337 - Dll4 Signaling Regulates Type I Alveolar Epithelial Cell Development in Mice

Publication Number: 1321.337

Dhanesh Sivadasan Bindu, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Heather Menden, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Guolun Wang, Cincinnati Children's Hospital Medical Center, CINCINNATI, OH, United States; Sherry M. Mabry, Children's Mercy, Kansas City, MO, United States; Jichao Chen, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States; Venkatesh Sampath, Children's Mercy Kansas City, kansas city, KS, United States

Background: Delta-like 4 (Dll4), a Notch ligand enriched in pulmonary endothelium, is essential for microvascular development and alveolarization. While its role in vascular patterning is well established, its influence on alveolar epithelial cell differentiation, specifically type I (AT1) and type II (AT2) cells remains unclear. We hypothesize that endothelial Dll4 deficiency disrupts AT1 and AT2 cell development through impaired endothelial-epithelial crosstalk, potentially mediated by angiocrine signaling.

Objective: To test whether endothelial Dll4 haploinsufficiency disrupts alveolar epithelial development via impaired angiocrine signaling.

Design/Methods: Lungs from P14 Dll4+/+ and Dll4+/lacZ mice were collected for immunohistochemistry, qRT-PCR, and scRNA-Seq to assess differential gene expression. Pulmonary endothelial cells were isolated using CD31-conjugated magnetic bead pull-downs, and RNA from these purified fractions was analyzed by qRT-PCR to validate candidate angiocrine factors identified by sCRNA-seq. DEGs were further analyzed using Qiagen Ingenuity Pathway Analysis (IPA) and the Enrichr platform (https://maayanlab.cloud/Enrichr/), with a focus on enriched biological processes and molecular functions.

Results: P14 Dll4+/+ and Dll4+/lacZ mice revealed reduced lung expression of differentiated alveolar type I (AT1) markers-AQP5, AGER, and PDPN-while the type II (AT2) marker SFTPC remained unchanged (Figure 1B). qRT-PCR confirmed downregulation of Ager and Pdpn transcripts in Dll4+/lacZ lungs, with no change in Sftpc (Figure 1C). To investigate impaired AT1 differentiation, we performed scRNA-Seq on P14 lungs from Dll4+/+ and Dll4+/lacZ mice (Figure 2A). Dll4 expression was restricted to Cap1 and Cap2 endothelial cells (Figure 2B), which showed altered angiocrine profiles, including Bmp6, Sema3c, Sema7a, Sparc, Sparcl1, and Timp3 in Dll4+/lacZ mice (Figure 2C). qRT-PCR validation in CD31-enriched endothelial cells confirmed changes in shared candidates such as Bmp6, Sema3c (Figure 2D&E). Pathway and GO analyses performed on AT1 cluster DEGs using Ingenuity Pathway Analysis revealed reduced viability and increased apoptosis in Dll4+/lacZ lungs (Figure 3A). GO enrichment showed dysregulation of ubiquitination, protein synthesis, and growth factor signaling, indicating disrupted angiocrine communication (Figure 3B&C).

Conclusion(s): Endothelial Dll4 is essential for AT1 cell development and alveolar maturation. Its haploinsufficiency disrupts angiocrine signaling from lung endothelial cells, compromising AT1 differentiation and homeostasis.

Dll4 haploinsufficiency impairs type I alveolar epithelial cell development in murine lungs
A) Schematic illustrating the breeding strategy used to generate Dll4 wild-type (Dll4+/+) and heterozygous (Dll4+/lacZ) mice. Genotyping confirmed Dll4 WT and Het offspring (n = 3 males and 3 females per genotype). B) Whole-lung immunostaining of P14 lungs showing AQP5(green), AGER (red), SFTPC(magenta) and DAPI (blue) ( N=6/group; 3 males and 3 females; Scale bar-20m). C) qRT-PCR analysis of whole-lung RNA from Dll4+/+ and Dll4+/lacZ mice at P14 showing relative expression of Dll4, Ager, Pdpn, Aqp5 and Sftpc with qRT-PCR. (n = 4 mice per group; *P < 0.05).

Dll4-driven endothelial-epithelial crosstalk may regulate alveolarization in the mouse lung
A) UMAP plot showing lung cell clusters from P14 Dll4+/+ and Dll4+/lacZ mice(n=3/group). Each cluster is color-coded and annotated. B) Expression levels of Dll4 across individual cell types in P14 Dll4+/+ and Dll4+/lacZ mice. C) Volcano plot of differentially expressed genes(DEGs) in Cap1 and Cap2 endothelial cells between P14 Dll4+/+ and Dll4+/lacZ mice. Green dots indicate upregulated genes, orange dots indicate downregulated genes, and red dots highlight candidate angiocrine factors. D) Schematic of the experimental workflow for isolating endothelial cells from P14 lungs using CD31-conjugated magnetic beads. E) qRT-PCR analysis of pulled down lung endothelial RNA from Dll4+/+ and Dll4+/lacZ mice at P14 showing relative expression of Bmp6, Sema3c and Timp3 with qRT-PCR. (n = 3 mice per group; *P < 0.05).

Pathway and Gene Ontology analysis of differentially expressed genes in AT1 cluster between P14 Dll4+/+ and Dll4+/lacZ mice
A) Ingenuity Pathway Analysis (IPA) summary of AT1 cluster DEGs. Blue nodes indicate predicted inhibition, orange nodes indicate predicted activation. The network suggests reduced cell viability and increased apoptosis in AT1 cells. B&C) Enrichr analysis (https://maayanlab.cloud/Enrichr/) of AT1 DEGs from Dll4+/+ and Dll4+/lacZ mice lungs at P14. Bar charts display the top 10 enriched terms from the GO_Biological_Process_2025 and GO_Molecular_Function_2025 libraries for significantly upregulated (B) and downregulated (C) genes, respectively. Colored bars represent terms with significant p-values ( < 0.05); asterisks (*) denote terms with significant adjusted p-values ( < 0.05).