LYMPHATIC RESEARCH AND BIOLOGYVolume 3, Number 3, 2005© Mary Ann Liebert, Inc.

Embryonic Lymphatic Development: Recent Advancesand Unanswered Questions

NATASHA L. HARVEY, Ph.D.

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INTRODUCTION

Despite the first description of the lym-phatic vasculature as “milky veins” cen-

turies ago,1 the field of lymphatic vascular de-velopment has remained vastly understudiedin comparison to the field of blood vascular de-velopment. This has been, in part, due to thelack of molecular tools available to distinguishand manipulate lymphatic vessels. The recentidentification of a select number of genes thatare important for lymphatic vascular develop-ment has greatly facilitated our ability to studythis important vascular system.2 The lymphaticvascular network is vital for the uptake and re-turn of fluid and proteins extruded from thebloodstream, for the absorption of lipids fromthe intestine, and as a conduit for trafficking ofcells of the immune system.

Dysfunctional lymphatic vessels are the hall-mark of a number of human diseases. These in-clude lymphatic vascular malformations, pri-mary congenital lymphedema syndromes, andsecondary lymphedema, arising in response tolymphatic vascular injury due to infection, ra-diation therapy, or tumor growth. The abroga-tion of lymphatic function due to any of theabove conditions can result in severe conse-quences, with painful and disfiguring swelling,fibrosis, and infection as common manifesta-tions of these diseases. Recent work has dem-onstrated that lymphatic vessels are exploitedas an important route of transport for somemetastatic tumor cells and that the production

of lymphangiogenic factors by tumor cells isable to promote tumor metastasis.3–5 Contri-butions to our knowledge of lymphatic vascu-lar development therefore have great potentialto aid our understanding of how lymphaticanomalies arise and to offer potential thera-peutic benefits for patients of lymphatic dis-eases and cancer. This review focuses on recentprogress in the field of embryonic lymphaticdevelopment and highlights some of the mostimminent questions remaining to be answered.

THE EMBRYONIC ORIGIN OF THELYMPHATIC VASCULATURE:

100 YEARS AGO

In the early 20th century, two theories of theorigin of lymphatic vessels were proposed; 1)that lymphatic vessels are derived from the em-bryonic venous system; and 2) that lymphaticvessels arise from the mesenchyme. Resultsfrom ink-injection studies led Florence Sabin6,7

to propose that the primitive lymph sacs, whichare first observed in close association withveins, are formed by endothelial cell buddingand sprouting from the veins. Sabin showedthat as development proceeds, a growing net-work of lymphatic vessels emerges from theprimary lymph sacs and eventually extendsthroughout the entire skin7 (Fig. 1). Isolatedlymphatic vessels were not observed using thismethod, suggesting that lymphangiogenesisproceeds progressively toward the periphery

Division of Haematology, Hanson Institute, Adelaide, South Australia.

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by sprouting from pre-existing lymphatic ves-sels. Sabin proposed that the jugular lymphsacs are derived from endothelial cells that budfrom the anterior cardinal veins and that the remaining lymph sacs originate from themesonephric vein and the veins in the dorso-medial edge of the Wolffian bodies.6,7 In simi-lar experiments, Heuer8 illustrated that theretroperitoneal lymph sac is the origin of thelymphatic vessels of the abdominal viscera.Studies using the dye-injection method to vi-sualize lymphatic vessels never detected lym-phatic vessels that were spatially isolated fromthe veins in, or from other lymphatic vessels,in this model. Until recently, these studies col-lectively provided the strongest evidence of avenous origin of the lymphatic vasculature.

In contrast, Huntington and McClure9 pro-posed that lymphatic vessels have a mesenchy-mal origin. Their analyses of serial sections fromdeveloping embryos led them to suggest thatlymphatic vessels form along the course of veinsbut are derived from the mesenchyme. Thisschool of thought proposed that lymphatic ves-sels secondarily establish connections to theveins and thus return lymph to the blood circu-lation (reviewed in Ref. 10). Although this viewof lymphatic development is less favored, evi-dence in support of a mesenchymal origin oflymphatic vessels has been obtained in birds.

Schneider and colleagues,11 proposed that lym-phatic vessels of the early wing buds of birdsare derived not only from sprouting of thelymph sacs, but also from angioblasts residentin the surrounding mesenchyme.

