81

Lymphology 33 (2000) 81-94

Anatomisches Institut der Albert-Ludwigs-UniversWit, Freiburg (JW,MS,MP,BC), Freiburg, Germany, and Molecular and Cellular Biology Laboratory, University of Helsinki (KA), Helsinki, Finland

ABSTRACT . Our studies demonstrate the importance of lymphangioblasts and Embryonic development of lymphatics lymphangiogenic growth factors in embryonic () in recent years has rarely lymphangiogenesis. been studied experimentally. Using an avian model, we showed that both intra- and extra­ About 8-9 decades ago the development embryonic blood vessels of chick and quail of embryonic lymphatics was studied embryos are accompanied by lymphatics. The intensively. Since then our knowledge in this lymphatics of the chorioallantoic membrane field has not appreciably increased, and it is (CAM) are drained by lymphatic trunks of the still unknown whether lymphatics derive by umbilicus and are connected to the posterior sprouting from veins, de novo from lymph . Intra-embryonic lymphatics are lymphangioblasts, or by both mechanisms. drained via paired thoracic ducts into the Recent studies, however, have shown that the jugulo-subclavian junction. The lymphatic Vascular Endothelial Growth Factor-C endothelial cells are characterized by the (VEGF-C) is a highly specific lymphan­ expression of Vascular Endothelial Growth giogenic growth factor (1,2). This observation Factor Receptors (VEGFR) -2 and -3. raises the possibility of studying new Application of VEGF-C, the ligand of these questions and perspectives. two receptors, on the differentiated CAM, Compared to the studies on the induces proliferation of lymphatic endothelial development of blood vessels, studies on the cells and formation of huge lymphatic sinuses. development of lymphatics are rare. This These lymphatics derive from pre-existing dichotomy has led in the last decades to use lymphatic endothelial cells, whereas, in early of the term "" to define the embryos lymphangioblasts are present in the process of blood vessel development, although . This phenomenon can be the expression "hemangiogenesis" is probably demonstrated by interspecific grafting more appropriate. This terminology, however, experiments between chick and quail embryos. interferes with the expression "hemangioblast," Together with the early lymph sacs, the which is generally used to define a precursor lymphangioblasts form the embryonic cell that gives rise to both blood cells and blood vessel endothelium. Perhaps the term "hemendothelioblast" would be more * Awarded the Presidential Prize at the XVII ISL accurate. In this paper, we will use the term Congress in Chennai, India (September 1999) "angiogenesis" for the formation of blood

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 82 vessels, and "lymphangiogenesis" to include posterior lymph sacs, and the unpaired are the mechanisms involved in the development the cisterna chyli and the retroperitoneal of lymphatics (reviews see: 3-5). (mesenteric) lymph sac. In the human, the Like developing blood vessels, the first subclavian lymph sac is an extension of the anlagen of lymphatics are solely made up of jugular lymph sac (6,8). Except for the endothelial cells. Development of lymphatics cisterna chyli, the lymph sacs become trans­ has been studied by injection methods, serial formed primarily into lymph nodes. The sections (6-8), and in living animals (9,10). lymph sacs are derived by fusion of lymphatic Nonetheless, given the lack of specific capillaries filled with stagnant blood, markers, the origin of lymphatic endothelial suggesting an origin from veins (21,22). The cells remains unknown. Thus, are they blood is removed into the veins when the derived from lymphangioblasts of the early lymphatics begin to function (9). mesenchyme (11,12), or from veins by We studied the development of the sprouting (6,13), or by both mechanisms (14)? lymphatics in chick and quail embryos. Blood vascular endothelial cells clearly derive Descriptive studies were performed with by multiple mechanisms; from angioblasts histological and immunohistochemical and hemangioblasts, and also from growth of methods, and in situ hybridization with early embryonic vessels (review see: 15). If VEGFR-2 and -3 probes. Two kinds of the assumption holds true that lymphatic experiments were then carried out: 1. Wing endothelial cells are derived from veins, and buds of early chick embryos were grafted grow exclusively by sprouting, then the homotopically into quail embryos, to study principal difference between angiogenesis whether the limb bud mesoderm already and lymphangiogenesis would reside in the contained lymphangioblasts, or, whether in absence of a lymphangioblastic cell lineage. contrast, the wing lymphatics were exclu­ This concept appears to be supported by the sively derived from sprouts of the jugulo­ fact that lymphatics develop much later than axillary lymph sacs. 2. The growth factor blood vessels. In the human, lymph sacs have VEGF-C was applied on the differentiated been found in 6-to 7-week-old embryos of chorioallantoic membrane (CAM) of 13 10-14 mm total length (8). This timing is day-old chick embryos to study its lymphan­ about 3 to 4 weeks after the development of giogenic potency. Our experiments showed the first blood vessels. In the chick, the deep that VEGF-C was a highly specific lymphatic system is first detectable during lymphangiogenic growth factor for VEGFR- day 4-5 of incubation (16), whereas the first 2- and -R3-positive lymphatic endothelial blood vessels are already seen after 1 day of cells. Our grafting experiments demonstrated incubation (17). It has been assumed, that there were lymphangioblasts in the early however, that besides the venous origin of avian mesenchyme. Therefore, embryonic lymphatics there are endothelial lined clefts lymphatics did not grow exclusively by that become integrated into the growing sprouts of the early lymph sacs, but also by lymphatic system (12,18,19). recruitment of locallymphangioblasts. Despite the uncertainty about the exis­ tence of multiple lymphatic anlagen, it is MATERIAL AND METHODS generally accepted that much of the lympha­ tic system is derived from endothelial lined Embryos lymph sacs, which are located immediately adjacent to veins. Studies on mammals have Fertilized chick and quail eggs were shown that there are eight lymph sacs; three incubated in a humidified atmosphere at paired and two unpaired (6,20). The paired 37.8°C. On various incubation days, normal anlagen are the jugular, subclavian and and experimental embryos were fixed

