The zygotic activation of genes involved in Drosophila embryonic pattern formation occurs within the first several hours of embryogenesis (1). Among these genes are segmentation factors, such as fushi tarazu (ftz) and even-skipped (eve), and homeotic genes such as Ultrabithorax (Ubx) and Antennapedia (Antp) (1). Mutations in these genes are embryonic lethal for ftz and eve, and larval lethal for Ubx and Antp. The genomic effects on transcription observed due to these mutations are best studied during the times when their gene products are active (2). The activity of proteins encoded by genes such as ftz and eve begins in late stage 4/early stage 5 of embryogenesis (about 2 h after fertilization). However, current methods for sorting wild-type from mutant embryos are based on markers, such as activation of green fluorescent protein (GFP), that cannot be scored effectively until stage 9 (about 5 h after embryogenesis begins and 3 h after FTZ and EVE protein activity in the epidermis) (3,4). An automatic embryo sorter capable of isolating embryos based on GFP fluorescence after embryonic stage 9 has previously been described (3,4); however the problem of identifying homozygous embryos in earlier stages has not been tackled, and thus expression profiling [i.e., using microarrays or reverse transcription PCR (RT-PCR) in early stages] has not been possible. To overcome this technical obstacle, we have developed a simple genotyping procedure for directly scoring the presence or absence of the GFP or lacZ balancer chromosomes in individual homozygous embryos. Establishing a linear RNA amplification protocol (5) has allowed us the use of <10 embryos to perform a microarray or RT-PCR experiments.

Drosophila lethal mutations are usually maintained as heterozygotes, in trans to balancer chromosomes that are used to suppress recombination, thus allowing stable strains heterozygous for mutant genes to be cultured easily (Figure 1A). ftz and eve mutations (6,7) were established in trans to balancer chromosomes carrying a transgene encoding GFP, while Ubx and Antp mutations were established in trans to balancer chromosomes carrying a transgene encoding the lacZ gene. The progeny of balanced ftz and eve embryos were hand-sorted under a dissecting microscope at stage 5 cellular blastoderm and were genotyped. To collect stage 7 embryos, stage 5 embryos were aged to that stage at 24°C and then genotyped. The progeny of balanced Ubx and Antp embryos were collected as early as stage 5 and were aged to stages 7 and 9 at 24°C and then genotyped.

Figure 1. Genetic crosses, genotyping, and RNA sample collections. (Click to enlarge)

Heterozygous flies carrying each mutation in the genes ftz, eve, Ubx, and Antp (Figure 1 A, G) were allowed to lay eggs on grape-agar plates for 1 h. The laid embryos of each genotype contained a mixed population: one-quarter homozygotes for the mutation and lacking the GFP or lacZ chromosome (Figure 1A, boxed genotype G/G), half heterozygotes with one copy of the GFP or lacZ chromosome, and one-quarter homozygous for the balancer chromosome, and therefore with two copies of GFP or lacZ (Figure 1 A). The collected embryos were rapidly dechorionated in 100% bleach for 3 min, and stage 5 single embryos (8) were identified under a light microscope. For genotyping, each embryo was directly transferred into a 0.2-mL PCR tube containing 14.5 L extraction buffer (100 mM Tris-Cl, pH 8.2, 1 mM EDTA, and 25 mM NaCl). Twenty to twenty-five embryos were handled as one batch, each in a single tube. Every individual embryo was homogenized using a pipettor tip. Eleven microliters from the single-embryo extract were stored individually in 96-well plate wells, each containing 30 L TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) to preserve RNA (Figure 1B, step 1), and each batch was directly stored in -20°C. Proteinase K (Sigma-Aldrich, St. Louis, MO, USA) was added to the remaining 3.5 L extract to a final concentration of 200 g/mL (from a stock of 2 mg/mL), and samples were then incubated at 28°C for 30 min, followed by 95°C for 2 min (Figure 1B, step 1). After incubation with proteinase K, the extract was used in PCR containing 5 L 10× PCR buffer supplemented with 15 mM MgCl₂ (Promega, Madison, WI, USA), 0.5 mM mixture dNTP (final concentration 0.125 mM for each nucleoside), 20 pmol each GFP forward and reverse primers (or lacZ primers), 20 pmol each X primer, and 1 U Taq DNA polymerase (see Table 1 for the primers) in a total reaction volume of 25 L. The X primers were designed to specifically amplify a genomic fragment of the CG9650 gene on the X chromosome as a positive control. The PCR conditions were as follows: one cycle of 95°C for 3 min, 35 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and one cycle of 72°C for 10 min. Ten micro-liters each PCR were run on a 1.2% agarose gel [0.5× Tris-acetate-EDTA (TAE) buffer]. Embryos were directly scored for the presence or absence of GFP or lacZ chromosomes (indicated by the presence of the X control band and absence of GFP or lacZ specific band) (Figure 1B, step 2). Homozygous mutant extracts preserved in TRIZOL were then pooled (Figure 1B, step 3), and total RNA extraction, amplification, and microarray expression profiling or RT-PCR could then be performed using standard protocols. Table 2 shows all the collections made from the different genotypes studied for one replicate, the total number of embryos assayed, and the amounts of RNA obtained before and after amplification. The expected one-quarter percentage of homozygous embryos to the mutation was observed in all collections made. The amounts of RNA obtained after each total RNA extraction were enough to perform one round of RNA amplification (1–2 g total RNA are needed for one round of RNA amplification) (Table 2). The amounts after RNA amplification were enough to perform at least three micro-array hybridizations (5 g amplified RNA are needed for labeling to perform one microarray hybridization) (Table 2).

Table 1. PCR Primers for Genotyping (Click to enlarge)

Table 2. Collections and Genotyping Results from Different Genotypes of One Replicate (Click to enlarge)

Our method for the extraction of enough amounts of RNA from homozygous Drosophila embryos during very early stages of pattern formation will be helpful to supplement many expression studies aimed at studying factors activated during this important time in development. Furthermore, our protocol is potentially helpful for workers in other systems where marked chromosomes are available or being developed.