Genome-wide identification of Hand2 target regions in mouse embryos using dRMCE, a new genetic tool

Limb bud development is a paradigmatic model to study the molecular signals that orchestrate cell growth and behaviour. Anterior-posterior patterning of the limb bud mesenchyme is dependent on the secreted ligand Sonic hedgehog (Shh). Shh expression in the posterior limb bud mesenchyme defines the zone of polarizing activity (ZPA) and controls cell survival and proliferative expansion during limb bud outgrowth. The bHLH transcription factor Hand2 binds to the limb-specific far-upstream Shh enhancer termed ZPA regulatory sequence (ZRS) and is essential for Shh activation. With the exception of the ZRS, no other direct Hand2 target regulatory regions and genes have been identified. Given that Hand2 is also required for development of the heart and neural crest derivatives, determining the genome-wide range of Hand2 target regions in mouse embryos will contribute to the understanding of underlying gene-regulatory networks. We decided to insert an epitope tag into the endogenous Hand2 protein to be able to precisely determine the range of Hand2 target sequences by ChIP-seq analysis. However, as genetic engineering of the Hand2 locus by homologous recombination is very inefficient, we developed dRMCE to re-engineer the Hand2 conditional allele. In doing so, we realized that dRMCE is compatible with thousands of conditional alleles and allows highly efficient custom-modification of the endogenous locus. dRMCE allowed me to rapidly generate a mouse model encoding an epitope tag within the endogenous Hand2 protein, which permits highly sensitive detection and localization of endogenous Hand2 in differentiated ES cells and embryonic tissues. We successfully used this fully functional epitope-tagged Hand2 protein to identify the large range of Hand2 target sequences in mouse embryonic tissues using a ChIP-seq approach. Our results indicate that Hand2 interacts with Gli3 and Tbx regulatory sites in limb buds and binds to a minimal ZRS element associated with human point mutations that cause polydactyly. I show that Hand2 is required for the development of the proximal skeleton of the hindlimb, likely by interacting directly with a Tbx4 enhancer. Furthermore, I describe the Hand2 target range associated with essential regulators of cardiac or craniofacial development. Thus, my approach begins to provide insight into the regulatory gene networks regulated by Hand2 during embryogenesis

Startups’ roads to failure

The role of a relatively small cadre of high-tech startup firms in driving innovation and economic growth has been well known and amply celebrated in recent history. At the same time, it is well recognized that, while the overall contribution of startups is crucial, the high-risk and high-reward strategy followed by these startups leads to significant failure rates and a low ratio of successful startups. So, it is curious to notice that literature tends to focus on successful startups and on quantitative studies looking for determinants of success while neglecting the numerous lessons that can be drawn by examining the stories of startups that failed. This paper aims to fill this gap and to contribute to the literature by providing a repeatable and scalable methodology that can be applied to databases of unstructured post-mortem documents deriving startup failure patterns. A further and related contribution is the analysis carried out with this methodology to a large database of 214 startup post-mortem reports. Descriptive statistics show how the lack of a structured Business Development strategy emerges as a key determinant of startup failure in the majority of cases

The cis-regulatory architecture of a gene desert controlling pleiotropic Shox2 expression and cardiac pacemaker development

Trabajo presentado en Weinstein Meeting, celebrado en Marsella (Francia) del 12 al 14 de mayo de 2022

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.NM has been funded by SNF grant 320030E-164245 and ERC Consolidator grant 2018 819717. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Benoît Zuber is supported by SNF grant 179520 and ERA-NET NEURON grant 185536. M.O. was supported by SNF grant PCEFP3_186993.S

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development

Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation

Walk-ins seeking treatment at an emergency department or general practitioner out-of-hours service: a cross-sectional comparison

Background Emergency Departments (ED) in Switzerland are faced with increasing numbers of patients seeking non-urgent treatment. The high rate of walks-ins with conditions that may be treated in primary care has led to suggestions that those patients would best cared for in a community setting rather than in a hospital. Efficient reorganisation of emergency care tailored to patients needs requires information on the patient populations using the various emergency services currently available. The aim of this study is to evaluate the differences between the characteristics of walk-in patients seeking treatment at an ED and those of patients who use traditional out-of-hours GP (General Practitioner) services provided by a GP-Cooperative (GP-C). Methods In 2007 and 2009 data was collected covering all consecutive patient-doctor encounters at the ED of a hospital and all those occurring as a result of contacting a GP-C over two evaluation periods of one month each. Comparison was made between a GP-C and the ED of the Waid City Hospital in Zurich. Patient characteristics, time and source of referral, diagnostic interventions and mode of discharge were evaluated. Medical problems were classified according to the International Classification of Primary Care (ICPC-2). Patient characteristics were compared using non-parametric tests and multiple logistic regression analysis was applied to investigate independent determinants for contacting a GP-C or an ED. Results Overall a total of 2974 patient encounters were recorded. 1901 encounters were walk-ins and underwent further analysis (ED 1133, GP-C 768). Patients consulting the GP-C were significantly older (58.9 vs. 43.8 years), more often female (63.5 vs. 46.9%) and presented with non-injury related medical problems (93 vs. 55.6%) in comparison with patients at the ED. Independent determining factors for ED consultation were injury, male gender and younger age. Walk-in distribution in both settings was equal over a period of 24 hours and most common during daytime hours (65%). Outpatient care was predominant in both settings but significantly more so at the GP-C (79.9 vs. 85.7%). Conclusions We observed substantial differences between the two emergency settings in a non gate-keeping health care system. Knowledge of the distribution of diagnoses, their therapy, of diagnostic measures and of the factors which determine the patients' choice of the ED or the GP-C is essential for the efficient allocation of resources and the reduction of costs

Cosmic Rays and the Search for a Lorentz Invariance Violation

This is an introductory review about the on-going search for a signal of Lorentz Invariance Violation (LIV) in cosmic rays. We first summarise basic aspects of cosmic rays, focusing on rays of ultra high energy (UHECRs). We discuss the Greisen-Zatsepin-Kuz'min (GZK) energy cutoff for cosmic protons, which is predicted due to photopion production in the Cosmic Microwave Background (CMB). This is a process of modest energy in the proton rest frame. It can be investigated to a high precision in the laboratory, if Lorentz transformations apply even at factors $\gamma \sim O(10^{11})$. For heavier nuclei the energy attenuation is even faster due to photo-disintegration, again if this process is Lorentz invariant. Hence the viability of Lorentz symmetry up to tremendous gamma-factors - far beyond accelerator tests - is a central issue. Next we comment on conceptual aspects of Lorentz Invariance and the possibility of its spontaneous breaking. This could lead to slightly particle dependent ``Maximal Attainable Velocities''. We discuss their effect in decays, Cerenkov radiation, the GZK cutoff and neutrino oscillation in cosmic rays. We also review the search for LIV in cosmic gamma-rays. For multi TeV gamma-rays we possibly encounter another puzzle related to the transparency of the CMB, similar to the GZK cutoff. The photons emitted in a Gamma Ray Burst occur at lower energies, but their very long path provides access to information not far from the Planck scale. No LIV has been observed so far. However, even extremely tiny LIV effects could change the predictions for cosmic ray physics drastically. An Appendix is devoted to the recent hypothesis by the Pierre Auger Collaboration, which identifies nearby Active Galactic Nuclei - or objects next to them - as probable UHECR sources.Comment: 81 pages, 15 figures, some points extended and improved, references adde