Tube development & epithelial matrix biology in C. elegans
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Overview of Research
Most of the organs in our body are composed of tubes that transport vital nutrients and waste and serve as important gatekeepers between us and the outside environment. Many diseases are essentially “plumbing problems” in which these tubes clog, leak or collapse.
Our lab’s research utilizes the nematode C. elegans as a model system for studying the mechanisms that build, shape and stabilize epithelial tubes.
Multicellular Tubes: The Vulva
Most tubes in the body are made up of multiple different cells whose apical surfaces face a common lumen. The shapes and sizes of the individual cells will determine the overall diameter and shape of the tube.
We use the C. elegans vulva to study multi-cellular tube development and shaping. The vulva contains 22 cells arranged to form 7 stacked rings. Cells within each ring have different identities and shapes. Both EGF-Ras-ERK and Notch signaling play important roles in patterning vulva cell fates.
How does the luminal matrix assemble and shape tubes? Most tubes secrete various proteoglycans, glycoproteins and lipoproteins into their developing lumens. Examples in mammals include the vascular glycocalyx, lung surfactant, and the mucus-rich linings of the gut and upper airway. There is a growing appreciation of the importance of this luminal matrix or apical Extracellular matrix (aECM) in development and disease. However, relatively little is known about how aECMs assemble within lumens or the mechanisms by which they shape developing tubes. Furthermore, aECMs are very difficult to visualize and study in most systems because they are transparent by light microscopy and destroyed by most standard fixation approaches used for immunofluorescence.
We’ve identified components of an early C. elegans aECM that shapes developing epithelia, including the vulva tube. Many of these components belong to conserved protein families also found in mammalian ECMs. We can visualize these components in live worms using fluorescent tags inserted into the endogenous loci. We can also visualize the luminal matrix in preserved samples processed for electron microscopy using high pressure freezing. Our foundational studies have shown that the vulva luminal matrix is extremely complex and dynamic, and that the 7 different cell types produce and assemble different parts of this matrix. We are now poised to address many questions related to how the various components traffic to their correct locations and assemble to form these beautiful patterns.
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Unicellular Tubes: The Excretory System
The canal and duct are seamless (doughnut-like) tubes. The pore is a seamed (canoli-like) tube.
Organs are made up of tubes with different sizes and shapes. The tiniest tubes, such as many mammalian capillaries, are unicellular, with the lumen actually inside the cell. More than half of all capillaries in the brain and in the renal glomeruli are unicellular tubes. Capillary defects are associated with cardiovascular diseases, stroke and age-associated dementia, and are a devastating side effect of diabetes, but little is known about how narrow capillaries are formed or protected.
We use the C. elegans excretory system to study unicellular tubes. This organ system contains 3 unicellular tubes that form in different ways and take on very different shapes.
Below are some of the questions we’re addressing.
How does auto-fusion promote seamless tube growth and shaping?
Receptor Tyrosine Kinase (RTK) signaling patterns cell fates in most tubular organs. We showed that EGF-Ras-ERK signaling promotes excretory duct identity and controls many aspects of this unicellular tube’s morphology through a key target, the transmembrane fusogen AFF-1.AFF-1 fuses plasma membranes to convert a unicellular “seamed” tube into a “seamless” tube that lacks adherens junctions or tight junctions along its length. AFF-1 then mediates additional intracellular membrane-merging events that grow and shape the tube – specifically, we’ve proposed that AFF-1 promotes endocytic scission to allow transcytosis of membrane from the basal to apical surfaces. We are studying this role of AFF-1 and other vesicle trafficking pathways that allow seamless tubes to adopt very complex, elongated shapes.
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How does the luminal extracellular matrix shape and protect narrow tubes?
Luminal matrix appears especially important for shaping the body’s narrowest tubes. We’ve identified several types of apically localized matrix proteins or lipophilic cargo binding proteins that are required to shape and protect the narrow duct and pore tubes, and to prevent them from bursting or leaking. We’ve also identified suppressor mutations that “fix” these tube problems. Current studies are examining links between lipid transporters and luminal matrix organization, and taking advantage of C. elegans‘ unique advantages to visualize the luminal matrix and understand its assembly and disassembly.
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C, D. Excretory tube junctions in a newly hatched larva.
E. In the absence of LET-653, the excretory duct and pore tubes dilate or collapse.
What controls tube delamination and trans-differentiation?
The excretory system is also an excellent model for studying junction remodeling and epithelial fate plasticity. At a specific stage of development, the “G1” excretory pore tube delaminates from the organ, loses epithelial identity, re-enters the cell-cycle and generates two neuronal daughters. Another cell (“G2”) takes its place as the pore. The lab has identified mutants that perturb delamination, which should provide insight into mechanisms that trigger identity change and allow junction remodeling and withdrawal.
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Sundaram Lab at UPenn 