Plexin-B1 plays a redundant role during mouse development and in tumour angiogenesis
© Fazzari et al; licensee BioMed Central Ltd. 2007
Received: 09 February 2007
Accepted: 22 May 2007
Published: 22 May 2007
Plexins are a large family of transmembrane receptors for the Semaphorins, known for their role in the assembly of neural circuitry. More recently, Plexins have been implicated in diverse biological functions, including vascular growth, epithelial tissue morphogenesis and tumour development. In particular, PlexinB1, the receptor for Sema4D, has been suggested to play a role in neural development and in tumour angiogenesis, based on in vitro studies. However, the tissue distribution of PlexinB1 has not been extensively studied and the functional relevance of this receptor in vivo still awaits experimental testing. In order to shed light on PlexinB1 function in vivo, we therefore undertook the genomic targeting of the mouse gene to obtain loss of function mutants.
This study shows that PlexinB1 receptor and its putative ligand, Sema4D, have a selective distribution in nervous and epithelial tissues during development and in the adult. PlexinB1 and Sema4D show largely complementary cell distribution in tissues, consistent with the idea that PlexinB1 acts as the receptor for Sema4D in vivo. Interestingly, PlexinB1 is also expressed in certain tissues in the absence of Sema4D, suggesting Sema4D independent activities. High expression of PlexinB1 was found in lung, kidney, liver and cerebellum.
Mutant mice lacking expression of semaphorin receptor PlexinB1 are viable and fertile. Although the axon collapsing activity of Sema4D is impaired in PlexinB1 deficient neurons, we could not detect major defects in development, or in adult histology and basic functional parameters of tissues expressing PlexinB1. Moreover, in the absence of PlexinB1 the angiogenic response induced by orthotopically implanted tumours was not affected, suggesting that the expression of this semaphorin receptor in endothelial cells is redundant.
Our expression analysis suggests a multifaceted role of PlexinB1 during mouse development and tissue homeostasis in the adult. Nonetheless, the genetic deletion of PlexinB1 does not result in major developmental defects or clear functional abnormalities. We infer that PlexinB1 plays a redundant role in mouse development and it is not strictly required for tumour induced angiogenesis.
Plexins are a highly conserved family of single pass transmembrane receptors which, in mammals, comprises nine genes grouped into four subfamilies (A thru D) based on sequence homology . They are characterized by a conserved sequence, the "sema domain", a structural domain that mediates protein-protein interaction, and phylogenetically links the Plexins to the Semaphorins and the Scatter Factor Receptors . The intracellular domain is highly conserved among the Plexins but does not share striking homology with other known proteins. Although the mechanisms of Plexin-mediated signalling have not been well understood, they are known to impinge on cytoskeletal dynamics and on cell adhesion, e.g. by regulating monomeric GTPases of Rho and Ras families .
Plexins were initially characterized for their role in axon guidance, where they function, either alone or in complex with the Neuropilins, as semaphorin receptors. Subsequently, Plexins were shown to control immune response and angiogenesis, and more recently were proposed to orchestrate tissue morphogenesis and cancer progression . We have previously identified PlexinB1 as the receptor for Sema4D. Upon ligand binding, PlexinB1 regulates cytoskeletal remodeling (via Rac [5, 6], PDZ-RhoGEF , p190 RhoGAP ), integrin activation (via R-Ras , PI3K ), MAPK signalling , cytosolic tyrosine kinases (Src, PYK2) , and can trigger the activation of the tyrosine kinase receptors Met and ErbB-2 [13, 14].
It was also demonstrated that PlexinB1 can mediate axon outgrowth  and endothelial cell migration . In addition, its ligand Sema4D is a potent angiogenetic factor [16, 17], possibly involved in tumour induced angiogenesis .
PlexinB1 mRNA expression has previously been shown in the nervous system  and in diverse tissues at E14 of murine development ; on the other hand, detailed information on the expression of Sema4D mRNA outside the nervous system is missing. Notably, PlexinB1 and Sema4D expression at protein level by immunohistochemistry has not been reported yet. Furthermore, little is known about the expression of Sema4D and PlexinB1 in the adult tissues.
In spite of extensive studies performed on Plexins of A subfamily, molecular genetic studies to address the functional relevance in vivo of Plexins-B are currently lacking. Here we report our analysis of PlexinB1 deficient mice, including functional studies to test the relevance of this semaphorin receptor in tumour induced angiogenesis.
