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.BBSRC grant BB/P009336/1 in Cambridge (MA and EC) BBSRC grant BB/L020092/1 in Leeds (ADP) Trinity College JRF to Erik Clark in Cambridge EMBO Long term fellowship to Erik Clark in Harvar

Modeling of gap gene regulatory networks in Drosophila with the account of the gene modular structure (in Russian)

Genes are frequently regulated in complex manners, necessitating modelling approaches which go beyond simple (linear) gene-to-gene interactions and address the modularity of cis-regulatory regions and alternate transcription initiation sites. In particular, sharp expression patterns (peaks or stripes) indicate that gene regulation involves nonlinear transcription factor kinetics (beyond first-order). We propose a methodology for approaching this problem, using the example of the multiple cis-regulatory modules (CRMs) and two transcripts (P1 and P2) found in the Drosophila hunchback (hb) and (CD1 and CD2)in Kruppel (Kr) genes, the genes expressed along the anteroposterior axis of the embryo. The positional characteristics of the mRNA expression patterns are studied for their sensitivity to the variation of the input factors and initial conditions.Comment: in Russia

An Omega-Based Bacterial One-Hybrid System for the Determination of Transcription Factor Specificity

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome

Protein-protein interactions mediated by Cys2His2 zinc-fingers

The C2H2 zinc finger motif is a compact ~ 30 amino acid molecular recognition domain that comprises a beta-hairpin followed by an alpha-helix. These domains are typically found as tandem arrays that mediate specific interactions with various macromolecules including DNA, RNA and other proteins. Although very well characterized as a DNA-binding domain, relatively little is currently understood about the molecular details of protein-protein interactions mediated by C2H2 ZFs. The Ikaros and Hunchback transcription factor family provides an ideal model system for studying ZF mediated protein-protein interactions. Ikaros, the founding member of this family is defined as a classical C2H2 ZF protein composed of a cluster of four C2H2 ZFs at the N-terminus and two additional C2H2 ZFs at the C-terminus. While the N-terminal ZFs are involved in specific DNA recognition, the C-terminal domain (termed as Dimerization Zinc Finger or DZF domain) has been shown to mediate the homo- and hetero-typic interactions. In this thesis, the DZF domains found in the Ikaros and Hunchback transcription factor family have been examined using a combination of genetic, biochemical and functional assays. To test if protein-interacting C2H2 ZFs can be used to create novel protein-protein interaction specificities, libraries of synthetic DZFs were constructed by shuffling individual C2H2 ZFs from DZF domains found in the human Ikaros and other related transcription factors. Using a bacterial-based selection system, we identified synthetic heterodimeric DZFs that can mediate activation of a reporter gene in bacterial cells. These protein-protein interaction domains can also be used to reconstitute a synthetic bi-partite activator in the nucleus of a human cell, which results in transcriptional activation of the endogenous VEGF-A gene. In addition, these synthetic two-finger domains can be linked together to create more extended protein-protein interaction interfaces. Analysis of the interaction specificities of these domains led to the discovery of a novel anti-parallel interaction mode for the DZF domain. The homo-typic interaction mediated by different DZF domains was examined in greater detail using mutational analysis. These studies narrowed down residues that are likely to be important for dimerization mediated by the Hunchback DZF domain. To obtain further information about the physical and chemical interaction surface we attempted to purify active peptides consisting of different DZF domains for X-ray crystallography. Although highly purified DZF peptides were obtained, various attempts to refold these peptides into active domains resulted in the formation of aggregates consisting of the various DZFs. Based on findings in the bacterial and cell culture systems, we started exploring if Hunchback dimerizes in Drosophila melanogaster using its DZF domain and if dimerization is essential for the function of the protein. Constructs encoding the full-length Hunchback protein harboring various natural and modified DZF domains were generated and expressed in transgenic flies. These transgenics were used to perform functional in vivo studies of the Hunchback DZF domain in Neuroblast specification during Drosophila melanogaster development. We confirmed previous studies that the C-terminal domain in Hunchback is important for maintaining the function of Hunchback in specifying early-born temporal identity in Drosophila neural stem cell lineages. Importantly, our results indicate that this domain can be functionally replaced with a heterologous (i.e., non fly) DZF domain, suggesting that the importance of the DZF domain is due to its ability to mediate dimerization

