Screening assay for blood vessel maturation inhibitors

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.     

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.     

Six3 is required for ependymal cell maturation

Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.     

KLF2 transcription factor modulates blood vessel maturation through smooth muscle cell migration

Vasculogenesis, angiogenesis, and maturation are three major phases of the development of blood vessels. Although many receptors required for blood vessel formation have been defined, the intracellular signal transduction pathways involved in vascular maturation remain unclear. KLF2(-/-) embryos fail to develop beyond 13.5 days because of a lack of blood vessel stabilization. The molecular mechanism of KLF2 function in embryonic vascular vessels is still largely unknown. Here we show a normal development pattern of endothelial cells in KLF2(-/-) embryos but a defect of smooth muscle cells at the dorsal side of the aorta. This phenotype results from arrested vascular maturation characterized by the failure of mural cells to migrate around endothelial cells. This migration defect is also observed when platelet-derived growth factor-B (PDGF) controlled migration is studied in murine embryonic fibroblast (MEF) cells from KLF2(-/-) animals. In addition, KLF2(-/-) MEFs exhibit a significant growth defect, indicating that KLF2 is required to maintain the viability of MEF cells. The PDGF signal is mediated through the Src signaling pathway, and a downstream target of KLF2 is sphingosine 1-phosphate receptor 1. These studies demonstrate that KLF2 is required for smooth muscle cell migration and elucidate a novel mechanism involving communication between PDGF and KLF2 in vascular maturation.     

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.     

Peroxiredoxin 6 is required for blood vessel integrity in wounded skin

Peroxiredoxin 6 (Prdx6) is a cytoprotective enzyme with largely unknown in vivo functions. Here, we use Prdx6 knockout mice to determine its role in UV protection and wound healing. UV-mediated keratinocyte apoptosis is enhanced in Prdx6-deficient mice. Upon skin injury, we observe a severe hemorrhage in the granulation tissue of knockout animals, which correlates with the extent of oxidative stress. At the ultrastructural level endothelial cells appear highly damaged, and their rate of apoptosis is enhanced. Knock-down of Prdx6 in cultured endothelial cells also increases their susceptibility to oxidative stress, thus confirming the sensitivity of this cell type to loss of Prdx6. Wound healing studies in bone marrow chimeric mice demonstrate that Prdx6-deficient inflammatory and endothelial cells contribute to the hemorrhage phenotype. These results provide insight into the cross-talk between hematopoietic and resident cells at the wound site and the role of reactive oxygen species in this interplay.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa -induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt⁻). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.     

Signal transduction genes required for heterocyst maturation in Anabaena sp. strain PCC 7120

How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.     

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.     

Proteasome assembly triggers a switch required for active-site maturation

The processing of propeptides and the maturation of 20S proteasomes require the association of beta rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes. In contrast, mutations affecting interactions with residues of the S2-S3 loop allow the assembly of full, but activity impacted, proteasomes. The crystal structure of the inactive H3 mutant, Phe145Ala, shows that the S2-S3 loop is displaced from the position observed in wild-type proteasomes. These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch.     

p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture

The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B‐cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell‐type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73‐deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730–747, 2016     

Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif

Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes. Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes. Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi. Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin. A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined. Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes. The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes. On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation.     

NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation

NK T cells are a lymphocyte lineage that is selected by CD1d and is characterized by the ability to rapidly secrete large amounts of both IFN-gamma and IL-4 after TCR stimulation. Using reactivity to CD1d tetramers to define presumptive NK T cells, several NK T cell progenitor populations were characterized based upon NK marker expression and CD4 vs CD8 expression. The earliest populations were found to be negative for NK markers and could proliferate to IL-7, while mature NK T cells did not. The NK1.1(-) NK T cell progenitors were capable of up-regulating NK1.1 when transferred in vivo. Upon stimulation, the NK1.1(-) populations secrete IL-4, but little IFN-gamma. As the cells mature and up-regulate NK1.1, they acquire the ability to secrete IFN-gamma. Finally, the Tec family tyrosine kinase Itk is necessary for optimal NK1.1 up-regulation and hence final maturation of NK T cells. The itk(-/-) mice also display a progressive decrease in NK T cells in older animals, suggesting a further role in peripheral maintenance.     

Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.     

Novel mechanisms for maturation of chloroplast transfer RNA precursors

Despite the prokaryotic origins of chloroplasts, a plant chloroplast tRNA precursor is processed in a homologous in vitro system by a pathway distinct from that observed in Escherichia coli, but identical to that utilized for maturation of nuclear pre-tRNAs. The mature tRNA 5' terminus is generated by the site-specific endonucleolytic cleavage of an RNase P (or P-type) activity. The 3' end is likewise produced by a single precise endonucleolytic cut at the 3' terminus of the encoded tRNA domain. This is the first complete structural characterization of an organellar tRNA processing system using a homologous substrate. In contrast to eubacterial RNase P, chloroplast RNase P does not appear to contain an RNA subunit. The chloroplast activity bands with bulk protein at 1.28 g/ml in CsCI density gradients, whereas E.coli RNase P bands as ribonucleoprotein at 1.73 g/ml. Chloroplast RNase P activity survives treatment with micrococcal nuclease (MN) at levels 10- to 100-fold higher than those required to totally inactivate the E.coli enzyme. The chloroplast system is sensitive to a suppression of tRNA processing, caused by binding of inactive MN to pre-tRNA substrate, which is readily overcome by addition of carrier RNA to the assay.