PlexinB1 and Sema4D expression during embryo development and in the adult
In order to gain further evidence of PlexinB1 expression at the protein level, we generated specific anti-PlexinB1 antibodies (see Additional file 2 for specificity controls), which we used to analyze further embryonic and adult tissues.
The immunohistochemical analysis of PlexinB1 and Sema4D expression revealed an intriguing distribution in developing organs characterized by epithelial branching morphogenesis, including the kidney, the lung and the pancreas. During epithelial branching morphogenesis, tissues are shaped by the outgrowth and branching of tubular epithelial structures, which invade the surrounding mesenchyme under the control of growth factors and guidance cues. We found that, at an early stage, PlexinB1 is expressed in the mesenchyme surrounding epithelial tubules in kidney and lung (Figure 1B, D) and in the pancreas (not shown), while Sema4D is expressed by epithelial cells (Figure 1C, E).
In the adult kidney, we found an elevated expression of PlexinB1 in distal tubules and collecting ducts (Figure 2B), while Sema4D displays a complementary expression in the pars convoluta of proximal tubules (Figure 2C).
In the mouse liver, we could not clearly detect PlexinB1 expression during early embryonic stages (not shown), though we found a high expression of PlexinB1 in adult hepatocytes (Figure 2D). Surprisingly, we could not detect Sema4D expression in the liver at any stage (not shown), suggesting Sema4D-independent activities of PlexinB1 in this tissue.
Generation of PlexinB1 null mutant
Mendelian distribution of PlexinB1 deficient mice in offsprings
PlexinB1 deficient mice do not display major developmental defects
PlexinB1 mutant mice did not show any detectable difference in size and were undistinguishable from their wild type (WT) littermates; in addition, their feeding behaviour and weaning appeared to be regular, they developed normally and did not show defects in reproduction or fertility. Moreover, we measured some of the main blood parameters (i.e. white blood cells counts, red blood cells counts and values, haemoglobin concentration, platelet count) and found them to be normal (data not shown). Thus we undertook a histological analysis of embryonic and adult tissues in PlexinB1 null mice.
In the kidney PlexinB1 is highly expressed; however, the histological analysis of mutant mice did not reveal any gross defect or abnormality in tubules or nephrons (compare Figure 5B to 5E). These specialized structures develop as the result of an intricate dialogue between the epithelial cells and the surrounding mesenchyme, and these results implicate that PlexinB1 is not essential during nephrogenic induction. Furthermore, the number of renal glumeruli were counted and found to be similar in WT and mutant mice (Figure 5G). Because significant defects in kidney function may not implicate gross histological abnormalities and because PlexinB1 is also expressed in the adult, we performed blood tests on two major indicators of renal function, creatinine and urea levels, which turned out to be comparable in normal and mutant mice (see Tab. 2).
Axonal growth cones from PlexinB1 mutants fail to collapse in response to Sema4D
PlexinB1 is dispensable for tumour-induced angiogenesis
Sema4D is a potent angiogenetic factor in vivo [16, 17] and it is released by many tumour cells [; and our unpublished data]. Furthermore, it was shown that inhibiting the expression of Sema4D in human head and neck carcinoma cells leads to reduction of tumour burden and vascularity in a mouse xenograft model .
In fact, the so called "angiogenic switch", i.e. the ability of tumour cells to recruit new vessels in order to provide blood supply, is a basic step in solid tumour growth and metastatic dissemination. Without overcoming this rate-limiting step, most tumours cannot grow over 2 mm in diameter, nor metastasize. PlexinB1 is known to transduce Sema4D signals in vitro and it is expressed in HUVEC endothelial cells and in the tumour vessels in vivo [16–18], however its requirement in tumour angiogenesis has not been clearly established.
To scrutinize further vessel growth within the primary tumours, we stained tissue sections with an antibody against CD31 endothelial marker, and analyzed them by fluorescence microscopy. We could not reveal morphological differences between tumour vessels in mutant and WT mice. In addition, we quantified total vessel area and vessel density (the number of vessels per mm2) in tumour sections. In both cases, we did not measure a significant change in PlexinB1 -/- compared to WT mice.
Plexins were originally characterized for their role in the wiring of neural network as receptors for Semaphorin guidance cues. However, accumulating lines of evidence later prompted the investigators to reappraise and revise the biological role of these molecules. First, it was found that many Plexins are widely expressed outside the nervous system. Moreover, as the signal transduction mechanisms start to be uncovered, it is now clear that Plexins can impinge on a range of signaling pathways potentially regulating cell adhesion, cell proliferation and differentiation.
