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Determining Dpr and DIP Protein Expression in Drosophila Neural Circuits
Rebekah Rashford, UMBC, HHMI, MARC Emily sales, Mentor Doe lab UO, Institute of Neuroscience Summer Program for Undergraduate Research August 4, 2016
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Neurons Communicate Through Synapses
Cell Body Dendrite Axon Terminal *Synapse* Axon
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Circuit Formation is Critical for Proper Nervous System Function
B C *Axon Guidance* *Synapse Specificity*
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Axon Guidance Synapse Specificity
B C *Axon Guidance* *Synapse Specificity* Axon searches for correct area location Axon establishes connection with partner neuron
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Dpr and DIP Families Have Unique Binding Interactions
Dpr Family 12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 DIP-δ DIP-α DIP-β DIP-ζ DIP-ε DIP-γ DIP-ι DIP-θ DIP-η Add references to this slide for in-vitro binding assays and previous work Dpr Interacting Protein (DIP) Family DIP Dpr Carillo et al. (2015) Defective proboscis extension response (Dpr) Dpr-Interacting protein (DIP)
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Synaptically Coupled A08a and Dbd Neurons Used As Model Neurons
Dbd sensory neuron A08a interneuron 10 synapses
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Which Dpr and DIP proteins are expressed by the A08a and Dbd neurons?
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Genetic Tools Allow for the Visualization of Neurons and Dpr and/or DIP Expression
Visualize a08a neuron Visualize Dpr/DIP expression Look for co-localization of a08a and Dpr::GFP
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A08a Interneuron Expresses dpr8 and DIP-delta
A08a neuron Dpr 8::GFP Colocalization A08a neuron DIP-delta::GFP Colocalization
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A08a Expresses dpr8 and DIP-delta
12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 DIP-δ DIP-α DIP-β DIP-ζ DIP-ε DIP-γ DIP-ι DIP-θ DIP-η Dpr Family Dpr Interacting Protein (DIP) Family
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Dbd Sensory Neuron Expresses dpr9 and dpr16
Dbd neuron Dpr9::GFP Colocalization Dbd neuron Dpr16::GFP Colocalization
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Dbd Expresses dpr9 and dpr16
12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 DIP-δ DIP-α DIP-β DIP-ζ DIP-ε DIP-γ DIP-ι DIP-θ DIP-η Dpr Family Dpr Interacting Protein (DIP) Family
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Conclusions So far, A08a and dbd neurons do not express Dpr/DIP binding partners 12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 DIP-δ DIP-α DIP-β DIP-ζ DIP-ε DIP-γ DIP-ι DIP-θ DIP-η A08a Dbd 12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 DIP-δ DIP-α DIP-β DIP-ζ DIP-ε DIP-γ DIP-ι DIP-θ DIP-η
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Conclusions So far, A08a and dbd neurons do not express Dpr/DIP binding partners Some Dpr/DIP::GFP proteins are weakly expressed
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Future Directions Finish determining Dpr/DIP expression in a08a and dbd neurons Test function of Dpr and DIP proteins using RNAi to knockdown expression and cDNA to misexpress Dpr and DIP proteins
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Thank you! SPUR Peter O’Day Marilyn Drennan HHMI MARC/UMBC Dr. Doe
Emily Sales Matt Clark Pat & Casey Doe Lab SPUR Peter O’Day Marilyn Drennan HHMI MARC/UMBC NSF REU Site P rogram in Molecular Biosciences at the University of Oregon: NSF DBI/BIO )
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Questions?
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Which Dpr and DIP proteins are expressed on synaptically coupled neurons?
a08a interneuron axon of dbd sensory neuron
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Which Dpr and DIP proteins are expressed by the A08a and dbd neurons?
Dbd sensory neuron A08a interneuron A 10 synapses Posterior view
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} } } X UAS-RFP ; 26F05-Gal4 Dpr/DIP::GFP Gal4 TF GFP 26F05 Gal4
Dpr or DIP GFP (a08a neuron) } RFP UAS RFP
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Dpr DIP η θ ι γ 12 10 6 8 9 21 11 13 16 19 20 14 17 15 1 2 3 4 5 7 ε In order to determine if a Dpr or DIP protein is expressed, co-localization of the two signals must be observed. ζ β α δ
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Lack of Co-Localization Indicates Neuron Does Not Express Protein
RFP-A08a GFP-dpr13
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Lack of Co-Localization Indicates Neuron Does Not Express Protein
RFP signals of a08a interneuron Only see background of GFP signal
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Determining dbd Expression
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Genetic Tools Allow for the Visualization of Neurons and Dpr and/or DIP Expression
Visualize a08a neuron Visualize Dpr/DIP expression 26F05 Gal4 (a08a neuron) Dpr or DIP GFP UAS RFP Look for co-localization of a08a and Dpr::GFP
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