Drosophila myogenesis as a model for studying cis-regulatory networks : identifying novel players and dissecting the role of transcriptional repression

Recent studies have identified in vivo binding profiles of key mesodermal regulators across the Drosophila melanogaster genome. Many of the occupied sites lie in the vicinity of loci encoding yet other transcription factors. The analyzed cis- regulatory modules drive expression in a variety of complex spatio-temporal patterns that cannot be explained by the binding of the core regulators alone. Thus there clearly are additional, unknown transcription factors in the regulatory network that governs the process of embryonic mesoderm specification and muscle differentiation. In order to identify novel myogenic regulators in a systematic way, and thereby enrich the underlying network, I initiated a molecular screen to uncover new players. Candidate putative transcription factors were prioritized based on their expression in mesoderm and on available ChIP-on-chip and expression profiling data. Their role in myogenesis was subsequently assayed using Drosophila deficiency lines whose phenotypes were analyzed with a muscle-specific marker. Altogether, 67 different deficiency and loss-of-function lines were used individually or in combination to delete 46 transcription factors with mesodermal expression. In 21 of the 46 cases, the mutant embryos displayed specific defects in the development of one or more muscle types. One pair of partially overlapping deficiencies placed in trans showed a failure in myoblast fusion, a process that gives rise to muscle syncytia from mononucleated myoblasts. The corresponding deleted candidate gene was MED24, a subunit of the Mediator complex, which is a general co-activator of transcription. Muscle-specific knockdown of MED24 or MED14, another subunit of the complex whose deletion by deficiency lines phenocopies that of MED24, leads to lethality. To establish whether MED24 and MED14 are indeed involved in muscle development, I generated smaller deletion lines using FRT-mediated recombination. While deletion of MED14 does not affect myogenesis, embryos deficient for MED24 display supernumerary mononucleated myoblasts. Both small deletion lines were then combined together to detect possible redundancy that could obscure the requirement of MED14 and MED24 in muscle development. Another candidate transcription factor within the myogenic network based on ChIP-on-chip experiments is the transcriptional repressor Tramtrack69 (Ttk69). Ttk69 is expressed in the primordium of visceral and, more transiently, somatic muscle. In ttk69 mutant embryos, homozygous for a loss-of-function allele, myoblast fusion is delayed, the myoblasts aggregate in clusters, and fail to migrate towards the ectodermal attachment sites. Two distinct myoblast populations, a founder cell and multiple fusion competent myoblasts, contribute to each muscle fibre. As revealed by immunohistochemistry and in situ hybridization, in ttk69 mutants there are significantly more founder cells formed while the number of fusion competent myoblasts is decreased. Consistently, ectopic expression of Ttk69 in the founder cells, but not fusion competent myoblasts, gives rise to severe myoblast fusion defects. These phenotypic analyses suggest a model where Ttk69 is required for specification of fusion competent myoblasts and in its absence, their conversion to a founder cell-like fate may occur. According to the proposed model, Ttk69 would repress founder cell genes within the fusion competent myoblasts. To determine whether this holds true on a global scale, I performed a high-resolution ChIP-on-chip experiment in 6-8 hour wild type embryos. Indeed, Ttk69 binding was significantly enriched in the vicinity of founder cell-specific genes as compared to fusion competent myoblast-specific genes. ChIP-on-chip data generated for Lame duck, a transcriptional activator essential for fusion competent myoblast determination, showed the opposite tendency. It therefore appears that proper specification of fusion competent myoblast identity requires both positive input from Lame duck and inhibition of founder cell-specific genes by Ttk69. These findings advance our limited knowledge about the role of transcriptional repression within the myogenic regulatory network. Finally, I re-evaluated the role of Snail, a well-established transcriptional repressor involved in early mesoderm specification and gastrulation. Multiple observations suggested that Snail may also play a positive role in regulating mesodermal genes. To investigate this possibility, I performed luciferase assays with previously characterized mesodermal enhancers and showed that Snail can elevate their activation levels. In one case, this ability of Snail was suppressed upon mutagenesis of putative Snail binding motifs, both in cell culture and in vivo. Moreover, expression of the enhancers and their associated genes is significantly reduced in snail mutant embryos. Snail thus seems to play a dual role in repressing non-mesodermal genes, but also in contributing to the activation of some early mesodermal genes

Combinatorial binding leads to diverse regulatory responses:Lmd is a tissue-specific modulator of Mef2 activity

Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation

Qualitative Dynamical Modelling Can Formally Explain Mesoderm Specification and Predict Novel Developmental Phenotypes

International audienceGiven the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. Here, we delineate a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature. The four main tissues derived from mesoderm correspond to alternative stable states. We demonstrate that the model can predict known mutant phenotypes and use it to systematically predict the effects of over 300 new, often non-intuitive, loss- and gain-of-function mutations, and combinations thereof. We further validated several novel predictions experimentally, thereby demonstrating the robustness of model. Logical modelling can thus contribute to formally explain and predict regulatory outcomes underlying cell fate decisions

Key signalling pathways and markers genes involved in mesoderm specification.

<p>A, B: In situ hybridizations for Tin and Bin during mesoderm specification at stages 8 and 9–10. Tin is implicated in the formation of VM and H, while Bin participates only in the development of VM. Initially, the expression of Tin is mainly due to Twist activation. Later, Tin expression needs the presence of Dpp, Tin itself, in combination with Pan. C: Graphical representations of the main pathways activated by signals coming from the ectoderm, encompassing target transcription factors and cross-regulations underlying the specification of VM, H, FB and SM. In the absence of these factors, these tissues do not form or are severely reduced. Black and light grey arcs denote active and inactive regulations, depending on stage or tissue. Normal and blunt end arrows denote activations and inhibitions, respectively.</p

Simulations of known genetic perturbations.

<p>The results of selected simulations of loss-of-function (lof), gain-of-function (gof) mutations, and of combination thereof are shown in the form of coloured square vignettes, along with references to articles presenting matching data. The first vignette (top left) correspond to the wild type situation, with VM, H, FB and SM presumptive territories coloured in blue, red, green and orange, respectively. In the following vignettes, the coloration of the four presumptive territories are modified to reflect the absence or important markers, or the combination of markers associated with different tissues. Wg lof leads to the loss of Wg/Slp domain, resulting in an expansion of the En/Hh domain; consequently, the model correctly predicts the loss of H along with a potential perturbation of SM (yellow domains). Dpp lof leads to the loss of dorsal derivatives (VM and H), along with an expansion of FB. Dpp gof leads to an expansion of VM at the expense of FB, along with a perturbation of SM. Tin lof shows a loss of dorsal tissues, while Bap lof exhibits only the loss of VM. Finally, the combination of Wg gof and Hh lof leads to a dorsal expansion of H, along with a loss of FB, while the combination of Dpp gof, Hh gof and Wg lof leads to an expansion of VM in the whole mesoderm.</p