Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Collaborative Specialization

Developmental Biology

Supervisor

Damjanovski, Sashko

Abstract

Proper cell-cell and cell-extracellular matrix (ECM) interactions are vital for cell migration and patterning of the vertebrate embryo. Matrix metalloproteinases (MMPs) and their inhibitors, reversion-inducing cysteine-rich proteins with Kazal motifs (RECK) and tissue inhibitors of metalloproteinases (TIMPs), are all differentially expressed during embryogenesis to regulate such ECM remodeling events and cell interactions. While TIMPs are a family of 4 secreted proteins that share overlapping substrate specificities of MMPs, RECK is unique in that it is a membrane-anchored MMP inhibitor that is embryonic lethal in mice. I used Xenopus laevis as a model organism to investigate the role of RECK as a regulator of ECM turnover during development. The X. laevis RECK sequence was compared to a breadth of vertebrate and a few invertebrate RECK amino acid sequences. The X. laevis RECK amino acid sequence was found to be highly conserved with other RECK proteins. RECK knockdown in X. laevis embryos resulted in neural tube closure failure and axial defects, in part due to altered mRNA levels of MT1-MMP, MMP-2, and TIMP-2. Upon examination of RECK, MT1-MMP, and TIMP-2 protein localization in different tissues throughout early X. laevis development, I found that all 3 proteins showed highly similar localization patterns, particularly in the dorsal-ventral differentiation of the neural tube. To further investigate RECK regulation of MMP activity in vitro, I used X. laevis A6 cells to knockdown, overexpress, and shed RECK from the cell surface. I demonstrated that changes in RECK levels (overexpression and cell surface shedding) that may reduce the ability of the cell to remodel the ECM are compensated for by increases in MT1-MMP and MMP-2 levels and changes in ERK signaling. Altogether, these results support a role for RECK in the regulation of ECM remodeling and tissue patterning during X. laevis development.


Summary for Lay Audience

All cells secrete or release materials around themselves, allowing tissues to form. In multicellular organisms such as ourselves, some of these materials form a network (the extracellular matrix or ECM) in which the cells anchor themselves to. In adults, the ECM provides stability and a safe environment in which our cells can carry out their functions - muscle cells contract, nerve cells communicate, and so on. But the situation is different in a developing embryo. As animals grow from a fertilized egg, cells need to move. In adults, when cells move, it is often associated with a disease state, such as cancer cells metastasizing. Thus, understanding how cell movement is regulated is essential to both embryogenesis and adults. For cells to move around, the ECM network that surrounds them often has to be broken down and then remade. The molecules involved in this breakdown are called matrix metalloproteinases (MMPs), and one of their roles is to cleave the ECM. Their activity has to be tightly regulated as excessive ECM degradation is detrimental. Therefore, there are 2 other molecules, tissue inhibitors of metalloproteinases (TIMPs) and reversion-inducing cysteine‑rich protein with Kazal motifs (RECK), whose roles are to control MMPs. RECK is a protein found on the surface of cells and stops cells from moving by preventing MMPs from cleaving components of the ECM. RECK has been identified as a crucial player during embryogenesis, though exactly why RECK is essential to development remains unclear. My research examined the role of RECK during frog development. I found that reducing RECK levels in frog embryos caused abnormal development, particularly in the formation of the spinal cord. These defects were in part due to improper ECM degradation. Additionally, I found that RECK is associated with MMPs even after cells migrate, at a time when embryonic cells are beginning to specialize, particularly into nerve cells. Thus, in addition to regulating MMPs when cells are migrating, RECK also plays a role after ECM breakdown to signal to and help cells function. Overall, my study supports the role for RECK as an important regulator of cell movement during embryogenesis.

Download

Share

COinS