The Extracellular Matrix as a Regulator of Branching Morphogenesis

Abstract

During mammalian development, branching morphogenesis generates branched tissues as diverse as the kidney, lung, and mammary gland. Despite the ubiquity of branched tissues, it remains unclear how the geometry of these structures is regulated during normal development. This dissertation investigated how the extracellular matrix (ECM) regulates branching morphogenesis during pubertal development of the mouse mammary gland. We used quantitative image analysis, 3D-printed culture models, and computational models of tissue morphogenesis to investigate how the orientation and distribution of type I collagen regulates the global pattern of the developing mammary epithelium.

To investigate how type I collagen affects mammary epithelial branching, we developed a 3D-printed culture model. We found that 3D printing of collagen-Matrigel inks can be used to spatially control the geometry and alignment of type I collagen fibers over length scales relevant to the mammary gland. We observed that collagen fiber alignment was regulated by the extent of molecular crowding in the printing ink, as well as by the shear and extensional flows present in the printing nozzle.

Next, we investigated an alternative approach for engineering aligned networks of type I collagen. Using evaporating droplets, we demonstrated that Marangoni flows can orient type I collagen fibers over centimeter-scale areas. By incorporating human skeletal muscle cells into these networks of collagen, we demonstrated that cell orientation and differentiation can be patterned.

Finally, we characterized the pattern of the epithelium and the distribution of type I collagen in the developing mouse mammary gland. We observed that the mammary epithelium is preferentially oriented along the long axis of the developing gland during pubertal development. Furthermore, we found that local accumulation of collagen-rich ECM constrains the angle of epithelial bifurcation and regulates the global bias in epithelial orientation.

Together, this dissertation describes two approaches for engineering networks of type I collagen over physiologically relevant length scales. In addition, by combining engineered networks of collagen with quantitative analysis of mammary glands, we investigated how type I collagen regulates mammary epithelial branching in vivo. Our findings may provide further insight into the role of the ECM in sculpting the architecture of other branched tissues.