Figure 6

Ectopic Sema7Asec diffusive cue provides neural guidance in vivo. (A) A diagrammatic overview depicts the generation of a transgenic animal that expresses the Sema7Asec protein ectopically under the control of a thermally inducible promoter. Larvae with ectopic myotomal-integration near the network of sensory arbors (red rectangle) were imaged to analyze arbor morphology. (B) A schematic drawing of a sensory arbor from a heat-shocked larva depicts an extended axonal projection (arrowhead) that reaches toward the myofibers expressing the ectopic Sema7Asec protein. Parameters that quantitate the accuracy of the extended axonal projections toward the ectopic Sema7Asec sources are denoted. (C) In a micrograph of an ectopically expressing Sema7Asec (orange) larva, the sensory arbor (magenta) extends two aberrant axonal projections. One elongates (cyan arrowhead) along the somite boundary to reach and contact an ectopically integrated muscle progenitor cell (white dashed line) and the other (red arrowhead) reenters the posterior lateral-line nerve while following a second ectopic source. The through-focus scans (i?iii) from the epidermis to the dermomyotome and the three-dimensional (3D) surface reconstruction (iv) reveal the intimate contact between the aberrant sensory arbor (arrowheads) and the muscle progenitor cell. (D) In a micrograph of an ectopically expressing Sema7Asec (orange) larva, the sensory arbor (magenta) extends a single aberrant axonal process (cyan arrowhead) to reach ectopically integrated myofibers (white dashed line). The through-focus scans (i?iii) from the epidermis to the dermomyotome and the 3D surface reconstruction (iv) reveal the proximal association of the aberrant sensory arbor (arrowheads) to the myofibers. Melanocytes (yellow arrowhead) along the horizontal myoseptum intermittently block the visibility of the lateral-line nerve. (E) An injected, but not heat-shocked, control larva does not express ectopic Sema7Asec and does not show aberrant projection from the sensory arbor. (F) A plot demonstrates the accuracy of 18 extended axonal arbors in finding ectopic Sema7Asec sources. Each circle represents a single ectopic integration event. The two pairs of numbers represent the minimal and maximal lengths of the projection path (black) and its corresponding source path (green). (G) A plot quantitates the distribution of projection-proximity length from 18 ectopic integration events. (H) A schematic drawing of a section of the lateral-line nerve between two neuromasts from a heat-shocked larva depicts a few extended neurites (magenta) reaching toward the cells expressing the ectopic Sema7Asec protein (orange). The Sema7Asec integration site (blue rectangle) with the sensory axonal projections was imaged for ten hours to visualize axonal dynamics. (I) Micrographs (i-iii) at three distinct times in the time-lapse video microscopy show directed branching (cyan arrowheads) of the sensory neurites from the lateral-line nerve toward the ectopic Sema7Asec sources. Each panel depicts the ectopic integration site and the associated sensory neurites from three different planes, XY, XZ, and YZ. White arrowheads in XZ and YZ planes highlight the close association of the sensory axons with the ectopic Sema7Asec sources. The t=0 hr denotes the onset of imaging, which is two hours post the beginning of heat shock. (J) The micrographs depict in pseudocolored trajectories the dwell times of the sensory neurites (left) at the Sema7Asec sources (right) for ten hours. MPC, muscle progenitor cell; scale bars, 20 µm; means ± SEMs.