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 83 overnight. They were dehydrated and hybridization sites were photographed onto embedded in paraffin wax for immunohisto­ Kodak Ectachrome and Agfa Ortho film. chemistry and in situ hybridization studies, The VEGFR-3 sense controls did not reveal or in Epon resin for semi- and ultra-thin a specific signal (26). sectioning. Immunohistochemistry Hybridization of paraffin sections Endothelial cells of quail embryos were Normal and experimental embryos were stained with the QHl antibody (17; Develop­ fixed overnight at 4°C in Serra's fixative (23). mental Studies Hybridoma Bank, Iowa City, The samples were dehydrated, embedded in IA). Staining was performed according to the paraffin wax and 8pm sections mounted onto indirect peroxidase method as described silanized slides. The sections were post-fixed previously (27). In order to stain all quail in 4% paraformaldehyde solution (PFA) and, cells, we used the anti-quail antibody QCPN in older specimens, treated with proteinase K (DSHB). The antibody was diluted 1:500. and refixed in 4% PFA. The quail VEGFR- The secondary antibody was peroxidase­ 3/Quek2 and VEGFR-2/Quekl mRNA conjugated goat-anti-mouse Ig (Sigma, riboprobes was a generous gift from Dr. Anne Deisenhofen, Germany), diluted 1:300. DAB Eichmann (Nogent-sur-Marne). Quek2 has was used as chromogen. For staining of the 70% identity with the humanflt4 (VEGFR-3) media of vessels, an anti-smooth muscle gene (24). The 1500 bp probe was cloned into a-actin (aSMA) antibody (Sigma) was used, pcDNAlAmp (Invitrogen, San Diego). diluted 1:5000. Secondary antibody and Linearization was performed with HindUI chromogen were the same as described above. and XhoI. The 3kb VEGFR-2/Quekl probe was linearized with HindUI and SphI. The VEGFR-3IQuek2 and QHl double staining sense and antisense riboprobes (24-26) were labeled as described before with digoxigenin In several specimens we combined RNA labeling kit as recommended VEGFR-3 in situ hybridization and QHl (Boehringer, Mannheim, Germany). A immunofluorescence. For this purpose, in situ hybridization mixture of 40% formamide, hybridization on paraffin sections was 25% 20X SSC, 1% Denhardt's solution, 1% performed as described above, with slight tRNA, 1% herring sperm DNA, 2% labeled modifications. The sections were not treated sense or antisense probe and 30% DEPC­ with proteinase K. Furthermore, the anti­ treated water was prepared. Sixty pI of digoxigeninlAP antibody and the QHl hybridization mixture was placed on each antibody were applied simultaneously; slide and the slides were incubated overnight diluted 1:4000 and 1:2000, respectively. at 65°C. After standard washing, the location Thereafter, the alkaline phosphatase reaction of the digoxigenin was detected using a was performed as usual. When the blue 1:4000 solution of an alkaline phosphatase­ reaction product in the lymphatics was visible conjugated anti-digoxigenin antibody in the sections, the alkaline phosphatase (Boehringer) in a blocking agent (1% bovine reaction was stopped with 1% EDTA in serum albumin in malate buffer) at 4°C alkaline phosphatase buffer. The slides were overnight. The antibody was detected using rinsed with A. dest. and PBS and the sections BCIP/NBT (Boehringer) in alkaline were blocked with 1% BSA, and again phosphatase buffer for 3-5 days, revealing a incubated with QHl antibody, 1:2000. After blue reaction product. The background was several washings with PBS, the secondary stained with nuclear fast red and the slides Cy3-conjugated goat-anti-mouse antibody mounted. Representative photographs of the (Dianova, Hamburg, Germany) was applied;

