Abstract
The signaling pathway associated with the formation and looping of the early Zebrafish gut is poorly understood. The present study uses a chemical genetics approach to examine aspects of the signaling pathway thought to be involved in the process of gut looping in the Zebrafish. A chemical genetics approach is one that uses small molecules known to modulate the activities of specific proteins in a signaling pathway in order to study their role tissue or organ morphogenseis. Compounds thought to be involved in gut looping used in the present study included rho kinase III inhibitor (rockout), blebbistatin which acts as a reversible inhibitor of non‐muscle myosin II, retinoic acid (RA) and DEAB. Effective maximum working concentrations that allowed for hatching of Zebrafish larvae were determined to be 5 uM DEAB, 0.5 uM RA, 1‐2 uM Blebbistatin and 10 uM Rockout. Gut looping was visualized in 30‐32 hpf embryos utilizing WISH for Fox A3 that recognizes early endoderm tissue of the gut tube and with Zebrafish gut‐GFP mutants (Tg(XlEef1a1:GFP). In addition, IHC staining for laminin, pan‐cadherin, B‐catinin, PKC and smooth muscle actin was utilized for visualization of the polarity, orientation and movement of the endodermal and mesodermal cell layers of the early and later stage Zebrafish gut tube. WISH and gut‐GFP mutant experiments demonstrated that 10 uM rockout but not 2 uM blebbistatin resulted in an inhibition of gut looping in 30‐32 hour old Zebrafish embryos. This work was performed in the lab of Dr. Nannette Nascone‐Yoder and supported by NSF award # 0642012.
Published Version
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