Fig. 4: Presence of MusD retroviral-like particles is associated with AER cell death.

a, Anti-Gag-MusD whole-mount immunofluorescence on E10.5 Dac1J/Dac1J embryos showing embryo (left), forelimb (middle) and AER (right). n = 5 out of 5 biological replicates were confirmed. FL, forelimb; HL, hindlimb; BA, branchial arches; MHB, midbrain–hindbrain boundary. b, TEM analysis on E11.0 Dac1J/Dac1J AER cells. Three different cells are shown. Green arrowheads and dotted lines indicate single and aggregated VLPs, respectively. n = 5 biological and n = 2 technical replicates. C, cytoplasm; N, nucleus; ER, endoplasmic reticulum. c, TEM analysis after immuno-gold labeling with anti-Gag-MusD on E11.0 Dac1J/Dac1J AER cell shows cytoplasmic aggregates of GAG. n = 2 biological replicates and n = 6 technical replicates were confirmed. d, TEM analysis on E11.0 wild-type and Dac1J/Dac1J forelimbs. Zoomed-in view (right) shows the structure of an AER cell. Red asterisk indicates an apoptotic body. e, Anti-cleaved-Caspase-3 whole-mount immunofluorescence on E11.0 wild-type and Dac1J/Dac1J forelimb and AER. n = 4 out of 4 (wild-type) and n = 6 out of 6 (Dac1J/Dac1J) biological replicates were confirmed. f,g, Anti-γH2AX whole-mount immunofluorescence on wild-type and Dac1J/Dac1J E10.5 forelimbs (f) and E11.0 forelimbs and AER (g). n = 3 out of 3 biological replicates were confirmed. h, Schematic representation of wild-type and Dac1J/Dac1J limbs at E10.5, E11.0, E11.5 and E18.5. Although the AER of the limb bud is not morphologically affected in the Dac1J/Dac1J at E10.5, VLPs are expressed, and we detected increased DNA damage. Half a developmental day later, more DNA damage and apoptosis occur. By E11.5, most of the AER cells have been eliminated by cell death. Consequently, at E18.5, the autopod of the Dac1J/Dac1J embryos is severely affected by a lack of metacarpal structures, but the stylopod and zeugopod have developed similarly to the wild-type embryos. Styl., Stylopod; Zeug., Zeugopod; Aut., Autopod.