PMC

Axonal projections of aCC and RP2 visualized with an RN2-Gal4 driver expressing UAS-tau-LacZ (magenta) in flat-mounted stage 16 embryos. To better localize and trace the motor nerve exit of these motoneurons, embryos were also stained with anti-Fas2 antibody (green). Anterior is shown on top in all panels and partial genotypes are indicated on the left of each panel.
(A) aCC and RP2 axons in over 90% of hemisegments in eve mosaic embryos fail to exit the CNS and some of these axons cross the midline; a phenotype that is absent in wild-type aCC and RP2 neurons (M).
(B–E) Individual re-expression in eve mutant aCC and RP2 motoneurons of UAS-nrg (B), UAS-Fas2 (C), UAS-beat Ia (D), or UAS-unc-5 (E).
(F–J) Simultaneous re-expression of two membrane molecules: UAS-beat Ia and UAS-nrg (F), UAS-beat Ia and UAS-Fas2 (G), UAS-beat Ia and UAS-unc-5 (H), UAS-nrg and UAS-unc-5 (I), or UAS-Fas2 and UAS-unc-5 (J).
(K and L) Combinatorial re-expression of UAS-unc-5, UAS-beat Ia, and UAS-nrg (K) or UAS-unc-5, UAS-beat Ia, and UAS-Fas2 (L) in eve mutant aCC and RP2 restore exit and fasciculation in 66% and 69% of hemisegments, respectively.
(N) Quantification of total exit for aCC and RP2 in different genetic backgrounds divided between dual exit (either fasciculated or not) of both motoneurons and single exit of either one of them per hemisegment. Single re-expression of any individual gene leads to almost exclusively single exit. Single or dual re-expression of both CAMs (Nrg of Fas2) leads only to single exit in 26% of the hemisegments.
(O) Quantification of dual exit in different backgrounds in which two or more genes are reintroduced in eve mutant aCC and RP2. Dual exit is divided between fasciculated or nonfasciculated exit. The ratio of fasciculated to unfasciculated exit increases when a CAM is re-expressed with one or both guidance receptors (unc-5 or beat Ia). From 25%/21% fasciculated/unfasciculated when unc-5 and beat Ia are reintroduced to 66%/13% or 69%/11% fasciculated/unfasciculated when both receptors are reintroduced with Nrg or Fas2, respectively. Data are represented as mean ± SEM.

Conceptual illustration of the EXIT circuit. The EXIT circuit comprises a control module and an exit module. The two modules can be assembled into one or more plasmids. Activation of the homing endonuclease gene (HEG) will result in cleavage of the control and exit modules, thereby eliminating the host plasmids

Ten days of IH increases hippocampal HIF1a and disrupts Barnes maze performance in wild-type mice but not in HIF1a+/−. A, left, Representative digitized Western blotting images for HIF1a (103 kDa) and PCNA (40 kDa) in hippocampal nuclear protein fractions from control (n = 4) and IH₁₀ (n = 4). Right, Quantification of HIF1a protein normalized to PCNA revealed that nuclear HIF1a was increased in IH₁₀ when compared with control (p = 0.019). B, Total latency to exit the Barnes maze during three training sessions in control (n = 10) and in IH₁₀ (n = 11). Each blue (control) and red (IH₁₀) line represents an individual performance during training. Training to the exit was conducted over three sessions. Each session was separated by 24 hours. C, Left, During the probe trial, the distance traveled to initially enter the exit zone was shorter in control when compared with IH₁₀ (p = 0.048). Right, Latency to initial entry was smaller in control as well (p = 0.034). D, Heat maps of the mean entry probability across all false exits (1–19) and the exit zone during probe trial for the control and IH₁₀. Comparison of entry probability into the exit zone during the probe trial reveals that control has a greater probability for entering the exit zone when compared with IH₁₀ (p = 0.004). E, Left, Representative digitized Western blotting images HIF1a and PCNA in hippocampal nuclear protein fractions from 0-HIF1a^+/− (n = 4) and 10-HIF1a^+/− (n = 4). Right, Quantification of HIF1a protein normalized to PCNA revealed that nuclear HIF1a is similar between 0-HIF1a^+/− and 10-HIF1a^+/− (p = 0.84). F, Total latency to exit the Barnes maze during three training sessions in 0-HIF1a^+/− (n = 7) and in 10-HIF1a^+/− (n = 8). Each gray (0-HIF1a^+/−) and yellow (10-HIF1a^+/−) line represents an individual performance during training. All experimental groups exhibit decreased total latency over the course of training. G, Left, In HIF1a^+/−, the distance initial to initial entry into the exit zone was similar between 0-HIF1a^+/− and 10-HIF1a^+/− (p = 0.55). Right, Latency to initial entry into the exit zone during the probe trial were similar between 0-HIF1a^+/− and 10-HIF1a^+/− (p = 0.39). H, Heat maps of the mean entry probability into all zones during the probe trial for 0-HIF1a^+/− and 10-HIF1a^+/−. Entry probability was similar between 0-HIF1a^+/− and 10-HIF1a^+/− (p = 0.21); *p < 0.05; **p < 0.01; N.S., p > 0.05.

Nek8445-depleted cells misposition or lack exit sites. Control morpholino and Nek8445 knockdown cells were stained for tubulin (6-11B-1 antibody; green) and ESP8855-3HA (red). White arrowheads mark the caudal axoneme exit site and are oriented with the trajectory of the exiting axoneme. (A) Typical exit site organization in control cells. (B) Knockdown cell with typical teardrop shape retains exit sites at the expected position. (C) Mispositioned caudal axoneme exit site observed in 10% of rounded knockdown cells (n = 58). Note that these images are maximum projections; the caudal axoneme exit site in this cell is clearly positioned above the adjacent posterolateral axoneme exit site (cyan arrowhead) when viewed in the 3D image stack. (C′) Shows maximum projections of the ventral and dorsal halves of the Z-stack to show that these exit sites are displaced in Z similar to the cell with the arrowhead in . (D) Complete lack of a caudal axoneme exit site is observed in 59% of rounded knockdown cells (n = 58). Scale bar = 5 µm.