Here we demonstrate, by immunohistochemical analysis, the expression of PlexinB1 receptor and of its ligand Sema4D in diverse epithelial tissues and in the nervous system, suggesting that PlexinB1 might play multiple functional roles in vivo, during development and in the adult stage. In fact, this is the first extensive analysis, in the mouse embryo and in the adult, of PlexinB1 and Sema4D tissue distribution undertaken so far.
Previous expression studies and experiments in vitro had highlighted a potential role for secreted semaphorins and their receptors during tubular morphogenesis in the lung  and in the kidney , and it was hypothesized that these guidance cues may define permissive and restricted regions for cell migration. Interestingly, based on our expression analysis, Sema4D-PlexinB1 signaling might have a role in this function, for instance at the boundaries between different cell populations. Moreover, PlexinB1 can regulate the activation of Met , a tyrosine kinase receptor known to mediate tubular morphogenesis in fetal lung and kidney . In spite of these facts, the genetic deletion of PlexinB1 did not lead to morphological or functional abnormalities, indicating that the function of this semaphorin receptor is redundant in development. Notably, developing kidney and lung also express PlexinB2 , another putative Sema4D receptor . Although we observed that PlexinB2 binds to Sema4D with lower affinity than PlexinB1 (J.P. and L.T., unpublished observation), it is conceivable that PlexinB2 can compensate for the lack of PlexinB1 in these organs. In fact, by measuring PlexinB2 mRNA levels in tissues with real time PCR, we found significant expression in liver and kidney, which did not increase in PlexinB1-deficient mice (not shown).
These findings are in line with that found in previously reported mouse mutants of semaphorins or plexins expressed in developing lung (such as Sema3A , Sema3F , PlexinA1 ), which also lack to show obvious abnormalities of these organs [22, 30]. Thus, this scenario suggests a high level of redundancy in semaphorin signaling during branching morphogenesis and in the maintenance of tissue homeostasis in lung and kidney. Moreover, since additional guidance cues are known to regulate these developmental processes, it is conceivable that the absence of semaphorin signals may be compensated by functionally related morphogenetic factors such as the Netrins and the Slits (for an extensive review see ).
In contrast to the lung and the kidney, PlexinB1 expression in the liver is not consistent with a function as receptor for Sema4D, since we could not detect significant expression of this semaphorin in the mouse liver at any stage. This may suggest that hepatocytes or other liver cells express as yet unidentified alternative ligands for PlexinB1. Furthermore, since it was shown that Plexins can associate in receptor complexes on the cell surface [13, 14] or regulate homophilic cell-cell adhesion [32, 33], it is also possible that PlexinB1 acts in the maintenance of liver tissue architecture by functionally regulating other receptors or homologous plexins.
It was previously shown that Sema4D-PlexinB1 signalling can collapse the growth cone of hippocampal neurons in culture . Interestingly, we have observed loss of responsiveness to Sema4D in PlexinB1-deficient neurons derived from our mutants. On the other hand, our histological analysis revealed that the overall morphology, cell composition and neural connections in the hippocampus do not display obvious abnormalities in the absence of PlexinB1, suggesting a compensation of Sema4D function in development.
In the cerebellum, PlexinB1 is highly expressed in Purkinje (Pk) cells during the postnatal development. Notably, these neurons do not express PlexinB2 or CD72, which are lower affinity receptors for Sema4D [19, 21]. Incidentally, it was shown that Pk cells express PlexinB3 , but we previously demonstrated that this receptor cannot bind Sema4D . PlexinB1 distribution in the cerebellum appears consistent with the traditional role of Plexins as receptors for neurite outgrowth inhibitory cues. In fact, during the first postnatal days, Purkinje axons show exuberant sprouting of collateral branches that are eventually pruned during cerebellar development . The final shaping of Pk axons is associated with the axonal myelination mediated by the olygodendrocytes that express Sema4D . However, our analysis shows that Pk axons are correctly myelinated and the pruning of excessive branches occurs normally in the PlexinB1 mutant mice. It is noteworthy that PlexinB1 in Pk cells is also present in the perikaryon and dendrites. In addition, the protein shows different levels of expression in Pk cell subsets, corresponding to morphofunctional modules of the cerebellar cortex . Together with the precise distribution of Sema4D in the molecular layer, these observations suggest that Sema4D-PlexinB1 signalling may play a role in regulating the patterning and plasticity of intracortical connectivity, although this may be compensated during cerebellar development by alternative pathways.
