The formation of functional epithelial tubules is a central feature of many organ systems. Although the process of tubule formation by epithelial cells is well-studied, the way in which tubules connect with each other (i.e. anastomose) to form functional networks both in vivo and in vitro is not well understood. A key, unanswered question in the kidney is how the renal vesicles of the embryonic kidney connect with the nascent collecting ducts to form a continuous urinary system. We performed a ligand-receptor pair analysis on single cell RNA-seq data from embryonic mouse kidney tubules undergoing anastomosis to select candidates that might mediate this process in vivo. This analysis identified hepatocyte growth factor (HGF), which has known roles in cell proliferation, migration, and tubulogenesis, as one of several possible candidates. To test this possibility, we designed a novel assay to quantitatively examine epithelial tubule anastomosis in vitro using epithelial spheroids with fluorescently-tagged apical surfaces to enable direct visualization of anastomosis. This revealed that HGF is a potent inducer of tubule anastomosis. Tubule anastomosis occurs through a proliferation-independent mechanism that acts through the MAPK signaling cascade and matrix metalloproteinases (MMPs), the latter suggestive of a role in extracellular matrix turnover. Accordingly, treatment of explanted embryonic mouse kidneys with HGF and collagenase was sufficient to induce kidney tubule anastomosis. These results lay the groundwork for investigating how to promote functional interconnections between tubular epithelia, which have important clinical implications for utilizing in vitro grown kidney tissue in transplant medicine.
Keywords: anastomosis; development; epithelial tubules; hepatocyte growth factor; interconnection; kidney; spheroids.