, 2009). Cell counts were performed on 10–20 sections from three to five embryos in each instance using ImageJ software. Details are provided in Supplemental Experimental Procedures. In situ hybridization and immunostaining experiments were carried out as described (Rao and Sockanathan, 2005). Confocal images were acquired with a Zeiss LSM 5 PASCAL microscope. The mGde2 (680 bp) and find more mErr3 (776 bp) in situ probes were generated from the 3′UTR region of each gene. Bright-field images were captured on a Zeiss Axioskop2

microscope. Details of antibodies are provided in the Supplemental Experimental Procedures. TUNEL analysis was carried out using the ApopTag fluorescein in situ apoptosis detection kit (Chemicon S7110). Whole-mount GFP staining was performed as described ( Huber et al., 2005) and eGFP-labeled motor axons were visualized in projections of confocal Z stacks (500–700 μm). Cell-cycle analyses were performed as described (Yan et al., 2009). Briefly, BrdU (100 mg/kg body weight) was injected intraperitoneally (i.p.) into pregnant females 30 min OSI744 and 16 hr prior to embryo harvest for estimation of S phase and cell-cycle exit indices, respectively. To assess S phase, we calculated proliferation index as BrdU+/Ki67+ cells where Ki67 marks all cycling cells, we calculated cell-cycle exit index as BrdU+Ki67−/BrdU+Ki67+,

and we calculated mitotic index as Mpm2+/Ki67+ where Mpm2 marks cells in mitosis (Chenn and Walsh, 2002). 4-OHT injections were performed as described (Badea et al., 2003). Briefly, 4-OHT (Sigma) was dissolved in ethanol

at a concentration of 20 mg/ml and was stored in aliquots at −80°C. Aliquots were emulsified new in five volumes of sunflower seed oil, centrifuged under vacuum to remove the ethanol, and delivered as a single i.p. injection. Motor neuron cell size measurements were performed on Z series confocal projection images of the LMC at L1–L4 levels. Area measurements were performed using the LSM5 Image Examiner software, and distribution histograms were constructed for each animal by grouping cell body cross-sectional areas into 20 μm bins. Average histograms were fit to dual Gaussian distributions using OriginPro8.5 (OriginLab). From the fitted distributions, average cross-sectional area and standard deviation (SD) of the small- and large-size MN populations were estimated. The threshold cutoff size for the small population was estimated as the average (μ) + 2 SD (σ) of the fitted small population distribution in control animals of similar age. We thank Jeremy Dasen, Thomas Jessell, and Ben Novitch for antibodies, Zachary T. Bitzer for technical assistance, Goran Periz for antibody manufacture, members of the Sockanathan laboratory for discussions, and Alex Kolodkin and Ye Yan for comments on the manuscript. This work was funded by grants from the Muscular Dystrophy Association and from the NIH (NINDS RO1NS046336).