Figure 5. Abundance, differentiation, and apoptosis of motor neurons in Fz3^−/− embryos, and the effects of suppressing apoptosis.

(A) Diagram showing the pattern of transcription factor expression in LMC_L and LMC_M motor neurons. SC, spinal cord; D, dorsal; V, ventral. (B and B′) Islet1 and Foxp1 expression in spinal motor neurons in cross sections of E11.5 Fz3^+/− and Fz3^−/− lumbar spinal cords. Continuous white line, lateral edge of the spinal cord. The broken white line encircles LMC_M motor neurons. L, lateral; M, medial. Scale bar, 100 µm. (C and C′) The number of LMC_L (Islet1⁻/Foxp1⁺) and LMC_M (Islet1⁺/Foxp1⁺) motor neurons per 14 µm frozen section in E12.5 Fz3^+/− and Fz3^−/− lumbar spinal cords. Motor neurons were counted and averaged from 12 serial sections from each of three pairs of embryos, with adjacent counted sections separated by four uncounted sections. (D–E′′) Motor neuron apoptosis visualized with Islet1/2 and cleaved Caspase3 immunostaining in cross sections of E11.5 Fz3^+/− (arrows) and Fz3^−/− (arrowheads) lumbar spinal cords. Scale bar, 500 µm. (F–G′′) Reduced LMC volume in the Fz3^−/− lumbar spinal cord visualized by comparing Islet1/2 and Foxp1 immunostaining in cross sections of E12.5 Fz3^+/− (arrows) and Fz3^−/− (arrowheads) lumbar spinal cords. Scale bar, 500 µm. (H–L) Cell death in VII^th (H–I′), and XII^th (J–K′) cranial motor nuclei (arrows and arrowheads) visualized with cleaved Caspase3 and NF immunostaining in horizontal sections of E12.5 Fz3^+/− and Fz3^−/− embryos. (H′–K′) show cleaved Caspase3 immunostaining with DAPI counterstaining for (H–K). (L) planes of section. Scale bar, 100 µm. (M–R′) NF immunostaining of whole-mount forelimbs (M–O′) and hindlimbs (P–R′) from E12.5 Bax^+/−, Fz3^−/−;Bax^−/−, and Fz3^−/−;Bax^+/− embryos. For each pair of panels, the inset in the upper panel is enlarged in the lower panel. Arrows and arrowheads indicate the dorsal nerve. Depth is color coded as in Figure 4B–F′. Scale bars: (R), 500 µm; (R′), 100 µm.

DOI: http://dx.doi.org/10.7554/eLife.01482.012

Figure 5—figure supplement 1. Precocious motor neuron death in Fz3^−/− spinal cord is blocked by loss of Bax.

(A–B′′) Cell death in the ventral spinal cord at the brachial level visualized by cleaved Caspase3 and NF immunostaining on cross sections of E11.5 Fz3^+/− (A–A′′) and Fz3^−/− (B–B′′) embryos. In contrast to the ventrolateral spinal cord, there is no effect of Fz3 loss on cell death in the DRG. Scale bar, 500 µm. (C–D′′) Cell death in the ventral spinal cord at the lumbar level visualized by cleaved Caspase3 and NF immunostaining on cross sections of E12.5 Fz3^+/− (C–C′′) and Fz3^−/− (D–D′′) embryos. In contrast to the ventrolateral spinal cord, there is no effect of Fz3 loss on cell death in the DRG. Scale bar, 500 µm. (E–G′′′) Caspase3 and Islet1/2 immunostaining on cross sections from E12.5 Fz3^+/− (E–E′′′), Fz3^−/− (F–F′′′), and Fz3^−/−;Bax^−/− (G–G′′′) lumbar spinal cords. Boxed regions in (E–G) are enlarged in (E′′′–G′′′). Loss of Bax suppresses cell death in both the DRG and spinal cord. Scale bars: (G), 500 µm; (G′′′), 100 µm.

DOI: http://dx.doi.org/10.7554/eLife.01482.013