Skip to main content
. 2021 Feb 15;12:1026. doi: 10.1038/s41467-021-21173-9

Fig. 5. Emergence of PN subtypes during development.

Fig. 5

a Three molecularly distinct clusters of E16.5 PNs shown by tSNE. b Dot plot illustrating examples of marker genes in each cluster. c In vivo validation of the three E16.5 PN populations by RNAscope using the identified markers (magenta) and PN markers Pvalb and Runx3. Scale bar: 20 µm. d Strategy to retrogradely label Ia-PNs in E16.5 embryos. e Left: transverse section of spinal cord 1 h post-injection stained for PV and CHAT, showing the injection site in the motor neurons area. Right: transverse section of spinal cord over night (ON) post-injection stained for PV and CHAT, showing the retrogradely labeled pre-motor spinal interneurons. Scale bars: 100 µm. f DRG section from embryo 6 h post-injection stained for PN markers PV and RUNX3, showing the retrogradely labeled Ia-PNs. Scale bar: 50 µm. Scale bar of the micrograph: 20 µm. g Retrogradely labeled DRG section stained for PN marker RUNX3 (immunostaining) and subtype marker Tnfaip8l3 (RNAscope). Right: Proportion of the three PN populations among all PNs (Pvalb+/Runx3+) and retrogradely traced PNs from experiment in d (n = 4 embryos for Tnfaipl3 and Doc2b, n = 3 embryos for Vstm2b). Scale bar: 20 µm. Scale bar of the micrograph: 20 µm. h Strategy to genetically trace the Doc2b+ PNs lineage (TMP: trimethoprim). i P8 DRG section from Doc2bddCre;Ai14 mice injected with TMP at E16.5. The sections were stained for PN marker PV and postnatal Ib-PN marker BRN3C. The staining of BRN3C in gray scale is pseudo-coloring from blue. Scale bar: 20 µm. Right: proportion of Ia/II-PNs and Ib-PNs labeled by RFP, respectively, presenting enriched tracing of Ib-PNs (n = 2 animals); dots represent values from individual animals. j Spatial distribution of Doc2b+ PNs in representative DRG of E16.5 embryos. Data are presented as mean ± SEM (n = 3 animals). Data for Th. and Lum. regions represent an average of the proportion observed in segments T2-T12 and L1-L5, respectively. k Correspondence between PN types and the clusters identified by scRNAseq at E16.5. l tSNE of PNs depicting molecularly distinct clusters at P5. m Box and whisker plots showing the marker expression of 4 II-PN subtypes at P5, while Ia-PN subtypes were indistinguishable by their subtype markers at this stage (e.g., Runx1, Calb1). Lower and upper hinges: first and third quartiles; the horizontal line: median; the whiskers extend to the value no further than 1.5 * IQR from the hinge; large dots: outliers. For the box plots, Cl.3 (n = 108), Cl.4 (n = 96), Cl.5 (n = 14), Cl.6 (n = 11). n Correspondence between PN types and the clusters identified by scRNAseq at P5. Source data are provided as a Source Data file.