The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling.

The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.

Contributions of cell behavior to geometric order in embryonic cartilage.

During early development, cartilage provides shape and stability to the embryo while serving as a precursor for the skeleton. Correct formation of embryonic cartilage is hence essential for healthy development. In vertebrate cranial cartilage, it has been observed that a flat and laterally extended macroscopic geometry is linked to regular microscopic structure consisting of tightly packed, short, transversal clonar columns. However, it remains an ongoing challenge to identify how individual cells coordinate to successfully shape the tissue, and more precisely which mechanical interactions and cell behaviors contribute to the generation and maintenance of this columnar cartilage geometry during embryogenesis. Here, we apply a three-dimensional cell-based computational model to investigate mechanical principles contributing to column formation. The model accounts for clonal expansion, anisotropic proliferation and the geometrical arrangement of progenitor cells in space. We confirm that oriented cell divisions and repulsive mechanical interactions between cells are key drivers of column formation. In addition, the model suggests that column formation benefits from the spatial gaps created by the extracellular matrix in the initial configuration, and that column maintenance is facilitated by sequential proliferative phases. Our model thus correctly predicts the dependence of local order on division orientation and tissue thickness. The present study presents the first cell-based simulations of cell mechanics during cranial cartilage formation and we anticipate that it will be useful in future studies on the formation and growth of other cartilage geometries.

A pan-tissue survey of mosaic chromosomal alterations in 948 individuals.

Genetic mutations accumulate in an organism's body throughout its lifetime. While somatic single-nucleotide variants have been well characterized in the human body, the patterns and consequences of large chromosomal alterations in normal tissues remain largely unknown. Here, we present a pan-tissue survey of mosaic chromosomal alterations (mCAs) in 948 healthy individuals from the Genotype-Tissue Expression project, augmenting RNA-based allelic imbalance estimation with haplotype phasing. We found that approximately a quarter of the individuals carry a clonally-expanded mCA in at least one tissue, with incidence strongly correlated with age. The prevalence and genome-wide patterns of mCAs vary considerably across tissue types, suggesting tissue-specific mutagenic exposure and selection pressures. The mCA landscapes in normal adrenal and pituitary glands resemble those in tumors arising from these tissues, whereas the same is not true for the esophagus and skin. Together, our findings show a widespread age-dependent emergence of mCAs across normal human tissues with intricate connections to tumorigenesis.

A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia.

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

Polymorphic parasitic larvae cooperate to build swimming colonies luring hosts.

Parasites have evolved a variety of astonishing strategies to survive within their hosts, yet the most challenging event in their personal chronicles is the passage from one host to another. It becomes even more complex when a parasite needs to pass through the external environment. Therefore, the free-living stages of parasites present a wide range of adaptations for transmission. Parasitic flatworms from the group Digenea (flukes) have free-living larvae, cercariae, which are remarkably diverse in structure and behavior.1,2 One of the cercariae transmission strategies is to attain a prey-like appearance for the host.3 This can be done through the formation of a swimming aggregate of several cercariae adjoined together by their tails.4 Through the use of live observations and light, electron, and confocal microscopy, we described such a supposedly prey-mimetic colony comprising cercariae of two distinct morphotypes. They are functionally specialized: larger morphotype (sailors) enable motility, and smaller morphotype (passengers) presumably facilitate infection. The analysis of local read alignments between the two samples reveals that both cercaria types have identical 18S, 28S, and 5.8S rRNA genes. Further phylogenetic analysis of these ribosomal sequences indicates that our specimen belongs to the digenean family Acanthocolpidae, likely genus Pleorchis. This discovery provides a unique example and a novel insight into how morphologically and functionally heterogeneous individuals of the same species cooperate to build colonial organisms for the purpose of infection. This strategy bears resemblance to the cooperating castes of the same species found among insects.5.

hPSCs-derived Schwann cells serve for disease modeling and drug discovery.

In this issue, Majd et al.1 derive Schwann cells from human pluripotent stem cells (hPSCs), which can be used to study Schwann cell development and physiology and model diabetic neuropathy. hPSC-derived Schwann cells possess the molecular features of primary Schwann cells and are capable of myelination in vitro and in vivo.

The peripheral nervous system.

The peripheral nervous system (PNS) represents a highly heterogeneous entity with a broad range of functions, ranging from providing communication between the brain and the body to controlling development, stem cell niches and regenerative processes. According to the structure and function, the PNS can be subdivided into sensory, motor (i.e. the nerve fibers of motor neurons), autonomic and enteric domains. Different types of neurons correspond to these domains and recent progress in single-cell transcriptomics has enabled the discovery of new neuronal subtypes and improved the previous cell-type classifications. The developmental mechanisms generating the domains of the PNS reveal a range of embryonic strategies, including a variety of cell sources, such as migratory neural crest cells, placodal neurogenic cells and even recruited nerve-associated Schwann cell precursors. In this article, we discuss the diversity of roles played by the PNS in our body, as well as the origin, wiring and heterogeneity of every domain. We place a special focus on the most recent discoveries and concepts in PNS research, and provide an outlook of future perspectives and controversies in the field.

Directionality of developing skeletal muscles is set by mechanical forces.

Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin.

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

Evolutionary origin of the neural tube in basal deuterostomes.

