Fig 2: scRNAseq and pseudotime analysis of the developing somatosensory system.a UMAP embedding representation of the single-cell RNA sequencing dataset, annotated by embryonic day. b RNA Velocity vectors projected onto the UMAP embedding, indicating differentiation directionality. c Differentiation trajectories inferred in a semi-supervised way on Diffusion space using ElPiGraph, revealing 12 main clusters represented within two main trajectories (branches A and B), 10 branches and 3 bifurcations (1, 2 and 3) on branch A. List of genes is provided in the Source data file. d, e Most significant biological aspects extracted using pagoda2 indicate cell state changes from cycling (Sox10+ and Sox2+) to post-mitotic cells (d) and from non-neuronal to neuronal cells (e) (Isl1+ and Tubb3+). f PC score from gene set related to GO term “positive regulation of cell migration” (GO:0030335) subtracted by the PC score for “negative regulation of cell migration” (GO:0030336) indicates a transition from migratory neural crest progenitors to settling neuronal populations. g UMAP plots of selected genes distributed along the trajectories and among cells states represented in (c–e). h Transcriptomic dynamic during neurogenesis and neuronal specification show that the different states account for the differentiation of sensory sub-classes that can be distinguished based on their specific expression of neurotrophic factors receptor and transcription factors (Ntrk1, Ntrk2, Ntrk3, Ret, Runx1 and Runx3). i E12.5 DRG sections immunostained for markers highlighted in (h) representing major sensory subpopulation at the trajectories endpoints and quantification (n = 3-5). Scale bar, 20 µm. Data are presented as mean values ± SEM. j Hierarchical bifurcation model of NCCs-derived sensory neurons differentiation within Branch A based on our scRNAseq data analysis (color code and cluster identity according to panel h). Mixed color squares reflect the potential fate choice that the lineage retains at the corresponding developmental point.

Fig 3: Activation of TrkC and not of TrkB in A10 neurons. Western blots for phosphorylated Trk protein (P-Trk) and synaptophysin were performed using lysates of A10 neurons (left panel) and H9 neurons (right panel). Left panel: BDNF, TrkB-activating antibody ZEB85 and the TrkB polyclonal antibodies used for Western blot (Figure 1) and immunostaining (Figure 2) all failed to generate any detectable Trk phosphorylation signal in A10 neurons while NT3 did. Right panel: The same three TrkB activators used in the left panel at the same concentrations all induced a robust Trk phosphorylation signal. All treatments were for 1 h

Fig 4: Immunostaining of H9 and A10 neurons. TrkB, TrkC and Tuj1 immunostaining of H9 and A10 neurons. Rabbit 488 and Goat 488 secondary antibody controls were obtained by omitting incubation with the primary antibodies. Note that the goat secondary antibody showed a strong background staining primarily over cell bodies. A10 neurons did not show any staining of neuronal processes with the TrkB antibody. Apparent cell body staining was also seen with the anti-goat secondary antibody only. The mean numbers ± SEM of Tuj1-positive neurons were 72.9 ± 3.1 and 72.8 ± 1.6 for H9 and A10 cells, respectively, 68.9 ± 1.9 and 73.7 ± 2.0 for H9 and A10 neurons stained with TrkC, and 72.7 neurons with TrkB. Twenty random fields were counted in each case using two different coverslips. Low magnification, phase-contrast images of H9 and A10 neurons illustrate examples of neuronal cultures at 30 days and the red arrows point to DAPI-positive dead cells. Scale bar is 20 µm for immunostaining images and 200 µm for phase-contrast images

Fig 5: Differential expression of TRKC in PSNs prior to the cell death period. a Scheme of our working hypothesis. b, c Temporal fate mapping of TRKC PSNs by 4-OHT induction. TrkCCreER mice allow temporary activation of CreER in the TRKC+ cells 2 h after 4-OHT injection21,22. Immunostaining for PV, RFP and RUNX3 on E17.5 DRG sections (c) and graph showing distribution of PV+/RUNX3+ PSNs among the TOM+ cells (n = 4). Scale bar: 20 µm. d Quantification of PSNs at C5 and C7. ***P < 0.001, one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test (n = 2–3). The window of PSNs cell death is shown. e TRKC expression in E11.5 ISL1+ (and RUNX3+, whose staining is not shown for more visibility) DRG neurons. Scale bar: 50 µm. f TRKC levels in PSNs of e illustrated by color coding; dark blue indicates the lower and red the higher TRKC levels. From here, all observations are done at brachial levels (C5–8). g Distribution of TRKC levels in PSNs from e. h Distribution of TRKC levels in PSNs in E11.5 DRG neurons (from g). The data exhibit a Poisson-like distribution (one representative animal), with the mean used to define the two different categories of TRKC intensity (TRKCHigh and TRKCLow). i Projection of seven images of RUNX3+/TRKC+ PSNs from one brachial DRG; dots indicate TRKC-labeled neurons and color codes reveal TRKC intensity as shown in h. j Projection image of smFISH for pan Ntrk3 and Ntrk3 full length (FL) transcripts in E11.5 DRG, visualized at high magnification in (1) and (2) (images show full projection); right panel shows color coding of Ntrk3 FL levels in red; the brighter, the higher levels. k Distribution of the number of Ntrk3 FL molecules in E11.5 DRG neurons by smFISH, normalized to pan Ntrk3 (Ntrk3 FL represent 68% of all Ntrk3 transcripts). l TrkCCreER;R26tdTOM mice were injected at E9.75 with 4-OHT and analyzed at E11.5 (n = 3). m, n Frequency distribution (m) and pie chart (n) of TOM+/TRKC+ neurons from l according to their level of TRKC intensity. Source data are available as a Source Data file