Human Molecular Genetics, 2003, Vol. 12, No. 10 1179-1185
DOI: 10.1093/hmg/ddg123
© 2003 Oxford University Press
FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome
1University of Arizona, Departments of Surgery, Pathology and Pediatrics, Tucson, Arizona, USA, 2University of Michigan, Departments of Human Genetics and Pediatrics, Ann Arbor, Michigan, USA, 3Department of Biochemistry, Hamamatsu University, Hamamatsu, Japan, 4Oxford University Department of Pathology, Oxford, UK and 5Medical Genetics Branch, NHGRI, Bethesda, Maryland, USA
Received January 23, 2003; Accepted March 11, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lymphedema-distichiasis (LD) (OMIM 153400) is a rare autosomal-dominant condition characterized by pubertal onset of lower limb lymphedema and an aberrant second row of eyelashes arising from the meibomian glands. In some patients cardiac, skeletal and other defects coexist. We previously identified inactivating, nonsense and frameshift mutations in the forkhead transcription factor FOXC2 in affected members of LD families. To further delineate the relationship of FOXC2 deficiency to the clinical (and lymphangiodysplastic) phenotype in this syndrome, we performed dynamic lymphatic imaging and immunohistochemical examination of lymphatic tissues in mice heterozygous (+/-) for a targeted disruption of Foxc2. Adult heterozygote mice characteristically exhibited a generalized lymphatic vessel and lymph node hyper plasia and rarely exhibited hindlimb swelling. Retrograde lymph flow through apparently incompetent interlymphangion valves into the mesenteric nodes, intestinal wall and liver was also observed. In addition, Foxc2 +/- mice uniformly displayed distichiasis. We conclude that Foxc2 haploinsufficient mice mimic closely the distinctive lymphatic and ocular phenotype of LD patients. Furthermore, the craniofacial, cardiovascular and skeletal abnormalities sometimes associated with LD have previously been shown to be fully penetrant in homozygous Foxc2 null mice. This Foxc2 mutant mouse thus provides an ideal model for exploring molecular mechanisms and physiologic events in mesenchymal differentiation associated with lymphatic growth and development and the clinical abnormalities seen in human LD syndrome.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lymphedema-distichiasis syndrome (LD) (OMIM 153400) is a highly penetrant autosomal-dominant disorder, which typically manifests as distichiasis (double row of eyelashes arising from the meibomian glands) at birth and bilateral lower limb lymphedema at puberty. Associated features may include cardiovascular anomalies (e.g. tetralogy of Fallot), cleft palate, ptosis, hydrops fetalis, cystic hygroma and bone defects (1,2). In contrast to the more common Milroy lymphedema syndrome, where peripheral lymphatic vessels are aplastic or hypoplastic, the lymphatic channels, as visualized on conventional oil contrast lymphography, have been described as hyperplastic, a pattern seen in less than 10% of all cases of primary lymphedema (3,4).
We previously identified inactivating mutations in the FOXC2 winged helix transcription factor gene, located at 16q24.3, in families with LD (5). Additional FOXC2 mutations have now been identified in more than 170 affected individuals in 45 families (2,5–9). All but two mutations are truncating type mutations in one allele; the other two are missense in the conserved forkhead DNA-binding domain, strongly suggesting that haploinsufficiency of FOXC2 leads to the abnormal phenotype (2,5–9).
Mice with targeted inactivation of the Foxc2 gene have been previously reported by two laboratories (10,11). Homozygous null animals die embryologically after day E13.5 up to shortly after birth. The phenotype includes cardiac and skeletal abnormalities, such as cleft palate. Heterozygotes have been reported to be overtly normal (10–12) or to display variable anterior segment ocular abnormalities when the mutation is on a 129BS background (13). Although abnormalities of the lymphatic system have not previously been observed, they would likely not have been evident without specialized imaging techniques. We therefore examined the phenotypic effects of Foxc2 haploinsufficiency on the lymphatic system by directly and functionally visualizing the peripheral and central lymphatic vessels and regional lymph nodes through vital dye lymphangiography. Sections of lymphatic vessels, lymph nodes and initial lymphatics in skin, small intestine and liver were histologically studied. In addition, ocular examinations were performed, including microscopic study of the eyelids for distichiasis. We observed both highly abnormal lymphatics, displaying a generalized hyperplasia, with occasional hindlimb lymphedema, as well as bilateral distichiasis in Foxc2+/- mice. The dual phenotypic features of a hyperplastic lymphatic system and distichiasis coincide with those defining the human LD syndrome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Foxc2 heterozygous mice appeared grossly normal except for ocular abnormalities and an increased amount of brown fat deposits. Specifically, they displayed no effusions (chylous or non-chylous) and, except for 2/25 mice with hindlimb swelling and 2/25 different mice with periorbital swelling, no peripheral edema. Phenotypic features are summarized in Table 1. Distichiasis was uniformly noted under a dissecting microscope (Fig. 1A and B) and confirmed in histological sections of the eyelids, which showed aberrant growth of lashes arising from the sebaceous meibomian glands (Fig. 1C and D). In five of 36 heterozygotes examined, the eyes appeared cloudy. Of these, two mice had focal corneal abrasions, and three had unilateral cataracts, which were confirmed by histologic examination (not shown). In three different heterozygotes, only a vestigial dysplastic ocular remnant was identified unilaterally, associated with a deformity of the orbit (not shown).
