Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system

Abstract

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

Figure 1

Strategies for knockdown of maternal and zygotic transcripts. (A) Depletion of a maternal transcript following expression of shRNAs in the female germline. The maternal Gal4 driver (blue) activates shRNAs (red), which deplete target transcripts (green). (B) Depletion of a zygotic transcript by loading the embryo with maternally derived shRNAs. (C) Depletion of a zygotic transcript following zygotic activation of shRNAs by maternally loaded Gal4 protein. Strategies A and B correspond to F₂ phenotypes in Table 1 while strategy C corresponds to an F₁ phenotype.

Figure 2

Embryonic phenotypes associated with knockdown of Mat and Zyg genes. For nos, dl, bcd, tor, and dpp-F2, mat-tub-Gal4>>UAS-shRNA mothers were crossed to UAS-shRNA males. All phenotypes resemble strong classic alleles. dpp-F1 embryos were obtained from crossing mat-tub-Gal4 females to UAS-shRNA males. For Kr, twi, hh, ftz, and wg, mat-tub-Gal4>>UAS-shRNA mothers were crossed to males heterozygous for a strong mutant allele of the target gene. Only a subset (see text) of embryos from these crosses had cuticle phenotypes. The phenotypes for twi, hh, and wg resemble classic mutants. For Kr, the main defect is the absence of the A2 segment (arrowhead), which is a smaller gap than seen in classic mutant embryos. The same phenotype was observed with two shRNA lines (GL01322 and GL01324). For ftz, the embryos are missing two anterior segments, a weaker phenotype than is seen in classic mutant embryos. Description of the mutant phenotypes and references for each gene tested can be found at http://flybase.org/. WT refers to a wild-type cuticle. The “i” superscript refers to the RNAi-induced phenotypes.

Figure 3

Zygotic phenotypes revealed by the expression of zygotic shRNAs by maternally loaded Gal4 protein. For some genes, high rates of F₁ lethality and specific embryonic phenotypes were detected when maternal-Gal4 females were crossed to UAS-shRNA males. These included armadillo (arm), Notch (N), domeless (dome), shotgun (shg), myospheroid (mys), upheld (up), and Histone deacetylase 3 (Hdac3). Additional shRNA lines associated with F₁ phenotypes are listed in Table 1.

Figure 4

Embryonic phenotypes associated with rolled and hunchback shRNAs. (A) rolled. When MTD-Gal4>>UAS-shRNA-rl (GL215) females were crossed to WT males, the embryos showed differentiated cuticles with terminal defects (A1). However, when crossed to UAS-shRNA-rl homozygous males, all embryos show poor cuticle development (A2). These phenotypes reflect the role of Rl/MAPK in the Tor and EGFR RTK pathways, respectively (see text). (B) hunchback. Embryos from MTD-Gal4>>UAS-shRNA-hb mothers crossed to WT fathers are missing the T2 and T3 thoracic segments, while abdominal segmentation is normal (B1). B2 shows the head of embryo in B1. Note that the dorsal bridge (DB) is present and appears normal. When we crossed MTD-Gal4>>UAS-shRNA-hb females to WT males, we could not distinguish between embryos with zero or one copy of the UAS-shRNA-hb transgene. Similarly, when we crossed MTD-Gal4>>UAS-shRNA-hb females to UAS-shRNA-hb homozygous males, we could not distinguish between embryos with one or two copies of the UAS-shRNA-hb transgene; all three classes of embryos resembled the one shown in B1 and B2. Together these results demonstrate that zygotically expressed shRNAs do not contribute meaningfully to this phenotype. However, when MTD-Gal4>>UAS-shRNA-hb mothers were crossed to hb[12]/+ males, half of the embryos showed a more severe phenotype (B3). In addition to lacking T2 and T3, these embryos lack the A1 abdominal segment and head structures (B4). Computational representation of hb mRNA (maternal and zygotic) in situ hybridizations in mid blastoderm stage embryos. The arrow indicates the shift in the anterior expression domain, and the arrowhead indicates that the posterior pattern has not shifted (B5). mRNA expression domain boundaries in embryos from MTD-Gal4>>UAS-shRNA-hb females (B6). The vertical lines show the posterior boundary of the anterior expression domain and both boundaries of the posterior domain for each class. The posterior expression domain is unchanged in the hb RNAi embryos, while the anterior pattern shifts anteriorly by 10% egg length. Error bars indicate standard deviations. For WT n = 11, for hb RNAi n = 9.

Figure 5

Embryonic phenotypes associated with Mat&Zyg genes. F₂ embryonic phenotypes of embryos derived from maternal-Gal4>>UAS-shRNA females crossed to UAS-shRNA males. Details on the shRNA lines associated with F₂ phenotypes can be found in Table 1 and the text.

Figure 6

Model for gene knockdown using the “maternal-Gal4–shRNA” system. Maternally deposited shRNAs can deplete early zygotic transcripts only modestly, in most cases not enough to reveal a phenotype (red acting on cyan). Zygotically activated shRNAs can effectively deplete target transcripts when they are expressed before the target is activated (orange acting on green). Early patterning genes escape knockdown because maternally loaded shRNAs lose efficacy over time, and zygotically expressed shRNAs are activated too late. Maternal–zygotic transition (MZT).