How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation

Abstract

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, we have conducted a comprehensive systems-level examination of the regulatory architecture underlying pair-rule stripe formation. We find that runt (run), fushi tarazu (ftz) and odd skipped (odd) establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, we observe long-range cis-regulation across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, our study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo.

Fig. 1.

Regulation in the pair-rule gene network. (A) The mode of cis-regulatory control of primary versus secondary pair-rule genes according to the traditional model. (B) Overview showing the cis-elements and the patterns they produce for all pair-rule genes. Elements discovered in our work are framed in black. (C) Cross-regulatory relationships among the (expanded) group of primary pair-rule genes, based on ectopic expression studies conducted by Krause and colleagues (Manoukian and Krause, 1992; Manoukian and Krause, 1993; Nasiadka and Krause, 1999; Saulier-Le Drean et al., 1998), as well as loss-of-function analyses (Carroll and Vavra, 1989; Ingham and Gergen, 1988). The green arrow indicates activation and line thickness represents strength of regulation. (D) The positional code defined by phase-shifted pair-rule stripes within a two-segment unit, based on expression data from Myasnikova et al. (Myasnikova et al., 2001). Circles represent single nuclei.

Fig. 2.

Expression dynamics of the pair-rule genes. (A) Schematics and high-magnification in situ hybridization images visualizing the four morphologically distinct phases of cellularization used to stage Drosophila embryos. Time is indicated in minutes. Initially small and spherical (phase 1), the nuclei elongate during phase 2; the plasma membrane then begins to ensheath the nuclei (phase 3), extending past them and closing to form cells during phase 4 (Lecuit and Wieschaus, 2000). Embryos are shown from the end of phases 1-3 and from the middle of phase 4. (B) RNA in situ hybridization of wild-type embryos using pair-rule gene antisense probes, for each of the four phases depicted in A. In these and all subsequent figures, lateral views of whole-mount embryos are shown, with anterior to the left and dorsal up. The stripes are labeled for phase 1, as the patterns are first forming. See also Fig. S1 in the supplementary material. (C) Expression of lacZ reporter genes for the primary pair-rule gene 7-stripe elements; shown are the eve late element (Fujioka et al., 1995), the run_(−3) element (see Fig. 3), the ftz LacC element (Hiromi et al., 1985) and odd_basal–1&–10 element (see Fig. 4B). Individual stripes expressed during phase 2 are labeled. The full 5 kb run 7-stripe element similarly shows early expression with complex dynamics for individual stripes (Klingler et al., 1996).

Fig. 3.

Dissection of the cis-regulatory region of run. (Top) The genomic region surrounding the Drosophila run locus, with annotated transcripts and Stubb free energy profiles as indicated. Boxes below the free energy profiles for maternal/gap input (black) depict the positions of previously known cis-elements (gray) (Klingler et al., 1996); cis-elements identified in this study that produce striped expression are shown in blue and those that do not in white. (Bottom) The RNA expression patterns of selected cis-element reporter constructs for phases 1 and 3. The run_(−16) element partially overlaps the known element for, and drives expression in, stripe 1. For the run_(+30) element, the inset in the top part of the figure shows the free energy profile for KR (brown); note the separate peaks discernible within the element. Curly brackets indicate the position of the KR expression domain.

Fig. 4.

Dissection of the cis-regulatory regions of ftz and odd. (A) Dissection of Drosophila ftz. Constructs, including rescue constructs (green), are from Calhoun and Levine (Calhoun and Levine, 2003) and Hiromi et al. (Hiromi et al., 1985). (B) Dissection of odd, with constructs tested in search of the 7-stripe element as indicated. Note that the staining for the transgenic line containing the −10 element alone takes unusually long to develop, indicating that the element drives weak expression. Single-factor free energy profiles are shown for selected pair-rule genes. See Fig. 3 for further description of layout.

Fig. 5.

Pair-rule gene mutant analysis. Transcript patterns of all pair-rule genes in wild type (wt) and pair-rule gene mutants in Drosophila phase 3 blastoderm embryos. Genotypes are arrayed by row, transcript patterns by column. For eve mutant embryos, the position of the fused stripe 1/2 domain of slp1 is indicated by a curly bracket.

Fig. 6.

Binding site composition and genomic organization of stripe-specific cis-elements. (A) The spatial correlation of cis-element expression domains (dark gray) with the repressive gap factor input they receive. HB, blue; KR, brown; KNI, magenta; GT, green; TLL, purple; the strength of input is represented by the color intensity. Elements are grouped according to the central gap factors that provide the dominant repressive input. Expression patterns of gap factors are shown above [FRDWT 10% strip, time class 14A 4, data from Myasnikova et al. (Myasnikova et al., 2001)]. (B) Scatter plots showing the relationship between strength of predicted input (x, Stubb integrated profile value) and distance from the center of the gap factor domain to the proximal stripe border (y; % EL, percentage egg length). The results of linear regression analysis are indicated. The data points for the full h 1+5 element and the run 2+7 element are shown in gray, and for the separable h stripe 1 and 5 elements in gray with a black border. (C) Genomic organization of stripe-specific cis-elements within the pair-rule gene loci, with color-coding based on the classification shown in A. See also Fig. S2 and Tables S1, S2 and S3 in the supplementary material.