P/Def flies are viable and fertile
As the generation of a mutant did not lead to a phenotpye, a deficiency line with full deletion of the FMNL gene (Figure 60) and a fly line with a P-element inserted into the 5’ UTR of FMNL (Figure 61) were used for further analysis.
Figure 60 Genetic map of FMNL-deficiency.
Genetic locus of FMNL (frl). The deletion of the used deficiency line Df(3L)ED4528 is indicated with a black line. Taken from flybase.org.
Both lines are homozygous lethal, however, the P-Element line carries a second lethal, as it was homozygous viable and fertile after cleaning of the chromosome by recombination. However, it was not possible to hold homozygous flies as a stock. Also, the deficiency line was homozygous lethal, as other essential genes were also deleted. Crossing both together, resulted in flies carrying one allele of the P-element insertion and one allele deficient for 3L:14,030,141-14,070,123, from now on named as P/Def flies. These flies were viable and fertile but could also not be kept as a stock.
Embryos from P/Def flies express less FMNL
To analyze the expression of FMNL in P/Def embryos, a quantitative qPCR was done with wild type and embryos from P/Def flies. For further analysis it has to be considered, that embryos from crosses of P/Def females with P/Def males can be P/Def (1/3), P/P (1/3) or Def/Def (1/3). Figure 62 shows the PCR cycle number of amplified cDNA, that was needed to reach a critical threshold measured by fluorescence intensity of the incorporated fluorophore for FMNL and actin, a housekeeping gene that was used as reference. The cDNA was prepared by reverse transcription after RNA extraction from embryonic lysates from overnight embryo collections. The cycling number was in logarithmic scale and inversely proportional to the quantity of cDNA, that was reversely transcribed from RNA extracted from embryonic lysates. The negative control H20 without cDNA gives high cycling numbers of ~34 for FMNL and ~35 for actin, this is also the case for the RNA controls, in which
Figure 61 Genetic map of P-Element insertion into the FMNL locus.
Genetic locus of FMNL (frl) is shown as well as it’s transcripts. The transgenic insertion site of P{GSV7}GS23052 is marked by a red circle. Taken from flybase.org.
wild type or P/Def RNA was added to control for DNA contamination of the RNA extraction. As the cycling numbers for actin with 15.7 for wild type and 15.2 for p/Def embryos respectively, were nearly the same, the cycle numbers for FMNL for wild type and P/Def embryos could directly be compared. In wild type 22.8 cycles were needed to reach the threshold for FMNL, as in P/Def the cycling number was 25.6, showing that embryos from P/Def flies express 23 times less FMNL-RNA than wild type embryos.
Embryos from P/Def flies show a reduced viability
To analyze the effect of less FMNL-RNA expression in P/Def flies, the viability at different developmental stages was compared with wild type embryos.
Again, embryos from wild type and from crosses of P/Def females and P/Def males were used for the analysis and hatched larva, pupa and adults were counted (Figure 63). From 202 wild type embryos 11 embryos died (5.4 %) during development, whereas 81 embryos of 202 embryos from P/Def flies died (40.1 %). Starting with 193 wild type larvae, 9 larvae died before pupation (4.7 %) and 92 larvae of 121 larvae from P/Def flies died (76 %) and from 194 wild type pupae 2 did not hatch (1.0 %), whereas 8 pupae died of 29 starting
Figure 62 Quantitative real time PCR of p/Def and wild type embryonic extracts.
Cycle numbers (logarithmic scale) are plotted for samples as indicated. FMNL: Primers against FMNL; Actin: Primers against actin (positive control); WT: cDNA sample from wild type embryos; p/Def: cDNA sample from P/Def embryos; H20: negative control with water instead of cDNA; RNA WT: Total RNA extracted from wild type embryos; RNA P/Def: Total RNA extracted from P/Def embryos. Numbers are averaged from two replicates.
pupae of embryos from P/Def embryo (27.6 %) Taken together, embryos, larva and pupa from crosses of P/Def females with P/Def males show increased lethality. Also, for this analysis it has to be considered, that embryos of crosses from P/Def females and P/Def males have different allelic combinations as described before and it is not clear if lethality comes from the second lethal of the P-element line, deficiency of essential genes or reduced FMNL expression.
P/Def flies showed reduced fitness
Although P/Def flies were viable and fertile, it was not possible to keep them as a stock. To test the general fitness of these flies a negative geotaxis assay was performed. Flies with normal locomotor activity have the tendency to move against gravity. This ability decreases with aging or with reduced locomotor capability (Benzer, 1967). To be able to compare fitness one-day old flies were used for both genotypes and P/TM3 flies were used as control. The assay was performed by transferring single anesthetized flies to plastic tubes with a diameter of around 1 cm and after waking up, flies were shaken down to the bottom and the distance they crawled up was measured at three timepoints (Figure 64). While P/TM3 flies crawled up to ~15 cm after 30 seconds, P/Def flies reached only ~1 cm. Also, after 1:30 min, P/Def could only reach ~1.5 cm, indicating that they show less locomotor activity and less fitness.
Figure 63 Viability assay of wild type and P/Def flies.
Lethality of wild type (blue) and P/Def (red) during embryonic, larval und pupal development. Lethality rates are shown above the bars.