Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 6

Figure 6. Kinesin-2 promotes NSC reactivation and targets E-cad to the NSC-neuropil contact sites

A) Larval brains at 16 h ALH from wild-type (yw) and E-cadR69 were analyzed for EdU incorporation, and larval brains were labeled with EdU, Dpn and Mira.

B) Quantification graph of the percentage of qNSCs with primary protrusion in wild-type (yw), E-cadR69 and E-cadR69.Ubi-p63E-E-cad cells. GFP at 16 h ALH. Larval brains were labeled with Dpn and Mira (not shown). n=19 BL for control; n=23 BL for E-cadR69; n=11 BL for E-cadR69.Ubi-p63E-E-cad BL. ****p<0.0001; p=0.2690 (ns).

C) Quantification graph of NSC diameter at 16 h ALH from wild-type (yw), E-cadR69 and E-cadR69.Ubi-p63E-E-cad.GFP. NSCs were marked by Dpn and Mira. n=110 NSCs for control; n=131 NSCs for E-cadR69; n=114 NSCs for E-cadR69.Ubi-p63E-E-cad. ****p<0.0001; p=0.8151 (ns).

D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=10 BL for control; n=20 BL for per E-cadR69. ****p<0.0001.

E) Larval brains at 24 h ALH from the wild-type control (yw) and klp64Dk5h/Df, kap3V6 and kap3V6; g-kap3#11 were stained with EdU, Dpn, and Mira.

F) Quantification graph of NSCs with primary protrusion per brain lobe for genotypes in (E) as well as klp64Dk5h, kap3V5, and kap3V6; g-kap3#31. n=26 BL for control; n=16 BL for klp64Dk5h; n=7 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=7 BL for kap3V6; n=6 BL for kap3V6; g-kap3#11; n=7 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.9998 (ns); control vs. kap3V6; g-kap3#31, p>0.9999 (ns).

G) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (E) as well as klp64Dk5h, kap3V5, and kap3V6; g-kap3#31, which were not shown in (E). n=33 BL for control; n=15 BL for klp64Dk5h; n=11 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=10 BL for kap3V6; n=10 BL for kap3V6; g-kap3#11; n=10 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.9713 (ns); control vs. kap3V6; g-kap3#31, p>0.9999 (ns).

H) Quantification graph of PH3-positive NSCs (Dpn+, Mira+) per brain lobe for wild-type control (yw), klp64Dk5h klp64Dk5h/Df, kap3V5, kap3V6 and kap3V6; g-kap3#11, and kap3V6; g-kap3#31 larval brains. n=26 BL for control; n=16 BL for klp64Dk5h; n=7 BL for klp64Dk5h/Df; n=20 BL for kap3V5; n=7 BL for kap3V6; n=6 BL for kap3V6; g-kap3#11; n=6 BL for kap3V6; g-kap3#31. ****p<0.0001; control vs. kap3V6; g-kap3#11, p=0.0977 (ns); control vs. kap3V6; g-kap3#31, p=0.1658 (ns).

I) Larval brains at 24h ALH from wild-type control (yw), klp64Dk5/Df, and kap3V5 were analyzed E-cad at the NSC-neuropil contact sites, and NSCs were labeled with Dpn and Msps.

J) Quantification graph of the fold change of basal E-cad intensity in qNSCs with primary protrusion for genotypes in (I). n=85 NSCs for control; n=40 NSCs for klp64Dk5/Df; n=48 NSCs for kap3V5. ****p<0.0001..

K) A working model. Microtubule arrays in the primary protrusion of Drosophila qNSCs are predominantly acentrosomal with plus-end-out orientation. Msps regulates microtubule growth and orientation in quiescent NSCs. Msps and Kinesin-2 target E-cadherin to the NSC-neuropil contact for NSC reactivation.

Data information: Arrows in A and E indicate EdU-negative NSCs. In (B-D, F-H, J), data are presented as mean ± SD. In (B-D, F-H, J), statistical significances were determined by one-way ANOVA with multiple comparisons. Scale bars: 10 µm.