THE EMBRYONIC ORIGIN OF THELYMPHATIC VASCULATURE: TODAY

The strongest support for Florence Sabin’sproposal that lymphatic vessels arise from theveins during embryogenesis came from stud-ies of mice harboring a targeted inactivation ofthe homeobox gene Prox1.12 Prox1 was the firstgene identified to be essential for developmentof the lymphatic vasculature.12 Prox1-null em-bryos die at approximately E14.5 and displaystriking edema due to a complete absence oflymphatic vessels. Analysis of early stagemouse embyros illustrated that Prox1 is first ex-pressed in a polarized subpopulation of endo-thelial cells in the cardinal veins and that thesecells ultimately bud, proliferate, and migratefrom the veins to form the lymph sacs and sub-sequently, the lymphatic vascular network2,12

(Fig. 2). In Prox1-null embryos, the endothelialcells that initially bud from the cardinal veinsnever acquire a lymphatic phenotype; insteadthey maintain a default blood vascular pheno-type.13 This indicated that Prox1 is essential tospecify the fate of lymphatic endothelial cellsand that embryonic lymphatic progenitor cellsoriginate in the veins. Whether the embryonicveins are the sole source of lymphatic endo-thelial progenitor cells in mammals remains tobe established.

GROWTH FACTORS IMPORTANT FOREMBRYONIC LYMPHATIC

DEVELOPMENT

Vascular endothelial growth factor receptor-3(VEGFR-3/Flt4) was one of the first lymphaticendothelial cell markers characterized.14 Duringembryonic murine development, VEGFR-3 isfirst expressed in angioblasts of head mes-enchyme and in endothelial cells of the dorsalaorta, cardinal vein, and allantois.14,15 As devel-opment progresses, the expression of VEGFR-3

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FIG. 1. The growth of lymphatic vessels in pig embryosvisualized by the ink injection method of Sabin.7 (A) Em-bryo 3 cm in length. (B) Embryo 4.3 cm in length. (C) Em-bryo 5.5 cm in length. Note the appearance of a progres-sively sprouting lymphatic vascular network, initiallyfrom the jugular lymph sacs (A), and later from pre-ex-isting vessels (C). Reproduced from Ref.7 with permissionfrom John Wiley & Sons, Inc.

A B C

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becomes more restricted to the venous and ini-tial lymphatic endothelium; by adulthood, ex-pression of this marker is restricted largely tolymphatic endothelial cells.14–16

The precise role of VEGFR-3 in embryoniclymphatic development has not been assessed,because inactivation of the VEGFR-3 gene inmice results in early embryonic lethality.15

These embryos display cardiovascular failureand defects in vascular remodeling and matu-ration,15 and typically die before the lymphaticvasculature is established. The identification of

missense mutations in VEGFR-3 in patientswith hereditary lymphedema17,18 suggestedthat VEGFR-3 plays an important role in thedevelopment of the lymphatic vasculature.

Additional evidence demonstrating the im-portance of VEGFR-3 signaling in lymphaticdevelopment and function has been providedby results from studies in which vascular en-dothelial growth factor C (VEGF-C), a ligandof VEGFR-3, induced hyperplasia of the lym-phatic vessels in the skin of transgenic mice.19

Analogous studies using a mutant VEGF-C

EMBRYONIC LYMPHATIC DEVELOPMENT 159

FIG. 2. Embryonic development of the lymphatic vasculature in the mouse. (A) At E11.5, lymphatic endothelial pro-genitor cells, seen here by immunostaining with an antibody to Prox1 (green), are visible in a polarized population ofendothelial cells within, and budding from, the cardinal veins. An antibody to the pan-endothelial marker CD31 (red)was used to visualize all endothelial cells. CV, cardinal vein; DA, dorsal aorta; H, heart. (B) The population of cellsbudding from the veins is visualized here by X-gal staining of an E11.5 Prox1�/� embryo (arrow). (C) By E16.5, anearly complete network of lymphatic vessels is visible in the dermal layer of embryos. Note the similarity to Sabin’soriginal drawing (Fig. 1C). Lymphatic vessels were visualized by X-gal staining of an E16.5 Prox1�/� embryo. Allpanels N. Harvey and G. Oliver, unpublished results.

TABLE 1. SUMMARY OF GENES IMPORTANT FOR EMBRYONIC LYMPHATIC DEVELOPMENT IN MOUSE

Gene class Gene Function Reference

Transcription factors Prox1 LEC fate specification 12, 13FoxC2 Valve formation/inhibition of 34

pericyte recruitmentNet LV integrity 36

Growth factors/receptors VEGF-C LEC sprouting/migration 22Nrp2 Formation of superficial LVs 24Ang2 LV patterning and integrity 26

Adhesion/migration Podoplanin LEC migration/adhesion 38Integrin �9 LV integrity 40

Signaling mediators Syk Separation of lymphatic/ 42SLP-76 blood vascular networks 42

Although there is little doubt that many additional genes will be important for lymphatic vascular development,this table summarizes those that are currently known and for which models of gene inactivation exist.

