Christer Betsholtz's projects in developmental genetics

Mechanisms of angiogenesis and vascular permeability: the role of G-protein coupled receptors

Konstantin Gaengel, Colin Niaudet, Barbara Lavina-Siemsen, Marco Castro, Alberto Alvarez

 cdn5 expression in retina
 Photo: Barbara Lavina-Siemsen

We previously identified a core set of 58 gene transcripts expressed specifically (and quite universally) in endothelial cells. This set of genes included some 20 well-established endothelial markers, many of which are known to play critical roles in vasculogenesis and angiogenesis.

Interestingly, however, approximately half of the 58 gene transcripts had not been previously implicated in vascular biology. Many of them are highly interesting as candidate novel regulators of angiogenesis since they 1) are highly endothelial-specific in their expression, and 2) encode proteins
predicted to play a role in cell signaling, such as GPCRs.

In our current research program, we are investigating cellular and molecular mechanisms involved in angiogenesis, with focus on new regulators and regulatory processes involved in vascular morphogenesis, stabilization and barrier formation.

PDGF signalling during organ development – perivascular brain fibroblasts and alveolarization of the lung

Johanna Andrae

The platelet-derived growth factors (PDGFs) are known mediators in several developmental processes. Our main focus is on processes that are dependent on proper signalling through the tyrosine-kinase receptor PDGFRa and the two ligands PDGF-A and PDGF-C. We ask questions like – What are the characteristics of the cells that express PDGFRa? Where are PDGFRa+ cells located in relation to the ligand-expressing cells? What happens to those cells in the absence of PDGF, or if they are over-stimulated? We are interested to know how different cells contribute to a specific tissue organization.

Green perivascular fibroblasts along
a blue blood vessel in the brain,
surrounded by orange astrocytes.

In general, the PDGFR is expressed on mesenchymal cells whereas the ligands are secreted from adjacent epithelial, neuronal or muscle cells. This is true in both the central nervous system and in the lung, which we are specifically interested in.

Perivascular brain fibroblasts are a group of relatively undescribed cells, located along certain blood vessels in the brain. These PDGFRa+ cells express several fibroblast markers and are situated outside of endothelial cells, but inside of the astrocytic endfeet. Their biology, origin and functional role is still to be described, but our hypothesis is that PDGF signaling is important and that the cells are needed for the vascular system.

Lung alveolarization is the developmental process where the small, functional units in the lung are formed. In the absence of PDGF-A, the alveolarization process is negatively affected. In the adult lung, there are several reasons to believe that PDGFRa+ cells have other functions. We hypothesize that PDGF signalling is needed in the stem cell niche if the lung is exposed to an injury.

All cell types use specific molecular signals to communicate with each other, and knowing the normal signalling pathways is often crucial to understand potential pathological mechanisms.

Pericyte biology and markers

Bongnam Jung, Michael Vanlandewijck, Koji Ando

Pericytes are essential for development and stabilization of the vascular networks. These cells also regulate capillary blood flow, and are a component of the neurovascular unit that controls blood-brain permeability. In addition, immune, phagocytic and contractile functions are assigned to pericytes.

Genetic mutation and cell-based studies have demonstrated pericyte engagement in physiological functions and in diseases, including vascular/ organ development, wound healing, scarring, fibrosis and tissue remodeling. For example, PDGF-B or PDGFRβ deficient mice die perinatally exhibiting vascular dysfunction due to a lack of pericyte investment around blood vessels, suggesting the critical role of PDGFB/R signaling in vascular maturation.

Although the biological significance of pericytes is appreciated, a lack of pericyte-specific markers has hampered in-depth study on their origin, presence and function during physiological and pathological processes. To date, existing pericyte markers, such as PDGFRβ, NG2, desmin and CD13, cannot distinguish pericytes from vascular smooth muscle cells (vSMCs) or other mesenchymal cells. The expression patterns of these markers also vary between species, developmental stages and tissues. Therefore, 1) pioneering a reliable, pericyte-specific marker and 2) characterizing known marker expression in a timely- and organ- specific manner are necessary for proper analysis of pericyte biology in health and disease.

We take advantage of the double fluorescent transgenic mouse model, PDGFRβ-EGFP/NG2-dsRed, to study pericyte expression in embryonic and adult mouse tissues by immunofluorescence staining and imaging. Further, we use these mice to FACS pericytes for deep sequencing-based transcriptional profiling to investigate not only novel and specific pericyte markers but also transcriptional differences in pericytes from various organs.

Utilizing the PDGF knock out mice crossed to the NG2-dsRed mouse, we hope to address the precise mechanism of PDGFs in regulation of pericyte function, and differential behavior of pericytes throughout development and in adulthood.

Zebrafish models

Koji Ando

Despite the biological importance, we still don't know exactly how pericytes develop and how pericytes regulate vascular development and homeostasis. We are expecting that our novel transgenic zebrafish lines expand our current knowledge regarding pericytes.

Zebrafish brain vasculature at
2 months.

We have succeeded in establishing several fluorescent transgenic zebrafish lines, which allow us to monitor in vivo mural cell behavior including pericytes. Importantly, by utilizing these lines, we found that fundamental Pdgfb/Pdgfrb signalling regulating mural cell development/dynamics, the roles of mural cells in vascular development/maintenance, and gene expression pattern in mural cells appear conserved
among vertebrate.

Zebrafish trunk vasculature at 5 days

Especially, taking the advantages of rapid development, transparency or ex utero development in zebrafish, we are exploring the molecular mechanisms underlying pericyte development. Besides, we are trying to make the lineage of pericytes clear by utilizing cre/lox-mediated lineage tracing system or direct observation during the development. As well as high-resolution live imaging to study cellular behaviour and interactions with endothelial cells, we also apply reverse genetic approaches to elucidate the molecular mechanisms important for pericyte development and functions.

Zebrafish trunk vasculature at 20 days

For the genetic screening, in addition to mouse pericytes, we have launched the project for the FACS based deep sequencing-based transcriptional profiling of zebrafish pericytes utilizing novel fluorescent transgenic zebrafish lines. By comparing gene expression profile of pericytes between zebrafish and mouse, we are trying to find out highly conserved pericyte specific genes and perform reverse genetic screening for these selected genes in order to know the conserved mechanisms of pericyte development and functions.