acquisition of many lymphatic markers [8, 9].
But despite the overwhelming evidence for the role of Prox1 as a lymphatic master regulator, it is still entirely unclear which molecular mechanism triggers the transcription of Prox1 during the differentiation of the first LECs and, equally important, how lymphatic expression of the transcription factor is maintained throughout life. With respect to the first part of this question at least, a study by Peter Koopman and co-workers (François et al. ) published recently in Nature adds an important piece to the puzzle, by elucidating the role of the transcription factor Sox18 in the regulation of Prox1.
Mutations in the gene for Sox18 are known to be responsible for the naturally occurring mouse mutants of the ragged allelic series . Ragged mutations affect the coat hair and also cause vascular malfunctions that result in chylous ascites and edema. In humans, dysfunction of Sox18 is likely to contribute to the development of the hypotrichosis-lymphedema-telangiectasia syndrome .
Sox18: just one day of fame?
The study by the Koopman lab raises the question of whether Sox18 is the ultimate lymphatic master switch. Clearly, Sox18 is part of an essential decision process upstream of Prox1. However, in contrast to Prox1, Sox18 is neither indispensable nor likely to act single-handedly during lymphatic differentiation, as is indicated by the normal lymphatic development in Sox18-knockout mice on an outbreed background. Here, the related transcription factors Sox7 or Sox17 might compensate for the loss of Sox18, and it could prove revealing to test the C57/Bl6 mouse for defects in one of these genes. Furthermore, François et al.  demonstrate abundant Sox18 expression in embryonic blood vessels and blood vessels of the newborn mesentery and skin. This pattern of expression indicates that Sox18 alone cannot be sufficient for the specification of lymphatic vessels and points to the existence of an indispensable, but as yet unidentified, ally of Sox18 during lymphatic specification.
Intriguingly, and in contrast to Prox1, persistent expression of Sox18 is not necessary for the maintenance of lymphatic identity. Obviously, both genes define two different classes of master switch during tissue specification. Sox18 appears to act as an inducer of the lymphatic program in the early embryo and apparently becomes dispensable thereafter. Prox1 rather exerts a sustaining function and its constant presence is necessary for the maintenance of the lymphatic program. The nature of the signals required for this later phase of Prox1 expression are unclear, but one possibility is that Prox1 might stimulate its own promoter either directly or via intermediate transcriptional targets.
Lymphatic endothelial cell plasticity
More recently, unexpected plasticity of lymphatic endothelial cells has been reported. Johnson et al.  used tamoxifen-inducible Cre-mediated, and therefore temporally controlled, inactivation of the Prox1 gene in mice to study the role of Prox1 in lymphatic vessels at various developmental stages. Loss of Prox1 from venous lymphatic precursors resulted in prominent edema and scattered blood-filled vessels at mid-gestation, reminiscent of constitutive Prox1-knockout mice  (Figure 1d). Similarly, targeting of the Prox1 gene during later steps of lymphatic development in the embryo led to the presence of blood in the superficial lymphatics of the developing skin and in the mesenteric lymphatics.
In keeping with the proposed function of Prox1 as a master regulator of lymphatic differentiation, loss of Prox1 expression was accompanied by the loss or downregulation of other lymphatic markers such as podoplanin, CCL21 (SLC) and Lyve1. Concomitantly, markers characteristic for the endothelium of blood vessels, such as endoglin or CD34, were upregulated, and perivascular cells positive for smooth muscle α-actin, a characteristic feature of blood vessels but not of lymphatic capillaries, covered the mutant lymphatic vasculature . Previous work has shown that the endothelial cells of lymphatic capillaries (also termed initial lymphatics) are connected by discontinuous, button-like junctions, which presumably facilitate the uptake of cargo from the extracellular space . Loss of Prox1 compromised the lymph-specific distribution of the junctional adhesion molecule VE-cadherin and consequently impaired the formation of button-like junctions. However, the continuous and zipper-like junctional pattern seen in the endothelium of blood vessels was not reacquired in Prox1 mutants, suggesting that LEC dedifferentiation in these mutants is incomplete or deregulated. Nevertheless, the sum of the findings argues for a change in vessel identity and the partial adoption of a blood-vessel-like phenotype by the dedifferentiated lymphatics.
