Role of the transcription factor TBX3 in liver development

In multicellular organisms, cell intrinsic and extrinsic factors control the balance between the number of cells and their differentiation state, to grow and function normally during development, homeostasis, and repair after injury. Multiple types of growth factors are involved in this process and regulate the cell division cycle by activating downstream intracellular transcription factors. The mammalian liver grows rapidly during postnatal development to become the largest gland in the body. Wnt growth factors are necessary for this process; however, the mechanism of Wnt function in liver growth is not known. Liver growth occurs through hepatocyte proliferation and polyploidization. Early during postnatal growth, hepatocytes proceed through all phases of the cell division cycle and give rise to daughter cells. In the second stage of liver growth, subsets of hepatocytes replicate their genome but do not divide and instead become large polyploid cells. To identify the role that Wnt signals play in liver growth, we focused on one of its targets, T-box3 (Tbx3), which is a transcriptional repressor. The T-box family of genes and their frequent interactions with the Wnt signaling pathway are reviewed in Chapter 1. In the liver, Wnt ligands are secreted by endothelial cells of the central veins. Hepatocytes in the proximity of central veins are Wnt-responsive and express Tbx3. We found that deletion of Tbx3 during liver growth caused hyper-polyploidization of hepatocytes. Through Chromatin-IP sequencing, we discovered that Tbx3 binds and represses two target genes, E2F7 and E2F8, which are mitotic inhibitors and necessary for hepatocyte polyploidy. To confirm that Tbx3 acts by repressing E2F7 and E2F8 we deleted all three genes simultaneously and found that the balance of polyploidy in the liver was restored. Additionally, through lineage tracing we showed that Tbx3-expressing hepatocytes proliferate to contribute to liver growth. Experiments on cultured primary hepatocytes further revealed that Wnt and Tbx3 are necessary for the proliferative activity of hepatocytes. Together, the work presented in Chapter 2 describes a function and mechanism of Wnt signaling in liver growth. We show that local Wnt signals promote hepatocyte progression through mitosis via Tbx3, which binds and represses E2F7 and E2F8 factors. Being at the forefront of metabolism and toxin removal, hepatocytes are constantly exposed to harmful molecules. Consequently, the liver has evolved an extensive capacity for renewal and repair. To assess how loss of Tbx3 or hyper-polyploidy affects liver function, in Chapter 3 we challenged livers lacking Tbx3 with different types of injuries and dietary regimens. While loss of Tbx3 did not create drastic defects in recovery, it led to tissue scarring and anomalies in hepatocyte nuclear contents, indicative of defects in cell division. Finally, to further examine the developmental role that Wnt and Tbx3 play, we turned to embryonic livers, where Wnt signaling is necessary for growth and Tbx3 marks the hepatic progenitors. We used lineage tracing techniques to show that Tbx3+ hepatic progenitors are bipotential and give rise to hepatocytes and biliary cells in vivo. We identified the Wnt ligands expressed in the embryonic liver and found that while initially they are expressed uniformly throughout the liver lobes, by e15.5 when the liver vascular network forms, Wnt2, Wnt9b and Tbx3 become localized to the central vein zones and the liver periphery, to maintain a pool of cells in their progenitor state. Future studies will explore how the vascular network guides such patterning in the liver and whether areas of high Wnt activity are zones of cell proliferation and tissue growth. The Wnt signaling pathway plays many roles in tissue growth and patterning. By identifying Tbx3 as its downstream agent in the liver, we have uncovered a mechanism of cell-cell communication and gained insights into how the cell division cycle can be regulated by growth factors. Meanwhile, we have also encountered many unanswered questions upon which we ponder throughout this thesis

Keywords: Developmental Biology.
Advisor: Nusse Roel
Fuller Margaret T.
Kim Seung K.
O'Brien Lucy Erin