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 84 diluted 1:200. The slides were rinsed in PBS CAM-assay and mounted with Moviol (Hoechst, Frankfurt a. M., Germany). The sections Fertilized eggs of the White Leghorn were studied with Zeiss Axioskop using chick (Gallus gallus) were incubated at brightfield and fluorescence illumination. 37.8°C and 80% humidity. On day 4 of development, a window was made into the Grafting experiments egg shell. The embryos were checked for normal development and the eggs sealed with In order to study the origin of the cellotape. They were further incubated until lymphatics in the wing, the distal part of the day 13. Thermanox coversIips (Nunc, wing bud of 3 - 3.5 day-old quail embryos Naperville, IL) were cut into discs of about was replaced by a corresponding wing bud of 5 mm in diameter. Sterile and salt-free a HH stage 21-22 chick embryo (28), as growth factors (VEGF-A, VEGF-C) were described previously (29). The quail embryos dissolved in distilled water and about were reincubated until day 10. The chimeric 3.3 pg/5 pI were pipetted on the discs. After wings were fixed in Serra's solution, air-drying, the inverted discs were applied on embedded in paraffin and sectioned serially CAM. After three days, the specimens were at 8 pm. The sections were studied with the fixed in 3% glutaraldehyde and 2% QH1, QCPN and uSMA antibodies, with formaldehyde and rinsed in 0.12 M sodium VEGFR-3/Quek2 in situ hybridization, and cacodylate buffer. They were photographed with Quek2 and QH1 double staining. with a stereomicroscope (Wild M8) and embedded for semi- and ultrathin sectioning Ultrastructure according to standard procedures. Controls were performed with a great number of Specimens were fixed in 3% glutaralde­ different proteins. hyde and 2% formaldehyde in 0.12 M sodium cacodylate buffer, postfixed with 1% osmium Injection method solution, immersed with uranyl acetate and embedded in Epon resin (Serva, Heidelberg, The lymphatics of 10 to 16 day-old chick Germany). Semithin (0.75 pm) and ultrathin and quail embryos were perfused with (70 nm) sections were cut with an Ultracut S glutaraldehyde/formaldehyde fixative. Two (Leika, Bensheim, Germany). Semithin milliliters of Merkox-blue (Norwald, sections were stained with AzurBlNileblue. Hamburg, Germany) were mixed with 25 pI Ultrathin sections were studied with an EM of accelerator and injected into the lympha­ 10 (Zeiss, Stuttgart, Germany). tics of the CAM or the umbilicus using fine glass needles and a micromanipulator. Proliferation studies RESULTS Proliferation of cells was monitored with the BrdU/anti-BrdU method (30) after Lymphatics of chick embryos were application of VEGF-C on CAM of 13-day­ documented by injecting tracer (Merkox-blue) old chick embryos. One or two days later, the into the umbilical lymphatic trunks (2,32). embryos were incubated with 100 pI of a By this means, the deep and superficial parts 40mM BrdU solution (Sigma) for 45 min. The of the posterior lymph sacs of day 10 chick specimens were then fixed with ethanol embryos can be demonstrated. In day 16 containing 3% acetic acid. Paraffin sections chick and day 14 quail embryos, it is possible were stained as previously described (31). to demonstrate the lymphatics of the trunk. From the umbilical region the tracer flows

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Fig. 1. Intra-lymphatic injection of Merkox-blue. Left - Illustration of the lymphatic capillaries surrounding a vein of the CAM of a day 16 chick embryo. X20; Right - Illustration of the paired thoracic duct (D) and the lymphatic plexus surrounding the abdominal of a day 14 quail embryo. XI5.

