Unique functions for Notch4 in murine embryonic lymphangiogenesis

Area of Science:

• Developmental Biology
• Molecular Biology
• Vascular Biology

Background:

• Embryonic dermal lymphatic development is crucial for organismal health.
• Notch signaling is a conserved pathway with complex roles in lymphangiogenesis, capable of both inhibition and promotion.
• Distinct roles of Notch receptors in lymphatic development are not fully elucidated.

Purpose of the Study:

• To investigate the differential functions of Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis.
• To understand how DLL4-Notch interactions influence lymphatic endothelial cell (LEC) behavior and gene expression.
• To elucidate the specific contributions of Notch4 to lymphatic vessel formation and maturation.

Main Methods:

• Analysis of gene expression (NOTCH1, NOTCH4, DLL4, Hes/Hey) in embryonic dermal lymphatics and LECs.
• Functional assays using VEGF-A and VEGF-C treatments on LECs.
• Phenotypic analysis of Notch4-null mice at embryonic stages (E14.5, E16.5).
• In vitro wounding assays to assess LEC migration.
• Inhibition of canonical Notch signaling using dominant-negative MAML1 (DNMAML) in Prox1+ LECs.

Main Results:

• Actively growing lymphatics express NOTCH1, NOTCH4, and DLL4, indicating Notch pathway activity.
• DLL4 activation of Notch in LECs induces specific Notch effectors and lymphatic genes, differentially regulated by Notch1 and Notch4.
• VEGF-A/C treatment upregulates Dll4 and differentially regulates Notch1 and Hes/Hey genes.
• Notch4 deficiency leads to increased closure of lymphangiogenic fronts, reduced vessel caliber, and decreased branching.
• Notch4 activation significantly suppresses LEC migration more than Notch1.
• Inhibition of canonical Notch signaling increases lymphatic density and LEC proliferation, similar to Notch1 loss.
• Loss of Notch4 does not impact canonical Notch signaling in LECs.

Conclusions:

• Notch4 signaling and canonical Notch signaling exert distinct functions in orchestrating embryonic dermal lymphangiogenesis.
• Notch4 plays a dominant role in suppressing LEC migration and regulating vessel caliber and branching.
• Canonical Notch signaling, particularly involving Notch1, is associated with LEC proliferation.
• The findings reveal a complex, context-dependent role for different Notch receptors in vascular development.