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In 22 of 25 heterozygotes examined, EBD (Evans blue vital dye) lymphography displayed, to a varying but usually striking degree, an increase in the number and caliber of the peripheral and central deep lymphatic collectors and trunks and in the superficial dermal lymphatics, which were arranged in a plexiform network (Fig. 2A–H). The number and size of lymph nodes were also generally increased throughout the body in 23/25 heterozygotes examined (Fig. 2C–H). Occasionally, a light blue halo surrounded isolated segments of the lymphatic trunks, suggesting localized areas of heightened permeability to the protein-bound dye not seen in wild-type littermates (data not shown). A distinctly abnormal finding was observed in 20/25 heterozygous mice, where EBD-stained lymph refluxed retrograde from the cisterna chyli into dilated lymphatic channels within the hepatic hilum (Fig. 3), mesentery (intrinsic lymphatic contractions intact), mesenteric lymph nodes and the intestinal wall through visible, yet apparently incompetent, interlymphangion valves. The thoracic duct, the principal cardinal lymphatic collector, was generally (23/25 mice) normal in location and caliber as well as chyle-containing and EBD-stained. Although somewhat friable on dissection, the duct was patent throughout its length without obstruction, including proximal to its entry into the left subclavian vein. In two of 25 mice, the thoracic duct was hyperplastic with multiple tortuous bifurcations.
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Histology of regional lymph nodes revealed an increased number of massively dilated lymphatic vessels in the capsule (Fig. 4A and B). With immunohistochemical analysis, the lymphatic specific hyaluronan receptor LYVE-1 antibody highlighted prominent lymphatic sinusoidal dilation (lymphangiectasia), often occupying up to
50% of the nodal volume (Fig. 4C and D). Smaller caliber lymphatic channels in the hilum were also strongly LYVE-1 positive, but large caliber lymphatic trunks were negative, a lymphatic staining pattern also seen in the comparatively much smaller lymphatics of wild-type controls.
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310 µm). (See text for further explanation.)No ocular (0/12 mice) or lymphatic abnormalities (0/30 mice) were found in any of the wildtype +/+ littermate or non-littermate controls examined by the same methods from the same breeding stock.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Despite an explosion of knowledge during the past decade regarding the genes, proteins, and events involved in vasculogenesis and angiogenesis, all but a few of these studies have been directed at the growth and development of the blood vasculature. Much less attention has been paid to the lymphatic vasculature. As a consequence, relatively little is known about the molecular events involved in normal development of the lymphatic system or abnormal (lymphangiodysplastic) growth that underlies the overt manifestation of lymphatic circulatory failure, namely, lymphedema (14). Hence, the genes responsible for hereditary lymphedema and corresponding animal models have been of great interest to the field due to their potential for providing insights into the molecular mechanisms of lymphatic development and associated abnormalities.