into both directions towards the embryo and caval vein, an anastomosis between right and into the chorioallantoic membrane (CAM), left thoracic duct can be observed. The indicating that there are no or only a small arterial branches emerging from the number of valves within the lymphatics. In abdominal aorta are also accompanied by the CAM both the arterial and venous vessels lymphatics. Among these, the external iliac are accompanied by lymphatics. Arteries and artery supplies the upper thigh, whereas the arterioles are accompanied by a pair of ischial artery is the main vessel of the leg. lymphatics and a dense plexus of lymphatics The injection medium also fills the is located around the large veins (Fig. 1, left). lymphatics that accompany the branches of The intraembryonic, abdominal and thoracic the thoracic aorta, the celiac and the cranial lymphatics can be injected up to the point mesenteric artery. The cranial mesenteric where the paired thoracic duct reaches the artery supplies the small intestine and the paired cranial caval vein. Dense lymphatic . A very dense plexus of lymphatics plexuses can be observed around both arteries accompanies this artery and can be traced and veins. Numerous lymphatics surround into the yolk sac. The cranial mesenteric vein the abdominal aorta (Fig. 1, right) and its is located beside the artery and is also caudal extension, the median sacral artery. ensheathed by lymphatics. The caudal The paired thoracic duct is the continuation mesenteric vein which drains the cloacal of the lymphatics of the abdominal aorta region and the rectum is also surrounded by (Fig. 1, right). Before reaching the cranial numerous lymphatics. The lymph hearts,

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Fig. 2. In situ hybridization of a paraffin section of a day 10 quail embryo with a VEGFR-3 probe. Upper-The endothelium of the aorta (A) is negative, accompanying lymphatics (L) are positive. X30; Lower-The endothelium of both the aorta (A) and the lymphatics (L) is positive. X30. originally observed in the chick by Budge (33) embryos VEGFR-3 is a marker of the and Sala (34), are located in the dermis of the lymphatics of the body wall, the limbs and sacrococcygeal transition, and derive from the most internal organs. In the lung, where superficial part of the posterior lymph sacs. maturation takes place very late, there is The lymphatic and the blood vascular prolonged expression of VEGFR-3 in the endothelium of quail embryos can be stained blood vessels. The same expression pattern with the QH1 antibody (17). This antibody can be observed during maturation of the does not differentiate between the two types extraembryonic tissues, such as the CAM. In of endothelial cells, but it is specific for the contrast, VEGFR-2 is expressed in both the quail and does not stain any chick cells. endothelium of arteries and veins and of However, a distinction between blood vessels lymphatics of day 10 quail embryos (Fig. 2, and lymphatics can be made by in situ lower). Application of VEGF-A (which binds hybridization with VEGF receptor probes. In to the VEGF receptors -1 and -2) on the the differentiating tissues of avian embryos, CAM of day 13 chick embryos induces the VEGFR-3 is a highly specific marker of expression of VEGFR-2 but not R-3 in the lymphatic endothelial cells (Fig. 2, upper). In blood vascular capillaries. This growth factor early embryos, however, VEGFR-3 is induces angiogenesis but not expressed in both blood and lymph vessels, lymphangiogenesis in the CAM (data not but it is switched off in blood vessels during shown). The combination of the VEGF tissue maturation. Therefore, in day 10 quail receptors -2 and -3 is characteristic of the

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Fig. 3. Application of VEGF-C on the differentiated CAM of day 13 chick embryos. After 3 days the specimens were fixed and intra-lymphatic injection of Merkox-blue was performed. Upper - Note the circular lymphatic sinus (arrows) in the application area. X5; Lower - Higher magnification of the specimen in Fig. 5 showing the high density of lymphatics in the application area. X30.

lymphatic endothelial cells. The ligand of in the circular application area (Fig. 3). The these two receptors is VEGF-C. Application circular distribution of the growth factor is of VEGF-C on the CAM of day 13 chick due to the method of drying of the protein embryos induces lymphangiogenesis (Figs. solution on the carrier disc. The histological 3-5), and only a mild angiogenic side effect studies reveal the thin endothelial lining of can be observed. Injection of Merkox-blue the lymphatics induced by VEGF-C (Fig. 4). into the lymphatics shows that the newly Proliferation studies with the BrdUlanti­ developed lymphatic sinuses are found only BrdU method show that in the differentiated

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Fig. 4. Semi-thin section of the specimen shown in Fig. 3. Note the numerous lymphatic capillaries (L) which are filled with Merkox-blue. X130.

