Leonor Gouveia

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.

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, Pdgfaex4 COIN, 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 Pdgfaex4 COIN-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 Pdgfaex4 COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4 COIN-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.

A role for PDGF-C/PDGFRα signaling in the formation of the meningeal basement membranes surrounding the cerebral cortex

ABSTRACT Platelet-derived growth factor-C (PDGF-C) is one of three known ligands for the tyrosine kinase receptor PDGFRα. Analysis of Pdgfc null mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFRα ligands might obscure additional functions. In search of further developmental roles for PDGF-C, we generated mice that were double mutants for Pdgfc−/− and PdgfraGFP/+. These mice display a range of severe phenotypes including spina bifida, lung emphysema, abnormal meninges and neuronal over-migration in the cerebral cortex. We focused our analysis on the central nervous system (CNS), where PDGF-C was identified as a critical factor for the formation of meninges and assembly of the glia limitans basement membrane. We also present expression data on Pdgfa, Pdgfc and Pdgfra in the cerebral cortex and microarray data on cerebral meninges. Summary: Developmental expression of Pdgfc and Pdgfra is necessary for the development of meninges and the glia limitans basement membrane. Failure of these processes leads to neuronal over-migration.

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‐α (PDGFRα) 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 PDGFRα 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 PDGFRα, 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 (Pdgfaex4‐COIN‐INV‐lacZ, Pdgfctm1Nagy, and Pdgfratm11(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 PDGFRα expression was located to different mesenchymal cell populations. The detailed characterization of gene expression provides a comprehensive map of PDGFRα signaling in lung cells, opening up for a better understanding of the role of PDGF signaling during lung development.

Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.

PDGF-A signaling is required for secondary alveolar septation and controls epithelial proliferation in the developing lung

ABSTRACT Platelet-derived growth factor A (PDGF-A) signaling through PDGF receptor α is essential for alveogenesis. Previous studies have shown that Pdgfa−/− mouse lungs have enlarged alveolar airspace with absence of secondary septation, both distinctive features of bronchopulmonary dysplasia. To study how PDGF-A signaling is involved in alveogenesis, we generated lung-specific Pdgfa knockout mice (Pdgfafl/−; Spc-cre) and characterized their phenotype postnatally. Histological differences between mutant mice and littermate controls were visible after the onset of alveogenesis and maintained until adulthood. Additionally, we generated Pdgfafl/−; Spc-cre; PdgfraGFP/+ mice in which Pdgfra+ cells exhibit nuclear GFP expression. In the absence of PDGF-A, the number of PdgfraGFP+ cells was significantly decreased. In addition, proliferation of PdgfraGFP+ cells was reduced. During alveogenesis, PdgfraGFP+ myofibroblasts failed to form the α-smooth muscle actin rings necessary for alveolar secondary septation. These results indicate that PDGF-A signaling is involved in myofibroblast proliferation and migration. In addition, we show an increase in both the number and proliferation of alveolar type II cells in Pdgfafl/−; Spc-cre lungs, suggesting that the increased alveolar airspace is not caused solely by deficient myofibroblast function. Summary: PDGF-A is needed during alveolar formation in the lung to promote migration and proliferation of myofibroblasts and to control the growth of alveolar epithelial type II cells.

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

In Fig. 1b of this Article, ‘Csf1r’ was misspelt ‘Csfr1’. In addition, in Extended Data Fig. 11b, owing to an error during figure formatting, the genes listed in the first column shifted down three rows below the first gene on the list, causing a mismatch between the gene names and their characteristics. These errors have been corrected online, and the original Extended Data Fig. 11b is provided as Supplementary Information to the accompanying Amendment.

PDGFRα signaling is required for alveolar development in the mouse lung

to adult, there are morphologically striking changes in the pupa regulated by a finely metabolism, these changes include the degradation, remodeling, and neogenesis of tissues. The immobile silkworm pupa live in the cocoon which protects it from unfavorable situations such as desiccation and predation, but, after the completion of metamorphosis the adult moth must escape from its cocoon. At this time, Cocoonase is secreted by which can hydrolyze and soften the cocoon end, then the silk moth can escape from the cocoon after eclosion. This pierced and moth urine stained cocoon cannot be used for reeling, so pupae are killed by heating or the cocoon are stored in the refrigerator before eclosion because the pupal stage is so short (approximately two weeks) to the silk industry. To reduce the labor and energy required for cocoon processing, drying and storage, we expect to knock out the cocoonase gene by CRISPR/CAS9 system for preventing silk moth escaping from cocoon. CRISPR/CAS9 system is a member of gene editing technologies with high specificity and adaptability as well as easy utility, it is the most widely used technology currently. CRISPR/CAS9 system can be used by different methods in silkworm: (1) injecting of mRNA of CAS9 and gRNA into embryo; (2) constructing transgenic lines of CAS9 drove by constitutive promoter or spatio-temporal specific promoter; (3) establishing stably expressed-CAS9 cell lines of silkworm for further study of mechanism at the cellular level. We have constructed cas9 and gRNA (target cocoonase) transgenic line respectively which have been verified in cellular level. The Cocoonase-free silkworm stain can be obtained by crossing two transgenic lines. We expect this silkworm stain can achieve our expectations, meeting production requirements.