A novel reporter allele for monitoringDll4expression within the embryonic and adult mouse

Abstract

Canonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, precisely defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized β-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.

SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis

“…Overall, these results demonstrate the critical role of SOX17 in the specification of definitive lympho-myeloid hematopoiesis and DLL4 + CXCR4 + AHE from hPSCs. Although both DLL4 and CXCR4 are considered markers of AHE ( Chong et al, 2011 ; Yamamizu et al, 2010 ), DLL4 expression has been found in the arterial vessels of the yolk sac and aorta ( Duarte et al, 2004 ; Herman et al, 2018 ; Robert-Moreno et al, 2005 ), while Cxcr4 expression was detected in the aorta and vitelline/umbilical arteries ( McGrath et al, 1999 ; Werner et al, 2020 ) (i.e., vasculature harboring precursors capable of maturing into definitive HSCs) ( Dzierzak and Medvinsky, 1995 ; Gordon-Keylock et al, 2013 ), but not in the yolk sac ( McGrath et al, 1999 ; Venkatesh et al, 2008 ; Werner et al, 2020 ). Thus, SOX17 is the most essential factor for the formation of AHE with the CXCR4 + phenotype typical of HE with HSC potential in vivo .…”

Notch3 signalling and vascular remodelling in pulmonary arterial hypertension

“…Little is known about Notch ligand expression specific to the pulmonary vasculature. Both Dll1 [49] and Dll4 [50] are expressed during embryogenesis in endothelial cells of mouse lung. Jag1 is expressed in the endothelium of pulmonary vessels during development and after birth [45] and is also present in PASMCs [46].…”

Analyzing the effect of cell rearrangement on Delta-Notch pattern formation

“…An example of Delta-Notch pattern formation on a one-dimensional line is the expression pattern of Delta-like ligand 4 (Dll4) mRNA in retinal endothelial cells, as reported in [8,13,14]. Delta-like ligand 4 (Dll4) is a Notch ligand that is expressed mainly in the blood vessels, specifies the tip cells of growing vessels, and promotes arterialization [8].…”

“…We used a one-dimensional model because it is tractable analytically, and the distinct salt and pepper pattern of Delta-Notch expression has been reported in endothelial cells which are aligned one-dimensionally [8,13,14]. We assume that the number of cells is sufficiently large so that we can use periodic boundary conditions.…”

“…The Delta-Notch system is a well-studied cell-cell communication system that generates cellular scale periodic patterns [1][2][3][4][5][6][7][8]. Delta and Notch are, respectively, cell surface ligands and receptors.…”