No abstract

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), e.g. enhancers, and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.

factor. Our loss-and gain-of-function studies, as well as the observed genetic interactions among Dorsocross, tinman and pannier, suggest that co-expression of these three genes in the cardiac mesoderm, which also involves crossregulation, plays a major role in the specification of cardiac progenitors. After cardioblast specification, the Dorsocross genes are re-expressed in a segmental subset of cardioblasts, which in the heart region develop into inflow valves (ostia). The integration of this new information with previous findings has allowed us to draw a more complete pathway of regulatory events during cardiac induction and differentiation in Drosophila. 4912 Bodmer, 2003), at a time when the segmentally distributed cardiac progenitors coalesce into a continuous band of cells. However, one important and direct consequence of Wg signals is the induction of sloppy paired (slp1 and slp2) in striped domains in the early mesoderm (Riechmann et al., 1997;Lee and Frasch, 2000). The slp genes encode forkhead domain repressor factors, which prevent the induction of visceral mesoderm regulators by Dpp that would otherwise interfere with cardiac induction in the presumptive cardiogenic areas (Zaffran et al., 2001). Research article DevelopmentIn addition to excluding these inappropriate regulators from the cardiogenic areas, wg is thought to promote the formation of both pericardial and myocardial progenitors also in a direct fashion. Indeed, such a mechanism operates during the specification of one specific subset of pericardial cells, termed Eve-PCs, and the induction of the even-skipped (eve) gene in progenitors of these cells requires binding sites for the Wg effector Pangolin (Pan, also known as dTCF) in combination with binding sites for Tinman and the Dpp-effectors Mad and Medea (Halfon et al., 2000;Knirr and Frasch, 2001;Han et al., 2002). Hence, the combinatorial inputs from Wg, Dpp and the cardiogenic competence factor Tinman are directly integrated at the level of the enhancer of a pericardial regulatory gene. Based upon the available genetic data on cardioblast specification, it is likely that an analogous integration of Wg and Dpp signals also occurs during the induction of certain early-acting myocardial regulatory genes. However, clear candidates for common targets of Wg and Dpp in myocardial development have not been described.Our current study identifies the Dorsocross T-box genes as crucial new components that mediate the combinatorial activities of Wg and Dpp during early steps of myocardial induction. The Dorsocross (Doc) locus encodes a cluster of three genes, Doc1, Doc2 and Doc3, that are closely related in terms of their T-box sequences and embryonic expression patterns (Reim et al., 2003). Our previous work has identified important roles of these genes during amnioserosa development and epidermal patterning of the embryo (Reim et al., 2003). We now show that, within the mesoderm, the Doc genes have an essential role for the formation of cardioblasts and a subset of pericardial cells. The indu...

The NK homeobox gene tinman (tin) is required for the specification of the cardiac, visceral muscle and somatic muscle progenitors in the early dorsal mesoderm of Drosophila. Like its vertebrate counterpart Nkx2.5, the expression of tin is maintained in cardiac cells during cardiac maturation and differentiation; however, owing to the complete lack of a dorsal vessel in tin mutant embryos, the function of tin in these cells has not been defined. Here we show that myocardial cells and dorsal vessels can form even though they lack Tin, and that viable adults can develop, as long as Tin is provided in the embryonic precardiac mesoderm. However, embryos in which tin expression is specifically missing from cardial cells show severe disruptions in the normal diversification of the myocardial cells, and adults exhibit severe defects in cardiac remodeling and function. Our study reveals that the normal expression and activity of Tin in four of the six bilateral cardioblasts within each hemisegment of the heart allows these cells to adopt a cell fate as 'working' myocardium, as opposed to a fate as inflow tract (ostial) cells. This function of tin involves the repression of Dorsocross (Doc) T-box genes and, hence, the restriction of Doc to the Tin-negative cells that will form ostia. We conclude that tin has a crucial role within myocardial cells that is required for the proper diversification, differentiation, and post-embryonic maturation of cardiomyocytes, and we present a pathway involving regulatory interactions among seven-up, midline, tinman and Dorsocross that establishes these developmental events upon myocardial cell specification.