The purpose of this study was to characterize Foxc2 +/- mice for lymphatic and eye abnormalities as a possible model for lymphedema-distichiasis syndrome in humans and for abnormalities of lymphatic development. We have found that Foxc2 mutant mice closely mimic the unusual and distinctive hyperplastic lymphangiodysplastic phenotype accompanied by distichiasis seen in humans with lymphedema-distichiasis syndrome. In humans with LD, with inactivating mutations in the FOXC2 gene, both lymphedema and distichiasis are highly penetrant, occurring in 77 and 95% of heterozygous individuals, respectively (8). Lymphedema, with variable expression, usually appears around puberty, although fetuses with non-immune hydrops fetalis have been reported, as well as adults with onset after adolescence (2,8). Associated cardiac and skeletal abnormalities, particularly cleft palate, are occasionally seen, but are much less penetrant (2,8). There are few reported observations of the underlying lymphatic system abnormalities in LD-affected individuals and none in those individuals not exhibiting overt lymphedema. On direct invasive conventional lipid contrast lymphography, a generalized increase in number and size of peripheral and more central lymphatic collecting trunks and regional lymph nodes (termed ‘bilateral lymphatic hyperplasia’) has been reported in eight LD patients with lymphedema (3,4). Kinmonth also noted retrograde truncal lymph reflux as a radiographic feature in patients with the relatively uncommon lymphatic hyperplastic form of hereditary lymphedema (3).
Our study documents that lymphatic abnormalities, specifically hyperplasia, and distichiasis are both highly penetrant in the Foxc2 haploinsufficient mouse, seen in 92 and 100% of heterozygotes, respectively, thus representing one of the instances where a genetically engineered heterozygous mouse knockout closely mimics the phenotype of a dominant human disease. Whereas we did not observe any gross cardiac or skeletal defects in any of the 25 Foxc2 +/- mice examined, mice homozygous for Foxc2 deficiency die between E13.5 and shortly after birth and do show abnormalities of the heart, aorta, palate and vertebrae (10–12), features strikingly similar to those that are occasionally seen in human LD. The most commonly observed cardiac abnormality is interruption of the aortic arch and ventricular septal defect (10,12). Because our study was limited to 25 Foxc2 +/-mice, it is possible that similar cardiac, skeletal or other abnormalities associated with LD syndrome in humans may also manifest in heterozygous mice at a low frequency and penetrance. More detailed imaging techniques would be required to rule out subtle defects.
Although it has been known since the early 1900s that the lymphatic system develops from the embryonic anterior and posterior lymph sacs, controversy persists regarding whether these lymphatic sacs and their peripheral and central connections arise from centrifugal sprouting of central veins or centripetally from tissue mesenchymal lymphangioblasts or, more likely, a combination of both processes as recent molecular evidence now suggests (15–17). Lymphatic growth is under the control of a poorly understood sequence of events and interactions involving transcription factors, including FOXC2 and PROX-1 and growth factor ligand–receptor pairs of the vascular endothelial growth factor (VEGF) and angiopoietin families (16). VEGF-C and VEGF-D are lymphatic growth factors, and VEGFR-3 is their high affinity receptor on lymphatic endothelial cells. The expression of Vegfr-3 is seen beginning at day E8.5 in the developing mouse blood vessels but becomes largely restricted to lymphatics after days E10–16 (18). Mice homozygous for inactivating mutations in Vegfr-3 die at day E9.5 with severely defective blood vessel development leading to fluid accumulation in the pericardial cavity and cardiovascular failure (19). Recently, the Chy mouse, heterozygous for an inactivating missense mutation in the Vegfr-3 kinase domain, has been suggested as a model for human Milroy disease, where a subpopulation of affected individuals shows mutations on VEGFR-3 (20–22). These mice show hypoplasia of the cutaneous lymphatic vessels, lymphedema of the limbs, and chylous ascites after birth, the former but not the latter feature characteristic of human Milroy syndrome.
The Tie receptors and their ligands, Ang1 and Ang2, (angiopoietin-1 and -2) function in an agonistic fashion in remodeling the preformed blood vasculature. They are also implicated in lymphatic development as reflected in the Ang2 knockout mouse (23). Mice homozygous for Ang2 inactivating mutations show a lymphatic phenotype (resembling several hereditary human syndromes) of chylous effusions, peripheral lymphedema, intestinal lymphangiectasia and marked lymphatic vessel and lymph node aplasia or hypoplasia; the primitive hyaloid vasculature of the eye also fails to regress (23). Genetic rescue with Ang1 corrects the lymphatic, but not retinal, defect, suggesting that Ang1 and Ang2 have distinct functions in the blood vascular system but redundant roles in lymphatic development.
