A simple smiFISH pipeline to quantify mRNA at the single-cell level in 3D.

Techniques allowing the precise quantification of mRNA at the cellular level are essential for understanding biological processes. Here, we present a semi-automated smiFISH (single-molecule inexpensive FISH) pipeline enabling quantification of mRNA in a small number of cells (∼40) in fixed whole mount tissue. We describe steps for sample preparation, hybridization, image acquisition, cell segmentation, and mRNA quantification. Although the protocol was developed in Drosophila, it can be optimized for use in other organisms. For complete details on the use and execution of this protocol, please refer to Guan et al.1.

Post-transcriptional regulation of transcription factor codes in immature neurons drives neuronal diversity.

How the vast array of neuronal diversity is generated remains an unsolved problem. Here, we investigate how 29 morphologically distinct leg motoneurons are generated from a single stem cell in Drosophila. We identify 19 transcription factor (TF) codes expressed in immature motoneurons just before their morphological differentiation. Using genetic manipulations and a computational tool, we demonstrate that the TF codes are progressively established in immature motoneurons according to their birth order. Comparing RNA and protein expression patterns of multiple TFs reveals that post-transcriptional regulation plays an essential role in shaping these TF codes. Two RNA-binding proteins, Imp and Syp, expressed in opposing gradients in immature motoneurons, control the translation of multiple TFs. The varying sensitivity of TF mRNAs to the opposing gradients of Imp and Syp in immature motoneurons decrypts these gradients into distinct TF codes, establishing the connectome between motoneuron axons and their target muscles.