Whether LC projections to the SC are duplicated is unknown as the mapping of the LC to the SC is hindered by the small size and specific sub-nuclei organization of the LC.
Furthermore, the expression of either GFP, full-length N-WASP, or VCA domain in GAPDH siRNA myofibers did not interfere with peripheral localization of nuclei, triad organization, or myofiber thickness (Fig 5A, E G, and Supplementary Fig S4B and C).
After 10 days of differentiation in the presence of agrin, we found that expression of the VCA domain of N-WASP in Amph2 exon 3 or exon 11 siRNA myofibers restored peripheral localization of nuclei, triad organization as well as myofiber thickness to the same level as the GAPDH siRNA (Fig 5A, B, E G and Supplementary Fig S4A C).
Finally, we tested the role of N-WASP on peripheral positioning of nuclei and triad organization during myofiber differentiation.
We found that expression of TdT-VCA in Amph2 siRNA myofibers also restored peripheral localization of nuclei and triad organization (Supplementary Fig S4E and F).
Instead, they became oval with centrally located nuclei in a nest-like structure, indicative of impairment in the nuclei positioning and/or cytoskeleton organization.
Principle neurons in the telencephalon are organized into layers with distinct circuit and ensemble functions that can be surmised simply based on the location of the nuclei but the anatomical organization of the striatum has proven more challenging.
After the 48 h culture period, nuclear spreads were made and stained with Scp3 to detect meiotic stages, as represented in Fig. 2B. Figure 2C shows that the cell population cultured under SM underwent a dramatic increase (10 fold) in the percentage of nuclei with typical meiotic organization of Scp3 onto chromosomes (35.7%±14.3) with respect to cells left at unit gravity (3.60%±2.00).
Altogether, these results suggest a role for N-WASP in peripheral nuclei positioning and triad organization, probably mediated by N-WASP interaction with amph2.
In order to quantify the emergence and disappearance of different types of centromere organization we categorized nuclei with fewer than twelve foci as "clustered", nuclei with twelve to twenty foci as "paired" and nuclei with more than twenty as "dispersed" (Fig. 1A).
Interestingly, the ROM2 associated defects in cell morphology, location of nuclei, and actin and microtubule organization were not observed in cells grown at temperatures below 37°C.
