Delta-like 4 is required for pulmonary vascular arborization and alveolarization in the developing lung.

Sheng Xia,Michael F Nyp,Thomas Korfhagen,Daniel P Heruth,Sherry M Mabry,Venkatesh Sampath,Heather L Menden,Jeffrey Johnston,Nick Townley

doi:10.1172/jci.insight.134170

Abstract

The molecular mechanisms by which endothelial cells (ECs) regulate pulmonary vascularization and contribute to alveolar epithelial cell development during lung morphogenesis remain unknown. We tested the hypothesis that delta-like 4 (DLL4), an EC Notch ligand, is critical for alveolarization by combining lung mapping and functional studies in human tissue and DLL4-haploinsufficient mice (Dll4+/lacz). DLL4 expressed in a PECAM-restricted manner in capillaries, arteries, and the alveolar septum from the canalicular to alveolar stage in mice and humans. Dll4 haploinsufficiency resulted in exuberant, nondirectional vascular patterning at E17.5 and P6, followed by smaller capillaries and fewer intermediate blood vessels at P14. Vascular defects coincided with polarization of lung EC expression toward JAG1-NICD-HES1 signature and decreased tip cell-like (Car4) markers. Dll4+/lacZ mice had impaired terminal bronchiole development at the canalicular stage and impaired alveolarization upon lung maturity. We discovered that alveolar type I cell (Aqp5) markers progressively decreased in Dll4+/lacZ mice after birth. Moreover, in human lung EC, DLL4 deficiency programmed a hypersprouting angiogenic phenotype cell autonomously. In conclusion, DLL4 is expressed from the canalicular to alveolar stage in mice and humans, and Dll4 haploinsufficiency programs dysmorphic microvascularization, impairing alveolarization. Our study reveals an obligate role for DLL4-regulated angiogenesis in distal lung morphogenesis.

Highlights

Mammalian lung development progresses sequentially through the embryonic, pseudoglandular, canalicular, saccular, and alveolar phases [1]
To confirm delta-like 4 (DLL4) antibody staining, we performed X-gal staining on frozen lung sections of Dll4+/lacZ CD1 pups, where 1 Dll4 allele is replaced by the lacZ reporter [11]
We focused on VEGFA and VEGF receptor 2 (KDR) in VEGF/KDR pathway, DLL4, JAG1, Notch intracellular domain (NICD), and HES1 in Notch pathway

Summary

Introduction

Mammalian lung development progresses sequentially through the embryonic, pseudoglandular, canalicular, saccular, and alveolar phases [1]. The molecular mechanisms regulating distal lung vascularization, a requisite for alveolarization, are not fully understood, limiting our understanding of defective vascularization in BPD. Vasculogenesis and angiogenesis are distinct mechanisms that govern vascular network development. Based on vascular casts and electron microscopy of mouse lungs demonstrating initial discontinuity, followed by fusion of proximal and distal lung vasculature, deMello et al suggested that vasculogenesis is important for peripheral pulmonary blood vessel development [6, 7]. Parera et al mapped the endothelial cell–specific Tie promoter during lung development and suggested that angiogenesis is required for distal lung vascular arborization [8]. The key endothelial cell (EC) molecular players that regulate angiogenesis and pulmonary vascular arborization required for normal alveolar development remain unknown

Methods

Results

Conclusion