Honors Capstone Project
Date of Submission
Dr. Katharine Lewis
Dr. Melissa Pepling
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Biology | Marine Biology
Located in the vertebral column, the human spine is responsible for regulating body movements and receiving sensory input about pain and touch. Currently, few treatments for neurological diseases and spinal cord injuries exist, partly because we know little about how a fully functioning spinal cord is constructed. As such, studying spinal cord development, specifically neuronal specification and patterning, should be useful for developing better treatments for people with spinal cord injuries and diseases. Zebrafish are a prime model organism for studying neuronal specification because their transparent embryos develop outside the mother, allowing us to easily examine gene expression, cell movements and cell morphology during development . Furthermore, the zebrafish spinal cord has few types of interneurons compared to mammals, and each interneuron type can be recognized by its distinct morphology.
I focused on V1 cells, which form in the ventral spinal cord and are functionally similar in all vertebrates. In zebrafish, V1 cells develop into CiAs, or Cicumferential Ascending interneurons, which control movement and sensory gating. Several transcription factors are expressed consistently in all vertebrate V1 cells, and my research focused on Lhx1a, Lhx1b and Lhx5. As a result of findings in mice, I predicted that knocking down these transcription factors would result in neurotransmitter deficits and potentially compromise movement ability. Additionally, a main focus of my project became assaying various experimental strategies for knocking-down Lhx1a, Lhx1b and Lhx5. These included injecting reagents into 1-4 cell stage embryos and taking advantage of a lhx1b mutant fish line. For the injections, I used morpholinos (MOs), antisense agents that either interfere with RNA transcription to protein or with RNA splicing. I also used RNA constructs which should act as either dominant activators or dominant repressors. Using in situ hybridization, I then tried to assess the impact on neurotransmitters throughout the spinal cord and specifically within CiAs.
I successfully identified a PCR and restriction enzyme digest method for identifying lhx1b mutants. This was exciting as it was a completely novel method for identifying these fish. Furthermore, my results demonstrated that homozygous lhx1b mutants are viable which was previously unknown. My injection results demonstrate that lhx1b and lhx5 splice-blocking MOs are also an effective tool for knocking-down the function of these two genes. In contrast, the lhx1a MOs that I tried were not effective. My RNA injection results were inconclusive and I determined that a higher concentration was probably needed to impact Lhx1 and Lhx5 function.
Balakirsky, Samantha, "Investigating the Roles of Lhx1a, Lhx1b, and Lhx5 in Zebrafish Spinal Cord Interneuron Specification and Function" (2012). Honors Capstone Projects - All. 135.
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