Lymphatic endothelial cell plasticity in health and disease
The study by Johnson et al.  adds to the view that differentiated tissues may retain a surprising degree of plasticity. Continued expression of Prox1 is required for maintaining LEC differentiation even in the adult. Conversely, lost expression or dysfunction of Prox1 might be potentially relevant for certain human diseases such as hereditary lymphedema syndromes, in which malformed lymphatic vessels are seen [18–20]. Moreover, neoplastic endothelial cells in angiosarcoma and Karposi's sarcoma express both BEC and LEC markers and lack a clear identity [21–23]. Future work will have to address whether Prox1 plays any part in these or other disease conditions.
The physiological reasons for the remarkable plasticity of the lymphatic endothelium also remain unclear. Are there any circumstances during development, growth or tissue regeneration that might trigger the reprogramming of LECs and their incorporation into blood vessels? Equally enigmatic is the question of how the dedifferentiation of LECs leads to the presence of blood cells within the mutant lymphatic vessels. This defect is observed in a number of mouse mutations affecting lymphatic differentiation [20, 24–26], and indicates the presence of aberrant connections between blood vessels and lymphatic vessels. An important issue to be resolved is whether such connections are formed by cell-cell interactions between now identically specified Prox1-deficient endothelial cells at sites of closest proximity. Alternatively, dedifferentiated LECs might respond to the same tissue-derived guidance signals as BECs, so that sprouts and growing vessels will make contact.
Owing to the availability of targeted mouse mutations and increasingly refined genetic tools that allow the timed and tissue-directed precise deletion of genes, the regulation of blood vessel specification and differentiation is slowly unfolding. Interestingly, endothelial differentiation appears to entail an unexpected degree of reversibility, which may be encouraging news for future attempts towards therapeutic intervention and regeneration.
We thank the Max-Planck-Society and the University of Münster for support.
- Saharinen P, Tammela T, Karkkainen MJ, Alitalo K: Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol. 2004, 25: 387-395. 10.1016/j.it.2004.05.003.View Article
- Cueni LN, Detmar M: New insights into the molecular control of the lymphatic vascular system and its role in disease. J Invest Dermatol. 2006, 126: 2167-2177. 10.1038/sj.jid.5700464.View Article
- Adams RH, Alitalo K: Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol. 2007, 8: 464-478. 10.1038/nrm2183.View Article
- Oliver G, Detmar M: The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev. 2002, 16: 773-783. 10.1101/gad.975002.View Article
- Oliver G: Lymphatic vasculature development. Nat Rev Immunol. 2004, 4: 35-45. 10.1038/nri1258.View Article
- Wigle JT, Oliver G: Prox1 function is required for the development of the murine lymphatic system. Cell. 1999, 98: 769-778. 10.1016/S0092-8674(00)81511-1.View Article
- Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD, Jackson DG, Oliver G: An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 2002, 21: 1505-1513. 10.1093/emboj/21.7.1505.View Article
- Hong YK, Harvey N, Noh YH, Schacht V, Hirakawa S, Detmar M, Oliver G: Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn. 2002, 225: 351-357. 10.1002/dvdy.10163.View Article
- Petrova TV, Mäkinen T, Mäkelä TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, Ylä-Herttuala S, Alitalo K: Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002, 21: 4593-4599. 10.1093/emboj/cdf470.View Article
