White matter predicts functional connectivity in premanifest Huntington's disease.

OBJECTIVES: The distribution of pathology in neurodegenerative disease can be predicted by the organizational characteristics of white matter in healthy brains. However, we have very little evidence for the impact these pathological changes have on brain function. Understanding any such link between structure and function is critical for understanding how underlying brain pathology influences the progressive behavioral changes associated with neurodegeneration. Here, we demonstrate such a link between structure and function in individuals with premanifest Huntington's. METHODS: Using diffusion tractography and resting state functional magnetic resonance imaging to characterize white matter organization and functional connectivity, we investigate whether characteristic patterns of white matter organization in the healthy human brain shape the changes in functional coupling between brain regions in premanifest Huntington's disease. RESULTS: We find changes in functional connectivity in premanifest Huntington's disease that link directly to underlying patterns of white matter organization in healthy brains. Specifically, brain areas with strong structural connectivity show decreases in functional connectivity in premanifest Huntington's disease relative to controls, while regions with weak structural connectivity show increases in functional connectivity. Furthermore, we identify a pattern of dissociation in the strongest functional connections between anterior and posterior brain regions such that anterior functional connectivity increases in strength in premanifest Huntington's disease, while posterior functional connectivity decreases. INTERPRETATION: Our findings demonstrate that organizational principles of white matter underlie changes in functional connectivity in premanifest Huntington's disease. Furthermore, we demonstrate functional antero-posterior dissociation that is in keeping with the caudo-rostral gradient of striatal pathology in HD.

Development of lymphatic vessels: tumour lymphangiogenesis and lymphatic invasion.

In human solid cancer, the lymph node status is the most important prognostic indicator for the clinical outcome of patients. Follow-up data has shown that about 80% of metastasis follows an orderly pattern of progression via the lymphatic network while about 20% systemic metastasis occurs, bypassing the lymphatic system. Over the past few years, advances have been made in understanding the cellular and molecular aspects of physiological lymphangiogenesis and tumour-induced lymphangiogenesis, and the majority of studies point out to a positive correlation between tumour-induced lymphangiogenesis and lymphatic metastasis. However, the impact of intra- and peritumoural lymphatics on the tumour biology and the first steps of lymphatic metastasis, i.e. the invasion of tumour cells into the lymphatic vessels, are not well understood. We will give an outline of i. the physiological process of lymphangiogenesis, ii. tumour-induced lymphangiogenesis and lymphatic metastasis, iii. lymphatic invasion and the common pathways of tumour-lymphangiogenesis and lymphatic invasion. The growing interest in this topic has brought up a number of new molecular players in the field, which may provide the basis for a rational therapy against the process of lymphatic dissemination of tumour cells.

The lymphatic vascular system: secondary or primary?

It has generally been accepted that the blood vascular system is primary and the lymphatic vascular system secondary. Diseases of the blood vascular system are the leading cause for mortality and morbidity in developed nations. In contrast, lymphedema is seldom life-threatening and can generally be well-managed by combined physiotherapy. During ontogeny, the blood vessels and the heart develop much earlier than the lymphatic vessels. However, there is growing evidence that the first vascular system occurring during ontogeny and phylogeny has lymphatic functions. Defense mechanisms are crucial for all organisms irrespective of their size. Macrophages precede the emergence of erythrocytes during ontogeny, and their circulation in the hemolymphatic (more accurately, lymphohematic) system of insects, which do not possess erythrocytes, shows that the lymphatic function is primary whereas the nutritive function is secondary, needed only in larger organisms. In molluscs and arthropods, which have an open vascular system, hemocyanin has both oxygen transporting and defense functions. In vertebrates, the early blood vessels have structural characteristics of lymphatics and express the lymphendothelial receptor flt-4 (Vascular Endothelial Growth Factor Receptor-3). Later, flt-4 becomes restricted to the definitive lymphatics, which are either formed from the primary vessels or from mesodermal lymphangioblasts. The primary lymphatic function has become overruled by the nutritive function in blood vessels of larger animals. The circular movement of cells is driven by a blood heart, which, however, is not an unique organ. Lymph hearts are present in lower vertebrates, still develop transiently in birds, and are vestigial in the contractile lymphangion which "circulates" immune cells. We conclude that the definitive lymphatics are perhaps secondary in mammals, but the blood vascular system seems to develop on the basis of an ancestral lymphatic system with lymph hearts.

The homeobox transcription factor Prox1 is highly conserved in embryonic hepatoblasts and in adult and transformed hepatocytes, but is absent from bile duct epithelium.

