Figure 4.

Macrophages provide the death signal to E12 but not to E13 motoneurons. A, Scheme of dissection of an asymmetric explant in which the most proximal part of the somite was left in contact with one ventral horn (same abbreviations and representation as in Fig. 2A). B, Macrophages and motoneurons simultaneously labeled with anti-Iba1 (green) and anti-islet1/2 (red) antibodies on a 36-somite (E12) rat embryo section passing through the brachial region. Motoneuron areas (mn), somites (som), and dorsal root ganglia (drg, only visible on one side) are outlined (dotted line). Note that numerous macrophages are within the somite, and some of them (arrows) are also detected in the proximal somite and occasionally within the neural tube (asterisks). C1-D2, Asymmetric explants cultured for 2 d without (C1, C2) or with (D1, D2) the macrophage-specific toxin IB4sap. Triple labeling identifying Iba1 (C1, D1), islet1/2 (C2, D2, red) immunoreactivities and TUNEL (C2, D2, green) are shown. The position of the somite is indicated by a large arrow, and Iba1-positive macrophages are indicated by small arrows. E, F, Quantification of macrophages (E1, F1) and motoneurons (E2, F2) on same sections from E12 (E1, E2) and E13 (F1, F2) explants cultured for 2 d. E12-ct, Explants cultured without somite; ct-asym-O, ct-asym-A, asymmetric explants cultured without IB4sap, ventral horn opposite (ct-asym-O) and adjacent (ct-asym-A) to the somite; IB4sap-O, IB4sap-A, asymmetric explants cultured with IB4sap, ventral horn opposite (IB4sap-O) and adjacent (OB4sap-A) to the somite. Note that in E12 explants, the somite prodeath effect is abolished in IB4sap-treated explants, which contain almost no macrophages. In E13 explants, the elimination of macrophages has no effect on motoneuron survival. ^***p < 0.0001, Student's t test. Numbers of explants quantified in each condition are indicated on the histograms. Scale bars: B, 250 μm; C1-D2, 100 μm.