Figure 4. The Conventional Marker Gene Chx10 Labels V2a Types along the Rostrocaudal Axis that Emerges during Embryonic Development.

(A) Chx10 immunostaining in Chx10:Cre; tdTomato animals at P1. In cervical segments, a subset of Tom+ V2a interneurons lacked Chx10 immunostaining (dashed circles). At lumbar levels Chx10 was detected in most Tom+ V2a interneurons. Among the Chx10+ V2a interneurons, we observed signal intensity differences (asterisks indicate low Chx10). 20-μm cryosections are shown. Scale bar, 50 μm.

(B) Quantification of type I (Chx10+) and type II (Chx10−) V2a IN numbers along the rostrocaudal axis (n = 6 animals).

(C) Spatial distribution of type I (Chx10+) and type II (Chx10−) V2a interneurons in cervical and lumbar segments (n = 6 animals). Contour maps revealed medial bias for type I relative to type II.

(D) Time course of Chx10 protein expression. Chx10 immunostaining was conducted from embryonic day (E)11.5–E14.5 and P70 animals. 12-μm cryosections at E11.5 and E14.5 and 20-μm cryosections at P70 are shown. Scale bars, 50 μm at E11.5 and 14.5 and 100 μm at P70.

(E) Ratio of Chx10+ V2a interneurons along the rostrocaudal axis. Since the lengths of the spinal cords are different at different developmental stages, Foxp1 immunostaining was conducted to identify brachial/cervical and lumbar segments (data not shown).

(F and G) EdU was injected into E10.5–E13.5 pregnant females, respectively, and spinal cords were collected at P0 to identify V2a interneuron birth dates (F). Type II V2a interneurons were born at an earlier time point (G). One-way ANOVA with post hoc Dunnett’s test, E10.5 versus E11.5, **p < 0.01 and E10.5 versus E12.5, ***p < 0.001. 20-μm cryosections are shown. Scale bar, 50 μm.

(H) Summary of V2a diversification. Type I and type II interneurons emerge progressively from immature postmitotic V2a interneurons between E12.5 and E14.5, and the diversification is maintained into adulthood.