Michèle Crozatier

The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing

BACKGROUNDnThe secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing.nnnRESULTSnWe show that short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF), the activity of which is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes our understanding of the sectorial organisation of the Drosophila wing.nnnCONCLUSIONSnCol, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix (HLH)-containing transcription factors, is a mediator of the short-range organising activity of Hh in the Drosophila wing. Our results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signalling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates.

Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier.

Drosophila immune response involves three types of hemocytes (‘blood cells’). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells.

collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain

BACKGROUNDnSegmentation of the Drosophila embryo is based on a cascade of hierarchical gene interactions that is initiated by maternal morphogens; these interactions define spatially restricted domains of zygotic gene expression within the blastoderm. Although the hierarchy of the segmentation genes that subdivide the trunk is well established, the patterning of the head is less well understood. Seven head segments can be assigned on the basis of metameric patterns of segment-polarity gene expression and internal sensory organs. The domains of expression of head gap-like genes broadly overlap, with their posterior margins out of phase by one segment. Taken together with the lack of pair-rule gene expression in the head, these observations led to the suggestion that head gap genes act in a combinatorial manner, determining head segmental borders and segmental identity at the same time.nnnRESULTSnWe have identified a new Drosophila gene, collier (col), whose expression at the blastoderm stage is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, possibly parasegment O. Reduction of col activity in early gastrula embryos by antisense RNA expression results in a specific lack of head structures derived from these segments. The expression of col coincides with a mitotic domain, which supports the proposal that cells in this domain undergo a concerted mitotic and differentiation program that is orchestrated at the transcriptional level. Col is an ortholog of mammalian early B-cell factor/Olfactory-1. These proteins define a new family of transcription factors that contain a helix-loop-helix dimerization motif and a new type of DNA-binding domain that is highly conserved during evolution.nnnCONCLUSIONSnHere we describe Col, the first Drosophila member of a new family of transcription factors. Col may act as a second-level regulator of head patterning. The structural conservation of Col during evolution raises the questions of its conservation of function in head specification and its interactions with other factors conserved between insects and vertebrates.

The metazoan history of the COE transcription factors. Selection of a variant HLH motif by mandatory inclusion of a duplicated exon in vertebrates

BackgroundThe increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. Transcription factors (TFs) involved in regulation of gene-specific expression are key players in the evolution of metazoan development. The low complexity COE (Collier/Olfactory-1/Early B-Cell Factor) family of transcription factors constitutes a well-suited paradigm for studying evolution of TF structure and function, including the specific question of protein modularity. Here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.ResultsCOE proteins display a modular organisation, with three highly conserved domains : a COE-specific DNA-binding domain (DBD), an Immunoglobulin/Plexin/transcription (IPT) domain and an atypical Helix-Loop-Helix (HLH) motif. Comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral COE DBD was built from 7 separate exons, with no evidence for exon shuffling with other metazoan gene families. It also confirms the presence of an ancestral H1LH2 motif present in all COE proteins which partly overlaps the repeated H2d-H2a motif first identified in rodent EBF. Electrophoretic Mobility Shift Assays show that formation of COE dimers is mediated by this ancestral motif. The H2d-H2a α-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. We put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.ConclusionThree main features in the history of the coe gene family can be inferred from these analyses: (i) each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. (ii) There exists a single coe gene copy per metazoan genome except in vertebrates. The H2a-H2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. (iii) This duplication provides an interesting example of counter-selection of alternative splicing.

cDNA sequence and expression pattern of the Drosophila melanogaster PAPS synthetase gene: a new salivary gland marker

PAPS synthetase is a bifunctional enzyme containing both ATP sulfurylase and APS kinase activities required for the biosynthesis of PAPS, the sulfate donor in sulfation reactions. Here we report the sequence of the Drosophila melanogaster PAPS synthetase, the first gene implicated in the sulfation pathway to be described in that organism, and the characterization of its specificity of expression in embryos. Whole-mount in situ hybridization reveals that DmPAPSS is a novel salivary gland marker. At the end of embryogenesis, expression of DmPAPSS is also observed at the entry and exit of the gut and the posterior spiracles. We discuss the possibility that the pattern of expression of the DmPAPSS gene might reflect a major role for sulfation in mucus biosynthesis at the end of Drosophila embryogenesis.

Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis.

Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling.

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.

Neither Too Much Nor Too Little: Reactive Oxygen Species Levels Regulate Drosophila Hematopoiesis

Owing to their fast diffusion, reactive oxygen species (ROS) are important tissue signalling components. High levels of ROS are generally considered as deleterious to cells. In vivo experiments in the Drosophila hematopoietic organ now challenge this view by showing that high ROS levels are intrinsically required to prime myeloid-like progenitors to differentiate.

Interspecific comparison of Drosophila serendipity δ and β: Multimodular structure of these C2H2 zinc finger proteins

The Drosophila serendipity (sry) β and δ genes, which resulted from a gene duplication event, provide an interesting model for the evolutionary diversification in structure and function of C2H2 zinc finger proteins. We examined here the divergence of the sry β and δ proteins over an estimated period of 45 million years by comparing their predicted sequences in D. melanogaster, D. pseudoobscura, and D. subobscura. Between orthologs, i.e., pairs of either sry β or sry δ, the NH2-proximal region delineated by pairs of C-X2-C motifs and the DNA-binding finger domain are highly conserved. Sequence conservation operates over the entire finger domain, including the links separating adjacent fingers, even though each has a unique sequence different from the widespread TGEKP motif. In contrast, the sequence of the central acidic region has extensively diverged and differs between species in the number of amino acids, probably because of slippagedriven mutations. The NH2-terminal region and fingers 1, 5, and 6 differentiate the sry β and δ proteins while zinc fingers 2, 3, and 4 are virtually identical in these two paralogs. A nuclear localization signal of the SV40T antigen type, preceded by a potential CKII phosphorylation regulatory site, is conserved in sry δ but not found in sry β. The interspecific conserved regions correlate well with the positions of zygotic lethal mutations in the D. melanogaster sry δ protein. Furthermore, P-element transformation experiments show that a transgenic copy of the D. pseudoobscura sry δ gene rescues the sry δ mutant phenotype. Convergence of genetic and structural data on the sry proteins supports a multimodular function and mode of evolution of these C2H2 finger proteins.

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. Summary: Chromatibody is a chromatin-binding single-domain antibody, derived from llama nanobodies, that can be used as a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.