Generation of a Zebrafish Transgenic Line for Manipulating Notochord Patterning

Generation of a Zebrafish Transgenic Line for Manipulating Notochord Patterning

Meghana and graduate student mentor in lab

For the past two and a half years, I have been studying zebrafish notochord segmentation in the Bagnat lab. After taking Biology 201 in the fall semester of my sophomore year, I became fascinated with molecular biology and genetic engineering technology. The Bagnat lab, which specializes in zebrafish spine and gut formation, caught my attention due to its groundbreaking research in notochord and spine segmentation and its applicability to humans. In particular, I was interested in engineering new genetic tools that would aid in the discovery of cellular and molecular mechanisms behind segmentation. Therefore, my research culminated in the generation of a novel transgenic line of zebrafish that allows us to specifically manipulate a particular cell population in the notochord. Essentially, the notochord is an embryonic tissue that acts as the scaffold for the spine in all vertebrates. In the zebrafish notochord, the outer cell layer, or sheath, segments into alternating domains that instruct the formation of spinal intervertebral discs (IVDs) and vertebral bodies. Mature IVDs express a gene called col9a2 while the vertebral bodies overlay sheath cell segments expressing another gene called entpd5a. Thus, the notochord plays a crucial role in the patterning of the spine. To understand segmentation, my lab had previously generated a transgenic line of zebrafish, col9a2-QF2, that allows us to manipulate gene expression specifically in the col9a2+ cells using the Q transcriptional regulatory system. However, since col9a2 expression becomes confined to only the IVDs in mature fish, we were unable to do the same in entpd5a+ cells. Therefore, I generated a complementary entpd5a-QF2 transgenic line that would allow us to manipulate gene expression in entpd5a+ cells. To do this, I designed a recombinant plasmid with the QF2 sequence downstream of the entpd5a gene and used CRISPR/Cas9 to insert it into the genome of zebrafish embryos. I then selected fish with successful integration of the plasmid using fluorescent and confocal imaging, and I was eventually able to identify fish with the stably expressing, inheritable entpd5a-QF2 gene. Overall, this transgenic line opens the door to a plethora of experiments that will allow us to investigate patterning mechanisms and help us understand the role of the notochord in spine malformations. While successfully generating this line was a huge accomplishment for me, I’m more proud of how much I’ve grown as a curious researcher and a resilient person throughout my time in my lab. Generating my plasmid was a process marked by trial and error - sometimes the ligation into the plasmid failed, the transformation was unsuccessful, or the CRISPR injection didn’t work. While the repeated failures were often frustrating, I knew I had to analyze each one, redesign my approach, and try again. I learned to be patient and persistent with my research; science is messy, and you have to quickly adapt and stay positive throughout the process. Finally, my passion for this project pushed me to write a thesis for Graduation with Distinction in Biology. I constantly wrote and rewrote my paper to explain terminology in depth and make it accessible to the broader biology audience. This process has shown me that you must intimately understand a subject in order to explain it in its simplest form, and it helped me hone my writing skills. Overall, my research has taught me to think creatively and communicate effectively, and I’m so grateful for this experience that has shaped my time at Duke.

Images: Meghana and her mentor in lab, and Meghana at her thesis defense