RNA Profiling of FAC-Sorted Neurons From the Developing Zebrafish Spinal Cord.

In this report, we describe a successful protocol for isolating and expression-profiling live fluorescent- protein-labelled neurons from zebrafish embryos. As a proof-of-principle for this method, we FAC-sorted and RNA-profiled GFP-labelled spinal CiA interneurons and compared the expression profile of these cells to those of post-mitotic spinal neurons in general and to all trunk cells. We show that RNA of sufficient quality and quantity to uncover both expected and novel transcription profiles via Affymetrix microarray analysis can be extracted from 5,700 to 20,000 FAC-sorted cells. As part of this study, we also further confirm the genetic homology of mammalian and zebrafish V1 interneurons, by demonstrating that zebrafish V1 cells (CiAs) express genes that encode for the transcription factors Lhx1a and Lhx5. This protocol for dissociating, sorting and RNA-profiling neurons from organogenesis-stage zebrafish embryos should also be applicable to other developing organs and tissues and potentially other model organisms. Fifteen samples were analysed in total, all at 27 hours post fertilization (hpf). Five biological replicates were performed for all spinal neurons (isolated from Tg(elavl3:EGFP)), six biological replicates were performed for V1 (CiA) spinal interneurons (isolated from Tg(pax2a:GFP)), and four biological replicates were performed for all trunk cells (FAC-sorted trunk cells).