Supplementary Figure 4: NA2 and NA4-5 neurons specialize postnatally; PROX1 labels actively proliferating neuroblasts in the SG. | Nature Neuroscience

Supplementary Figure 4: NA2 and NA4-5 neurons specialize postnatally; PROX1 labels actively proliferating neuroblasts in the SG.

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

Supplementary Figure 4

(a) Double immunohistochemistry for RET and TRKA on P3, P5, P6, P7, P8 and P11 SGs from wild-type mice shows emergence of RET+TRKA+ neurons during postnatal development. Quantification of the proportion of TRKA+RET+ neurons in the postnatal SG. TRKA+RET+ but not TRKA-only+ neurons exhibit a selective soma area growth (b, P5 vs P6: t(4) = 17.06, P <0.0001; P6 vs P7: t(4) = 10.40, P = 0.0005; P7 vs P8: t(4) = 7.294, P = 0.0019; P8 vs P11: t(4) = 1.474, P = 0.2145; P11 vs Adult: t(4) = 1.452, P = 0.2202; unpaired t test, n=3) and soma size (c, P5 vs P6: t(4) = 6.384, P = 0.0031; P6 vs P7: t(4) = 6.446, P = 0.0030; P7 vs P8: t(4) = 6.361, P = 0.0031; P8 vs P11: t(4) = 2.166, P = 0.0962; P11 vs Adult: t(4) = 1.456, P = 0.2190; unpaired t test, n=3. All data are presented as mean ± s.e.m.). (d-g) EMNs markers are largely absent before birth. (d) Triple immunohistochemistry for FST, a marker of NA1-NA3 neurons, NPY and TH on SG sections from E18.5 and P11 WT mice. FST+ neurons are increased at P11, compared to E18.5. (e) Quantification of (d): (t(4) = 15.9, P < 0.0001, unpaired t test, n=3. Data are presented as mean ± s.e.m.). (f-g) Double immunohistochemistry for TYRO3, a marker of NA4-5 neurons (f) or CNTN6 (g), a marker of NA4 cells, and TH on SG sections from E18.5 and P11 WT mice. Expression of both markers is absent at E18.5 but present at P11. (h) Triple immunohistochemistry for PROX1, TH and VACHT on SG sections from E12.5 to P11 WT mice. The proportion of PROX1+TH+ neuroblasts decreases during development. (i) Quantification of (h). n = 2-3 per stage, data are presented as mean ± s.e.m.. (j, k) Triple immunohistochemistry for PROX1 and EdU and KI67 (j) or PH3 and ISL1 (k) on SG sections from P0 wild-type mice. Most or all PROX1+ cells were KI67+. PROX1+ cells incorporate EdU and contain PH3, which is consistent with proliferation. (l) Double immunohistochemistry for PROX1 and EdU on SG sections from E12.5, E15.5 and E18.5 wild‑type mice showing that PROX1+ cells incorporate EdU at all analyzed stages. (m) Quantification of (l). n = 2-3 per stage, data are presented as mean ± s.e.m.. (a), (f), (g), (j) (o) and (q) are representative images from experiments carried out on at least two animals. Scale bars: 10 μm in (a); 20 μm in (h), (d), (f), (g) and (j-l).

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