JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), we observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, our data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

Defasciculation and collapse of longitudinal connectives is widely spread. Scale bar 10 μm. b) Self cross-correlation matrix of (a). Scales and colormap as in Figure 1. In puc B48 , single 3D nodes per segment are consistently developed (purple double headed arrows). c) Image cross-correlation score along the AP axis for (b) (n = 8 embryos). Segmental d) VNC length in µm. Significant differences in length were detected between the wild type and puc alleles (wild type, (grey) n = 11; puc E69 , (green) n = 9; puc B48 , (light green) n = 15). Data are presented as mean ± SD. Parametric Student t-tests were employed. *, p = 0.0135 for puc E69 and ****, p < 0.0001 for puc B48 .
Source data are provided as a Source Data file. 8 9

Supplementary Figure 4. JNK activity and cell death in puc mutants
a) Single (left and right) and combined (center) channels highlighting the expression of Fas 2 (left -green) and anti-P-JNK (right-magenta) of a stage 17 wild type embryo. Scale bar 10 μm. b) Equivalent as (a) for a stage 17 puc E69 embryo. Scale bar 10 μm. c) Equivalent as (a) for a stage 17 puc B48 embryo. Scale bar 10 μm.
In mutant embryos ((b) and (c)) a significant fraction of the P-JNK signal remained in or around cell bodies (yellow arrowheads) and is not just found in axons, as in wild type. d) Graph displaying the averaged Integrated Density per segment for the P-JNK signal (wild type, (grey) n = 10; puc E69 , (green) n = 11; puc B48 , (light green) n = 4). Data are presented as mean ± SD. Parametric Student t-tests were employed. Significant differences in P-JNK levels were detected between the wild type and both mutant conditions (****, p < 0.0001, for puc E69 and puc B48 ). e) From left to right, stage 17 heterozygous and homozygous puc E69 embryo stained for Fas 2 (green) and Dcp1 (magenta), which highlights those cells in the process of apoptosis. Scale bar 10 μm.
Source data are provided as a Source Data file.