Fig. 5 . Proliferation studies with the BrdUlanti-BrdU method two days after application of VEGF-C on the CAM of day 13 chick embryos. Note the numerous BrdU-positive nuclei (arrows) of the lymphatic capillaries (L). X130.

CAM of control embryos, labeling of In early embryos, the first histologically lymphatics is an extremely rare event. detectable anlagen of the lymphatics are the However, after application of VEGF-C, a so-called lymph sacs (Fig. 6). In order to high labeling index of lymphatic endothelial determine the origin and growth of the cells is found (Fig. 5). In the differentiated lymphatics in early embryos, we replaced CAM, VEGF-C induces proliferation and distal wing buds of 3.5 day-old quail embryos growth of pre-existing lymphatics. by corresponding wing buds of chick

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Fig. 6. Staining of the endothelial cells of a day 6.5 quail embryo with the QHl antibody illustrating the aortic arch (A), the jugular vein (V) and the jugular lymph sac (L). X30.

embryos, as described recently (29). The zeugopodium) were mostly of chick origin, experiment was performed to determine but quail endothelial cells were regularly whether the lymphatics of the wing are integrated into the endothelial lining of exclusively derived by sprouts of the jugulo­ arteries, veins and microvessels. We also axillary lymph sacs, or whether lymphangio­ performed VEGFR-3/Quek2 and QHl double blasts are present in the early wing bud. staining of serial sections of the chimeric The embryos were reincubated until day 10. wings. With this method it was possible to In 25 out of 80 experiments, chimeric wings identify both the lymphatics by their with grossly normal morphology could be VEGFR-3 expression and, at the same time, harvested. Serial sections of these wings were the origin of the endothelial cells by the QHl further analyzed and showed that the signal. The results showed that the vascular pattern was generally normal in all lymphatics in the distal parts of the chimeric specimens. Arteries and veins could be wings were made up of both chick and quail identified by their localization and the endothelial cells (Fig. 7). Similar to the differences in the thickness of the media normal wings, the lymphatics accompanied (data not shown). The chimeric nature of the the main blood vascular routes such as the experimental wings was studied with the deep radial artery and the basilic vein. The QCPN antibody, which stains the nucleus of chimerism of the endothelium showed that quail cells. As could be expected, the nerves, the distal wing buds of day 3.5 chick embryos which are derived from the neural tube and already contained lymphangioblasts that were the neural crest, were of quail origin, whereas integrated into the growing lymphatics of the most connective tissue cells were of chick wing. origin (data not shown). The origin of the vascular endothelium was studied with the DISCUSSION QHl antibody. These studies showed that the vessels in the distal wing (autopodium and Congenital hypoplasia of the lymphatics

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Fig. 7. VEGFR-3 in situ hybridization (left) and QH1 immunofluorescence (right) double staining of a paraffin section of the forewing of a day 10 quail/chick chimera. Note that the endothelium of both the interosseous artery (arrowhead) and the lymphatics (arrows) are chimeric; quail endothelial cells being QH1-positive, chick endothelial cells QH1-negative. X150. and failure of lymphatics to regenerate result resonance imaging and lymphangioscinti­ in primary and secondary lymphedema graphy (44). However, most markers that (25,36). Abnormal proliferation of lymphatic have been used to stain blood vascular endothelial cells may be the key process in endothelial cells also stain lymphatic lymphangioma, lymphangiosarcoma and endothelium, and only quantitative but no Kaposi sarcoma (37). The fact that the consistent qualitative differences have been lymphatic system has gained less attention found (35). probably resides in the methodical problem of In recent years, some endothelial receptor detection of the lymphatics. In routine tyrosine kinases involved in growth and histology, lymphatics are difficult to recog­ differentiation of embryonic blood vessels and nize and to distinguish from blood vessels. lymphatics have been found, including the The absence of a basal lamina which can be vascular endothelial growth factor receptor stained in blood vessels with antibodies (VEGFR) family. The three VEGF receptors against type IV collagen and laminin has (fltl, KDR,f/t4 named VEGFR-1, -2 and -3, been used to distinguish between the two respectively) are almost exclusively expressed types of vessels (38). Antibodies with high in endothelial cells (25,26,45-47). The ligand, specificity for rat and mouse lymphatic VEGF-A, binds VEGFR-1 (fltl) and -2 endothelium have been produced (39-41). (KDR,f/kl, Quekl) with high affinity and Furthermore, 5' nucleotidase activity of induces angiogenesis (blood vessel develop­ lymphatic endothelium has been observed in ment) (for review: see 5). This interaction is histochemical studies (42). Analysis of accompanied by upregulation of VEGFR-2 in lymphatics is also possible by radiological blood vascular endothelial cells, whereas the lymphangiographical methods in vivo (37). lymphatics are not affected (48). The latter is Injection of fluorescent dyes has been used in an unexpected result because at least one of microlymphangiography (1,43), and the ligands, namely VEGFR-2, is expressed in lymphatics can also be studied by magnetic lymphatic endothelium. The expression of

