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1.  Pkd1 regulates lymphatic vascular morphogenesis during development 
Cell reports  2014;7(3):623-633.
Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by well-studied signaling and transcriptional mechanisms. Less well understood is the ongoing elaboration of vessels to form a network. This involves cell polarisation, coordinated migration, adhesion, mixing, regression and cell shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. We found a mutation in polycystic kidney disease 1a to be responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial sprouting of lymphatic precursors is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice also has no effect on sprouting of precursors but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.
PMCID: PMC5005109  PMID: 24767999
2.  A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis 
Angiogenesis  2016;19(4):463-486.
Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34+CD45dim hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34+CD45dimDSG2+ progenitor cells are multi-potent and pro-angiogenic in vitro. Using a ‘knockout-first’ approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2lo/lo) and observed that, in response to reduced levels of Dsg2: (i) CD31+ ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell–cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.
Electronic supplementary material
The online version of this article (doi:10.1007/s10456-016-9520-y) contains supplementary material, which is available to authorized users.
PMCID: PMC5026727  PMID: 27338829
DESMOGLEIN-2; Endothelial progenitor cells; Endothelium; Hematopoietic stem and progenitor cells; Mouse models of neoangiogenesis
3.  GATA2 is required for lymphatic vessel valve development and maintenance 
The Journal of Clinical Investigation  2015;125(8):2979-2994.
Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.
PMCID: PMC4563742  PMID: 26214525
4.  Deciphering the roles of macrophages in developmental and inflammation stimulated lymphangiogenesis 
Vascular Cell  2012;4:15.
Lymphatic vessels share an intimate relationship with hematopoietic cells that commences during embryogenesis and continues throughout life. Lymphatic vessels provide a key conduit for immune cell trafficking during immune surveillance and immune responses and in turn, signals produced by immune lineage cells in settings of inflammation regulate lymphatic vessel growth and activity. In the majority of cases, the recruitment and activation of immune cells during inflammation promotes the growth and development of lymphatic vessels (lymphangiogenesis) and enhances lymph flow, effects that amplify cell trafficking to local lymph nodes and facilitate the mounting of effective immune responses. Macrophages comprise a major, heterogeneous lineage of immune cells that, in addition to key roles in innate and adaptive immunity, perform diverse tasks important for tissue development, homeostasis and repair. Here, we highlight the emerging roles of macrophages in lymphangiogenesis, both during development and in settings of pathology. While much attention has focused on the production of pro-lymphangiogenic stimuli including VEGF-C and VEGF-D by macrophages in models of inflammation including cancer, there is ample evidence to suggest that macrophages provide additional signals important for the regulation of lymphatic vascular growth, morphogenesis and function.
PMCID: PMC3444946  PMID: 22943568
Lymphangiogenesis; Macrophages; Monocytes; Development; Inflammation; VEGF-C; VEGF-D
5.  In Vitro Assays Using Primary Embryonic Mouse Lymphatic Endothelial Cells Uncover Key Roles for FGFR1 Signalling in Lymphangiogenesis 
PLoS ONE  2012;7(7):e40497.
Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.
PMCID: PMC3391274  PMID: 22792354
6.  Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity 
PLoS ONE  2010;5(2):e9377.
The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.
Methodology/Principal Findings
We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.
These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.
PMCID: PMC2826422  PMID: 20186345
7.  Prox1 maintains muscle structure and growth in the developing heart 
Development (Cambridge, England)  2008;136(3):495-505.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
PMCID: PMC2655234  PMID: 19091769
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy

Results 1-7 (7)