Drosophila lacking the Cdk5 activator, p35, display defective axon guidance, age-dependent behavioral deficits and reduced lifespan

Abstract

The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. We show here that Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. We further show that, while viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan.

Figure 1. Genomic region of p35–21C

A. Schematic showing a ca. 12 Kb region surrounding the D-p35 gene (also called p35–21C). Predicted genes (Flybase: <ftp://flybase.org/flybase> are depicted as boxes, with the direction of transcription indicated with an arrow. Positions of P[w+] transposons discussed in the text are indicated with inverted triangles. Horizontal brackets delineate sequences that are deleted by the indicated p35 mutations; hatched box delineates genomic sequences included in a transgene (referred to as Tn[p35⁺]) that rescued all p35 mutant phenotypes that were analyzed. B-D. Lateral views of embryos subjected to in situ hybridization with a p35 probe and visualized by alkaline phosphatase histochemistry. Panels display stage 17 embryos of the indicated genotypes: (B) wild type; strong labelling is observed in the CNS (arrow) and peripheral sense organs (bracket), (C) p35⁻, (D) p35^20C; Tn[p35⁺]. Anterior is to the left and dorsal to the top in all embryos in this and all other figures. Apparent difference in staining intensity between Panels B (wild type) and D (rescued mutant) reflect sample-to-sample variation in histochemical staining; in parallel experiments we did not observe reproducible differences in p35 expression level between the wild type locus and the genomic transgene.

Figure 2. Axonal phenotypes in p35 mutant embryos

Stage 17 embryos were fixed, stained with anti-Fasciclin 2 antibodies and visualized with peroxidase histochemistry. Panel (A) shows Fasciclin 2-positive motonerves in two hemisegments of a wild type embryo; peripheral motonerves are labeled. Panels (B – E) show axonal aberrations in four different p35 mutant animals, and are typical of the range of phenotypes observed in the mutant. Each mutant panel shows a portion of a hemisegment at approximately the same scale as the wild type panel; color coded, dashed boxes on the wild type panel indicate the portion of the embryo displayed in the corresponding mutant panel. Circles and arrows highlight the defects in the mutants and the corresponding structures in the wild type; the nature of the defect is indicated below each panel. (B) Overextended ISN. (C) Missing SNa dorsal branch (lateral branch is not affected; #). (D) Missing SNa lateral branch (dorsal branch is not affected; *). E. Stalled TN. Abbreviations: ISN: intersegmental nerve, SNa: segmental nerve, “a” branch; ISNb: intersegmental nerve, “b” branch; TN: transverse nerve. (Note: TN in the more anterior segment of the wild type embryo of (C) was slightly damaged in dissection of the embryo.) F. Histogram displaying the distribution of patterning defects among the SNa, TN and ISN peripheral motonerves for two different p35 null excision alleles, p35^20C and p35^5D. N=180 hemisegments for each allele.

Figure 3. Reduced adult lifespan of p35 mutant flies

A. Flies of the indicated genotypes were maintained in parallel at 25° under optimal growth conditions. The number of surviving flies was tallied every other day and plotted as percent alive vs days after eclosion. Data from one typical experiment is shown. Df(2L)^J77 is a cytologically visible deficiency that uncovers D-p35; the p35 allele used for this experiment was p35^20C and P[100] is the P-element insertion that was used to generate p35^20C by imprecise excision. B. Average survivorship curves compiled from 13 independent experiments for w⁻ and w⁻; p35^20C, and 10 independent experiments for p35^20c; Tn[p35⁺] and p35^20c/Df(2L)^J77. Each experiment was performed as described for (A), except that for some experiments employing p35^20c; Tn[p35⁺] and p35^20c/Df(2L)^J77 survivorship was tallied every 5 days instead of every other day. Percent survival on a given day after eclosion was derived by averaging the fraction surviving on that day across all experiments for a given genotype. Horizontal black bars represent the standard error of the mean (SEM) for the time to 50% survival. For clarity, these bars are shown displaced slightly from the 50% level. Each experiment started with ≥100 flies of each genotype, except p35, experiment #5 (n=58) and experiment #7 (n=92). C. Summary statistics for the data from (B). The mean number of days to 50% survival was calculated from multiple experiments for each of the indicated genotypes and is indicated by the bar. Thin horizontal lines indicate ± 1 standard deviation for each lifespan; vertical hash marks indicate ± 1 SEM. Mean lifespan of w⁻ was not significantly different from that of p35⁻; Tn[p35⁺] and lifespan of p35^20C was not significantly different from p35/Df(2L)^J77, but in all pairwise comparisons, lifespan of genotypes lacking p35 was significantly different from that of p35⁺ genotypes (p < .05, ANOVA). Note that the “mean lifespan” reported in this panel is calculated as the mean of the medians of the separate experiments, whereas the mean survival curves in (B) are derived from compilation of all the datasets. Absolute time to “50% survival” is slightly different from these two calculations, but both support a conclusion of significant lifespan reduction in the mutant.

Figure 4. Progressive loss of motor function in p35 mutant flies

Populations of flies of the indicated genotypes were assayed weekly after eclosion for their ability to climb 5cm up the side of a vial within 10 seconds. The fraction that were successful at this task at each time point is indicated with a vertical bar. Each bar represents the average from 8 independent experiments. Standard error for each measurement displayed was ~5% (range 2–11%). Df(2L)^J77 is a cytologically visible deficiency that uncovers p35, p35⁻; Tn[p35⁺] is a null mutant bearing the wild type genomic locus as a transgene, and the p35 excision allele used for this experiment was p35^20C and is in a w⁻ genetic background. Dotted lines represent approximate interpolations of the data for the two p35⁺ genotypes (orange), and for the two p35⁻ genotypes (blue)