Fig 1.
The timecourse of bcd mRNA and protein accumulation during early embryogenesis.
Samples of embryos from wild type (wt) (left column) and pum null mutant females (right). (A) bcd and eve mRNAs visualized by in situ hybridization; the former is transcribed maternally and accumulates at the anterior, while the latter is transcribed zygotically and accumulates in seven stripes (in wt embryos) approximately in the regions of the embryos labeled above. pum mutant embryos lack posterior patterning activity and so the pattern of eve stripes 3–7 is altered. Approximate embryonic ages in hours post-fertilization are 0.75 (stage 2), 2.25 (early stage 5), 2.6 (mid-late stage 5). (B) Bcd (green) and Eve (red) proteins visualized by immunohistochemistry and confocal microscopy. Approximate embryonic ages are 1.75 hours (stage 4), 2.6 hours (mid-stage 5), and 3 hours (stage 6). Note that only a trace of Bcd is detectable at the onset of gastrulation (stage 6) under these conditions, but low levels of residual protein are readily visible at increased gain. (C) ocelliless (oc) mRNA; oc is a direct Bcd transcriptional target. (D) Timecourse of Bcd accumulation as revealed by Western blots, of hand-sorted samples, with embryonic age in hours above. The left lane is a negative control of 0–3 hour embryos from bcd6 females that produce no stable Bcd. As a loading control, the membrane was re-probed with an antibody to alpha-tubulin (below).
Fig 2.
The timecourse of bcd mRNA and protein accumulation in the absence of Nos activity.
bcd and eve mRNAs (left) and proteins (right) in embryos with no detectable nos activity, detected as described in Fig 1.
Fig 3.
Mutations in the bcd NRE abolish binding of Pum in vitro and in vivo.
(A) Sequence of the 45 nt wt bcd NRE, with the 4 substitutions in the mutant (mut) NRE above. Canonical Pum binding sites in shaded boxes. (B) Gel mobility shift experiments to assay Pum binding. An increasing concentration of the Pum RNA-binding domain (0, 0.25, 0.5, 1.0, 2.0, 4.0 μM in lanes 1–6 of each panel) was incubated with the RNAs indicated below and electrophoresed to separate bound and free RNA. The Pum binding site in the CycB NRE [19] is a positive control. (C) An in vivo assay for NRE-dependent regulation of bcd mRNA in embryos that have ectopic Nos at the anterior by virtue of maternal expression of a chimeric mRNA that is localized to the anterior pole of the embryo by its 3’-UTR (from bcd) and encodes wild type nos protein. Dark field micrographs reveal primarily the pattern of abdominal segmentation; arrows indicate segmental and embryonic polarity. All embryos were from females bearing the nos+—[bcd 3’-UTR] transgene that results in the accumulation of ectopic anterior Nos; in addition, the females either had no additional transgene (-) or a single copy of a wild type bcd NRE (+), bcd NRE(mut), or bcd NRE(Δ) transgene, as indicated.
Fig 4.
Bcd accumulation in embryos bearing a Pum-resistant bcd NRE (mut) mRNA.
(A) Bcd (green) and Eve (red) proteins in embryos from otherwise wild type females that bear a single copy of a bcd transgene with a wild type (+) (left) or mutant (mut) NRE (right). Embryonic stages as in Fig 1B. (B) Western blot of sorted samples (developmental age in hours post-fertilization above) of embryos bearing mRNA from either a bcd NRE(+) control transgene or the bcd NRE (mut) transgene, as indicated. On the left, the negative control sample of bcd6 mutant embryos reveals two proteins that cross-react with the anti-Bcd antibody, one of which serves as a convenient loading control for the blot. The left lane is a negative control of 0–3 hour embryos from bcd6 females that produce no stable Bcd. As a loading control, the membrane was re-probed with an antibody to alpha-tubulin (below). Note that the bcd transgenes encode wild type protein and therefore the Bcd detected on the blot is derived from both the endogenous bcd+ genes and the transgene.
Fig 5.
Viability of progeny developing from embryos with extra bcd mRNA.
(A) Phase contrast micrographs of representative head skeletons of embryos derived from females bearing two copies of the bcd transgenes indicated above. (B) Percentage of viable animals at three different developmental stages (labeled below and to the right) that arise from embryos of “two-copy” females bearing bcd NRE(+), NRE(mut), or NRE(Δ) transgenes, as indicated. Each entry is the average for three different trans-heterozygous pairs of transgenes, with error bars showing the S.D. The figure reports the percentage of fertilized eggs that hatch into viable larvae, pupate, and eclose to viable adulthood. With one exception, at each stage of development there is no significant difference among all pairwise comparisons by two-tailed t-test; the exception is a modestly significant (P = 0.04) difference in pupal viability between NRE(+) and NRE(Δ) animals.
Fig 6.
Pum- and Nos-mediated regulation of bcd mRNA in males.
(A) Relative levels of bcd mRNA in various samples, normalized to the housekeeping mRNA for ribosomal protein S2 (Rps2) and with the level in males set to 1. Values are the average of two independent experiments. Germline-free animals were second-generation escapers from tudor mutant females, as described in the text. (B) The level of bcd mRNA in various mutant males (black bars, mutant genotypes below), relative to the level in wt males (white bars, set to a value of 1.0). Full mutant genotypes are: pumMsc / pumET3, nosBN / Df, nosL7 / Df. The figure reports the value of the mean and the S.D. for four independent samples from qPCR experiments, measuring Rps2 mRNA to calculate ΔCt. By the two-tailed t-test, each mutant is significantly different from the wt control (P ≤ 0.0047). (C) The fertility of individual wild type (wt) and bcd 6 / bcd 12 null mutant males is shown in serial matings performed from 4–20 days post-eclosion. Red bars report the average for each genotype; by two-tailed t-test, there is no significant difference between the two genotypes at any time point; the smallest value of P is 0.16 at day 12. Note that the average number of progeny on day 12 from bcd mutant males is somewhat artificially depressed by two moribund animals that died shortly thereafter.