The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes¹. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo². Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos³. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na ⁺ ]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg ²⁺ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.

Myofibers are large multinucleated cells that have long thought to have a rather simple organization. Single-nucleus transcriptomics, spatial transcriptomics and spatial metabolomics analysis have revealed distinct transcription profiles in myonuclei related to myofiber type. However, the use of local tissue collection or dissociation methods have obscured the spatial organization. To elucidate the full tissue architecture, we combine two spatial omics, RNA tomography and mass spectrometry imaging. This enables us to map the spatial transcriptomic, metabolomic and lipidomic organization of the whole murine tibialis anterior muscle. Our findings on heterogeneity in fiber type proportions are validated with multiplexed immunofluorescence staining in tibialis anterior, extensor digitorum longus and soleus. Our results demonstrate unexpectedly strong regionalization of gene expression, metabolic differences and variable myofiber type proportion along the proximal-distal axis. These new insights in whole-tissue level organization reconcile sometimes conflicting results coming from previous studies relying on local sampling methods.

Background: Total-RNA sequencing (total-RNA-seq) allows the simultaneous study of both the coding and the non-coding transcriptome. Yet, computational pipelines have traditionally focused on particular biotypes, making assumptions that are not fullfilled by total-RNA-seq datasets. Transcripts from distinct RNA biotypes vary in length, biogenesis, and function, can overlap in a genomic region, and may be present in the genome with a high copy number. Consequently, reads from total-RNA-seq libraries may cause ambiguous genomic alignments, demanding for flexible quantification approaches. Results: Here we present Multi-Graph count (MGcount), a total-RNA-seq quantification tool combining two strategies for handling ambiguous alignments. First, MGcount assigns reads hierarchically to small-RNA and long-RNA features to account for length disparity when transcripts overlap in the same genomic position. Next, MGcount aggregates RNA products with similar sequences where reads systematically multi-map using a graph-based approach. MGcount outputs a transcriptomic count matrix compatible with RNA-sequencing downstream analysis pipelines, with both bulk and single-cell resolution, and the graphs that model repeated transcript structures for different biotypes. The software can be used as a python module or as a single-file executable program. Conclusions: MGcount is a flexible total-RNA-seq quantification tool that successfully integrates reads that align to multiple genomic locations or that overlap with multiple gene features. Its approach is suitable for the simultaneous estimation of protein-coding, long non-coding and small non-coding transcript concentration, in both precursor and processed forms. Both source code and compiled software are available at https://github.com/hitaandrea/MGcount.

In contrast to other mammals, the spiny mouse (Acomys) regenerates skin and ear tissue, which includes hair follicles, glands, and cartilage, in a scar-free manner. Ear punch regeneration is asymmetric with only the proximal wound side participating in regeneration. Here, we show that cues originating from the proximal side are required for normal regeneration and use spatially resolved transcriptomics (tomo-seq) to understand the molecular and cellular events underlying this process. Analyzing gene expression across the ear and comparing expression modules between proximal and distal wound sides, we identify asymmetric gene expression patterns and pinpoint regenerative processes in space and time. Moreover, using a comparative approach with nonregenerative rodents (Mus,Meriones), we strengthen a hypothesis in which particularities in the injury-induced immune response may be one of the crucial determinants for why spiny mice regenerate whereas their relatives do not. Our data are available in SpinyMine, an easy-to-use and expandable web-based tool for exploringAcomysregeneration-associated gene expression.