Ductal Tree Patterning During Branching Morphogenesis of the Submandibular Gland

Objectives: The development of ductal structures during branching morphogenesis of the mouse submandibular gland (SMG) relies on signals that specify ductal progenitors to set up a pattern for the ductal network. However, little is known about the early events that drive the development of ductal structures. Here, we searched to identify the earliest determinants of duct specification in the embryonic mouse submandibular gland (SMG).
Methods: Ex vivo cultures of embryonic mouse submandibular glands were coupled with functional perturbation of cell polarity regulators and imaged using high-resolution immunofluorescence imaging. Malformations were quantified using tracings of inverted grayscale microscopy images with Adobe Illustrator to measure bud circumference, clefts per bud, and buds per gland. Two sample t-tests were used to identify statistically significant (p<0.05) measurements.
Results: We identified F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of cellular asymmetry during duct development. These recruit apical polarity complex proteins aPKCz/Par3 and collaborate with ROCK signaling to set up apical-basal polarity of ductal progenitors and define the path of duct specification. Moreover, the motor protein myosin IIB, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. Inhibition of ROCK using Y-27632, myosin II using blebbistatin, or myosin light chain kinase with ML-7, resulted in bud and cleft malformations. SMGs treated with Y-27632, blebbistatin, and ML-7 had significantly larger but fewer buds, while glands treated with Y-27632 and blebbistatin displayed excessive clefting.
Conclusions: These studies identify cytoskeletal, junctional, and polarity proteins as early determinants of duct specification and the ductal tree patterning during embryonic development of the SMG. Moreover, they show that disruption of duct maturation by inhibition of ROCK and myosin II leads to defects in branching morphogenesis.