David Kosman

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.

RAPID PREPARATION OF A PANEL OF POLYCLONAL ANTIBODIES TO DROSOPHILA SEGMENTATION PROTEINS

Abstract We describe a method for rapidly raising a panel of high quality polyclonal antibodies from bacterially expressed proteins. Approximately 12/3 days of preparation is required per protein. One step that speeds up the procedure is the visualization of purified bands by precipitated sodium dodecyl sulfate (SDS). Antigenicity of the purified recombinant proteins may be increased by precipitation in double-distilled water. The results of using the serums obtained for fluorescent staining of Drosophila embryos are shown.

Stripe forming architecture of the gap gene system.

In this report, we show that gap genes encode exactly one set of pair-rule stripes, which occur in the native even-skipped position. The core of this work is a detailed analysis that shows how this conclusion follows from the arrangement of gap domains in the embryo. This analysis shows that: (1) pattern forming information is transmitted from gap to pair-rule genes by means of a nonredundant set of morphogenetic gradients, and (2) the stripe forming capability of the gap genes is constrained by the arrangement of these gradients and by the fact that each gap domain consists of a pair of correlated gradients. We also show that in the blastoderm, the regulatory sign of a transcriptional regulator is unlikely to change in a concentration dependent manner. The principal analytic tool used to establish these results is the gene circuit method. Here, this method is applied to examine hybrid data sets consisting of real gene expression data for four gap genes and hypothetical pair-rule expression data generated by translating native even-skipped data along the anterior-posterior axis. In this way, we are able to investigate the stripe forming capabilities of the gap gene system in the complete absence of pair-rule cross regulation. We close with an inference about evolutionary development. It is argued that the constraints on gap gene architecture identified here are a consequence of selective pressures that minimize the number of gap genes required to determine segments in long-germ band insects.

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. Developmental Dynamics 235:2949–2960, 2006.

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.

SEGMENTATION OF NUCLEI IN CONFOCAL IMAGE STACKS USING PERFORMANCE BASED THRESHOLDING

Combinatorial transcriptional fluorescent in situ hybridization (CT-FLSH) is a confocal fluorescence imaging technique enabling the detection of multiple active transcription units in individual interphase diploid nuclei. As improved combinatorial labeling methods allow simultaneous measurement of gene activities to expand from five genes in a single embryo or tissue section to upward of twenty genes, transforming image stacks into usable data becomes an increasingly labor in tensive task, in this paper we describe our progress towards a method for the computational analysis of confocal images from Drosophila melanogastar that involves the segmentation of the cell nuclei and of nascent transcription sites of specific genes. Using image processing and machine learning algorithms, we allow experimentalists to reiteratively tune and improve the analysis system to reflect biological reality

Experimental Determination of Drosophila Embryonic Coordinates by Genetic Algorithms, the Simplex method, and Their Hybrid

Modern large-scale functional genomics projects are inconceivable without the automated processing and computer-aided analysis of images. The project we are engaged in is aimed at the construction of heuristic models of segment determination in the fruit fly Drosophila melanogaster. The current emphasis in our work is the automated transformation of gene expression data in confocally scanned images into an electronic database of expression. We have developed and tested programs which use genetic algorithms for the elastic deformation of such images. In addition, genetic algorithms and the simplex method, both separately and in concert, were used for experimental determination of Drosophila embryonic curvilinear coordinates. Comparative tests demonstrate that the hybrid approach performs best. The intrinsic curvilinear coordinates of the embryo found by our optimization procedures appear to be well approximated by lines of isoconcentration of a known morphogen, Bicoid.

Fast redundant dyadic wavelet transform in application to spatial registration of the expression patterns of Drosophila segmentation genes

A 1D wavelet-based registration technique was applied to Drosophila segmentation gene expression data. We apply the fast redundant dyadic wavelet transform with a continuous basis as a method for extracting the ground control points. This transform was chosen for its basic properties, such as noise reduction and good localization of characteristic features.