Endothelial cells (ECs) require glycolysis for proliferation and migration during angiogenesis; however, the necessity for the mitochondrial respiratory chain during angiogenesis is not known. Here we report that inhibition of respiratory chain complex III impairs proliferation, but not migration of ECs in vitro by decreasing the NAD+/NADH ratio. To determine whether mitochondrial respiration is necessary for angiogenesis in vivo , we conditionally ablate a subunit of the respiratory chain complex III (QPC) in ECs. Loss of QPC decreases respiration, resulting in diminished EC proliferation, and impairment in retinal and tumor angiogenesis. Loss of QPC does not decrease genes associated with anabolism or nucleotides levels in ECs, but diminishes amino acid levels. Our findings indicate that mitochondrial respiration is necessary for angiogenesis, and that the primary role of mitochondria in ECs is to serve as biosynthetic organelles for cell proliferation.
The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature.
Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.
Holoprosencephaly (HPE) is defined as the incomplete separation of the two cerebral hemispheres. The pathology of HPE is variable and, based on the severity of the defect, HPE is divided into alobar, semilobar, and lobar. Using a novel hypomorphic Six3 allele, we demonstrate in mice that variability in Six3 dosage results in different HPE phenotypes. Furthermore, we show that whereas the semilobar phenotype results from severe downregulation of Shh expression in the rostral diencephalon ventral midline, the alobar phenotype is caused by downregulation of Foxg1 expression in the anterior neural ectoderm. Consistent with these results, in vivo activation of the Shh signaling pathway rescued the semilobar phenotype but not the alobar phenotype. Our findings show that variations in Six3 dosage result in different forms of HPE.
The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes.
SUMMARY Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP expressing ESCs, newly generated Six3−/− iPSCs and conditional null Six3delta/f;Rax-Cre ESCs we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay we determined that Six3-null cells exert a non-cell autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.
Although major progress in our understanding of the genes and mechanisms that regulate lymphatic vasculature development has been made, we still do not know how lumen formation and maintenance occurs. Here, we identify the Ras-interacting protein Rasip1 as a key player in this process. We show that lymphatic endothelial cell-specific -deficient mouse embryos exhibit enlarged and blood-filled lymphatics at embryonic day 14.5. These vessels have patent lumens with disorganized junctions. Later on, as those vessels become fragmented and lumens collapse, cell junctions become irregular. In addition, deletion at later stages impairs lymphatic valve formation. We determined that is essential for lymphatic lumen maintenance during embryonic development by regulating junction integrity, as loss results in reduced levels of junction molecules and defective cytoskeleton organization and We determined that Rasip1 regulates Cdc42 activity, as deletion of results in similar phenotypes to those seen following the loss of Furthermore, ectopic expression rescues the phenotypes in-deficient lymphatic endothelial cells, supporting the suggestion that Rasip1 regulates Cdc42 activity to regulate cell junctions and cytoskeleton organization, which are both activities required for lymphatic lumen maintenance.
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