Fig. 2: Validation of scRNA-seq data identifies ret+ neurons as pioneers.
From: Axon targeting of transcriptionally distinct pioneer neurons is regulated by retinoic acid signaling
A Schematic of a 30 hpf embryo, a stage utilized for FISH. Membranes are labeled by cldnB:memgfp transgene (green). Created in BioRender. Lab, N. (2025) https://BioRender.com/mdz3df6. B 3D reconstruction of individual pLL neurons by Imaris using membrane-tagged cldnB:memgfp fluorescence. Subsequently, binned fluorescent puncta are counted in each individual cell. C–E Representative single Z-slice confocal images through the pLL ganglion showing pairwise FISH of two genes from ret+ and ret- subclusters (ret and rpz5, nr2f2 and rpz5, or nr2f2 and ntrk3a) at 30 hpf. Note that genes from the same clusters are coexpressed, whereas genes from the ret+ and ret- subclusters are largely expressed in different cells. F–H Quantification of gene expression shown in 2C–E. Each dot corresponds to a single cell and the axes indicate binned gene expression levels. Each plot shows all cells across 10 embryos. Gene expression within the same pLL subcluster is strongly correlated, whereas gene expression between different pLL subclusters is not. I Heatmap shows pairwise Kendall tau-beta gene correlation coefficients based on examining expression of gene pairs by FISH. Empty squares represent untested probe combinations. J Z-projection of rpz5:mRuby pLLg. Cell membranes are marked by cldnB:memgfp transgene. Note that only a subset of dorsal pLL neurons is mRuby-positive at 40 hpf (n = 6). K Confocal image of the migrating pLL primordium from the same animal shown in (J). Note the presence of mRuby-positive axons embedded within the primordium (dashed outline) indicating that labeled pLL cells are indeed pioneer neurons. mRuby labeling dorsal to pLL primordium is muscle. All scale bars = 10 μm. All images scaled the same.
