Evolution of insect hindwing morphology: A comparative genomic analysis of targets of Hox protein Ultrabithorax
ABSTRACT: To gain insights into the species-specific events downstream of Ubx, we identified direct targets of Ubx from Apis by ChIP-seq and compared them with those of Drosophila at the level of both their function and regulation. While the majority of the targets are species-specific, the ontological distribution of all targets was similar amongst the two species. Interestingly, although considerable number of wing-patterning genes are retained as targets of Ubx over the past 300 millions years, many of them are differentially expressed only between wing and haltere in Drosophila and not between forewing and hindwing in Apis . Detailed bioinfomatics analysis and and experimental validation of enhancer sequences suggest that acquisition of binding sites for additional transcription factors without altering binding per se of Ubx could also contribute to the diversity in Ubx function.
Project description:In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300?millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.
Project description:Mutations in the <i>Ultrabithorax</i> (<i>Ubx</i>) gene cause homeotic transformation of the normally two-winged <i>Drosophila</i> into a four-winged mutant fly. <i>Ubx</i> encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. <i>Ubx</i> is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that <i>Ubx</i> acts by repressing the expression of two genes in the haltere, <i>Stubble</i> and <i>Notopleural</i>, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, <i>Ubx</i> induces expression of the <i>Tissue inhibitor of metalloproteases</i> in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.
Project description:Hox proteins have been proposed to act at multiple levels within regulatory hierarchies and to directly control the expression of a plethora of target genes. However, for any specific Hox protein or tissue, very few direct in vivo-regulated target genes have been identified. Here, we have identified target genes of the Hox protein Ultrabithorax (UBX), which modifies the genetic regulatory network of the wing to generate the haltere, a modified hindwing. We used whole-genome microarrays and custom arrays including all predicted transcription factors and signaling molecules in the Drosophila melanogaster genome to identify differentially expressed genes in wing and haltere imaginal discs. To elucidate the regulation of selected genes in more detail, we isolated cis-regulatory elements (CREs) for genes that were specifically expressed in either the wing disc or haltere disc. We demonstrate that UBX binds directly to sites in one element, and these sites are critical for activation in the haltere disc. These results indicate that haltere and metathoracic segment morphology is not achieved merely by turning off the wing and mesothoracic development programs, but rather specific genes must also be activated to form these structures. The evolution of haltere morphology involved changes in UBX-regulated target genes, both positive and negative, throughout the wing genetic regulatory network.
Project description:Hox genes encode a family of transcription factors that are key developmental regulators with a highly conserved role in specifying segmental diversity along the metazoan body axis. Although they have been shown to regulate a wide variety of downstream processes, direct transcriptional targets have been difficult to identify and this has been a major obstacle to our understanding of Hox gene function. We report the identification of genome-wide binding sites for the Hox protein Ultrabithorax (Ubx) using a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis. We identify 1,147 genes bound by Ubx at high confidence in chromatin from the haltere imaginal disc, a prominent site of Ubx function where it specifies haltere versus wing development. The functional relevance of these genes is supported by their overlap with genes differentially expressed between wing and haltere imaginal discs. The Ubx-bound gene set is highly enriched in genes involved in developmental processes and contains both high-level regulators as well as genes involved in more basic cellular functions. Several signalling pathways are highly enriched in the Ubx target gene set and our analysis supports the view that Hox genes regulate many levels of developmental pathways and have targets distributed throughout the gene network. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity with the Ubx binding profile. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and provide evidence of a link between Ubx/Hth binding and chromatin state at genes regulated by Polycomb silencing. Overall, we define a set of direct Ubx targets in the haltere imaginal disc and suggest that chromatin accessibility has important implications for Hox target selection and for transcription factor binding in general.
Project description:The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). In contrast to previous ChIP-chip studies carried out in Drosophila embryos, these binding studies reveal that there is a remarkable amount of tissue- and transcription factor-specific binding. Analyses of the putative target genes bound and regulated by these factors suggest that Ubx regulates many downstream transcription factors and developmental pathways in the haltere and T3 leg. Finally, we discovered additional DNA sequence motifs that in some cases are specific for individual data sets, arguing that Ubx and/or Hth work together with many regionally expressed transcription factors to execute their functions. Together, these data provide the first whole-genome analysis of the binding sites and target genes regulated by Ubx to specify the morphologies of the adult T3 segment of the fly.
Project description:Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilateral animals. Despite decades of research, mechanism by which Hox proteins select their targets and specify segmental identity remains elusive. To address this question we carried out whole genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. When mis-expressed in wing segment (T2) Ubx converts its identity to that of haltere segment (T3). We used CbxHm/+ wing discs ectopically expressing Ubx in the pouch region of discs to obtain chromatin. This helped us focus on targets of Ubx involved in pouch development without mixing with the targets involved in notum development. Polyclonal Ubx antibodies against N-terminal region (excluding homeodomain) were generated in our lab and used to pull down Ubx bound regions from CbxHm/+ wing discs. Mock DNA (No antibody) was used as control. Test Vs. Mock experiment. CbxHm/+ discs for Chromatin source. Biological replicates: 3 [Agilent two-color ChIP-on-Chip experiment,Organism: Drosophila melanogaster ,Drosophila Whole Genome ChIP-on-Chip Set 244K Microarray 1 of 2 (AMADID: 014816 and 014817)]
Project description:Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilaterian animals. Despite decades of research, the mechanisms by which Hox proteins select and regulate their targets remain elusive. We have carried out whole-genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. Direct targets identified include upstream regulators or cofactors of Ubx. Homothorax, a cofactor of Ubx during embryonic development, is one such target and is required for normal specification of haltere. Although Ubx bound sequences are conserved amongst various insect genomes, no consensus Ubx-specific motif was detected. Surprisingly, binding motifs for certain transcription factors that function either upstream or downstream to Ubx are enriched in these sequences suggesting complex regulatory loops governing Ubx function. Our data supports the hypothesis that specificity during Hox target selection is achieved by associating with other transcription factors.
Project description:Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.
Project description:Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. We have studied how the Drosophila Hox gene Ultrabithorax (Ubx) limits the size of the haltere, which, by the end of larval development, has approximately fivefold fewer cells than the wing. We find that Ubx controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp). Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes.
Project description:The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). This is a dataset generated by the Drosophila Regulatory Elements modENCODE Project led by Kevin P. White at the University of Chicago. This dataset was generated in collaboration with Richard S. Mann at Columbia University. It contains ChIP-chip data on Affymetrix Drosophila Tiling 2.0R arrays for multiple transcription factor antibodies in multiple Drosophila tissues. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Haltere or leg imaginal discs ChIPped for Ubx or Hth vs. input DNA from corresponding imaginal discs. For each combination of tissue and antibody, ChIP experiments have been performed and hybridized on Affymetrix Drosophila Tiling 2.0R arrays. At least 3 biological replicates for the ChIP sample have been hybridized.