Modelling and Simulation of GeneRegulatory Networks: Segmentation genes of Honeybee (Apis mellifera) embryos

The developmental process involves interactions between thousands and thousands genes. The reasons why, where, and when the genes are expressed can be revealed in the topology of a Gene Regulatory Network (GRN). In this study, we reconstruct a GRN which explains how the expression of gap-genes is initiated along the anterior-posterior axis of Honeybee embryos. We use gene-network-based methods to model these genes in four subdivisions of the anterior-posterior axis. Unlike Drosophila, few quantitative data are available for the segmentation genes of Honeybee embryos. However, we have enough qualitative data to start logically designing a network based on those data. Then, we extend the network considering the expression domain of genes, their functionality, and some assumptions. The generated network is tested using two ODE-based methods and taking into account the quantified form of the data. The methods are Hill-function-based method and gene-circuit method. Both methods create a set of ODEs that contains a number of unknown parameters. We run the simulated annealing optimisation to find the parameters including those that predict likely interactions occurring between the genes. Because the quantified data does not have enough number of time points, many possible solutions are obtained from the optimisation. This led us to reduce the number of unknown parameters considering genetic facts. In this way, the results derived from applying Hill-function-based method are not very encouraging as the results do not show consistency in several repeated experiments. However, the gene circuit method is successful and the results are more consistent after several repeats. The reason for this difference is that while the Hill-function-based method considers more biological details in its equations, the model also involves many parameters. However, applying two methods make the results strong enough to conclude the most likely interactions. Overall, our findings suggest a network whose interactions are testable. All the required data have been collected from the experiments done by Peter Dearden and his colleagues at the laboratory for Evolution and Development at the University of Otago

INVESTIGATING PAIR-RULE GENE ORTHOLOGS IN AN INTERMEDIATE GERM BEETLE, DERMESTES MACULATUS

Insects share a body plan based on repeating segments. Segmentation has been well characterized in Drosophila melanogaster, in which segments are established by a genetic hierarchy including gap, pair-rule and segment polarity genes. Pair-rule genes (PRGs) are a key class of segmentation genes as they are the first cohort of genes expressed in a periodic pattern. Segments are established simultaneously in Drosophila in early embryos, while most other insects add segments sequentially as the embryo elongates. Our goal is to understand molecular mechanisms controlling segment formation and to determine the extent of their conservation during evolution. Here, we established the hide beetle Dermestes maculatus, an intermediate germ developer, as a new model system for studying segmentation patterning. We first established a lab colony and studied early embryogenesis in Dermestes. All nine PRG orthologs were isolated using degenerate PCR and RACE, and their expression patterns were examined with in situ hybridization. Except for opa, all Dermestes PRG orthologs are expressed in PR-like striped patterns. Gene functions were tested using RNA interference (RNAi). We examined both hatched and unhatched larvae to uncover defects with different severities. Both Dmac-prd and -slp knockdown resulted in typical PR defects, suggesting that they are “core” PR genes. Dmac-eve, -run and -odd have dual roles in germ band elongation and in PR segmentation, as severe knockdown caused anterior-only, asegmental embryos while moderate knockdown resulted in PR-like defects. Elongated but asegmental germ bands resulted from Dmac-prd and -slp double knockdown, suggesting decoupling of germ band elongation and PR segmentation. Extensive cell death prefigured the cuticle patterns after knockdowns, seen long ago for Drosophila PR phenotypes, although disrupted cell mitosis was also observed after Dmac-eve knockdown. We propose that PRGs have retained basic roles in PR segmentation during the transition from short-to-long germ development and share evolutionary conserved functions in promoting cell viability. Finally, I also present detailed protocols on Dermestes lab rearing, embryo collection and fixation, in situ hybridization and RNAi. The technical information described here will provide useful information for other genetic studies in this new model system

Chromatin regulators and the determination of embryonic polarity in Drosphila

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1994.Includes bibliographical references.by Francisco Pelegri.Ph.D