Somatic mutations of plxnb2a and plxnb2b do not affect regenerative neurogenesis.
(A) RFLP and qPCR showing gRNA injection efficiency for plxnb1a and plxnb1b. For RFLP each lane represents one larva with and without digestion with the indicated restriction enzymes and with and without targeting these sites with gRNAs as indicated. Targeting the recognition sites with haCR gRNAs leads to efficient somatic mutation, as indicated by the almost complete resistance to digestion. Note a ~50% reduction in the RNA detected by qRT-PCR for either receptor. (B, C) HCR-FISH shows expression of plxnb2a (B) and plxnb2b (C) in a narrow domain in the spinal cord that does not change after lesion. Additional signal can be observed in the injury site after lesion for both genes (asterisks). (D, E) High magnifications of HCR-FISH for plxnb2a and plxnb2b show no detectable expression of plxnb2a at the level of ERGs (D), but of plxnb2b in her4.1:EGFP+ cells (E). (F) RFLP showing high gRNA injection efficiency for plxnb2a and plxnb2b. Each lane represents one larva with and without digestion with the indicated restriction enzymes and with and without targeting these sites with gRNAs as indicated. Targeting the recognition sites with haCR gRNAs leads to efficient somatic mutation, as indicated by the almost complete resistance to digestion. (G, H) haCR gene disruptions of plxnb2a (G, gControl: 5.6 cells per larva ± 1.26; gPlxnb2a: 5.5 cells per larva ± 1.00; Mann–Whitney U test: p = 0.6938) or plxnb2b (H, gControl: 6.9 cells per larva ± 0.83; gPlxnb2b: 5.9 cells per larva ± 0.73; Mann–Whitney U test: p = 0.3336) do not lead to changes in the number of newly generated motor neurons, compared to lesioned gControl-injected larvae. Each dot for qRT-PCR represents a pool of 50 larvae. β-actin was used as housekeeping gene. Raw data for qRT-PCR can be found in S5 and S6 Data. Error bars show SEM. Dotted lines delineate the position of the spinal cord. Scale bars: 100 µm (B, C), 25 µm (D, E). Data files for graphs available in S3 Data. Original gels with no adjustments can be found in S1 Raw Images.
|
