Fig 2: Temporal clonal analysis of single Sma+ cardiomyocytes during embryonic stages.a Scheme illustrating the genetic clonal tracing strategy using R26R-Confetti multicolor reporter mouse line crossed with tamoxifen-inducible Sma-CreERT2 mice. b Time course of 4-hydroxytamoxifen (4’OH-Tam) injections and analyses of independent unicolor clones represented on a time scale. Low doses of 4'OH induce low-frequency recombination. c 3D reconstructions of conductive and mixed clones induced at E7.75 and observed within Sma-CreERT2::R26R-Confetti opened-left ventricles at P21 of control (Ctrl) and Nkx2-5+/- left ventricle. Whole-mount immunostaining for Contactin-2 (CNTN2) is used to distinguish conductive cells (CNTN2+) as indicate by arrowheads. d Conductive versus mixed clones frequency evolution upon time-course inductions quantified in P21 control (Ctrl) and Nkx2-5+/- left ventricle. The progressive decrease of mixed clones frequency over time illustrates the kinetics of Sma+ cardiac progenitors segregation. e Diagram of the relative frequency of mixed clone sizes induced at E8.5 in P21 control and Nkx2-5+/- left ventricles. P value is derived from non-parametric Mann–Whitney t-test, **p < 0.01.

Fig 3: Dorsal commissural precrossing neurons express Nrp2 and PlxnA1, and their axons are responsive to Sema3B inhibition in a dosage-dependent manner. (A) Representative confocal images of a transverse wild-type mouse spinal cord section at E11.5 labeled with antibodies against PlxnA1 (blue) (Yoshida et al. 2006), Nrp2 (red), and TAG1 (green). White arrowheads illustrate the coexpression of PlxnA1, Nrp2, and TAG1 on precrossing axons in the dorsal spinal cord. Bar, 125 µm. (A') Higher-magnification images of the same spinal cord section shown in A illustrate colabeling of PlxnA1, Nrp2, and TAG1 in the ventral spinal cord and the ventral commissure. (FP) Floor plate. Bar, 75 µm. PlxnA1 antibody specificity is shown in Supplemental Figure 1. (B) Wild-type E10.5 spinal cord transverse section double labeled with antibodies against mouse PlxnA1 (left panel) and Nrp2 (middle panel) and merged (right panel). Bar, 60 µm. (C) Wild-type E12.5 spinal cord transverse section double labeled with antibodies against mouse PlxnA1 (left panel) and Nrp2 (middle panel) and merged (right panel). Bar, 250 µm. (D) E11.5 transverse spinal cord sections taken from wild-type and NrCAM-null animals and processed for immunolabeling against PlxnA1 and Nrp2. Dorsal commissural neuron cell bodies and their precrossing axons are labeled with white arrowheads and white arrows, respectively. Bar, 125 µm. (E) Schematic diagram illustrating the microdissection of E11 dorsal spinal cord explants for culture and analyzing precrossing axon outgrowth. (F) Representative dorsal spinal cord explant cultures challenged with increasing levels of Sema3B proteins. (G) Axonal outgrowth from dorsal spinal explants were normalized and are shown in percentages with respect to the untreated (100 ± 11.45) cultures. A decrease of >30% (67.54 ± 5.29), 95% (4.97 ± 1.22), and 100% (0.63 ± 0.02) in outgrowth was observed with 0.5 µg/mL, 1.0 µg/mL, and 2.0 µg/mL Sema3B, respectively. (H) Normalized axonal outgrowth of dorsal commissural precrossing axons treated with Sema3A and Sema3F at 1 µg/mL concentration. The graph shows percentages normalized to untreated controls (100 ± 10.55), Sema3A (113.6 ± 4.12), and Sema3F (125.6 ± 6.29). Data represent compiled means ± SEM from n = 4 independent explant cultures. Analysis of variance (ANOVA) followed by post-hoc Tukey test, (*) P < 0.05 compared with 0 µg/mL; (#) P < 0.05 compared with 0.5 µg/mL of Sema3B; (n.s.) not significant.

Fig 4: Early segregation of the central ventricular conduction system lineage in SMA+ cardiomyocytes. (A,A’) Whole-mount ventral and dorsal views of an E14.5 SmaCre/+::R26Rconfetti/+ heart with multicolor clusters induced by tamoxifen injection at E7.5. The four fluorescent reporters (GFP, YFP, RFP, and CFP) form independent clusters distributed side by side along the vertical axis in the apical interventricular region. (B) Section of an E14.5 SmaCre/+::R26Rconfetti/+ embryonic heart showing large vertical compact multicolor clones in the apical region of the IVS and scattered labeled cells in the upper part of the septum. (C) Hcn4 immunofluorescence on section of E14.5 SmaCre/+::R26Rconfetti/+ heart showing a heterogeneous contribution of labeled cells to the AVB and BB. (D) Example of clonal labeling in a P7 SmaCre/+::R26Rconfetti/+ heart with a small number of labeled clusters of which one RFP+ cluster is localized at the crest of the septum (arrow). Individual fluorescent reporters are presented in D’ (RFP), D’’ (GFP/YFP) and D’’’ (CFP). (E,G) Cntn2 immunofluorescence on sections from P7 SmaCre/+::R26Rconfetti/+ hearts to classify the nature these clusters. All cells in working clusters are negative for Cntn2 (E), few are positive in mixed clusters (E’) and all are positive for Cntn2 in conducting clusters (E’’). The distribution of these clusters is presented in the graph in the right panel (F). (G,G’,G”) Sections through independent conducting clones of different colors (YFP in G; RFP in G’ or GFP in G’’) showing co-labeling with Cntn2 in the AVB. (H) Table showing the distribution of mixed and conducting clones in different compartments of the central VCS.

Fig 5: Parallel fiber growth was enhanced in both Ythdf1 and Ythdf2 cKO mice. A,B) Representative images of A) YTHDF1 and B) YTHDF2 immunostaining in P15 cerebellum of A) Ythdf1 and B) Ythdf2 cKO, respectively. YTHDF1 or YTHDF2 was successfully eliminated in GCs while their expression in PCs was not affected. Scale bars represent 500 µm. C,D) Lengths of parallel fibers labeled by DiI were significantly increased in Ythdf1 and Ythdf2 cKO mice. The white arrowheads indicate the terminals of DiI-labeled PFs. Scale bars represent 100 µm. E,F) Quantification of parallel fiber (PF) lengths in (C) and (D). Data are expressed as box and whisker plots. In (E), ****p = 1.38E-05; for Ythdf1fl/fl mice, n = 36 confocal fields from 11 pups, for Ythdf1 cKO mice, n = 42 confocal fields from 11 pups. In (F), ****p = 2.29E-05; for Ythdf2fl/fl mice, n = 46 confocal fields from 13 pups, for Ythdf2 cKO mice, n = 43 confocal fields from 12 pups. All by unpaired Student's t test. G,H) Significantly higher Tag1 IF in the deep layer of cerebellar EGL of Ythdf1 and Ythdf2 cKO mice was detected. Scale bars represent 40 µm. I,J) Quantification of Tag1 IF intensity signals in (G) and (H). Data are expressed as box and whisker plots. In (I), ****p = 2.80E-06; n = 18 confocal fields for Ythdf1fl/fl mice, n = 15 confocal fields for Ythdf1 cKO mice. In (J), ****p = 2.02E-12; n = 26 confocal fields for Ythdf2fl/fl mice, n = 28 confocal fields for Ythdf2 cKO mice. All by unpaired Student's t test.