A B C

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molecule that bound only to VEGFR-3, and notto VEGFR-2, showed that signaling via VEGFR-3 was sufficient to induce the lymphatic vesselhyperplasia observed in these mice.20 There-fore, the mechanism of lymphatic vesselgrowth appeared to be confined to signalingthrough VEGFR-3.

The long suspected importance of VEGF-Cfor lymphatic development21 was recently con-firmed by the targeted inactivation of Vegfc inmice.22 Vegfc�/� embryos died prior to birthand exhibited pronounced edema due to a lackof lymphatic vessels. While lymphatic endo-thelial cell fate was specified in these embryos(Prox1-positive endothelial cells are present inthe cardinal veins), cell sprouting from theveins was arrested, resulting in a failure to formlymphatic vessels.22 Both VEGF-C and itsclosely related family member, VEGF-D, wereable to rescue the sprouting and guided mi-gration of lymphatic endothelial cells from theveins. Interestingly, the recent generation ofmice with a targeted inactivation of Vegfddemonstrated that VEGF-D is not required forembryonic development of the lymphatic vas-culature.23 Whereas previous evidence demon-strated that VEGF-D was able to promote lym-phangiogenesis,4 the absence of lymphaticabnormalities in Vegfd�/� mice suggested thateither VEGF-D was not required for embryoniclymphatic vascular development, or that an-other factor such as VEGF-C was able to com-pensate for the absence of VEGF-D by signal-ing through their common tyrosine kinasereceptor VEGFR-3.

The semaphorin receptor, neuropilin 2, is an-other molecule that has recently been shown tobe important for embryonic lymphatic devel-opment,24 possibly by virtue of the fact that itis able to act as co-receptor for VEGF-C.25 In asimilar manner to VEGFR-3, neuropilin 2 is ex-pressed at low levels in embryonic venous en-dothelial cells and at higher levels in lymphaticendothelial cells.24 Neuropilin 2-null mice dis-play an absence, or severe reduction selectivelyin the number of small lymphatic vessels andlymphatic capillaries during embryonic devel-opment. Interestingly, functional surface lym-phatic vessels were shown to regenerate inNrp2�/� mice postnatally,24 suggesting thatthey overcome a dependence on Nrp2-medi-

ated signaling. The development of large, col-lecting lymphatic vessels was unaffected inNrp2-null mice, a finding that indicates differ-ential control of large and small-caliber lym-phatic network development.

Targeted inactivation of the angiopoietin-2gene,26 illustrated an unexpected requirementfor this growth factor in the patterning and de-velopment of the lymphatic vasculature. Themajority of Ang2�/� pups died within a fewweeks of birth and displayed chylous ascitesand subcutaneous edema,26 indicative of se-vere lymphatic dysfunction. Investigation ofthe lymphatic vasculature in Ang2�/� mice re-vealed that the large lymphatic vessels weremispatterned and leaky, and were not in closeassociation with supporting smooth musclecells. Smaller lymphatic vessels in the intestineand ear were also disorganized in their patternand structure. These results demonstrated thatangiopoietin-2 is required to establish the cor-rect patterning of both small and large lym-phatic vessels and is also required for thesmooth muscle investment of large lymphaticchannels, critical to their integrity. Interest-ingly, angiopoietin-1 was able to rescue thelymphatic defects in Ang2�/� mice, suggestingthat Ang2 normally acts as an agonist of theTie2 receptor during lymphatic development.26

Ang1 has also recently been shown to pro-mote lymphatic endothelial cell proliferation,sprouting, and lymphatic vessel hyperplasia inadult mice.27,28 Ang1 may therefore also be im-portant for embryonic development of the lym-phatic vasculature, its role at this stage possi-bly obscured by the fact that the targetedinactivation of Ang1 results in early embryoniclethality due to defects in vascular remodel-ing.29 Another growth factor recently shown topromote lymphangiogenesis in an adult settingwas platelet derived growth factor BB (PDGF-BB).5

TRANSCRIPTION FACTORSIMPORTANT FOR LYMPHATIC

DEVELOPMENT

Besides the master transcriptional regulatorof lymphatic development, Prox1, a number ofadditional transcription factors have been

