Characterizing endocannabinoid system development in zebrafish and investigating cannabidiol-mediated downregulation of the Sonic Hedgehog Pathway

  • Author / Creator
    Son, Hae-Won
  • Cannabis is the most frequently used recreational drug during pregnancy. Nearly 4% of American women self-reported use during their pregnancies in 2014, which represents a 62% increase from 2002. The compounds in cannabis are mediated through interactions with the endocannabinoid system (ECS), which is well-studied for its role in synaptic plasticity and regulation of neural function, however, recent evidence suggests that the ECS is also responsible for regulating aspects of early development. Additionally, phytocannabinoids, such as cannabidiol (CBD), have been proposed to modulate signaling pathways outside of the ECS, including the Sonic Hedgehog (SHH) Pathway. As phytocannabinoids have the ability to pass freely through the placenta, it is critical to investigate the role of the ECS during development, as well as mechanisms of phytocannabinoid mediated-teratogenicity. In this thesis, two objectives were chosen to address both areas of research: 1) to characterize the expression of the ECS throughout early zebrafish development, and 2) identifying and assessing phytocannabinoid mediated downregulation of the Sonic Hedgehog (SHH) pathway. The ECS contains numerous receptors that bind cannabinoid ligands. Once bound, a response occurs in the cell that mediates a myriad of physiological responses, which may include developmental decisions. Despite these receptors playing a major role in controlling the activity of the ECS, little is known about spatial and temporal expression of these receptors during early development. Combining reverse-transcriptase PCR with in situ hybridizations, I compiled a developmental timeline of six key cannabinoid receptors; cb1, cb2, trpv1, trpa1a, trpa1b, and gpr55 to demonstrate spatial, temporal, and semi-quantitative expression of each receptor from the first six hours to three days of zebrafish development, which is roughly equivalent to two to ten weeks in human embryonic development. This timeline aims to provides a foundation at which further investigation of each receptor can be undertaken.
    Secondly, exposure of zebrafish embryos to cannabidiol (CBD) during the period of gastrulation has been shown to cause deformities to axial curvature, reduced body length, cardiac and yolk-sac edema, altered motor neuron development, and reduction in survival and hatching rates. Evidence suggests that cannabinoids are able to bind to, and decrease the activity of Smoothened (SMO), a receptor within the SHH pathway, which may be responsible for toxic and teratogenic effects that were observed following phytocannabinoid exposure. Co-exposing zebrafish embryos to both CBD and a known SMO agonist resulted in improvement of parameters including mortality and hatching rate, gross morphology, free-swimming locomotion, and expression levels of ptch2, a key regulatory receptor of the SHH pathway. Moreover, exposure cyclopamine, a known antagonist of SMO, resulted in similar morphological abnormalities compared to CBD, verifying that SMO downregulation is able to cause the teratogenic effects described previously.
    Taken together, this work provides a comprehensive overview of ECS expression during development, and is the first evidence of CBD exerting detrimental developmental effects through the SHH pathway in zebrafish.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.