Gene length may contribute to graded transcriptional responses in the Drosophila embryo.

An important question in developmental biology is how relatively shallow gradients of morphogens can reliably establish a series of distinct transcriptional readouts. Current models emphasize interactions between transcription factors binding in distinct modes to cis-acting sequences of target genes. Another recent idea is that the cis-acting interactions may amplify preexisting biases or prepatterns to establish robust transcriptional responses. In this study, we examine the possible contribution of one such source of prepattern, namely gene length. We developed quantitative imaging tools to measure gene expression levels for several loci at a time on a single-cell basis and applied these quantitative imaging tools to dissect the establishment of a gene expression border separating the mesoderm and neuroectoderm in the early Drosophila embryo. We first characterized the formation of a transient ventral-to-dorsal gradient of the Snail (Sna) repressor and then examined the relationship between this gradient and repression of neural target genes in the mesoderm. We found that neural genes are repressed in a nested pattern within a zone of the mesoderm abutting the neuroectoderm, where Sna levels are graded. While several factors may contribute to the transient graded response to the Sna gradient, our analysis suggests that gene length may play an important, albeit transient, role in establishing these distinct transcriptional responses. One prediction of the gene-length-dependent transcriptional patterning model is that the co-regulated genes knirps (a short gene) and knirps-related (a long gene) should be transiently expressed in domains of differing widths, which we confirmed experimentally. These findings suggest that gene length may contribute to establishing graded responses to morphogen gradients by providing transient prepatterns that are subsequently amplified and stabilized by traditional cis-regulatory interactions.

Dynamic control of positional information in the early Drosophila embryo.

Morphogen gradients contribute to pattern formation by determining positional information in morphogenetic fields. Interpretation of positional information is thought to rely on direct, concentration-threshold-dependent mechanisms for establishing multiple differential domains of target gene expression. In Drosophila, maternal gradients establish the initial position of boundaries for zygotic gap gene expression, which in turn convey positional information to pair-rule and segment-polarity genes, the latter forming a segmental pre-pattern by the onset of gastrulation. Here we report, on the basis of quantitative gene expression data, substantial anterior shifts in the position of gap domains after their initial establishment. Using a data-driven mathematical modelling approach, we show that these shifts are based on a regulatory mechanism that relies on asymmetric gap-gap cross-repression and does not require the diffusion of gap proteins. Our analysis implies that the threshold-dependent interpretation of maternal morphogen concentration is not sufficient to determine shifting gap domain boundary positions, and suggests that establishing and interpreting positional information are not independent processes in the Drosophila blastoderm.

Characterization of the Drosophila segment determination morphome.

Here we characterize the expression of the full system of genes which control the segmentation morphogenetic field of Drosophila at the protein level in one dimension. The data used for this characterization are quantitative with cellular resolution in space and about 6 min in time. We present the full quantitative profiles of all 14 segmentation genes which act before the onset of gastrulation. The expression patterns of these genes are first characterized in terms of their average or typical behavior. At this level, the expression of all of the genes has been integrated into a single atlas of gene expression in which the expression levels of all genes in each cell are specified. We show that expression domains do not arise synchronously, but rather each domain has its own specific dynamics of formation. Moreover, we show that the expression domains shift position in the direction of the cephalic furrow, such that domains in the anlage of the segmented germ band shift anteriorly while those in the presumptive head shift posteriorly. The expression atlas of integrated data is very close to the expression profiles of individual embryos during the latter part of the blastoderm stage. At earlier times gap gene domains show considerable variation in amplitude, and significant positional variability. Nevertheless, an average early gap domain is close to that of a median individual. In contrast, we show that there is a diversity of developmental trajectories among pair-rule genes at a variety of levels, including the order of domain formation and positional accuracy. We further show that this variation is dynamically reduced, or canalized, over time. As the first quantitatively characterized morphogenetic field, this system and its behavior constitute an extraordinarily rich set of materials for the study of canalization and embryonic regulation at the molecular level.

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster.

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system.

Computational identification of Ftz/Ftz-F1 downstream target genes.

Hox genes encode DNA binding transcription factors that regulate the body plans of metazoans by regulating the expression of downstream target 'realizator genes' that direct morphogenesis and growth. Although some Hox target genes have been identified, the code used by Hox proteins to select regulatory targets remains elusive. This failure is due, in part, to the overlapping and promiscuous DNA binding potential of different Hox proteins. The identification of cofactors that modulate Hox DNA binding specificity suggested that target site selection is specified by composite binding sites in the genome for a Hox protein plus its cofactor. Here we have made use of the fact that the DNA binding specificity of the Drosophila Hox protein Fushi Tarazu (Ftz) is modulated by interaction with its partner, the orphan nuclear receptor Ftz-F1, to carry out a computational screen for genomic targets. At least two of the first 30 potential target genes--apontic (apt) and sulfated (Sulf1)--appear to be bona fide targets of Ftz and Ftz-F1. apt is expressed in stripes within the Ftz domain, but posterior to engrailed (en) stripes, suggesting a parasegmental border-independent function of ftz. Ftz/Ftz-F1 activate Sulf1 expression in blastoderm embryos via composite binding sites. Sulf1 encodes a sulfatase thought to be involved in wingless (Wg) signaling. Thus, in addition to regulating en, Ftz and Ftz-F1 coordinately and directly regulate different components of segment polarity pathways in parallel.

Analysis of pattern precision shows that Drosophila segmentation develops substantial independence from gradients of maternal gene products.

We analyze the relation between maternal gradients and segmentation in Drosophila, by quantifying spatial precision in protein patterns. Segmentation is first seen in the striped expression patterns of the pair-rule genes, such as even-skipped (eve). We compare positional precision between Eve and the maternal gradients of Bicoid (Bcd) and Caudal (Cad) proteins, showing that Eve position could be initially specified by the maternal protein concentrations but that these do not have the precision to specify the mature striped pattern of Eve. By using spatial trends, we avoid possible complications in measuring single boundary precision (e.g., gap gene patterns) and can follow how precision changes in time. During nuclear cleavage cycles 13 and 14, we find that Eve becomes increasingly correlated with egg length, whereas Bcd does not. This finding suggests that the change in precision is part of a separation of segmentation from an absolute spatial measure, established by the maternal gradients, to one precise in relative (percent egg length) units.

Removal of background signal from in situ data on the expression of segmentation genes in Drosophila.

Here we present a method for the removal of nonspecific background signal from fluorescently localized expression patterns of Drosophila segmentation genes. Our algorithm for removal of background signal brings the data to a common standard form with zero background and removes systematic error in gene expression levels caused by the presence of background. The method is based on the discovery, reported here, that background is well fit by a very broad two-dimensional paraboloid. The paraboloid is determined from the area of the embryo in which a given gene is not expressed and the whole pattern is then normalized by this paraboloid to remove background from the entire embryo. The software implementing this algorithm is available from the authors.