Periodic Reporting for period 4 - INTEGRAL (Signal Integration by Gene Regulatory Landscapes) Reporting period: 2021-02-01 to 2022-01-31 Summary of the context and overall objectives of the project A source of fascination is the reproducibility and precision by which fertilized eggs develop into complex and functional organisms. Our group studies the gene regulatory networks (GRNs) that control and endow embryonic development with robustness to genetic and environmental perturbations. The precise spatial and temporal regulation of gene expression is one key feature of coordinated organ and tissues development, which is controlled by so-called cis-regulatory modules (CRMs). CRMs are located in the non-coding part of the genome and function either as enhancers or repressors of gene expression. Their analysis is of general importance as most congenital malformations and diseases are caused by mutations that alter CRMs and/or chromatin architecture rather than gene products. In addition, our study provides important insights into the changes in CRM activities and gene expression that have been postulated as drivers of diversification during vertebrate evolution. Our research provides fundamental insights into robustness and evolutionary plasticity of gene regulation. The developing limb of tetrapods (animals with four limbs) is a paradigm to study how gene regulation impacts morphogenesis. We have shown that a self-regulatory signaling system coordinately controls limb bud outgrowth and patterning of the limb skeleton and that the BMP antagonist Gremlin1 (Grem1) is essential to propagate this signaling system and for normal limb development. The ERC-funded research identifies several CRM enhancers embedded in a large genomic landscape and establishes how their interactions orchestrate Grem1 expression during mouse limb development. This analysis provides fundamental insights into how several CRM enhancers interact and integrate signaling inputs to regulate the spatio-temporal Grem1 expression. Genetic and molecular analysis identified a novel dual enhancer mode as CRM enhancers regulate Grem1 transcript levels in an additive manner, while their synergistic interactions provide the spatial regulation of Grem1 expression and digit development with robustness. During this analysis, we discovered that the Grem1 cis-regulatory landscape is deeply conserved in all vertebrates with paired appendages including evolutionary ancient fish. Functional analysis of two of the most conserved CRM enhancers revealed that their orthologues from basal fishes (Coelacanth and sharks) are strongly active in the mouse digit forming area, which is fascinating as it reveals that the regulatory circuits existed before fins evolved into limbs. Comparing CRM enhancer activities with Grem1 expression in limb buds from different mammalian and sauropsid species reveals the amazing evolutionary plasticity in Grem1 cis-regulation and expression, which correlates well with the evolutionary diversification of the limb skeleton in these species. This analysis indicates that the inherent robustness generated by the synergistic CRMs likely provided an evolutionary playground enabling diversification of enhancer activities during evolutionary adaptation of tetrapod limbs. In a parallel study we combined experimental with computational analysis of mouse limb and chicken wing buds to provide broad insights into how genome evolution impacted the cis-regulatory interactions and the spatio-temporal gene expression dynamics during diversification mouse limb and chicken wing development. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We used CRISPR/Cas9-mediated genome editing in combination with molecular analysis to assess the functional requirement of CRM enhancers for regulating Grem1 expression during mouse limb development. Inactivation of individual CRM enhancers had no significant effects on the spatial Grem1 expression and digit development. Instead, we uncovered a CRM enhancer network that regulates Grem1 expression by a dual mode: transcript levels are regulated in an additive manner while synergistic interactions among the CRM enhancers provide the spatially dynamic Grem1 expression with cis-regulatory robustness. Two of the essential CRM enhancers are deeply conserved from fish to mammals. This provided us with a unique opportunity to access the role of spatial regulation of Grem1 during evolutionary diversification of tetrapod limbs. The comparative analysis of CRM enhancers from different tetrapod species revealed significant differences in their spatial activities, which match the variation in Grem1 expression that prefigures the alterations in future digits. Unexpectedly, the conserved CRM enhancers from basal cartilaginous fishes display robust activities in the developing mouse hand plate, which revealed that the basic cis-regulatory circuits that control Grem1 expression in tetrapod limb buds was established before the emergence of limbs. These analyses shed light on how Grem1 might have been coopted during the fin-to-limb transition and uncovers the species-specific spatial variations in CRM enhancer activities and Grem1 expression during the vast evolutionary diversification of tetrapod limbs. The entire analysis was published as one comprehensive study: Malkmus, Ramos Martins et al (2021). Nature Commun 12, 5557 doi:10.1038/s41467-021-25810-1.Another main study concerns the integrative genome-wide analysis of gene regulation and expression during mouse limb and chicken wing development. This data-based computational analysis identified the synchrony between enhancer accessibility and gene expression in mouse forelimb buds, while stage-specific divergence was detected in chicken wing buds. Integration of these dataset with computational TF footprinting allowed construction of GRNs of interest. The in silico construction of TF target GRNs is of broad relevance as the necessary datasets can be generated with little material, which makes such analysis feasible for an increasing number of available non-model organisms: Jhanwar et al. 2021, Nature Commun 12, 5685 doi:10.1038/s41467-021-25935-3. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 1. The initial analysis of the Grem1 cis-regulatory landscape pointed to simple functional redundancy, but the analysis of combinations of CRM enhancer deletions reveals the dual role of CRM enhancers in regulating transcript levels and spatial Grem1 expression in limb buds. Most importantly the study establishes the synergistic nature of the CRM enhancer interactions that provide the spatial Grem1 expression and pentadactyly with robustness, which starts to reveal the molecular nature of developmental robustness.2. A second important discovery is the deep evolutionary conservation of the Grem1 cis-regulatory landscape in jawed vertebrates. The unexpected finding that CRM enhancers from basal fish are active in the mouse autopod and regulated by the same trans-regulatory inputs as their mouse orthologs points to the ancient origin of the trans/cis-regulatory circuits that control gene expression in paired appendages. 3. The comparative analysis of the temporal differences in open chromatin and gene expression at orthologous stages of mouse limb and chicken wing buds went way beyond its initial aim. In particular, ATAC-seq can be used for computational footprinting of TFs to profile their temporal interactions with DNA. Furthermore, candidate target GRN for specific TFs can be constructed in silico to identify both conserved and species-specific interactions. Finally this computational analysis provides a framework for future Evo-Devo studies. Grem1 CRM activity (blue) in a mouse embryo with a scheme of the signalling system
