Figure 3

Focal recombination obtained through genetic and mechanical approaches. (A, B) Genetic approach. Conditional induction of a caspase-dependent pro-apoptotic program in a neuronal subpopulation. Flat-mount embryonic spinal cord explants, from wild-type (A) or Dbx1-DTA strain (B), exposed for 96 h to 1 μl of 400 ng/μl H3NC. Activated-caspase III immunodetection showed induction of early steps of apoptosis only in transgenic explants exposed to the conjugate (B; arrows: representative positive cells, purple staining). The apoptotic marker was not induced in non-transgenic tissue (A). Likewise, vehicle solution did not induce significant apoptosis (see Additional file 4C). Red double arrow: floorplate (see also Additional file 3). (C-F) Mechanical approach. Focal conjugate delivery produced clusters of recombined cells. (C) H3NC-soaked beads (200 ng/μl) were deposited onto Z/AP cortical explants. hPLAP reporter activity was monitored 24 h later (purple precipitate). Inset, clusters of radiating positive cells. (D) In contrast, the drop assay using the same protein solution (200 ng/μl) on parallel explants led to widely dispersed reporter-expressing cells (inset, higher magnification). (E-F) Combination of genetic and focalized restriction of cell targeting. Detection of simultaneous reporters (β-Galactosidase, blue; GFP, brown; arrow) induced by H3NC (200 ng/μl) in TGZ cortical explants. In both C (top cluster of positive cells, asterisk) and F explants (center, asterisk), one bead was removed, exposing cells in contact with the beads. Arrows (E, F): representative nuclei surrounded by dense axon bundles, suggestive of neurons. For the whole figure, scale bar in A represents: A, B, E, 300 μm; C, D, 1000 μm; F, 150 μm.