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 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 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.

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.

Figure 2. Expression of Rdl (encoding a GABA receptor) and cac (encoding a voltage-gated calcium channel subunit) in the four T4 and T5 neuron subtypes (a,b,c,d) during pupal development. X axis shows developmental stage (hours after puparium formation). Y axis shows the count of transcripts per cell (mean ± SEM). — **Figure 2**. Expression of *Rdl* (encoding a GABA receptor) and *cac* (encoding a voltage-gated calcium channel subunit) in the four T4 and T5 neuron subtypes (a,b,c,d) during pupal development. X axis shows developmental stage (hours after puparium formation). Y axis shows the count of transcripts per cell (mean ± SEM).

**Figure 2**. Expression of *Rdl* (encoding a GABA receptor) and *cac* (encoding a voltage-gated calcium channel subunit) in the four T4 and T5 neuron subtypes (a,b,c,d) during pupal development. X axis shows developmental stage (hours after puparium formation). Y axis shows the count of transcripts per cell (mean ± SEM).

In addition, our lab has recently performed single-cell RNA sequencing of T4/T5 neurons at five different stages of pupal development (Hoermann et al., 2020). The analysis of this dataset revealed that each of the four anatomically distinct T4/T5 subtypes has a unique transcriptional profile during development. In particular, we found unique combinations of transcription factors, as well as unique combinations of cell-membrane proteins defining each T4/T5 subtype, and whose roles in the development of subtype-specific morphologies are under current investigation. Furthermore, our single-cell RNA sequencing of T4/T5 neurons provides a valuable resource for studying other interesting aspects of neuronal development, such as the molecular mechanisms of synaptic specificity, or the mechanisms regulating the expression of genes that influence the functional properties of T4/T5 neurons (Figure 2).

Sequencing data available

RNA sequencing datasets are available from the NCBI GEO archive under accession codes GSE77198 and GSE147987.

References

Hoermann N, Schilling T, Haji Ali A, Serbe E, Mayer C, Borst A, Pujol Martí J (2020) A combinatorial code of transcription factors specifies subtypes of visual motion-sensing neurons in Drosophila. Development, online 3. April 2020. DOI.

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.