Abstracts - Society for Developmental Biology

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A primary function of the liver is to produce bile and transfer it from hepatocytes to the biliary network, a complex series
of ducts comprising specialized epithelial cells (cholangiocytes). While the structure of the mature biliary network is well
characterized, the cellular and molecular mechanisms by which it differentiates and branches out to form a complex
network are poorly understood. Identifying regulators of morphogenesis is currently hindered by lack of an efficient and
accurate means of quantitating changes in network development. To overcome this barrier, we have combined threedimensional
confocal imaging with a novel computer-based algorithm to identify individual structures within cellular
networks and render them as skeletonized structures. After rendering the network as a series of discrete points, quantitative
data describing the dimensions of each structure within the network (intersections, connecting branches, terminal branches
etc...) and their interactions with each other are then computed. We also demonstrate the utility of this tool for
quantitatively tracking biliary network development in individual samples through time by coupling it to live multiphoton
confocal imaging. Furthermore, we demonstrate the practical utility of these tools through the identification of multiple
regulators of biliary morphogenesis through small molecule and forward genetic screens.
Program/Abstract # 134
Apical contraction of the actomyosin network initiates branching morphogenesis of the embryonic chicken lung
Kim, Hye Young; Nelson, Celeste M, Princeton University Chemical and Biological Engineering, United States
During development of the lung, branching morphogenesis sculpts the airway epithelium to maximize the surface area
available for gas exchange. The epithelial tube undergoes iterative rounds of morphogenetic routines including bud
formation, extension, and bifurcation to build the complex tree-like structure of the lung. Despite many biochemical signals
implicated in regulation of the branching process, little is known about the physical mechanisms that build the airways.
Here, we used the embryonic chicken lung to investigate remodeling of the actomyosin network and extracellular matrix
(ECM) during bud initiation. Lungs from 4-6-day-old embryos were immunostained and visualized using confocal
microscopy. 3D reconstruction of confocal stacks revealed that filamentous actin was enriched at the apical surface of the
emerging bud, and colocalized with phosphorylated myosin light chain. Moreover, inhibition of myosin contractility
blocked bud initiation, indicating that apical contraction of the actomyosin network is required to fold the epithelium into a
nascent bud. Furthermore, among several ECM proteins we found a distinct localization of tenascin-C (TNC). In addition
to the basement membrane, TNC was deposited preferentially in mesenchyme adjacent to the expanded and elongated
buds, but not in mesenchyme surrounding a newly forming bud. Together, our observations suggest that apical contraction
is the cellular machinery that induces nascent bud formation in the developing chicken lung, and TNC might indicate the
location of mechanical signals that act on the mesenchyme through the growing epithelial buds.
Program/Abstract # 135
Computational mechanobiology of peristalsis in embryonic lung
Lubkin, Sharon; Krishna, Kishore, North Carolina State University, Raleigh, United States
The lung is optimized for efficient transport of air in its lumen, yet it develops with a liquid-filled lumen. It is well
established that both prenatal occlusion and airway peristalsis (AP) increase branching morphogenesis, but the mechanisms
of their actions remain undetermined. Given that both occlusion and AP affect both transport and mechanics in the lung, it
is important to understand the mechanics and transport in the control and treatments. We present a study of the fluid-tissue
interactions of the pseudoglandular embryonic lung and its lumen contents. Our analysis suggests that some hypothesized
mechanosensing mechanisms may be irrelevant in the context of airway branching.
Program/Abstract # 136
Over-expression of receptors for advanced glycation end-products (RAGE) causes anomalous epithelial cell
survival and differentiation in the embryonic murine lung
Reynolds, Paul, Brigham Young University, United States
RAGE is a multi-ligand membrane receptor highly expressed in the developing lung and in inflammatory lung disease.
However, the contributions of RAGE to pulmonary organogenesis remain poorly characterized. In order to test the
hypothesis that RAGE misexpression affects lung morphogenesis, conditional transgenic mice were generated that overexpress
RAGE. Over-expression of RAGE throughout embryogenesis resulted in severe lung hypoplasia and perinatal
lethality. Flow cytometry and immunohistochemistry employing cell-specific markers demonstrated anomalies in key
epithelial cell types following RAGE up-regulation. Electron microscopy identified significant morphological disturbances
including blebbing of epithelium and loss of basement membrane integrity. Possible RAGE-mediated mechanisms leading
to the disappearance of pulmonary tissue were then evaluated. A time course of lung organogenesis demonstrated that
increased RAGE expression primarily alters lung morphogenesis beginning at E16.5. TUNEL immunostaining and blotting
for active caspase-3 confirm a shift toward apoptosis in lungs from RAGE over-expressing mice compared to controls.