Generation of the Plk4^Aa74 mutant allele. Previous studies of Plk4 have used the weak hypomorphic Plk4^c06612 allele (Bettencourt-Dias et al., 2005), so we generated a stronger allele (Plk4^Aa74) by imprecise excision of the Plk4^c06612 P-element. (A) The schematic shows the PLK4 genomic region, indicating the coding sequence (CDS; dark green), the UTRs (light green), the position of the original P{GSV1}GS3043 P-element insertion (red triangle), the position of the primers used to screen for imprecise excision of the P-element (purple), and the position of the Aa74 deletion (which deletes essentially the entire protein kinase domain; blue). (B) DNA gel shows the PCR products observed when these primers were used to amplify DNA from either WT flies or the homozygous Plk4^Aa74 deletion flies. Sequencing of the PCR products confirmed deletion of the 1,751-bp region indicated in A. MW stands for molecular weight marker as a unit label. (C) Photographs of adult flies taken without anesthesia (see also Video 4): the original Plk4^c06612 mutant flies can maintain a normal body posture and walk in a partially uncoordinated manner; Plk4^Aa74 flies are highly uncoordinated and appear to lack all proprioception, as is typical of flies lacking centrioles (Basto et al., 2006); GFP-Plk4 expression rescues the uncoordinated phenotype of Plk4^Aa74 mutant flies. (D) Micrographs show WT or Plk4^Aa74 third-instar larval brains, with DNA stained with Hoechst (blue) and centrioles revealed with GFP-PACT staining (green). Bar, 10 µm. (E) Bar chart quantifies the number of centrioles in mitotic larval brain cells of various genotypes (as indicated). n = 12 brains; n = 264 cells in toto for WT; 5 brains, 237 cells for Plk4^c06612; 7 brains, 355 cells for Plk4^Aa74; 4 brains, 143 cells for GFP-Plk4; Plk4^Aa74. Collectively, these data indicate that the Plk4^Aa74 allele behaves as a null or strong hypomorph.