Supplementary Figure 8: Premature cell cycle exit and differentiation of SG neurons to EMNs in PROX1-deficient mice. | Nature Neuroscience

Supplementary Figure 8: Premature cell cycle exit and differentiation of SG neurons to EMNs in PROX1-deficient mice.

From: Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control

Supplementary Figure 8

(a) Double immunohistochemistry for PROX1 and PHOX2B on SG sections from E14.5 control and Wnt1-Cre;Prox1f/f (Prox1cKO/cKO) mice. PROX1 is efficiently deleted in all SG neurons of Prox1cKO/cKO embryos, as shown by complete loss of PROX1 immunoreactivity in the mutant. (b) Double immunohistochemistry for PHOX2B and active CASPASE-3 (CASP3) on SG sections from E13.5 control and Prox1cKO/cKO mice. No apoptosis was detected in the SG, based on active CASPASE-3 staining, while some cells were positive in the dorsal root ganglion, which served as positive control tissue (inset). (c) Triple immunohistochemistry for KI67, EdU and PHOX2B on SG sections from E13.5 control and Prox1cKO/cKO mice shows a decreased proportion of proliferating cells in mutant mice. (d) Quantification of number of neurons per ganglion in E14.5 control and Prox1cKO/cKO embryos. (t(112) = 6.426, P < 0.0001, unpaired t test, 1 ganglion = 1 dot; 19 ganglia per animal were plotted, n=3). (e) EdU incorporation in control and Prox1cKO/cKO embryos. E13.5: t(3) = 6.587, P = 0.0071, unpaired t test, n=2 for control and n=3 for Prox1cKO/cKO; E14.5: t(4) = 6.659, P = 0.0026, unpaired t test, n=3; E18.5: t(4) = 5.084, P = 0.0071, unpaired t test, n=3. (f) Proportion of KI67+ neuroblasts in control and Prox1cKO/cKO embryos at E13.5. (t(3) = 5.845, P = 0.0100, unpaired t test, n=2 for control and n=3 for Prox1cKO/cKO). (g) Rate of cell division in control and Prox1cKO/cKO embryos at E13.5 (t(3) = 1.826, P = 0.1653, unpaired t test, n=2 for control and n=3 for Prox1cKO/cKO). All data are presented as mean ± s.e.m.. (h) Double immunohistochemistry for RET and TRKA on SG sections from E14.5 and E18.5 control and Prox1cKO/cKO reveals a massive increase of RET+TRKA+ neurons at both stages. (i) Triple immunohistochemistry for RET, NPY and TH on SG sections from E18.5 control and Prox1cKO/cKO mice. In control, NPY+TH+ neurons are RET-negative while in mutant mice, RET is upregulated in NPY+TH+ (e.g. NEM) neurons (arrowheads and insets). (j-m) Quantifications of (h) and (i). (j) Proportion of TRKA+ neurons at E14.5 and E18.5 in control and Prox1cKO/cKO mice. E14.5: t(9) = 4.552, P = 0.0014, unpaired t test, n=6 for control and n=5 for Prox1cKO/cKO; E18.5: t(4) = 13.12, P = 0.0002, unpaired t test, n=3. (k) Proportion of RET+ neurons at E14.5 and E18.5 in control and Prox1cKO/cKO mice. E14.5: t(9) = 2.366, P = 0.0422, unpaired t test, n=6 for control and n=5 for Prox1cKO/cKO; E18.5: t(4) = 4.525, P = 0.0106, unpaired t test, n=3. (l), TRKA+ soma size at E18.5 in control and Prox1cKO/cKO mice. t(4) = 6.072, P = 0.0037, unpaired t test, n = 3. (m), Proportion of NPY+ cells at E14.5 and E18.5 in control and Prox1cKO/cKO mice. E14.5: t(4) = 0.2445, P = 0.8189, unpaired t test, n=3; E18.5: t(4) = 0.1160, P = 0.9132, unpaired t test, n=3, All data are presented as mean ± s.e.m.). (n‑p) Artificial exposure to growth factors for 24 h induces precocious RET expression in explanted ganglia of Wnt1‑Cre;R26RTOMATO mice. Triple immunohistochemistry for RET (n) or CASPASE-3 (o) and TH and DAPI on SG sections from P1 Wnt1-Cre;R26RTOMATO mice. The number of RET+ but not CASPASE-3+ cells increases after 24h treatment with ARTN or NRTN (F(2, 8) = 2,782, P = 0.1210, one-way analysis of variance, n=4 for QVD and ARTN and n=3 for NRTN, data are presented as mean ± s.e.m.). (a) and (b) are representative images from experiments carried out on at least two animals. Scale bars: 20 μm in (a-c), (h), (i); 10 μm in (n), (o).

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