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

Neurons Communicate Through Synapses Cell Body Dendrite Axon Terminal *Synapse* Axon

Circuit Formation is Critical for Proper Nervous System Function B C *Axon Guidance* *Synapse Specificity*

Axon Guidance  Synapse Specificity B C *Axon Guidance* *Synapse Specificity* Axon searches for correct area location Axon establishes connection with partner neuron

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)

Synaptically Coupled A08a and Dbd Neurons Used As Model Neurons Dbd sensory neuron A08a interneuron 10 synapses

Which Dpr and DIP proteins are expressed by the A08a and Dbd neurons?

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

A08a Interneuron Expresses dpr8 and DIP-delta A08a neuron Dpr 8::GFP Colocalization A08a neuron DIP-delta::GFP Colocalization

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

Dbd Sensory Neuron Expresses dpr9 and dpr16 Dbd neuron Dpr9::GFP Colocalization Dbd neuron Dpr16::GFP Colocalization

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

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

Conclusions So far, A08a and dbd neurons do not express Dpr/DIP binding partners Some Dpr/DIP::GFP proteins are weakly expressed

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

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 P1460735rogram in Molecular Biosciences at the University of Oregon: NSF DBI/BIO )

Questions?

Which Dpr and DIP proteins are expressed on synaptically coupled neurons? a08a interneuron axon of dbd sensory neuron

Which Dpr and DIP proteins are expressed by the A08a and dbd neurons? Dbd sensory neuron A08a interneuron A 10 synapses Posterior view

} } } X UAS-RFP ; 26F05-Gal4 Dpr/DIP::GFP Gal4 TF GFP 26F05 Gal4 Dpr or DIP GFP (a08a neuron) } RFP UAS RFP

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

Lack of Co-Localization Indicates Neuron Does Not Express Protein RFP-A08a GFP-dpr13

Lack of Co-Localization Indicates Neuron Does Not Express Protein RFP signals of a08a interneuron Only see background of GFP signal

Determining dbd Expression

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