During the anterior−posterior fate specification of insects, anterior fates arise in a nonelongating tissue (called the "blastoderm"), and posterior fates arise in an elongating tissue (called the "germband"). However, insects differ widely in the extent to which anterior−posterior fates are specified in the blastoderm versus the germband. Here we present a model in which patterning in both the blastoderm and germband of the beetle Tribolium castaneum is based on the same flexible mechanism: a gradient that modulates the speed of a genetic cascade of gap genes, resulting in the induction of sequential kinematic waves of gap gene expression. The mechanism is flexible and capable of patterning both elongating and nonelongating tissues, and hence converting blastodermal to germband fates and vice versa. Using RNAi perturbations, we found that blastodermal fates could be shifted to the germband, and germband fates could be generated in a blastoderm-like morphology. We also suggest a molecular mechanism underlying our model, in which gradient levels regulate the switch between two enhancers: One enhancer is responsible for sequential gene activation, and the other is responsible for freezing temporal rhythms into spatial patterns. This model is consistent with findings in Drosophila melanogaster, where gap genes were found to be regulated by two nonredundant "shadow" enhancers.clock-and-wavefront | evolution | kinematic waves | cascade | enhancer switching R hythmic and sequential gene activity has been implicated in the spatial patterning of many embryonic structures. For example, a molecular clock mediates stripes of gene expression that delimit vertebrate somites (1-3), segments in short-germ arthropods (4-10), and lateral roots in plants (11,12). Aperiodic sequential activation of genes regulates the spatial patterning of Drosophila neuroblasts (13, 14) and the vertebrate neural tube (15). However, different strategies are used in each case to translate a temporal process into a spatial one. Two main mechanisms have been described: (i) one based on the continuous retraction of a steep gradient or boundary (usually called a "wavefront") and (ii) the other based on a static or nonretracting gradient. The "clock-and-wavefront" model exemplifies the first type, and was originally proposed in the context of vertebrate somitogenesis (16). In this model, an arrest front sweeps the tissue and freezes oscillations of a molecular clock into stripes. The "spatial and temporal gradient" model exemplifies the latter, which was proposed in the context of vertebrate neural tube development (15,(17)(18)(19). In this model, the concentration of and exposure time to a more or less static (nonretracting) gradient regulates the sequential activation of genes.Models that use a wavefront (henceforth called "wavefrontbased" models) are best suited for patterning elongating tissues, since axial elongation offers a natural mechanism for continuous and sustained gradient retraction. On the other hand, models that use a static gradient (he...

Evolution of -regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using in the wing and in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in , and in a cross-species context. Together, these advances will facilitate investigation of-evolution and morphological diversity in and other insects.

ABSTRACTset of open chromatin regions in each tissue and stage. Second, we established the 28 first reporter assay system that works in both Drosophila and Tribolium, using nubbin 29 in the wing and hunchback in the embryo as case studies. Together, these advances 30 will be useful to study the evolution of cis-language and morphological diversity in 31Tribolium and other insects. 32 33

Gap genes mediate the division of the anterior-posterior axis of insects into different fates through regulating downstream hox genes. Decades of tinkering the segmentation gene network of the long-germ fruit fly Drosophila melanogaster led to the conclusion that gap genes are regulated (at least initially) through a threshold-based French Flag model, guided by both anteriorly-and posteriorly-localized morphogen gradients. In this paper, we show that the expression patterns of gap genes in the intermediate-germ beetle Tribolium castaneum are mediated by a threshold-free 'Speed Regulation' mechanism, in which the speed of a genetic cascade of gap genes is regulated by a posterior gradient of the transcription factor Caudal. We show this by re-inducing the leading gap gene (namely, hunchback) resulting in the re-induction of the gap gene cascade at arbitrary points in time. This demonstrates that the gap gene network is self-regulatory and is primarily under the control of a posterior speed regulator in Tribolium and possibly all insects.

Gap genes mediate the division of the anterior-posterior axis of insects into different fates through regulating downstream hox genes. Decades of tinkering the segmentation gene network of Drosophila melanogaster led to the conclusion that gap genes are regulated (at least initially) through a threshold-based mechanism, guided by both anteriorly- and posteriorly-localized morphogen gradients. In this paper, we show that the response of the gap gene network in the beetle Tribolium castaneum upon perturbation is consistent with a threshold-free ‘Speed Regulation’ mechanism, in which the speed of a genetic cascade of gap genes is regulated by a posterior morphogen gradient. We show this by re-inducing the leading gap gene (namely, hunchback) resulting in the re-induction of the gap gene cascade at arbitrary points in time. This demonstrates that the gap gene network is self-regulatory and is primarily under the control of a posterior regulator in Tribolium and possibly other short/intermediate-germ insects.