VE-cadherin in arachnoid and pia mater cells serves as a suitable landmark for in vivo imaging of CNS immune surveillance and inflammation.

Meninges cover the surface of the brain and spinal cord and contribute to protection and immune surveillance of the central nervous system (CNS). How the meningeal layers establish CNS compartments with different accessibility to immune cells and immune mediators is, however, not well understood. Here, using 2-photon imaging in female transgenic reporter mice, we describe VE-cadherin at intercellular junctions of arachnoid and pia mater cells that form the leptomeninges and border the subarachnoid space (SAS) filled with cerebrospinal fluid (CSF). VE-cadherin expression also marked a layer of Prox1+ cells located within the arachnoid beneath and separate from E-cadherin+ arachnoid barrier cells. In vivo imaging of the spinal cord and brain in female VE-cadherin-GFP reporter mice allowed for direct observation of accessibility of CSF derived tracers and T cells into the SAS bordered by the arachnoid and pia mater during health and neuroinflammation, and detection of volume changes of the SAS during CNS pathology. Together, the findings identified VE-cadherin as an informative landmark for in vivo imaging of the leptomeninges that can be used to visualize the borders of the SAS and thus potential barrier properties of the leptomeninges in controlling access of immune mediators and immune cells into the CNS during health and neuroinflammation

Brain Abnormalities and Glioma-Like Lesions in Mice Overexpressing the Long Isoform of PDGF-A in Astrocytic Cells

BACKGROUND: Deregulation of platelet-derived growth factor (PDGF) signaling is a hallmark of malignant glioma. Two alternatively spliced PDGF-A mRNAs have been described, corresponding to a long (L) and a short (S) isoform of PDGF-A. In contrast to PDGF-A(S), the PDGF-A(L) isoform has a lysine and arginine rich carboxy-terminal extension that acts as an extracellular matrix retention motif. However, the exact role of PDGF-A(L) and how it functionally differs from the shorter isoform is not well understood.\ud \ud METHODOLOGY/PRINCIPAL FINDINGS: We overexpressed PDGF-A(L) as a transgene under control of the glial fibrillary acidic protein (GFAP) promoter in the mouse brain. This directs expression of the transgene to astrocytic cells and GFAP expressing neural stem cells throughout the developing and adult central nervous system. Transgenic mice exhibited a phenotype with enlarged skull at approximately 6-16 weeks of age and they died between 1.5 months and 2 years of age. We detected an increased number of undifferentiated cells in all areas of transgene expression, such as in the subependymal zone around the lateral ventricle and in the cerebellar medulla. The cells stained positive for Pdgfr-α, Olig2 and NG2 but this population did only partially overlap with cells positive for Gfap and the transgene reporter. Interestingly, a few mice presented with overt neoplastic glioma-like lesions composed of both Olig2 and Gfap positive cell populations and with microvascular proliferation, in a wild-type p53 background.\ud \ud CONCLUSIONS: Our findings show that PDGF-A(L) can induce accumulation of immature cells in the mouse brain. The strong expression of NG2, Pdgfr-α and Olig2 in PDGF-A(L) brains suggests that a fraction of these cells are oligodendrocyte progenitors. In addition, accumulation of fluid in the subarachnoid space and skull enlargement indicate that an increased intracranial pressure contributed to the observed lethality.\ud \u

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

Expression analysis of platelet-derived growth factor receptor alpha and its ligands in the developing mouse lung

Activation of the platelet-derived growth factor receptor-a (PDGFRa) signaling pathway is critically important during lung alveogenesis, the process in lung development during which alveoli are formed from the terminal alveolar sacs. Several studies have aimed to characterize the expression patterns of PDGFRa and its two ligands (PDGF-A and -C) in the lung, but published analyses have been limited to embryonic and/or perinatal time points, and no attempts have been made to characterize both receptor and ligand expression simultaneously. In this study, we present a detailed map of the expression patterns of PDGFRa, PDGF-A and PDGF-C during the entire period of lung development, that is, from early embryogenesis until adulthood. Three different reporter mice were analyzed (Pdgfa ex4-COIN-INV-lacZ, Pdgfc tm1Nagy, and Pdgfra tm11(EGFP) Sor), in which either lacZ or H2B-GFP were expressed under the respective promoter in gene-targeted alleles. A spatiotemporal dynamic expression was identified for both ligands and receptor. PDGF-A and PDGF-C were located to distinct populations of epithelial and smooth muscle cells, whereas PDGFRa expression was located to different mesenchymal cell populations. The detailed characterization of gene expression provides a comprehensive map of PDGFRa signaling in lung cells, opening up for a better understanding of the role of PDGF signaling during lung development

Characterization of platelet-derived growth factor-A expression in mouse tissues using a lacZ knock-in approach.

Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology

Characterization of Platelet-Derived Growth Factor-A Expression in Mouse Tissues Using a lacZ Knock-In Approach

Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfa(ex4COIN), which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfa(ex4COIN-INV-lacZ) allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfa(ex4COIN-INV-lacZ) allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfa(ex4COIN-INV-lacZ) recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology

Severe cerebellar malformations in mutant mice demonstrate a role for PDGF-C/PDGFR alpha signalling in cerebellar development

Formation of the mouse cerebellum is initiated in the embryo and continues for a few weeks after birth. Double-mutant mice lacking platelet-derived growth factor C (PDGF-C) and that are heterozygous for platelet-derived growth factor receptor alpha (Pdgfc(-/-); Pdgfra(GFP/+)) develop cerebellar hypoplasia and malformation with loss of cerebellar lobes in the posterior vermis. This phenotype is similar to those observed in Foxc1 mutant mice and in a human neuroimaging pattern called Dandy Walker malformation. Pdgfc-Pdgfra mutant mice also display ependymal denudation in the fourth ventricle and gene expression changes in cerebellar meninges, which coincide with the first visible signs of cerebellar malformation. Here, we show that PDGF-C/PDGFR alpha signalling is a critical component in the network of molecular and cellular interactions that take place between the developing meninges and neural tissues, and which are required to build a fully functioning cerebellum

PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands have been expressed under the same heart specific alpha-myosin heavy chain promoter. Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfra in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFR alpha agonists supports a role for endogenous PDGFRa agonists in the pathogenesis of cardiac fibrosis

Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart

Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFR alpha and PDGFR beta) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRa agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFR alpha-positive fibroblasts, PDGFR beta agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirusinduced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses.Radiosa Gallini and Jenni Huusko contributed equally to this work.</p