Molecular Characterization

Figure 1. Gene expression levels of Drosophila GABA receptors and voltage-gated calcium channels in the T4/T5 neuronal population. Reads per kilobase per million mapped reads (RPKM) values are plotted on a color scale ranging from 0 to 100.
Figure 2. Drosophila optic lobe at adult stage with GFP-labelled T4/T5 neurons (in green) exclusively innervating lobula plate layer 3. Neuropils are visualized by staining DN-cadherin (in magenta).

We have previously performed RNA sequencing and whole-transcriptome analysis on the T4/T5 neuronal population at adult stage to identify neurotransmitter receptors and ion channels that might underlie the computation of motion direction (Figure 1) (Pankova and Borst, 2016). In order to examine the spatial distribution of candidate neurotransmitter receptors and ion channels along T4 and T5 dendrites, we are currently following several experimental approaches, which include overexpression of fluorescently-tagged proteins (Raghu et al., 2009) and conditional tagging of endogenous genes using CRISPR/cas9-based genome editing (Pankova and Borst, 2017). In addition, we are using loss-of-function approaches to investigate the role of the candidate neurotransmitter receptors and ion channels on the computation of motion direction.

We have also generated transgenic lines that allow genetic access to the different T4/T5 neuron subtypes both during development and at adult stage (Figure 2). We are currently using these transgenic lines to profile the transcriptomes of the distinct T4/T5 neuron subtypes at different developmental stages. As a complementary approach, we are performing single-cell RNA sequencing of T4/T5 neurons. These experiments are helping us to identify candidate genes that might be involved in the emergence of subtype-specific morphological properties in T4/T5 neurons, which will be further tested by loss- and gain-of-function experiments.

References

Pankova K, Borst A (2016) RNA-Seq Transcriptome Analysis of Direction-Selective T4/T5 Neurons in Drosophila. PloS one 11:e0163986.

Pankova K, Borst A (2017) Transgenic line for the identification of cholinergic release sites in Drosophila melanogaster. The Journal of experimental biology 220:1405-1410.

Raghu SV, Joesch M, Sigrist SJ, Borst A, Reiff DF (2009) Synaptic organization of lobula plate tangential cells in Drosophila: Dalpha7 cholinergic receptors. Journal of neurogenetics 23:200-209.

Go to Editor View