Ongoing projects:

1. Genetic mechanism of JAGGED1 mutations in ALGS.

• Understanding the mechanism of how heterozygous JAGGED1 mutations leads to ALGS pathologies is critical for developing more informative animal models for ALSG. Cases of whole gene deletions of JAGGED1 indicate a haploinsufficient mechanism. However, current heterozygous Jagged1 mutant mouse models exhibits ALGS pathologies only in certain genetic backgrounds, limiting their utility for mechanistic studies and for testing potential therapeutics. About 80% of JAGGED1 mutations associated with ALGS lead to a truncation and loss of the transmembrane domain (ΔC-TM) which anchors the Jagged ligand to the cell surface. We aim to test the hypothesis that most of the heterozygous JAGGED1 mutation alleles implicated in ALGS can exert a dominant negative effect on Notch signaling (including Jagged2/Notch signaling), to consequently compromise biliary lineage specification and other Notch regulated processes.
• We are using the zebrafish vertebrate model to test whether these truncated forms can disrupt Notch signaling in a dominant negative manner. Given our founding that Jagged2 plays a redundant role in liver duct lineage induction, we will test whether Jagged1 transmembrane truncated ligands can disrupt both Jagged1/Notch and Jagged2/Notch signaling to lead to more severe pathologies.
• Because most of cases of ALGS are due to spontaneous mutations, it is imperative that we have a rapid approach to assess the potential function consequences of these JAGGED1 mutations to inform genetic counseling efforts. We are developing the zebrafish as a practical in vivo platform to functionally assess human JAGGED1 mutant alleles.

Our animal chimera studies demonstrate that normal liver cells (red) can rescue Jag/Notch signal and duct lineage specification (green/blue) in neighboring cells of livers lacking Jagged expression. Zhang/Gates et al., 2017, Nature Communications

Diagram of a normal Jagged1 ligand (top, wild-type) attached to the surface of one cell (plasma membrane in gray) with domains depicted. Most Jagged1 mutations in ALGS leads to a soluble form lacking the TM domain (bottom, ΔC-TM). Domain abbreviations: SP, signal peptide; MNNL, Notch ligand N-terminal; DSL, Delta/Serrate/Lag2; vWFC, von Willebrand factor type C; JSD, Jagged Serrate domain; TM, transmembrane; PDZL, PDZ ligand

Liver (hepatocytes, red) with regenerated duct cells (green) following resumption of endogenous Jagged expression.

In vivo fluorescent reporter of bilirubin. Live fish expressing UnaG, which fluoresces when bound to bilirubin (right).

Endocardial cells (green) of the heart outflow tract are Notch active (red/orange/yellow) in zebrafish.

2. Reversing ALGS defects.

• Developmental defects are generally thought to be permanent. However we are testing whether certain ALGS defects are an exception and can be reversed. We are finding that complete developmental agenesis of liver biliary cells due to loss of Jag/Notch signaling can be reversed if Jag/Notch signing is allowed to resume. We are investigating the origin of the regenerating duct cells, testing our hypothesis that these regenerated biliary cells arise from the transdifferentiated hepatocytes. To examine whether these regenerating biliary cells can restore liver duct structure and function, we are developing new genetic technologies to assess duct network assembly and evaluate in vivo levels of bilirubin, a metabolite accumulated in patients with biliary paucity. Given the potential reversibility of Jag/Notch defects and the amenability of the zebrafish vertebrate model for testing small molecules, we will perform a chemical screen to identify drugs that will help resolve ALGS pathologies.

3. Live Flourescent reporter for bilirubin.

• Liver duct paucity in ALGS patients lead to chronic cholestasis, a condition that causes elevated systemic bilirubin levels. High bilirubin levels in ALGS patients are a predictor of long-term outcome of liver disease. The ability to determine in real time whether a model animal has elevated bilirubin levels will be necessary to better understand the dynamic functional effect of biliary paucity and regeneration. An in vivo indicator of bilirubin levels would also yield unprecedented insight into possible levels differences among distinct tissues and organs. For example, it would be insightful to assess levels of bilirubin in the brain, because bilirubin molecules can cross the blood brain barrier, accumulate, and become neurotoxic. By expressing a protein (UnaG) that fluoresces upon bilirubin binding, we are assessing bilirubin levels in live zebrafish mutant models of ALGS. Particularly we are investigating how the dynamic state of loss or regenerating biliary duct cells can affect bilirubin levels.

4. Mechanism of Jagged function in liver, heart, and vasculature.

• With our discovery of new mechanisms for Jag/Notch signaling in liver duct cell development, we are continuing to leverage the unique genetic and imaging techniques available with the zebrafish vertebrate model system to rigorously explore the role of Jag/Notch signaling in the development and maintenance of other tissues affected in ALGS, particularly the heart and vascular system. Uncovering different new mechanisms of Jag/Notch function in these tissues will reveal new therapeutic potential for ALGS.