The absence of clear developmental defects in PlexinB1 mutants is consistent with that reported for Sema4D-deficient mice. In fact, the latter were only found to have minor defects in the immune response, likely attributed to defective CD72 signaling , which is an alternative low-affinity receptor for Sema4D expressed in lymphocytes.
Our expression analysis had revealed that PlexinB1 is importantly expressed in a number of adult tissues, potentially suggesting a physiological role beyond embryo development. To address this point, in addition to study tissue histology, we have performed blood tests to investigate major functions in PlexinB1-deficient mice. Our data show that liver and kidney functions were normal. In addition, we found that hematopoiesis and coagulation parameters were not affected (data not shown).
Furthermore, it was proposed that PlexinB1 expressed by endothelial cells may mediate the pro-angiogenic activity of Sema4D released by tumour cells . Tumour-induced angiogenesis is of fundamental importance in cancer progression; in fact, hampering the formation of new vessels is now a popular strategy to restrain tumour growth. In order to test the functional requirement for PlexinB1 in this process, we exploited B16 melanoma transplants, which are commonly used to study tumour growth and angiogenesis in the context of syngenic C57BL/6 mouse models. We found that B16 cells produce Sema4D and tested their ability to grow and induce angiogenesis in PlexinB1-deficient mice. Our results suggest that the angiogenic activity of Sema4D may be mediated not only by PlexinB1, but also via other receptors expressed in endothelial cells (e.g. PlexinB2, see Additional file 4, panel C). Future studies will tell whether other tumour models are equally capable to grow in a PlexinB1-deficient tissue microenvironment.
Collectively, our data concerning PlexinB1 and Sema4D expression suggest that PlexinB1 may carry out multiple functions during development and in the adult. Nonetheless, our analysis of PlexinB1 mutant mice did not show any major impairment in embryo development or differentiated functions in the adult, including the neo-angiogenetic response induced by tumour growth. We conclude that PlexinB1 plays a redundant role during development, in the maintenance of tissue homeostasis, and in tumour angiogenesis.
mRNA expression analysis
A human multiple tissue array (MTE™) was purchased from Clonetech laboratories (Cat. #775-1). It contains mRNA in amounts normalized for the expression of eight different housekeeping genes. The P32 radioactive labelled cDNA probe used for hybridization contained a 4 kb divergent sequence encoding plexin extracellular domain obtained by EcoRI restriction of human full length construct. The hybridisation was performed using Ultrahybe® solution (Ambion Austin, TX, US) according to the protocol provided by the manufacturer.
Plexin expression in Human Umbelical Vein Endothelial Cells (Cambrex) was detected by RT-PCR using gene-specific primer pairs provided by Applied Biosystem (Hs00182227_m1 for PlexinB1, and Hs00367063_m1 for PlexinB2).
mRNA In situ hybridyzation
Whole-mount in situ hybridization (ISH) was according to standard protocol. Briefly, embryos were fixed overnight at 4°C in 4% (w/v) paraformaldehyde in PBT (0.1% Tween 20 in PBS), progressively dehydrated in increasing concentrations of ethanol/PBT, stored at -20°C, then rehydrated in decreasing concentrations of ethanol/PBT and treated with proteinase K (10 μg/ml in PBS). They were then postfixed for 20 min in 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.1% Tween 20 and prehybridized 1 hr at 70°C in 1.3× SSC, 50% formamide, 2% Tween 20, 5% dextran, 5 mM EDTA, and 50 μg/ml yeast RNA.