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shown to be important for various aspects oflymphangiogenesis. Mutations in the forkheadtranscription factor FoxC2 have been describedin patients suffering from lymphedema-dis-tichiasis,30–32 suggesting that the transcrip-tional activity of this factor is likely to be im-portant for lymphatic vascular developmentand function. Indeed, the characterization ofFoxC2�/� mice revealed lymphatic abnormali-ties including aberrant lymphatic transport, in-creased numbers of lymph nodes, and the back-flow of lymph, together with distichiasis,indicating that these animals could be a suit-able mouse model of lymphedema-distichia-sis.33

The analysis of FoxC2�/� mice illustrated alikely mechanism for the lymphatic dysfunc-tion observed in lymphedema-distichiasis pa-tients. FoxC2�/� embryos displayed multiplelymphatic abnormalities; mispatterned lym-phatic vessels, abnormal pericyte investment oflymphatic vessels, and a lack of competentvalves in collecting lymphatic vessels, togetherresulting in generalized lymphatic dysfunc-tion.34 The abnormal recruitment of pericytesto lymphatic vessels normally devoid of thisstructural support was found to be mediatedby the increased production of platelet derivedgrowth factor b (Pdgfb) and endoglin,34 bothmolecules known to be associated with the re-cruitment of smooth muscle to blood vessels.Furthermore, an increased recruitment ofsmooth muscle to lymphatic capillaries wasfound in biopsy samples from patients suffer-ing from lymphedema-distichiasis.34 Two rolesfor FoxC2 in lymphatic vascular developmentcan therefore be concluded: 1) that FoxC2 is im-portant for the formation of valves in collect-ing lymphatic vessels; and 2) that FoxC2 is im-portant for the repression of molecules such asPdgfb and endoglin in lymphatic capillary en-dothelium, thereby preventing the aberrant re-cruitment of smooth muscle to these lymphaticvessels.

Two additional transcription factors that areimplicated in lymphatic development are thehomeobox factor SOX18, which is mutated inpatients suffering from hypotrichosis—lymph-edema—telangiectasia35 and the Ets-domaintranscription factor Net.36 Mice homozygousfor a hypomorphic mutation in Net die soon af-

ter birth due to respiratory failure, and displaychylothorax, the abnormal accumulation ofchyle in the thoracic cavity due to severe lym-phatic leakage from the thoracic vessels.36 Thegenes regulated by each of these transcriptionfactors that impact on lymphatic developmentremain to be characterized, although recentwork has revealed that one of the importanttarget genes regulated by Sox18 is the vascularcell adhesion molecule VCAM-1.37

MOLECULES IMPORTANT FORLYMPHATIC ENDOTHELIAL CELL

MIGRATION AND ADHESION

Very little is currently known regarding thecell surface molecules that regulate lymphaticendothelial cell adhesion and navigation. Thetransmembrane glycoprotein podoplanin maycontribute to both of these processes and to theformation of connecting lymphatics betweensuperficial and deep lymphatic plexi.38 Podo-planin is first expressed in lymphatic endothe-lial cells at approximately E11.0 as they com-mence budding and migration from thecardinal veins.38 Podoplanin-null mice displaydefects in lymphatic vessel pattern and func-tion and dilation of the cutaneous and submu-cosal intestinal lymphatic vasculature, culmi-nating in lymphedema.38 The mechanism ofpodoplanin mediation of lymphatic endothe-lial cell migration remains to be characterized.

Another candidate molecule possibly in-volved in lymphatic endothelial cell migrationis reelin, a protein known to be vital for the mi-gration and positioning of neurons in the CNS.Some patients suffering from an autosomal re-cessive form of lissencephaly due to mutationsin RELN also display congenital lymph-edema,39 indicating that reelin may have a rolein lymphatic vascular development in additionto that in brain development.

