Figure 3. Δ9-tetrahydrocannabinol (THC) regulates SCG10 expression in the fetal cerebrum.

A–A₂ Grey overlay indicates the origin of cortical tissues used for iTRAQ/mass spectrometry target discovery. Ontology classification of the 35 protein hits based on primary function assignments is shown in (A₁). Topmost modified protein targets are listed in (A₂). SCG10 was significantly down-regulated in THC-exposed fetuses (see also Supplementary Fig S3A).

B–B₂ Reduced SCG10 protein (B) and mRNA levels (B₁) in THC-exposed fetuses (E18.5) were verified by Western blotting and qPCR, respectively. Temporal profile of SCG10 mRNA expression during cortical development is shown in (B₂). Grey bars identify the hippocampus (HC). SCG10 protein and mRNA levels were normalized to Gapdh (B, B1) or TATA-binding protein (B₂), respectively.

C SCG10 protein is enriched in axonal pathways and accumulates in growth cone-like structures traversing the pallio-subpallial boundary by E14.5 (inset).

D–D₃ SCG10 was found co-distributed with CB₁Rs in pyramidal-like cells populating the cortical plate (cp) and corticofugal axons. Open rectangles denote the general localization of insets in (D₁) and (D₂). SCG10 co-existed with collapsing response mediator protein 2 (CRMP-2), a key mediator of appropriately targeted axonal extension (Goshima et al, 1995) in functional antagonism (Fukata et al, 2002) with SCG10 (D₁). High-resolution confocal imaging revealed CB₁Rs and SCG10 in close proximity in corticofugal axons (D₂). Quantitative analysis of the physical proximity relationship of CB₁Rs and SCG10 showed approximately 50% co-localization including overlapping and contacting signals (D₃) (> 100 proximity relationships per axon in n > 25 axons/group from ≥ 3 animal/group were determined).

Data information: Data were expressed as means ± s.e.m.; *P < 0.05. Scale bars, 250 μm (C), 50 μm (D), 20 μm (D₁), 5 μm (D₂) or 1 μm (D₃). For a list of abbreviations, see the Supplementary information.