Prox1 is a transcription factor with two highly conserved domains, a homeobox and a prospero domain. It has been shown that Prox1 knock-out mice die during early embryonic stages and display a rudimentary liver. We have studied the expression of Prox1 at RNA and protein levels in chick, rat, mouse and human liver and in transformed and non-transformed hepatic cell lines. Prox1 is expressed in early embryonic hepatoblasts and is still expressed in adult hepatocytes. Prox1 protein is located in the nuclei of hepatoblasts, which grow into the neighboring embryonic mesenchyme. The expression pattern in chick, mouse, rat and human embryos is highly conserved. Besides albumin and alpha-fetal protein, Prox1 belongs to the earliest markers of the developing liver. In adult liver, Prox1 is expressed in hepatocytes but is absent from bile duct epithelial and non-parenchymal cells (Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells and myofibroblasts). Isolated primary hepatocytes and hepatoma cell lines (HepG2, Hep3B) are Prox1 positive, whereas the immortalized murine liver cell-line MMH, which constitutively expresses the receptor c-met, is Prox1 negative. Transfection of MMH with Prox1 cDNA increases the expression level significantly as compared to control transfectants. In HepG2 and Hep3B, the Prox1 levels are even up to 100 times higher. Our studies show that Prox1 is a highly conserved transcription factor, expressed in hepatocytes from the earliest stages of development into adulthood and over-expressed in hepatoma cell lines. Its absence from bile duct epithelial cells suggests a function for the specification of hepatoblasts into hepatocytes. The genes controlled by Prox1 need to be studied in the future.

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.

Development of the avian lymphatic system.

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.

Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane.

It has generally been assumed that tumors do not induce lymphangiogenesis and only very recently animal models have been presented showing tumor-induced lymphangiogenesis. We have grown two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the chorioallantoic membrane (CAM) of chick and quail embryos. The suspended tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. When grown on the CAM of quail embryos intratumoral endothelial cells could be specifically stained with the QH1 antibody. In C6 gliomas the vascular pattern was more regular than in 10AS carcinomas. The vessels often grew radially into the glioma and many of them were invested by smooth muscle alpha-actin-positive periendothelial cells. Lymphatics, which were identified by vascular endothelial growth factor receptor-3 (VEGFR-3) in situ hybridization were absent from C6 gliomas, although a weak expression of the lymphangiogenic growth factor, VEGF-C, could be detected in the C6 cells by Northern blot analysis. In contrast, 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with BrdU-labeling rates of about 9% of lymphatic endothelial cells. Our studies demonstrate the heterogeneity of interactions of tumor cells with blood vessels and lymphatics and show that sufficient quantities and/or quality of lymphangiogenic growth factors are crucial for the induction of lymphatics in tumors.

Arterial identity of endothelial cells is controlled by local cues.

The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts.

The lymphatic endothelium has mostly been thought to be derived by sprouting from specialized veins. Recently it has been shown that mice deficient for the homeobox transcription factor Prox1 are practically devoid of lymphatics. We have studied the expression of Prox1 mRNA and protein in chick embryos and human fetuses. In the chick, Prox1 is expressed in specific compartments of all germ layers. In the ectoderm, it is found in the neural tube, trigeminal, spinal and sympathetic ganglia and the retina, and also in placodal structures such as the lens, olfactory, otic, facial, glossopharyngeal and vagal placodes, and the apical ectodermal ridge. In the endoderm, Prox1 is a marker of hepatocytes, bile duct and pancreatic epithelium. In the mesoderm, weak expression is observed in cardiomyocytes, and strong expression in lymphatic endothelium. Identical expression domains are found in 19-week-old human fetuses. In day 6.5 chick embryos, there are several sites of contact of lymphatics with the jugular vein, which has a mixed endothelium of Prox1-positive and -negative cells. The only non-lymphatic endothelial cells expressing Prox1 are found on the concave side of the cardiac valves. To further analyse development of lymphatics, we studied early chick embryos and observed scattered Prox1-positive cells in the dermatome, giving rise to Prox1-positive lymphatic networks during subsequent development. Furthermore, the anlagen of the posterior lymph sacs and the paired thoracic duct can already be observed in day-4 chick embryos. Our studies show that lymphatics develop much earlier than previously described, and they mostly do not seem to be derived by sprouting from veins. In contrast, lymphangioblasts are present in the deep and superficial compartments of the early mesoderm, independently giving rise to the deep and superficial lymphatics.

Induction of the blood-brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos.

Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood-brain barrier (BBB)-forming endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of chick embryos. We inoculated each 3-5 x 10(6) rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7 (but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin, studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy, vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated. Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential common functions of neurothelin in endothelial cells of the CNS and tumors are discussed.

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.

Active interaction of human A375 melanoma cells with the lymphatics in vivo.

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.