Brain microvessels supply billions of neurons and other brain cells with the oxygen and nutrients they need. Pathologies affecting brain microvasculature progress slowly and silently over a lifetime but have devastating consequences from decreased perfusion and vascular destabilization. Cerebral small vessel disease (CSVD) encompasses progressive heterogeneous changes in brain microvessels; it is the most common cause of vascular dementia and a significant contributor to stroke and cognitive decline (Østergaard et al., 2016). 25% of all strokes are the result of CSVD, yet effective targeted treatments remain elusive (Østergaard et al., 2016). This is in part due to the inability to both detect and assess progressive damage in the brain.
While there are several genes implicated in familial CSVD (i.e. NOTCH3, HTRA1, FOXC1, COL4A1, and COL4A2), there is a lack of suitable in vivo models for studying disease development and progression. Research has predominantly focused on NOTCH3, but over the last decade, FOXF2 has emerged as a risk locus for CSVD (Chauhan et al., 2016; Duperron et al., 2023). SNPs in the intergenic region between FOXF2 and FOXQ1 decrease FOXF2 expression and significantly increase stroke risk due to the variant decreasing the efficiency of ETS1 binding to a novel FOXF2 enhancer (Ryu et al., 2022).
Foxf2 promotes mural cell differentiation and vascular stability in the zebrafish brain and is expressed highly in brain pericytes (Ahuja et al., 2024 #1591); (Chauhan et al., 2016 #1403); (Reyahi et al., 2015 #1372); (Ryu et al., 2022 #1590). Brain pericytes interact closely with endothelial cells, contributing to extracellular matrix (ECM) deposition and blood-brain barrier (BBB) formation, in addition to providing vasoactivity and stability (Bahrami and Childs, 2020; Daneman et al., 2010; Dave et al., 2018; Stratman et al., 2009). In animal models, an absence of brain pericytes results in hemorrhages and accelerates vascular-mediated neurodegeneration (Bell et al., 2010; Wang et al., 2014). Foxf2 is clearly important for vascular stability across species, as loss of Foxf2 in mice and zebrafish leads to increased brain hemorrhage and alterations in brain pericyte numbers and differentiation (Chauhan et al., 2016; Reyahi et al., 2015; Ryu et al., 2022). Brain tissue from patients with aging-related dementias (i.e. post-stroke dementia, vascular dementia, Alzheimer’s disease) has reduced deep white matter pericytes and associated BBB disruption (Ding et al., 2020), suggesting that pericytes should be examined as mediators of CSVD progression in patients with FOXF2 deficiency.
We previously showed that complete loss of foxf2 in foxf2aca71; foxf2bca21 double-homozygous mutants in late embryogenesis leads to reduced brain pericyte numbers (Ryu et al., 2022). However, stroke susceptibility in humans is associated with reduced, but not absent, FOXF2 expression. Genome-wide association (GWA) indicates that carrying a minor allele of an SNP in a FOXF2 enhancer leads to reduced, but not absent, FOXF2 and is associated with stroke (Ryu et al., 2022). For this reason, we model CSVD using a zebrafish with reduced Foxf2 dosage using single homozygous foxf2a mutants. Zebrafish foxf2a and foxf2b genes are the result of genome duplication in zebrafish ~430 million years ago and have similar gene expression (Arnold et al., 2015; Chauhan et al., 2016). We have detected no difference in function between the two genes and, therefore, foxf2a loss of function may be similar to human heterozygous loss of FOXF2 function, a state that is observed in the population in GnomAD (Chen et al., 2024).
Strikingly, while pericytes in embryonic foxf2 mutants are clearly affected, foxf2 mutants can survive until adulthood, albeit with a reduced lifespan. How pericytes change across the lifespan while CSVD progresses is unknown. Here, we find that foxf2a mutants have significantly reduced brain pericyte numbers as embryos that do not recover over time. Pericytes in mutant embryos and larvae exhibit morphological abnormalities, including increased soma size, longer processes, and degeneration. We show that processes and soma in the adults are also abnormal, though their morphology differs over the lifespan. Although the initial pool of pericytes is smaller, mutants can regenerate pericytes after ablation. Our analysis suggests that foxf2 is required within pericytes to modulate numbers but also has a strong effect on morphology. We show that brain pericytes may contribute to the pathological progression of genetic CSVD, starting in embryonic development and continuing across the lifespan. Understanding the early developmental aspects of late-onset vascular conditions like CSVD will aid in the development of effective therapeutic strategies.