The Prox1 homeobox transcription factor is essential for early stages of lymphangiogenesis. Prox1 null mice die at day E14.5–15.0 from severe underdevelopment of the lymphatic, but not the blood vascular, system (17). Prox1 heterozygotes appear normal, but most die within 2–3 days of birth with edema and intestines filled with chyle (17). Early Prox1 expression is restricted to a subpopulation of embryonic venous endothelial cells required for lymphangiogenesis, suggesting that Prox1 is necessary for early sprouting of lymphatic vessels from veins and also for differentiation of blood vessel endothelium into the lymphatic endothelial cell phenotype (15,24). The transcription factor Net and neuropilin receptor NRP2 have also both been recently implicated in lymphatic development (25,26). Net nullizygous mutant mice die shortly after birth from massive chylothorax associated with pulmonary lymphangiectasia (25). Mice nullizygous for Nrp2 also die shortly after birth and show absence or marked reduction in small caliber lymphatic vessels including capillaries (26).
It is important to note that no other reported deficiency in developmental/growth and transcription factors produces a lymphatic hyperplastic phenotype similar to the Foxc2 deficiency syndrome in man or mouse. How Foxc2 functions in developmental pathways of the lymphatic system is not understood. Foxc2 is expressed in the developing mesodermal mesenchyme of the head, including the ocular drainage system, kidney and bones and appears to play a role in somite formation (27). By 8.5 d.p.c., it is also expressed in the developing heart, blood vessels, kidneys and limbs and is essential for normal development of the aortic arch and axial skeleton (10,11,28). It is highly expressed in adipose tissue postnatally where its expression is thought to be regulated by insulin and TNF
via a PI3K and EFK1/2 dependent pathway in adipocytes (29,30). The pattern of embryonic expression correlates with tissues affected in Foxc2 null mice and with the cardiac, skeletal and renal abnormalities associated with LD in humans. It is not currently known if Foxc2 is expressed in developing lymphatic endothelial cells. The downstream targets of Foxc2 are also unknown. Forkhead family transcription factors have been implicated in both transactivation and repression of target genes. Given that we have found a generalized hyperplasia of the lymphatic system in Foxc2 +/- mice, it is likely that abnormal Foxc2 expression functions to disturb the normal equilibrium between lymph vascular growth promoting and inhibiting genes thereby producing dysfunctional lymphatic growth during development. This haploinsufficient Foxc2 mouse model in conjunction with its homozygous null counterpart provide a rare opportunity to dissect the role of the Foxc2 transcription factor in multiorgan differentiation relative to lymphatic, ocular and cardiovascular development as well as to examine the effect of modifier genes on the resultant phenotype. Furthermore, these findings should have important implications for detection, evaluation and treatment of LD and related lymphedema-angiodysplasia syndromes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Breeding and genotyping
Haploinsufficient Foxc2tm1Miu mice (10) were bred on the C57BL6/J background at the University of Michigan and the University of Arizona under approved animal protocols. Genotyping was performed on tail-tip DNA using primers for the normal gene and the knockout insert of the phosphoglycerate kinase (PGK) promoter simultaneously, as described (10). The Foxc2 forward primer was 5'-CCAGTTGGTAACCTGGACTG-3', the Foxc2 reverse primer was 5'-CCAGTTCTTAGTCCCCCCAC-3', and the PGK antisense primer was 5'-GGATGTGGAATGTGTGCGAG-3'. These were used in a reaction with 50 mM KCl, 10 mM Tris–HCl, pH 9.0, 1.5 mM MgCl2, 200 µM of each dNTP, 10 pmol each primer, 1.25 unit Taq polymerase and 4% DMSO. Thermocycling at 95°C for 30 min, 59°C for 60 min, and 72°C for 60 min was repeated for 33 cycles. The PCR products were separated on 3% agarose gels with a 310 bp fragment from the intact Foxc2 gene and 200 bp fragment from the knockout allele.
Heterozygous (+/-) mice (age range 5.4–52.4 weeks) including both males and females were studied and the findings compared with wildtype (+/+) littermates and non-littermate controls acquired from the same breeding stock. All studies were carried out according to University Animal Care regulations. Anesthesia consisted of an IM injection of a 20:1:79 ketamine:xylazine:sterile saline mixture at 0.1 ml/10 g body weight.
