A timer gene network is spatially regulated by the terminal system in the Drosophila embryo.

In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete, and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

Time and space in segmentation.

Arthropod segmentation and vertebrate somitogenesis are leading fields in the experimental and theoretical interrogation of developmental patterning. However, despite the sophistication of current research, basic conceptual issues remain unresolved. These include: (i) the mechanistic origins of spatial organization within the segment addition zone (SAZ); (ii) the mechanistic origins of segment polarization; (iii) the mechanistic origins of axial variation; and (iv) the evolutionary origins of simultaneous patterning. Here, I explore these problems using coarse-grained models of cross-regulating dynamical processes. In the morphogenetic framework of a row of cells undergoing axial elongation, I simulate interactions between an 'oscillator', a 'switch' and up to three 'timers', successfully reproducing essential patterning behaviours of segmenting systems. By comparing the output of these largely cell-autonomous models to variants that incorporate positional information, I find that scaling relationships, wave patterns and patterning dynamics all depend on whether the SAZ is regulated by temporal or spatial information. I also identify three mechanisms for polarizing oscillator output, all of which functionally implicate the oscillator frequency profile. Finally, I demonstrate significant dynamical and regulatory continuity between sequential and simultaneous modes of segmentation. I discuss these results in the context of the experimental literature.

Arthropod segmentation.

There is now compelling evidence that many arthropods pattern their segments using a clock-and-wavefront mechanism, analogous to that operating during vertebrate somitogenesis. In this Review, we discuss how the arthropod segmentation clock generates a repeating sequence of pair-rule gene expression, and how this is converted into a segment-polarity pattern by 'timing factor' wavefronts associated with axial extension. We argue that the gene regulatory network that patterns segments may be relatively conserved, although the timing of segmentation varies widely, and double-segment periodicity appears to have evolved at least twice. Finally, we describe how the repeated evolution of a simultaneous (Drosophila-like) mode of segmentation within holometabolan insects can be explained by heterochronic shifts in timing factor expression plus extensive pre-patterning of the pair-rule genes.

Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors.

Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. We now show here that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression. In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. We propose that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis we demonstrate that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila.

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation.

Drosophila segmentation is a well-established paradigm for developmental pattern formation. However, the later stages of segment patterning, regulated by the "pair-rule" genes, are still not well understood at the system level. Building on established genetic interactions, I construct a logical model of the Drosophila pair-rule system that takes into account the demonstrated stage-specific architecture of the pair-rule gene network. Simulation of this model can accurately recapitulate the observed spatiotemporal expression of the pair-rule genes, but only when the system is provided with dynamic "gap" inputs. This result suggests that dynamic shifts of pair-rule stripes are essential for segment patterning in the trunk and provides a functional role for observed posterior-to-anterior gap domain shifts that occur during cellularisation. The model also suggests revised patterning mechanisms for the parasegment boundaries and explains the aetiology of the even-skipped null mutant phenotype. Strikingly, a slightly modified version of the model is able to pattern segments in either simultaneous or sequential modes, depending only on initial conditions. This suggests that fundamentally similar mechanisms may underlie segmentation in short-germ and long-germ arthropods.

Odd-paired controls frequency doubling in Drosophila segmentation by altering the pair-rule gene regulatory network.

The Drosophila embryo transiently exhibits a double-segment periodicity, defined by the expression of seven 'pair-rule' genes, each in a pattern of seven stripes. At gastrulation, interactions between the pair-rule genes lead to frequency doubling and the patterning of 14 parasegment boundaries. In contrast to earlier stages of Drosophila anteroposterior patterning, this transition is not well understood. By carefully analysing the spatiotemporal dynamics of pair-rule gene expression, we demonstrate that frequency-doubling is precipitated by multiple coordinated changes to the network of regulatory interactions between the pair-rule genes. We identify the broadly expressed but temporally patterned transcription factor, Odd-paired (Opa/Zic), as the cause of these changes, and show that the patterning of the even-numbered parasegment boundaries relies on Opa-dependent regulatory interactions. Our findings indicate that the pair-rule gene regulatory network has a temporally modulated topology, permitting the pair-rule genes to play stage-specific patterning roles.