Sense and control antisense ryboprobes were synthesized using T7 and SP6 polymerases with a kit for DIG labeling (Roche Diagnostics). The probes were amplified by PCR (for PlexinB1: sense GCT AAC AGC TGT GGC AAT CA, antisense GGG ACA TTG GAA GCT ATG GA; for Sema4D sense GTC TTC GTC CTC AGG TCT GC, antisense CGA CAG GTT GAA GAT GAG CA) and subcloned in TOPO™TA vector (Invitrogen). Hybridization was performed overnight with DIG-labeled riboprobes in the same buffer of prehybridization. Washes with hybridization buffer were followed by RNase A treatment (10 μg/ml in 0.5 M NaCl, 10 mM Tris, pH 7.5, and 0.1% Tween 20, 1 hr at 37°C), and subsequent washes with hybridization buffer at 65°C. Embryos were then blocked in MABT (0.1 M maleate, 0.15 M NaCl, and 0.1% Tween 20, pH 7.5) containing 20% sheep serum, and incubated overnight at 4°C with anti-DIG-alkaline phosphatase (AP)-conjugate (Roche Diagnostics) diluted 1: 2000 in MABT with 2% sheep serum. After extensive washes with MABT, revelation was performed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics) in 0.1 M NaCl, 50 mM MgCl2, 0.1% Tween 20, and 0.1 M Tris pH 9.5.
Histology and immunohistochemistry
Histology and immunohistochemistry were performed according to standard techniques. For the embryo and the epithelial tissues, samples were collected and fixed in 4% paraformaldehyde/PBS, embedded in paraffin and sectioned at 6 μm. Sections were deparaffinised and processed. For immunohistochemistry on paraffin sections, all primary antibodies were diluted in Tris buffered saline 0.1% Tween20, 5% FBS. Our anti-PlexinB1 rabbit polyclonal antibody (raised against the following epitope in the extracellular domain: RNLHLFQDGPGDNEC) was used 1:500; murine monoclonal anti-Sema4D (BD Transduction Laboratories, BD Biosciences, Palo Alto, CA) was used 1:100; anti Smooth muscle actin (Sigma) was diluted 1:500. Sections were incubated overnight with the primary antibody, washed, incubated for 1 hour with DakoCytomationEnVision+® System (Dako, CA, US) and diamminobenzidine staining was developed according to manufacturer instructions. Haematoxylin, eosin and reagents for PAS staining were purchased from Bio Optica (Milan, Italy) and used following the instructions of the producer. Paraffin sections were examined with LeicaDMLB light microscope connected to a LeicaDFC320 digital camera.
For the cerebellum the histology was performed as previously described . Briefly, deeply anaesthetized mice were transcardially perfused with 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.2). The brains were dissected, kept in the same fixative overnight and cyroprotected in 30% sucrose 0.12 M phosphate buffer. The cerebella were cut by freezing microtome in 30 μm thick sagittal or frontal sections and collected in Tris buffered saline (pH 7.4). Anti-Calbindin D-28K (CaBP; rabbit polyclonal; Swant, Bellinzona, Swizerland) was used 1:3000; anti-Myelin basic protein (MBP; mouse monoclonal; Sternberger monoclonal, Baltimore, MD, USA) was diluted 1:2000; anti-Parvalbumin (mouse monoclonal, Swant, Bellinzona, Swizerland) was diluted 1:2000; anti PlexinB1 monoclonal antibody was used 1:500. The antibodies were diluted in phosphate buffered saline (PBS) with 0.25% Triton X-100 with 0.2% normal serum of the species of the secondary antibody. For immunohistochemistry sections were incubated for 1 h with biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) reacted with the avidin-biotin-peroxidase method (Vectastain, ABC standard Kit; Vector Laboratories, Burlingame, CA, USA) and revealed using diamminobenzidine as a chromogen. For double immunofluorescence sections were incubated for 1 h with FITCH conjugated anti-rabbit antibody (1:200 in PBS-triton with 0.2% normal serum; Sigma). After rinsing, they were again incubated overnight at 4° biotinylated anti-mouse antibody (1:200; Vector Laboratories, Burlingame, CA, USA), followed by Texas Red conjugated streptavidin (1:200 in PBS-Triton; Molecular Probes, Eugene, OR, USA). Nissl staining was performed according to ordinary techniques. Cerebellar sections were examined with a Zeiss Axiophot light microscope and an Olympus Fluoview 300 confocal microscope.
Digital images were processed with Adobe Photoshop 6.0 to adjust contrast.
The number of glomeruli in the kidney was counted in sagittal sections taken in the middle of the kidney and counterstained with haematoxylin and eosin. Five sections were evaluated per mouse and three wild type mice were compared with three mutant mice.