- François M, Caprini A, Hosking B, Orsenigo F, Wilhelm D, Browne C, Paavonen K, Karnezis T, Shayan R, Downes M, Davidson T, Tutt D, Cheah KS, Stacker SA, Muscat GE, Achen MG, Dejana E, Koopman P: Sox18 induces development of the lymphatic vasculature in mice. Nature. 2008, 456: 643-647. 10.1038/nature07391.View Article
- Pennisi D, Gardner J, Chambers D, Hosking B, Peters J, Muscat G, Abbott C, Koopman P: Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat Genet. 2000, 24: 434-437. 10.1038/74301.View Article
- Irrthum A, Devriendt K, Chitayat D, Matthijs G, Glade C, Steijlen PM, Fryns JP, Van Steensel MA, Vikkula M: Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia. Am J Hum Genet. 2003, 72: 1470-1478. 10.1086/375614.View Article
- Pennisi D, Bowles J, Nagy A, Muscat G, Koopman P: Mice null for sox18 are viable and display a mild coat defect. Mol Cell Biol. 2000, 20: 9331-9336. 10.1128/MCB.20.24.9331-9336.2000.View Article
- Cermenati S, Moleri S, Cimbro S, Corti P, Del Giacco L, Amodeo R, Dejana E, Koopman P, Cotelli F, Beltrame M: Sox18 and Sox7 play redundant roles in vascular development. Blood. 2008, 111: 2657-2666. 10.1182/blood-2007-07-100412.View Article
- Sakamoto Y, Hara K, Kanai-Azuma M, Matsui T, Miura Y, Tsunekawa N, Kurohmaru M, Saijoh Y, Koopman P, Kanai Y: Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos. Biochem Biophys Res Commun. 2007, 360: 539-544. 10.1016/j.bbrc.2007.06.093.View Article
- Johnson NC, Dillard ME, Baluk P, McDonald DM, Harvey NL, Frase SL, Oliver G: Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity. Genes Dev. 2008, 22: 3282-3291. 10.1101/gad.1727208.View Article
- Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S, Vestweber D, Corada M, Molendini C, Dejana E, McDonald DM: Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med. 2007, 204: 2349-2362. 10.1084/jem.20062596.View Article
- Fang J, Dagenais SL, Erickson RP, Arlt MF, Glynn MW, Gorski JL, Seaver LH, Glover TW: Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am J Hum Genet. 2000, 67: 1382-1388. 10.1086/316915.View Article
- Kriederman BM, Myloyde TL, Witte MH, Dagenais SL, Witte CL, Rennels M, Bernas MJ, Lynch MT, Erickson RP, Caulder MS, Miura N, Jackson D, Brooks BP, Glover TW: FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet. 2003, 12: 1179-1185. 10.1093/hmg/ddg123.View Article
- Petrova TV, Karpanen T, Norrmén C, Mellor R, Tamakoshi T, Finegold D, Ferrell R, Kerjaschki D, Mortimer P, Ylä-Herttuala S, Miura N, Alitalo K: Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med. 2004, 10: 974-981. 10.1038/nm1094.View Article
- Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, Diem K, Weninger W, Tschachler E, Alitalo K, Kerjaschki D: Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol. 1999, 154: 385-394.View Article
- Hong YK, Foreman K, Shin JW, Hirakawa S, Curry CL, Sage DR, Libermann T, Dezube BJ, Fingeroth JD, Detmar M: Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus. Nat Genet. 2004, 36: 683-685. 10.1038/ng1383.View Article
- Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Mäkinen T, Elliman S, Flanagan AM, Alitalo K, Boshoff C: Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet. 2004, 36: 687-693. 10.1038/ng1384.View Article
- Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R, Mocsai A, Myers EE, Huang B, Jackson DG, Ferrari VA, Tybulewicz V, Lowell CA, Lepore JJ, Koretzky GA, Kahn ML: Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk. Science. 2003, 299: 247-251. 10.1126/science.1079477.View Article
- Backhed F, Crawford PA, O'Donnell D, Gordon JI: Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor. Proc Natl Acad Sci USA. 2007, 104: 606-611. 10.1073/pnas.0605957104.View Article
- Mäkinen T, Adams RH, Bailey J, Lu Q, Ziemiecki A, Alitalo K, Klein R, Wilkinson GA: PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev. 2005, 19: 397-410. 10.1101/gad.330105.View Article