Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. Here, we use Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. We show that miR-1 down-regulation in the heart leads to dilated cardiomyopathy (DCM), a DM1-associated phenotype. We combined in silico screening for miR-1 targets with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. We identify Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.

Gelsolin and dCryAB act downstream of muscle identity genes and contribute to preventing muscle splitting and branching in Drosophila.

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.

Developmental Expression and Functions of the Small Heat Shock Proteins in Drosophila.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance.

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.

Contribution of small heat shock proteins to muscle development and function.

Investigations undertaken over the past years have led scientists to introduce the concept of protein quality control (PQC) systems, which are responsible for polypeptide processing. The PQC system monitors proteostasis and involves activity of different chaperones such as small heat shock proteins (sHSPs). These proteins act during normal conditions as housekeeping proteins regulating cellular processes, and during stress conditions. They also mediate the removal of toxic misfolded polypeptides and thereby prevent development of pathogenic states. It is postulated that sHSPs are involved in muscle development. They could act via modulation of myogenesis or by maintenance of the structural integrity of signaling complexes. Moreover, mutations in genes coding for sHSPs lead to pathological states affecting muscular tissue functioning. This review focuses on the question how sHSPs, still relatively poorly understood proteins, contribute to the development and function of three types of muscle tissue: skeletal, cardiac and smooth.

Glycolysis supports embryonic muscle growth by promoting myoblast fusion.

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

Diversification of muscle types in Drosophila: upstream and downstream of identity genes.

Understanding gene regulatory pathways underlying diversification of cell types during development is one of the major challenges in developmental biology. Progressive specification of mesodermal lineages that are at the origin of body wall muscles in Drosophila embryos has been extensively studied during past years, providing an attractive framework for dissecting cell type diversification processes. In particular, it has been found that muscle founder cells that are at the origin of individual muscles display specific expression of transcription factors that control diversification of muscle types. These factors, encoded by genes collectively called muscle identity genes, are activated in discrete subsets of muscle founders. As a result, each founder cell is thought to carry a unique combinatorial code of identity gene expression. Considering this, to define temporally and spatially restricted expression of identity genes, a set of coordinated upstream regulatory inputs is required. But also, to realize the identity program and to form specific muscle types with distinct properties, an efficient battery of downstream identity gene targets needs to be activated. Here we review how the specificity of expression and action of muscle identity genes is acquired.

Specification and behavior of AMPs, muscle-committed transient Drosophila stem cells.

During development, transient stem cells play critical roles in the formation of specific tissues. Adult Muscle Precursors (AMPs) are at the origin of all adult Drosophila muscles and as we report here represent a novel population of muscle-committed transient stem cells. Similar to vertebrate muscle stem cells, AMPs keep Notch signaling active and express Enhancer of split m6 (E(spl)m6) gene, a read-out of Notch pathway. To get insights into AMP cell specification we performed a gain-of-function screen and found that the rhomboid-triggered Epidermal Growth Factor (EGF) signaling pathway controls both the specification and the subsequent maintenance of AMPs. Our findings are supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Interestingly, by targeting GFP to the AMP cell membranes we also demonstrated that AMPs send long cellular processes and form a network of interconnected cells. As revealed by laser ablation experiments, the main role of AMP cell connections is to maintain their correct spatial positioning.

Regulation and functions of the lms homeobox gene during development of embryonic lateral transverse muscles and direct flight muscles in Drosophila.

BACKGROUND: Patterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development. METHODOLOGY/PRINCIPAL FINDINGS: The newly described homeobox gene, termed lateral muscles scarcer (lms), which has yet uncharacterized orthologs in other invertebrates and primitive chordates but not in vertebrates, is expressed exclusively in subsets of developing muscle tissues. In embryos, lms is expressed specifically in the four lateral transverse (LT) muscles and their founder cells in each hemisegment, whereas in larval wing imaginal discs, it is expressed in myoblasts that develop into direct flight muscles (DFMs), which are important for proper wing positioning. We have analyzed the regulatory inputs of various other muscle identity genes with overlapping or complementary expression patterns towards the cell type specific regulation of lms expression. Further we demonstrate that lms null mutants exhibit reduced numbers of embryonic LT muscles, and null mutant adults feature held-out-wing phenotypes. We provide a detailed description of the pattern and morphology of the direct flight muscles in the wild type and lms mutant flies by using the recently-developed ultramicroscopy and show that, in the mutants, all DFMs are present and present normal morphologies. CONCLUSIONS/SIGNIFICANCE: We have identified the homeobox gene lms as a new muscle identity gene and show that it interacts with various previously-characterized muscle identity genes to regulate normal formation of embryonic lateral transverse muscles. In addition, the direct flight muscles in the adults require lms for reliably exerting their functions in controlling wing postures.

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway.

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as we demonstrate here, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, we show that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, we performed a gain-of-function screen with a special focus on lateral AMPs expressing the homeobox gene ladybird. Our data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, our results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance.

Muscle development and regeneration in normal and pathological conditions: learning from Drosophila.

The recent demonstration that, throughout evolution, many molecular mechanisms have been highly conserved is fundamental to the advancement of our knowledge on muscle development and regeneration. Research has provided new insights into genetic cascades governing early steps of embryonic myogenesis and the regeneration of adult muscle in normal and pathological conditions, thus revealing significant similarity of both processes. Here we provide a current view on genetic mechanisms underlying muscle regeneration with a special focus on regeneration processes that take place in diseased and aging human muscle. Through examples of Drosophila models of human muscular diseases, we discuss potential impact they might have on uncovering molecular bases and identifying new treatments of muscle disorders. Taking advantage of evolutionarily conserved aspects of muscle development and the relative ease by which molecular pathways can be uncovered and dissected in a simple animal model, the fruit fly, we provide a comprehensive analysis of muscle development in Drosophila. Importantly, identification of muscle stem cell like adult muscle precursors in Drosophila makes fruit fly an attractive model system for studying muscle stem cell biology and muscle regeneration. In support of this assumption, recent studies in our laboratory provide arguments that important insights into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors.

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions we performed muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search for direct Ladybird targets. Our data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, we identified bric-a-brac 2 gene as a new component of identity code and inflated encoding alphaPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells.

Shaping leg muscles in Drosophila: role of ladybird, a conserved regulator of appendicular myogenesis.

Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. Ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach.

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. We have developed a chromatin immunoprecipitation (ChIP)-chip strategy for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. We report the use of ChEST in Drosophila to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. Our approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome.

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells.

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.

Modifiers of muscle and heart cell fate specification identified by gain-of-function screen in Drosophila.

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors and are both implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, we have performed a gain-of-function (GOF) screen for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Amongst the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of our screen. Observed GOF phenotypes have led us to identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Amongst them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Our analysis of rac2 phenotypes clearly demonstrates that the altered mesodermal level of Rho-GTPase Rac2 can influence specification of a number of cardiac and muscular cell types including those expressing ladybird. Finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicate a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinases (RTKs) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types.

Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila.

In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors. Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes. To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown. We have analysed regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors. We show that deregulation of each of them alters the number of cells that express two other genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles. Specifically, we demonstrate that mutation of the muscle identity gene msh and misexpression of the heart identity gene lb lead to heart hyperplasia with similar cell fate modifications. In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve. Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells. As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted. Consistent with all these observations, we propose that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals. The cross-repressive interactions of identity genes are likely to ensure their localised expression over time, thus providing an essential element in establishing cell identity.