For vessel staining, tumour samples of similar volume (about 5 mm in diameter) were snap frozen in isopropanol at -60°, sectioned at the criostat and postfixed for 10' in 4% PFA. Sections were incubated with anti-CD31 (1:200 BD Pharmingen, USA) overnight, washed and incubated with anti-Rat Alexa 546 (1:500 Molecular Probes, USA). To reveal Sema4D expression in tumour samples by immunohistochemistry, tissue sections were post-fixed with acetone and incubated with monoclonal antibody clone BMA-12 (eBioscience), diluted 1:400.
Pictures were taken with a LeicaDM IRB microscope connected to a Leica DC350FX camera and analyzed with Metamorph 6.3 software (Molecular devices, USA).
Generation of PlexinB1 mutant mice
A cosmidic clone containing the murine PlexinB1 gene was obtained by screening a RZPD (Berlin, Germany) genomic library. A targeting vector was designed to flank exons 22 and 23 of PlexinB1 with two loxP sites. A neo gene flanked by two FRT sites and a LacZ reporter gene were placed in frame with exon 24. A 10 kb fragment was used as a 5' homology region, a 1 kb fragment was placed between the two loxP sites, and a 2 kb fragment was used as 3' homology region. Embryonic stem cells (W9.5) were transfected, cultured and selected for resistance against G418. Homologous recombined clones were identified by Southern blot analysis of EcoRI-digested DNA, using an external probe that was amplified by PCR (sense primer GGT CTG ACC CTG GAT ATG GA; antinsense primer CCA CCCTCT TTT ATG CCT GA). Chimeric mice were generated by injection of homologous recombined ES cell clones into blastocystes from C57BL/6 mice. Matings of male chimeras to C57BL/6 females yielded germline transmitted offsprings. Mice carrying the mutated PlexinB1 gene were crossed with C57BL/6 CRE deleter mice in order to achieve the excision of exons 22 and 23. The correct excision was checked by PCR (sense AAC ACC ATG TGT ATG CTG GAG AGG TCA GGG; antisense CAT CGC CTT CTA TCG CCT TCT TGA CG). Mice genotyping was carried out by PCR using a primer pair for the WT (sense AAC ACC ATG TGT ATG CTG GAG AGG TCA GGG; antisense GGG TCA CTG ATT CGT TTC TCA GAA CAC TGA C) and the same primers used to check CRE excision for the mutant allele.
Loss of PlexinB1 expression was confirmed in mutant mice by protein immunopurification and Western blotting using IC-2 antibodies, as previously described .
Cultures of hippocampal neurons
Primary hippocampal neurons, isolated from neonatal P1 mice, were cultured as previously described . Briefly, the hippocampi were isolated, trypsinized and cultured at low density in Neurobasal Medium supplemented with 2% B27 and 2% FCS. After 48 hours in culture neurons were stimulated for 1 hour with a purified preparation of Sema4D. Soluble Sema4D was purified from the conditioned media of cells transfected with a cDNA expression construct, generated by recombinant PCR as previously described .
Tumourigenesis assays in vivo
Sema4D expression in melanoma cells was detected using clone 30 antibody (BD-Transduction lab), by immunopurification and Western blotting experiments according to standard protocols . Tumourigenesis assays were performed as previously described . Shortly, 75000 B16 melanoma cells were diluted in PBS up to a final volume of 200 μl and injected subcutaneously into the right posterior flanks of 8-week old WT, heterozygous and homozygous PlexinB1 mutant mice. Tumour volume was calculated as described . At the end of the observation period, tumours were weighed. Superficial pulmonary metastases were counted under a stereoscopic microscope taking advantage of the black pigmentation of B16 melanoma cell line.
- Pk Purkinje:
WT wild type, MBP myelin basic protein
We gratefully acknowledge Stefania Artigiani for help in the cloning of the targeting construct, Fabienne Lamballe and Alessandro Ieraci for help with whole mount in situ hybridisation, Andrea Casazza for help with blood sample collection, and Lorena Capparuccia and Jose Sierra for sharing unpublished data on plexin and sema4D expression. Thanks to Raffaella Albano and Enrico Tenaglia for their precious help with animal care, and to Paolo Giacobini and Marika Crudelini for help and advise with histological analysis. We wish to thank Livio Trusolino for reading the manuscript and for helpful comments; Massimiliano Mazzone for sharing ideas and for the thoughtful discussion. This work was supported by the Italian Ministry for Research (PRIN-2005 project #2005055095, to L.T and F.R.) and by the Italian Association for Cancer Research (AIRC, to L.T. and P.M.C.). R.A.F. is an Investigator of the Howard Hughes Medical Institute.
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