Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1

Abstract

During angiogenesis, single endothelial cells (EC) specialize into tip cells that guide vessel sprouting towards growth factor gradients and instruct the adjacent vessel stalk. The balance between tip and stalk cells is regulated by endothelial Notch signalling through the expression of Notch ligand Delta-like 4 (Dll4) in tip cells, which suppresses a tip cell fate in adjacent stalk cells. Here we show, using genetic reporter and conditional deletion strategies, that myeloid cells regulate tip cell numbers and Dll4 expression via the Notch ligand Dll1 during vascular development in the retina. Dll1 is selectively expressed by a subpopulation of retinal myeloid cells, which progressively localizes to the sprouting vascular network. Conditional, myeloid-specific deletion of Dll1 impairs endothelial Dll4 tip-stalk gradient resulting in an increase of endothelial tip cells and EC filopodia, accompanied by an increase in vascular density and branching. In vitro , co-culture of human EC with monocyte-derived macrophages induced Dll1 upregulation in macrophages and Dll4 upregulation and an endothelial tip cell signature in EC. Furthermore, culturing human EC on recombinant DLL1 induced endothelial Dll4 expression and a tip cell program, indicating that changes are Dll1-dependent. Thus, myeloid cells regulate tip cell fate and angiogenesis through expression of Notch ligand Dll1.

Microglia Contribution to the Regulation of the Retinal and Choroidal Vasculature in Age-Related Macular Degeneration

“…The activation of MAS causes the upregulation of Notch1, Delta-like ligand 4 and Jagged1 expression, members of the Notch signaling pathway (Figure 1). Indeed, this signaling pathway has been implicated in microglia localization and interaction with endothelial cells during sprouting angiogenesis and on the regulation of the fate of tip cells [66,67].…”

“…members of the Notch signaling pathway (Figure 1). Indeed, this signaling pathway has been implicated in microglia localization and interaction with endothelial cells during sprouting angiogenesis and on the regulation of the fate of tip cells [66,67]. Interestingly, systemic inflammation in the neonatal period is known to impair vessel development by decreasing vessel extension, reducing capillary density and inducing localized overgrowth of abnormal retinal vessels [68].…”

Utility of LysM-cre and Cdh5-cre Driver Mice in Retinal and Brain Research: An Imaging Study Using tdTomato Reporter Mouse

“…Retinas of LysM-cre and Cdh5-cre reporter mice have been studied before with no specific reporting of neuronal expression. 14,[17][18][19][20][21]24 However, most of the previous stud-ies were conducted on pups. One interesting article that used both LysM-cre and Cdh5-cre mice crossed to floxed tdTomato mice showed a very similar expression pattern to our study.…”

“…Several recent retina studies have used the LysM-cre driver mouse for gene deletion in myeloid cells. [14][15][16][17][18][19][20][21][22][23][24][25] However, the use of LysM-cre mice to target genes in macrophages versus microglia has been a topic of major debate. Moreover, it has been reported recently that the LysM promoter is also expressed in brain neurons.…”

Vessel Enlargement in Development and Pathophysiology

“…However, both these reports were for embryos at stages much older than the ones that gave the results showing vessel enlargement by migration and/or fusion ( Chouinard-Pelletier et al, 2013 ). The retinal vasculature does form post-natally, when resident myeloid cells are present in the retina ( Haupt et al, 2019 ). Ablation of macrophages using chlodronate liposomes results in a dramatic loss of vascular density, which makes it difficult to assess whether vessel enlargement itself is affected ( Checchin et al, 2006 ).…”