While identifying genes necessary for organogenesis is important, having the appropriate cellular environment for cells to undergo morphogenesis is as important. Understanding the cell biology of zebrafish gut and liver development is the main interest of my thesis work. Claudins are junction proteins that are involved in the maintenance of polarity, the regulation of paracellular transport, and providing barrier functions. As such, they play a role in the establishment and maintenance of appropriate cellular environment by regulating the localization of different proteins or substances to their appropriate places. I have identified two claudins in my thesis work that are necessary for organ development.

During gut development, endodermal cells must rearrange themselves from an initial disorganized state to an organized radial pattern. I found that regulation of the dynamic localization of the adhesion proteins Cadherins may be important for mediating this process. After cells have arranged into a radial pattern, lumen that has formed at the apical region of these cells must coalesce to form a single lumen. We showed that the homeobox gene tcf2 transcriptionally controls the expression of claudin15 and Na+/K+ ATPase to regulate single lumen formation. Together, claudin15 and Na+/K+ ATPase create the appropriate electrochemical microenvironment to drive fluid accumulation. In turn, the fluid accumulation drives lumen coalescence. Subsequently, the single lumen must expand to form a functional gut tube. We provide evidence that apical membrane biogenesis is important for lumen expansion in gut development.

Another claudin, claudin15-likeb(cldn15lb), was found to have a dynamic expression pattern in the liver during development. Its initial expression in hepatocytes and biliary epithelial cells (BECs) becomes restricted to only the BECs at later stages of development. During biliary duct morphogenesis, BECs remodel to form a mature, functional network. Mutants of cldn15lb fail to remodel and form a disorganized biliary ductal network. Interestingly, these mutants do not have a severe polarity defect and can form tight junctions. The expression data and mutant phenotype suggest that cldn15lb may play a role in directing the remodeling process during biliary duct morphogenesis.

This thesis presents work toward understanding how cells within an embryo receive fate and positional information. It concentrates on the Nodal morphogen gradient, which specifies germ layer identity through differing levels of Nodal signaling. Although much progress has been made in understanding the components and outcomes of the Nodal signaling pathway, there is very poor quantitative understanding of how cells translate the duration and concentration of Nodal signaling into fate/positional information. To address this question, several new technologies were employed or developed.

Light sheet fluorescence microscopy was used to image Nodal signaling dynamics in vivo (Chapter 5). This new imaging technology allowed us to: determine which cells are exposed to the Nodal signal, quantitate the duration and level of this signaling, and directly correlate this with cellular response, i.e. which Nodal target genes are turned on and what germ layer fate is adopted. We will use this information to propose an initial model for how Nodal signaling is interpreted at a cellular level. We will test our model with additional data taken in the presence of an inhibitor, to access whether the system behaves as expected when the input is modified.

In order to develop finer control of the spatial and temporal aspects of perturbing a biological system, we simultaneously developed optogenetic tools for use in zebrafish embryos. Most optogenetic systems, especially those that control protein localization and expression, have been developed at the tissue-culture level and do not transfer directly to the multicellular organism level. We were successfully able to optimize two optogenetic systems for use in zebrafish, the phytochrome system for protein localization control (Chapter 2) and the LOV system for transcriptional control (Chapters 3 and 4).