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VEGFR-l has not as yet been studied in the mice (1). The mild angiogenic side effect may lymphatics, and no probe has yet been cloned be due to the relatively high amount of for the avian model. However, our own growth factor used in the CAM-assay. The unpublished data indicate that a homologous lymphatic endothelial cells of the CAM are gene is present in the chick. It can be thinner than those of blood vascular capil­ speculated that VEGF-A (and VEGF-C) laries, and they do not possess a continuous exert their effects by binding simultaneously basal lamina, which characterizes them as to two receptors. Thus, the combination of lymphatic capillaries. The lymphatics in the VEGFR-l and R-2 may be specific for the umbilical region of day 16 chick embryos are blood vascular endothelium, whereas the trunks that are invested by a thin layer of combination of VEGFR-2 and R-3 is smooth muscle _-actin-positive smooth characteristic of the lymphatic endothelium muscle cells (2). VEGF-C is a mitogen of the of differentiated tissues. It can also be lymphatic capillaries of the CAM as assumed that formation of receptor hetero­ demonstrated by the BrdU studies. It induces dimers is important for the transduction of hyperplasia of the preexisting lymphatics, the signal. and it may be conjectured that such an effect VEGF-C is a secreted, disulfide bonded may be useful in the treatment of patients homodimer which has been cloned from with lymphatic aplasia and hypoplasia. human prostatic carcinoma cells (49). It binds In differentiated tissues, lymphatics seem VEGFR-2 and -3 (jIt4, Quek2) with high to develop only from pre-existing ones. In affinity and induces lymphangiogenesis (5). contrast, a combination of different mecha­ The human VEGF-C gene comprises over 40 nisms is observed during the development of kb genomic DNA and consists of 7 exons. It embryonic lymphatics. In order to determine is located on chromosome 4q34 (50). VEGF-C the origin of the lymphatics in the wing, we is synthesized as a pre-pro-protein, and grafted distal wing buds (prospective auto­ becomes proteolytically processed both intra­ and zeugopodium) of 3.5 day-old chick and extra-cellularly (review see: 51). VEGF-C embryos homotopically into 3 - 3.5 day-old is highly expressed in the mesenchyme during quail embryos, making use of the quail/chick embryonic development (52), and is also marker system (55). The quail hosts were constitutively expressed in adult tissues. This reincubated until day 10, and normally expression suggests a role in both the developed chimeric wings were further development and the maintenance of the examined. In chick embryos, the jugular lymphatics (51). VEGFR-2 expression has lymph sacs develop during days 4-5 (16). been found in both blood vascular and Our grafting experiment was performed lymphatic endothelial cells during embryonic considerably before the development of the development of birds (26,48). Expression of jugulo-axillary lymph sacs, and we therefore VEGFR-3 has been observed in the endothe­ were able to exclude that the distal wing buds lium of blood vessels only during early of 3.5 day-old chick embryos already embryonic development. Expression then contained any derivatives of the lymph sacs. becomes restricted to lymphatic endothelial According to Sabin (6), all lymphatics of the cells in later stages of murine and avian wing developed from sprouts of the jugulo­ development (26,47). Immunohistochemical axillary lymph sacs. If this assumption was studies have confirmed that VEGFR-3 is a true, the distal chick wings in our experiment marker of lymphatics in differentiated tissues would have been invaded in a proximo-distal (53,54). Our studies show that VEGF-C is a direction by quail lymphatics. In order to highly specific lymphangiogenic growth identify the lymphatics in the chimeric wings, factor in the avian CAM. This effect has also we developed a double staining technique been observed in VEGF-C overexpressing consisting of VEGFR-3/Quek2 in situ

