Progressive confinement of high ERR2 levels to gamma motor neurons during postnatal development depends on muscle spindle-derived signals.

(A-C) Expression of ERR2 in most Isl1+ motor neurons in E10.5 mouse embryo thoracic neural tube (scale bar: 50 μm). (D-F) At P4 higher levels of ERR2 (D) have begun to be mostly confined to small NeuNlow or negligible (E) motor neurons, while many NeuNhigh motor neurons still retain relatively lower yet substantial ERR2 levels (compare D and E). (F) Fluoro-Gold (FG) tracing to visualize motor neurons. (G-H) At P14, high ERR2 levels (G) are further confined to small NeuNlow or negligible motor neurons (H) with high levels of FG incorporation (I). (J-K) At P21, high ERR2 levels (J) are further confined to small NeuNlow or negligible motor neurons (K) with high levels of FG retention (L), while ERR2 levels in NeuNhigh motor neurons have dropped further compared to earlier postnatal stages (compare D, G, and J). (M-Q) Scatter plots of ERR2 (red data points) and ERR3 (green) levels over soma sizes show increasing confinement of high-levels of ERR2 and ERR3 to a population of small motor neurons during postnatal development ages of P4 (n = 100), P14 (n = 189), and P21 (n = 159). (M-R) Scatter plots of ERR2 (red data points) and NeuN (blue) levels over soma sizes show increasing segregation of motor neurons into ERR2high, NeuNlow and ERR2low, NeuNhigh populations during postnatal development, while also revealing considerably cell-to-cell variability in relative expression levels of ERR2, ERR3, and NeuN. (SS’-XX’) Motor neurons fail to retain high ERR2 and ERR3 levels in mice with defective spindle development. (S-X) Control (transversal section of lumbar spinal cord of adult mouse): expression of high ERR2 (V) and ERR3 (W) levels by VACHT+ (X) NeuNlow or negligible (T) motor neurons (closed arrowheads). Open arrowheads: expression of ERR2 and ERR3 by subsets of VACHT− interneurons. Triangle: low ERR2 and ERR3 levels in some large motor neurons. Note: VACHT+ cytosol identifies motor neurons in lamina IX, while VACHT+ varicosities indicate cholinergic synapses or axons stemming from nonmotor neurons. (S’-X’) Egr3ko (Egr3−/−) transversal section of lumbar spinal cord of adult mouse: absence of high ERR2 (V’) and ERR3 (W’) levels in VACHT+ (X’) NeuNlow or negligible (T’) motor neurons. Open arrowheads: retention of ERR2 and ERR3 expression by VACHT− interneurons (compare V, W, and X). Triangles: retention of low ERR2 and ERR3 levels by large motor neurons (compare V, W, and X). n = # of neurons. Data for S4M-S4R Fig can be found under S2 Data. (TIF)