Spring 2016 BNFO 300 PTK7 protein domains involvement in PCP regulation of axon pathfinding of CoPA neurons in Zebrafish. Damien Islek:

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Spring 2016 BNFO 300 PTK7 protein domains involvement in PCP regulation of axon pathfinding of CoPA neurons in Zebrafish. Damien Islek:

The Human Brain: What makes us tick?  Understanding how the human nervous system makes connections during development is crucial to understanding the way the brain works and how we perceive external stimuli. brain-from-stem-cells/

 CoPA neuron in zebrafish  Wild Type 100% of CoPA axons travel anterior after crossing the midline.  Axon migration is dependent on Wnt mediated PCP pathway (Hayes et al 2013). Zebrafish primary commissural ascending neurons (CoPA)

Wnt mediated Planar Cell Polarity Pathway: Core Proteins Main action in this pathway is Wnt binding to Frizzled. In Lu et al. PTK-7 was found to be a novel regulator of this pathway in mice. Hayes et al reported in 2013 that mutant ptk7 zebrafish expressed similar defects as PCP core protein (Fz/Vang) gene mutants.

 Which domain of PTK7 is involved in regulating the PCP pathway which mediates CoPA neuron axon pathfinding in zebrafish? Experimental Question

 What domains of PTK7 are important for regulating the direction of axon growth in zebrafish CoPA neurons. My Experiment: PTK7 Rescue Start with zebrafish mutant TBX16. Express GFP in Copa neurons Microinject plasmid and mRNA of Tol2 transposon Grow zebrafish Observe direction of axon migration of neurons which express GFP and M- Cherry Use Zinc-Finger Nucelase technology to knockout PTK7 gene from TBX16 zebrafish Bread zebrafish generation of ptk7 mutant zebrafish Kawakami 2007

 In mutant PCP zebrafish axons randomly travel anterior or posterior (50/50 Anterior/Posterior)  If Rescue is successful, theoretically, all CoPA neurons which display Red and Green will have axons which travel anterior.  If this function can be rescued without a certain domain, then that domain must not be required for regulation of this pathway for correct axon migration. What are we looking for? AP

 PTK7 Full Length  Ptk7ΔICD  Ptk7EgfrTM  Ptk7ΔECD Experimental Groups: TBX16 Zebrafish: Express Green Fluorescent Protein in CoPA Neurons.

Plasmid Construction: Gateway Cloning cDNA PTK7 “ Step 1: PCR amplify cDNA of ptk7 with attB containing primers. Step 2: Transfer cDNA into entry vector through recombination BP Clonase Isolate the entry vector Transfer cDNA to destination vector through recombination LR Clonase Isolate the destination vector

Plasmids: Tol2 HSP-70 Ptk7 cDNA- M Cherry pA Tol2 HSP-70 Ptk7ΔICD- M Cherry pA Tol2 HSP-70 Ptk7ΔECD – M Cherry pA Tol2 HSP-70 Ptk7EgfrTM – M Cherry pA Tol2 Full Length PTK7 Deleted Intracellular Domain Deleted Extracellular Domain Substituted Trans- membrane Domain

Ptk7 mutant + hsp70: ptk7 FL -mCherry Prediction:

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