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 92 hybridization and QH1 immunofluorescence. MD, and the Department of Biological With this method we are able to stain Sciences, University of Iowa, Iowa City, lA, specifically the VEGFR-3-positive lympha­ under contract N01-HD-6-2915 from the tics, and to determine the origin of the NICHD. This study was supported by a grant lymphatic endothelium. In the distal parts of (Wi 1452/4-1) from the Deutsche the experimental wings, the lymphatics were Forschungsgemeinschaft. formed by both chick and quail endothelial cells; most of them of chick origin. These REFERENCES results were obtained for both the deep and the superficial lymphatics of the wing. This 1. Jeltsch, MM, A Kaipainen, V Joukov, et al: outcome tends to refute the pure venous Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276 (1997), 1423- origin theory of Sabin (6), as lymphatics of 1425. the wing were not exclusively derived by 2. Oh, SJ, MM Jeltsch, R Birkenhager, et al: sprouts of the jugulo-axillary lymph sac. In VEGF and VEGF-C: Specific induction of contrast, lymphatic anlagen were present in angiogenesis and lymphangiogenesis in the the distal wing buds of early avian embryos. differenciated avian chorioallantoic membrane. Dev. BioI. 188 (1997), 96-109. Together with the lymphatics derived by 3. Wilting, J, H Kurz, SJ Oh, et al: Angiogenesis sprouts of the early lymph sacs, the local and lymphangiogenesis: Analogous lymphangioblasts form the embryonic mechanisms and homologous growth factors. lymphatic system. We suggest that the In: Vascular Morphogenesis: In Vivo, In Vitro, complexity of mechanisms observed in In Mente. Little CD, V Mironov, EH Sage (Eds). Birkhauser, Basel, 1998,21-34. embryonic angiogenesis can also be found in 4. Wilting, J, H Neeff, B Christ: Embryonic embryonic lymphangiogenesis (for review lymphangiogenesis. Cell Tissue Res. 297 see: 3). The main difference seems to reside in (1999),1-11. the fact that lymphatics develop much later 5. Korpelainen, EI, K Alitalo: Signaling than blood vessels. Furthermore, lymphatic angiogenesis and lymphangiogenesis. Curro Opin. Cell BioI. 10 (1998),159-164. capillaries, which, in contrast to blood 6. Sabin, FR: The lymphatic system in human vascular capillaries, are not invested by a embryos, with a consideration of the system basal lamina and supporting cells such as as a whole. Am. J. Anat. 9 (1909), 43-91. pericytes, seem to depend on constitutive 7. Clark, EL: Injection and reconstruction of the jugular lymph "sac" in the chick. Anat. Rec. 6 expression of maintenance factors such as (1912a),261-264. VEGF-C. 8. Van der Putte, SCJ: The development of the lymphatic system in man. Adv. Anat. ACKNOWLEDGMENTS Embryoi. Cell BioI. 51 (1975), 1-60. 9. Clark, EL: General observations on early superficial lymphatics in living chick The authors are grateful to Dr. Anne embryos. Anat. Rec. 6 (1912b), 247-251. Eichmann for providing the VEGFR-2 and 10. Clark, EL, ER Clark: On the early R-3 probes. We thank Mrs. S. Antoni, Mr. G. contractions of the posterior lymph hearts in Frank, Mrs. L. Koschny, Mrs. U. Pein and chick embryos - their relation to the body Mrs. M. Schuttoff for their excellent technical movements. Anat. Rec. 8 (1914), 80-81. 11. Huntington, GS: The genetic interpretation of assistance, Mrs. Ch. Micucci for photographic the development of the mammalian lymphatic work, and Mrs. U. Uhl for typing of the system. Anat. Rec. 2 (1908), 19-46. manuscript. The QH1 and the QCPN 12. Kampmeier, OF: The value of the injection antibodies were obtained from the Develop­ method in the study of lymphatic mental Studies Hybridoma Bank, maintained development. Anat. Rec. 6 (1912), 223-233. 13. Ranvier, L: Developpement des vaisseaux by the Department of Pharmacology and lymphatiques. C R. Acad. Sci. 121 (1895), Molecular Sciences, Johns Hopkins 1105-1109. University School of Medicine, Baltimore, 14. Van der Jagt, ER: 1932. The origin and

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