School of Medicine
69 The Development of Astrocytes in Zebrafish and its Impact on Vanishing White Matter
Sarv Raafati
Faculty Mentor: Joshua Bonkowsky (Pediatrics, University of Utah)
A leukodystrophy is identified as a class of rare genetic diseases that attack the brain, spinal cord, and peripheral nerves, focusing on either the abnormal development or destruction of white matter, i.e., the myelin sheath. This destruction of the sheath leads to the dramatic decline in function of the nerves. Most patients with the disease present symptoms in infancy or early childhood, and can develop severe neurological deficits. One of the most common types of leukodystrophies is Vanishing White Matter disease (VWM), which has no treatment other then supportive care. The Bonkowsky lab has modeled VWM using zebrafish to try and study potential gene therapy treatments. Previous research in the field done by Dooves et al. though linked astrocytes of the central nervous system to VWM showing impaired growth of astrocytes in VWM was linked to disease progression. The aim of this project was to genetically prevent the expression of eIF2B5 within astrocytes and see how they were impaired in their in vivo development as a result. To create this model CRISPER Cas9 Vector was created with a Glast+ promoter in order to target and study the effect of the disease on the astrocytes. This vector was injected into wildtype zebrafish. These fish were then in crossed for two generations. The second generation (F2) was then subjected to a 10-day survival curve before completing an HRMA analysis of their DNA to see if any mutation was present. The survival curve done on the F2 generation fish had only 18 fish out of an original 851 over 10 days die. Comparing the survival curve of the F2 fish to that of that performed previously with Keefe et al’s work, if the mutation of eIF2B5 had been present a larger die off would be expected. This fact is further compounded and proven with the lack of editing shown in the HRMA results from these F2 fish. This indicates that the genetic mutation was not transgenetic and is not a desirable path to continue forward with for a genetic therapy target. Though this line of fish proved to be unfruitful in the attempt to make a successful animal model, future research though can build off the base laid out in this project and serve as a jumping off point for future studies.