Incongruence between transcriptional and vascular pathophysiological cell states

Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice.S

Visualization of vascular mural cells in developing brain using genetically labeled transgenic reporter mice

The establishment of a fully functional blood vascular system requires elaborate angiogenic and vascular maturation events in order to fulfill organ-specific anatomical and physiological needs. Although vascular mural cells, i.e. pericytes and vascular smooth muscle cells, are known to play fundamental roles during these processes, their characteristics during vascular development remain incompletely understood. In this report, we utilized transgenic reporter mice in which mural cells are genetically labeled to examine developing vascular mural cells in the central nervous system (CNS). We found platelet-derived growth factor receptor beta gene (Pdgfrb)-driven EGFP reporter expression as a suitable marker for vascular mural cells at the earliest stages of mouse brain vascularization. Furthermore, the combination of Pdgfrb and NG2 gene (Cspg4) driven reporter expression increased the specificity of brain vascular mural cell labeling at later stages. The expression of other known pericyte markers revealed time-,region-and marker-specific patterns, suggesting heterogeneity in mural cell maturation. We conclude that transgenic reporter mice provide an important tool to explore the development of CNS pericytes in health and disease

Defective endothelial cell migration in the absence of Cdc42 leads to capillary-venous malformations

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigate the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We find that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. While the loss of Cdc42 seem generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42 depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network.</jats:p

Defective endothelial cell migration in the absence of Cdc42 leads to capillary-venous malformations

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigated the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We found that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. Although the loss of Cdc42 seems generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42-depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network.status: publishe

Nmo phosphorylates Pk.

<p>(<b>A</b>) Prickle is phosphorylated by Nmo kinase but not dROK or Hpo kinases. Band shift assay of <i>in vitro</i> translated Pk (common region), Pan/dTCF and Myosin binding subunit (Mbs). The open arrowhead denotes the size of the unphosphorylated form (compared to no kinase lane). Note the band shift of Pk. Pan and Mbs serve as positive and negative controls, respectively. (<b>B</b>) Schematic of Pk constructs used in this study. The Pk protein scheme is shown above with the Pk-Sple N terminus (blue), short Pk N terminus as black line, PET domain (green) and three LIM domains (yellow). Constructs as denoted here are indicated by thick black lines: common (sequence shared between Pk and Pk-Sple isoforms); C-terminus; N-Δ1; N-Δ2 (both C-terminal deletions as indicated); C1; C2; Dom (PET and LIM domains); Sple N-terminus (Pk-Sple specific N-terminal extension); PET domain; M (Middle sequence) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#sec011" target="_blank">Methods</a> for sequence details). Full lines indicate fragments that are phosphorylated by Nmo, dashed lines indicate unphosphorylated fragments. (<b>C</b>) <i>In vitro</i> kinase assay gel using purified Nmo kinase and <i>in vitro</i> translated Pk fragments (from panel <b>B</b>). Upper panel; radiograph showing phosphorylation; autophosphorytion of Nmo is denoted by *N. Vang C-term is used as negative control (also [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.ref024" target="_blank">24</a>]). Common, C-term, N-Δ1, N-Δ2, and M fragments are phosphorylated by Nmo. Corresponding Coomassie-stained gel is shown below—full-length fragments of individual constructs are indicated by *.</p

Nmo acts with dTAK and Hipk to regulate Pk.

<p>(<b>A-B</b>) The phenotype of <i>sev</i>>Pk is enhanced in a <i>dTAK</i>^<i>179</i> heterozygous background (<b>A</b>), quantified in (<b>B</b>) (See also Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g005" target="_blank">5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g006" target="_blank">6</a> for <i>sev</i>><i>Pk</i> section examples). (<b>C-E</b>) Knockdown of <i>Hipk</i> (<b>D</b>) enhances the <i>sev</i>><i>Pk</i>, <i>>wIR</i> phenotype (<b>E</b>), similarly to knockdown of <i>nmo</i> (<b>C</b>). (<b>F</b>) Quantification of eye phenotypes in <b>C-E</b> (in each genotype <i>n</i>>200 and <i>P</i><0.005, and <0.0005 for *** and ****, respectively). (<b>G</b>) A schematic of the Pk protein showing location of the consensus Hipk phosphorylation site S880, within the consensus HE<b>S</b>PSR, along with the two clusters of Nmo phosphorylation sites.</p

Nmo limits Pk^Pk activity.