Integrins have been implicated in a diverserange of biological processes involving cell lo-comotion including embryonic development,wound healing, inflammation, and metastasis.Mice homozygous for a null mutation in inte-grin �9 die within 6–12 days after birth due torespiratory failure and display bilateral chy-lothorax.40 Interestingly, integrin �9 was ex-

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pressed transiently in the thoracic duct duringembryonic development,40 but continuously inthe adjacent aorta, suggesting that this mole-cule may be important both cell autonomouslyand nonautonomously for development of thethoracic duct. Two observations lend weight toa cell autonomous role of integrin �9 in lym-phatic endothelial cells: 1) regions of tissueedema and extravascular lymphocyte accumu-lation were not restricted to the thoracic duct,but were also present surrounding other lym-phatic vessels in the chest wall of �9�/� mice;40

and 2) the lymphangiogenic growth factorsVEGF-C and VEGF-D were shown to act as li-gands for integrin �9�141 and were able to pro-mote both the adhesion and migration of cellsexpressing integrin �9�1. The interruption ofthe VEGF—C/VEGF—D/VEGFR-3 signalingpathway may therefore be responsible for thelymphatic abnormalities observed in integrin�9 null mice. There seems little doubt that ad-ditional molecules important for the guidanceand navigation of lymphatic endothelial cellsduring lymphatic network development re-main to be identified.

HOW IS SEPARATION OF THELYMPHATIC AND BLOOD VASCULAR

NETWORKS MEDIATED?

Although derived from the embryonic veins,the lymphatic and blood networks remain sep-arated at all but one connection point: the junc-tion between the thoracic duct and the left sub-clavian vein. How is the separation of these twovascular networks effected? A signaling path-way that involves the tyrosine kinase Syk andthe adaptor signaling proteins SLP-76 andPLC�2 is required for the separation of the lym-phatic and blood circulatory systems.42 In micedeficient for Syk or SLP-76, abnormal connec-tions persisted between blood and lymphaticvessels, resulting in embryonic hemorrhagedue to the presence of blood within lymphaticvessels. These mice also displayed arteriove-nous shunting. The aberrant presence of bloodwithin the lymphatic vessels was conferred towild-type mice after the transplantation of Syk-or SLP-76–deficient bone marrow.42 Interest-

ingly, neither Syk nor SLP-76 appeared to beexpressed in the lymphatic endothelium, butcould be detected in a subpopulation of circu-lating hemopoietic progenitor cells.42 This ob-servation has led to two key hypotheses: 1) thata resident lymphatic progenitor cell exists inthe hemopoietic compartment; and 2) that thisprogenitor cell has defects in a signaling path-way required for the separation of lymphaticand blood vascular components. The precisesignaling pathways employed to mediate theseparation of embryonic connections betweenthe lymphatic and blood vascular networks re-main to be elucidated.

REMODELING OF THE LYMPHATICVASCULATURE POST EMBRYONIC

DEVELOPMENT

An unsuspected role for ephrinB2, known to be important for cardiovascular develop-ment,43,44 in lymphatic vascular development,and remodeling has recently been identified.45

Mice homozygous for a mutation targeted to thePDZ domain of ephrinB2 died within the firstfew weeks of birth displaying chylothorax.45 De-tailed characterization of the lymphatic vascula-ture in these mice revealed that the collectinglymphatic vessels were hyperplastic and devoidof valves, and that the remodeling of the dermallymphatic plexus was disrupted in these micesuch that the superficial lymphatic network waseither reduced, or absent.45 One of the next chap-ters in lymphangiogenesis research will no doubtbe to identify and characterize additional mole-cules that are important for lymphatic vascularremodeling in development and disease.

QUESTIONS OF THE MOMENT

The rapid and recent identification of newplayers in the field of embryonic lymphatic de-velopment has contributed greatly to our un-derstanding of lymphangiogenesis in healthand disease. In addition, the techniques and an-imal models recently generated will be integraltools employed to answer many of the ques-tions now posed to lymphatic biologists. Some

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of the most enticing questions remaining to beanswered include:

What is the identity of the signals requiredfor lymphatic endothelial cell fate specifica-tion?

What signals are important for the naviga-tion of embryonic lymphatic vessels?

Are lymphangioblasts present in mam-malian embryos?

What additional molecules are important forlymphatic and blood vasculature separation?Why do the visceral and superficial lymphaticnetworks differ in their response to lymphan-giogenic growth factors?

The answers to these questions, among oth-ers, will no doubt ensure that the field of em-bryonic lymphatic development will continueits exponential growth, and will ultimately re-sult in the development of therapeutics for pa-tients suffering from diseases of the lymphaticvasculature.

ACKNOWLEDGMENT

Many thanks to Dr. Guillermo Oliver for in-troducing me to the field of embryonic lym-phatic development and for hundreds of dis-cussions of all things lymphatic.

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Address reprint requests to:Natasha L. Harvey, Ph.D.

Florey Research FellowDivision of Haematology

Hanson InstituteFrome RoadP.O. Box 14Rundle Mall

Adelaide, South Australia, 5000

E-mail: natasha.harvey@imvs.sa.gov.au

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