Lymphatic system evaluation
Twenty-five heterozygous mice were weighed and examined for evidence of edema or serous effusions as well as other gross phenotypic abnormalities. Because non-invasive indirect lymphatic imaging is inadequate for delineating structures the size of murine lymphatic collectors, and tissue sections provide only static not functional information, direct dynamic lymphatic visualization was used as the procedure of choice. Evans blue dye (EBD) (50 µl of 0.5 g/dl), which is a vital dye that binds to tissue proteins and is selectively and exclusively absorbed from the interstitium by the initial lymphatic vessels, was injected intradermally into all four paws, tail tip, ears and snout serially to sequentially highlight the regional lymphatic systems and the central collection system. Under a dissecting microscope (Weck, Evergreen, CO, USA), the peripheral lymphatic vasculature of the mice was examined by sequential dissection of the popliteal, sacral, axillary and jugular areas. The EBD-stained central lymphatics were followed from the lumbar region proximally along the bifurcation of the aorta and inferior vena cava to the cisterna chyli. The mesentery was reflected and the usually milky, chyle-containing lymphatic trunks traced through the diaphragm into the thoracic cavity, where they formed the thoracic duct. The duct was visualized traveling alongside the azygous vein until its final entry into the left subclavian vein.
Following EBD studies, mice were sacrificed and tissues harvested for lymphatic examination. For histologic studies, samples were fixed in 4% paraformaldehyde and paraffin embedded. The tissues were stained with hematoxylin and eosin by standard protocols or boiled for 7 min in 0.1 M Tris–HCl, pH 9.0, 2 mM EDTA buffer for antigen retrieval and then incubated with antibody to LYVE-1, the lymphatic-specific receptor for glycosaminoglycan hyaluronan (31) (D. Jackson) for 30 min in a humid environment followed by biotinylated, secondary anti-rabbit antibodies (1:50; Vector Laboratories, Burlingane, CA). Streptavidin horseradish peroxidase (1:50; Vector Laboratories) and NovaRED (Vector Laboratories) color kit were used in combination with a methyl green (Vector Laboratories) counterstain for final visualization. Slides were viewed and photographs taken through a light microscope (American Optics).
Ocular evaluation
Twenty-four of the 25 +/- mice subjected to lymphatic study (and 14 +/+ littermate controls) were examined beforehand under the dissecting microscope for distichiasis and other ocular abnormalities. Selected eyes were subsequently processed as above for histological examination. An additional group of 12 heterozygous mice was examined solely for distichiasis and other ocular abnormalities.
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ACKNOWLEDGEMENTS |
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We thank Mitch Gillett, Department of Ophthalmology, University of Michigan, for assistance. This work was supported by NIH grants HL71206, R25RR15670 and Arizona Disease Control Research Commission Contract no. I-103.
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FOOTNOTES |
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* To whom correspondence should be addressed at: The University of Arizona College of Medicine, Department of Surgery, Lymphology Laboratories, 1501 N. Campbell Avenue, Room 4406, PO Box 245063, Tucson, AZ 85724-5063, USA. Tel: +1 5206266118; Fax: +1 5206260822; Email: lymph{at}u.arizona.edu
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 G. Belcaro, B. M. Errichi, M. R. Cesarone, E. Ippolito, M. Dugall, A. Ledda, and A. Ricci Lymphatic Tissue Transplant in Lymphedema--A Minimally Invasive, Outpatient, Surgical Method: A 10-Year Follow-up Pilot Study Angiology, March 1, 2008; 59(1): 77 - 83. [Abstract] [PDF]
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 K. N. Papanicolaou, Y. Izumiya, and K. Walsh Forkhead Transcription Factors and Cardiovascular Biology Circ. Res., January 4, 2008; 102(1): 16 - 31. [Abstract] [Full Text] [PDF]
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 N. W. Gale, R. Prevo, J. Espinosa, D. J. Ferguson, M. G. Dominguez, G. D. Yancopoulos, G. Thurston, and D. G. Jackson Normal Lymphatic Development and Function in Mice Deficient for the Lymphatic Hyaluronan Receptor LYVE-1 Mol. Cell. Biol., January 15, 2007; 27(2): 595 - 604. [Abstract] [Full Text] [PDF]
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 T. V. Karlsen, M. J. Karkkainen, K. Alitalo, and H. Wiig Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema J. Physiol., July 15, 2006; 574(2): 583 - 596. [Abstract] [Full Text] [PDF]
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 M Y M Ng, T Andrew, T D Spector, S Jeffery, and (representing the Lymphoedema Consortium) Linkage to the FOXC2 region of chromosome 16 for varicose veins in otherwise healthy, unselected sibling pairs J. Med. Genet., March 1, 2005; 42(3): 235 - 239. [Abstract] [Full Text] [PDF]
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 J.-C. Tille and M.S. Pepper Hereditary Vascular Anomalies: New Insights Into Their Pathogenesis Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1578 - 1590. [Abstract] [Full Text] [PDF]
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