The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila

Abstract

In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

Figure 1. TAGteam sites bind Zld and mediate transcriptional activation

(a) DNA sequence of the 91 bp zen enhancer (uppercase) plus surrounding sequences (lowercase). Base substitutions are in purple. (b) Schematic organization of the zld locus (CG12701; Flybase) with the two predicted transcription start sites, RB and RA. The P{RS3}UM8171-3 insertion site is between -661 and -660. The nucleotides deleted in zld²⁹⁴ and zld⁶⁸¹ are indicated as blank space between solid lines. (c) Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each section of the gel). The first lane in each section contains free probe. The second lane contains probe plus 10ng GST-ZldC, the third 30 ng GST-ZldC. (d) S2 cells were transfected with 0 ng (blue bar), 50 ng (red bar), or 100 ng (yellow bar) of plasmid expressing zld under control of the inducible metallothionein promoter, the zen91-lacZ or zen91m-lacZ reporter plasmids, and the luciferase control. Error bars, s.e.m.; n=3.

Figure 2. Maternal zld transcripts are lost as zygotic zld is activated in cycle 14

Wild-type (wt; a-d) and zld²⁹⁴ (e-j) ovaries (a) and embryos (b-j) were hybridized with zld (all but g) or bcd (g) RNA probes. (a) mid (left) and late-stage (right) egg chambers with zld transcripts in the nurse cells (nc) but not the columnar follicle cells that overlay the oocyte. (b) Unfertilized egg. (c) cycle 14 embryo undergoing cellularization. (d) late-stage embryo. (e) M⁺Z^- zld cycle 14 embryo. Maternal zld transcripts have disappeared. (f) M^- zld cycle 10-11 embryo. (g) M^- zld cycle 14 embryo has a normal bcd pattern. (h) M^-Z^- zld late cycle14 embryo showing anomalous distribution of cytoplasm (arrows). (i) M^-Z⁺ zld early cycle 14 embryo showing onset of zygotic zld expression. (j) M^-Z⁺ zld late cycle14 embryo showing abnormalities (arrows).

Figure 3. Maternal zld is required for cellularization

Confocal images of wt, zld^- (M^- zld²⁹⁴) and rescued (M^- zld²⁹⁴; otu-zld) embryos (as indicated) stained with phalloidin to detect F-actin, α-slam, or α-Nrt antibodies (green), and DAPI to detect DNA (red). In M^- zld embryos the cytoskeletal network is disorganized and quickly degenerates in early cycle 14 (d) accompanied by nuclear fallout (d’, arrow). Slam protein disappears in cycle 14 (f, f’), while Nrt accumulates apically (h, arrow). In M^- zld²⁹⁴; otu-zld embryos the cytoskeleton is organized (i) and cellularization proceeds (i’, arrow).

Figure 4. Zld plays a role in zygotic gene activation and maternal RNA degradation during the MZT

wt (left) and M^-Z^- zld²⁹⁴ (right) mitotic cycle 12-14 embryos were hybridized as indicated (309 = pri-miR-309). (k) Summary of expression profiles of 1-2 hr wt and M^-zld²⁹⁴ embryos. fold ch = fold change with respect to wt (genes absent in the array data are not included). (l) Percentage of genes for which there is expression data,, described as maternal (M), zygotic (Z) or both (MZ) in the down- (≥ 2 fold) and up-regulated (≥ 1.5 fold) gene sets.