<p>(<b>A</b>) Identification of Nmo phosphorylation sites. <i>In vitro</i> kinase assay with increasing amounts of M fragment (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g001" target="_blank">Fig 1</a>) in which the first or second cluster of MAPK consensus sites, (potential Nmo target sites), or both, are mutated. Positions of the Nmo S/T phosphorylation sites are indicated in above schematic of fragment with S and T, respectively (the S/T residues in Pk are for cluster 1: S515, S519, S595, and S599, and for cluster 2: T708, S725, T737, and S762). Upper panel; radiograph showing phosphorylated Pk fragments (arrowhead; * denotes Nmo autophosphorylation), lower panel; Coomassie-stained gel. Note that phosphorylation is significantly reduced only when both clusters are mutated. (<b>B-D</b>) Pk lacking both clusters of Nmo phosphorylation sites shows a stronger phenotype. <i>sevenless(sev)</i>-<i>Gal4</i> driven Pk overexpression (myc-Pk-WT; panel <b>B</b>) displays a gain of function phenotype with both flips and symmetrical clusters. The phenotype is more severe when a Pk construct in which both Nmo consensus site clusters have been mutated (myc-Pk-Mut; note increased symmetrical clusters, panel <b>C</b>) is expressed. Both transgenes are inserted in the same attP site and thus expressed at same levels; quantified in panel <b>D</b> (**** <i>P</i><0.00005 Chi-squared test, n>300). (<b>E-I</b>) Dose-dependent effect of Nmo on the <i>sev</i>-<i>Gal4</i> driven Pk gain-of-function phenotype. <i>sev</i>-driven Pk expression causes chirality defects (<b>E</b>). (<b>F-G</b>) Reduction of Nmo function through either one copy of the hypomorphic allele, <i>nmo</i>^<i>P/+</i> (<b>F</b>), or the null allele, <i>nmo</i>^<i>DB/+</i> (<b>G</b>) enhances these PCP defects, particularly number of symmetrical clusters (quantified in panel <b>I</b>); in contrast Nmo co-overexpression with Pk, (<i>sev>Pk</i>, <i>>Nmo</i>; panel <b>H</b>) suppresses the Pk-induced formation of symmetrical clusters (quantified in panel <b>I</b>; <i>P</i><0.0002, circle represents cluster with R-cell loss). (<b>I</b>) Quantification of chirality defects (****<i>P</i><0.0005, Chi-squared test n>300).</p

Nmo regulates Pk levels via proteasome-mediated degradation.

<p>(<b>A</b>) Loss of <i>nmo</i> phosphorylation sites increases the Pk protein level in eye discs. Lysates from myc-Pk-WT- or myc-Pk-Mut-expressing eye discs were immunoblotted using myc and γ-tubulin (control) antibodies. <i>w</i>^<i>1118</i> discs were used as a control. (<b>B</b>) Loss of <i>nmo</i> function increases Pk protein level in eye discs. The relative amount of EGFP-Pk protein in a <i>wt</i> or <i>nmo</i>^<i>DB/+</i> background was calculated and normalized to Arm levels. A representative blot is shown, data from four independent experiments are quantified in graph to the right. Reduction of <i>nmo</i> function increases the amount of EGFP-Pk protein, but not that of EGFP-Sple (see Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.s006" target="_blank">S6 Fig</a> for blot; paired t-test, * <i>P</i> = 0.038, ns <i>P</i>>0.05). (<b>C-F</b>) Eye phenotypes of <i>sevGal4</i>, <i>UAS-Pk</i> (<i>sev>Pk</i>) and <i>act-EGFP-Pk</i> co-expressing dominant negative (DN) proteasome components. The <i>sev</i>><i>Pk</i> phenotype (<b>C</b>) is enhanced by co expression of DNProsβ2 (<b>D</b>). The <i>act</i>-<i>EGFP-Pk</i> phenotype (<b>E</b>) is enhanced by <i>GMR>DNProsβ6</i> co-expression (<b>F</b>). Note the increase in symmetrical clusters. (<b>G</b>) Quantification of eye phenotypes (****<i>P</i><0.0001, Chi-squared test n>300). (<b>H</b>) Inhibition of proteasome function increases Pk protein level in eye discs. Lysates from eye discs expressing <i>act</i>-<i>EGFP-Pk</i> in either a <i>w</i>^<i>1118</i> or <i>GMR</i>> <i>DNProsβ6</i> background were immunoblotted using GFP and γ-tubulin (control) antibodies.</p