Bicoid cooperative DNA binding is critical for embryonic patterning in Drosophila

Abstract

Cooperative interactions by DNA-binding proteins have been implicated in cell-fate decisions in a variety of organisms. To date, however, there are few examples in which the importance of such interactions has been explicitly tested in vivo. Here, we tested the importance of cooperative DNA binding by the Bicoid protein in establishing a pattern along the anterior-posterior axis of the early Drosophila embryo. We found that bicoid mutants specifically defective in cooperative DNA binding fail to direct proper development of the head and thorax, leading to embryonic lethality. The mutants did not faithfully stimulate transcription of downstream target genes such as hunchback (hb), giant, and Krüppel. Quantitative analysis of gene expression in vivo indicated that bcd cooperativity mutants were unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis and displayed a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo.

Fig. 1.

Cooperative DNA-binding defect in the full-length Bicoid(K57R) mutant. Upper panels show representative gel shifts obtained by using cell extracts containing wild-type Bicoid or Bicoid(K57R). Labeled oligonucleotides carrying either two (SS) or three (SSS) strong (high-affinity) Bicoid binding sites (TCTAATCCC) were used. The single-bound (C1), double-bound (C2), and triple-bound (C3) complexes are indicated. Bicoid concentrations span 5 orders of magnitude. Lower graphs show the fraction of each major DNA-bound complex as a function of total Bicoid concentration. The curves are averages from two or more experiments. Higher-order species, which occur at high Bicoid concentrations (e.g., C4, C5, and C6) were not considered in this analysis.

Fig. 2.

bcd^K57R mutant embryos show head defects. Cuticle preparations of first-instar larvae are shown. Controls, bcd^E1/+ (a), and bcd^WT (b) show normal head development. A range of head defects occurs in bcd^K57R mutants (one copy of transgene). Two phenotypes are shown, mild head defects (c), which occur in approximately one-half of the progeny, and strong defects (d), which occur in approximately one-third of the progeny. In c, the solid arrow indicates reduced size of the dorsal arm, and the open arrow indicates disorganization of the labrum and epistomal sclerite.

Fig. 3.

Anterior hb expression is reduced in bcd^K57R mutants. In situ hybridization reveals hb mRNA expression patterns in blastoderm-staged embryos. Control wild-type (a), bcd^E1 heterozygous mutant (b), and bcd^WT (c) embryos all show the expected patterns of hb expression. Normal to mild phenotypes were observed in bcd^S35T mutants (d). Mild to strong defects in hb expression were observed for bcd^K57R mutants (e and f, respectively).

Fig. 4.

Quantitative analysis of hb expression in bcd cooperativity mutants. (a) Fluorescence immunostaining for Bcd, Eve, and Hb in control (bcd^E1/+)or bcd cooperativity mutant (bcd^K57R) embryos. In b–d, the relative Hb signal is plotted as a function of distance along the A-P axis, with each curve (color coded) representing data from an individual embryo staged ≈30 min after the onset of stage 14. In d, the data are from individual embryos double-stained for Bcd and Hb. In c and d, data for control embryos are also included. m is the average slope of the intensity of Hb staining for four to five embryos.

Fig. 5.

Expression of giant and kr are defective in bcd^K57R mutants. In situ hybridization reveals gt (a, c, e, and g) and Kr (b, d, f, and h) mRNA expression. Control embryos bcd^E1/+ (a) and bcd^WT (c) as well as bcd^S35T (e) show the expected patterns of gt expression. In contrast, more than half of the bcd^K57R (one copy of transgene) embryos show a severe reduction in the anterior (bcd-dependent) expression of gt (g). Kr expression is as expected in control embryos, bcd^E1/+ (b) and bcd^WT (d). In contrast, the Kr domain is expanded anteriorly in bcd^S35T and bcd^K57R embryos (one copy of each transgene) (f and h).