The central nervous system (CNS) of chordates, including humans, develops as a hollow tube with ciliated walls containing cerebrospinal fluid. However, most of the animals inhabiting our planet do not use this design and rather build their centralized brains from non-epithelialized condensations of neurons called ganglia, with no traces of epithelialized tubes or liquid-containing cavities. The evolutionary origin of tube-type CNSs stays enigmatic, especially as non-epithelialized ganglionic-type nervous systems dominate the animal kingdom. Here, I discuss recent findings relevant to understanding the potential homologies and scenarios of the origin, histology and anatomy of the chordate neural tube. The nerve cords of other deuterostomes might relate to the chordate neural tube at histological, developmental and cellular levels, including the presence of radial glia, layered stratification, retained epithelial features, morphogenesis via folding and formation of a lumen filled with liquid. Recent findings inspire a new view of hypothetical evolutionary scenarios explaining the tubular epithelialized structure of the CNS. One such idea suggests that early neural tubes were key for improved directional olfaction, which was facilitated by the liquid-containing internal cavity. The later separation of the olfactory portion of the tube led to the formation of the independent olfactory and posterior tubular CNS systems in vertebrates. According to an alternative hypothesis, the thick basiepithelial nerve cords could provide deuterostome ancestors with additional biomechanical support, which later improved by turning the basiepithelial cord into a tube filled with liquid - a hydraulic skeleton.

STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development.

AIMS: Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. METHODS AND RESULTS: To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. CONCLUSION: Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations.

scFates: a scalable python package for advanced pseudotime and bifurcation analysis from single-cell data.

SUMMARY: scFates provides an extensive toolset for the analysis of dynamic trajectories comprising tree learning, feature association testing, branch differential expression and with a focus on cell biasing and fate splits at the level of bifurcations. It is meant to be fully integrated into the scanpy ecosystem for seamless analysis of trajectories from single-cell data of various modalities (e.g. RNA and ATAC). AVAILABILITY AND IMPLEMENTATION: scFates is released as open-source software under the BSD 3-Clause 'New' License and is available from the Python Package Index at https://pypi.org/project/scFates/. The source code is available on GitHub at https://github.com/LouisFaure/scFates/. Code reproduction and tutorials on published datasets are available on GitHub at https://github.com/LouisFaure/scFates_notebooks. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The transcriptional portraits of the neural crest at the individual cell level.

Since the discovery of this cell population by His in 1850, the neural crest has been under intense study for its important role during vertebrate development. Much has been learned about the function and regulation of neural crest cell differentiation, and as a result, the neural crest has become a key model system for stem cell biology in general. The experiments performed in embryology, genetics, and cell biology in the last 150 years in the neural crest field has given rise to several big questions that have been debated intensely for many years: "How does positional information impact developmental potential? Are neural crest cells individually multipotent or a mixed population of committed progenitors? What are the key factors that regulate the acquisition of stem cell identity, and how does a stem cell decide to differentiate towards one cell fate versus another?" Recently, a marriage between single cell multi-omics, statistical modeling, and developmental biology has shed a substantial amount of light on these questions, and has paved a clear path for future researchers in the field.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

Developmental heterogeneity of embryonic neuroendocrine chromaffin cells and their maturation dynamics.

During embryonic development, nerve-associated Schwann cell precursors (SCPs) give rise to chromaffin cells of the adrenal gland via the "bridge" transient stage, according to recent functional experiments and single cell data from humans and mice. However, currently existing data do not resolve the finest heterogeneity of developing chromaffin populations. Here we took advantage of deep SmartSeq2 transcriptomic sequencing to expand our collection of individual cells from the developing murine sympatho-adrenal anlage and uncover the microheterogeneity of embryonic chromaffin cells and their corresponding developmental paths. We discovered that SCPs on the splachnic nerve show a high degree of microheterogeneity corresponding to early biases towards either Schwann or chromaffin terminal fates. Furthermore, we found that a post-"bridge" population of developing chromaffin cells gives rise to persisting oxygen-sensing chromaffin cells and the two terminal populations (adrenergic and noradrenergic) via diverging differentiation paths. Taken together, we provide a thorough identification of novel markers of adrenergic and noradrenergic populations in developing adrenal glands and report novel differentiation paths leading to them.

Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification.

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Schwann cell precursors represent a neural crest-like state with biased multipotency.

Schwann cell precursors (SCPs) are nerve-associated progenitors that can generate myelinating and non-myelinating Schwann cells but also are multipotent like the neural crest cells from which they originate. SCPs are omnipresent along outgrowing peripheral nerves throughout the body of vertebrate embryos. By using single-cell transcriptomics to generate a gene expression atlas of the entire neural crest lineage, we show that early SCPs and late migratory crest cells have similar transcriptional profiles characterised by a multipotent "hub" state containing cells biased towards traditional neural crest fates. SCPs keep diverging from the neural crest after being primed towards terminal Schwann cells and other fates, with different subtypes residing in distinct anatomical locations. Functional experiments using CRISPR-Cas9 loss-of-function further show that knockout of the common "hub" gene Sox8 causes defects in neural crest-derived cells along peripheral nerves by facilitating differentiation of SCPs towards sympathoadrenal fates. Finally, specific tumour populations found in melanoma, neurofibroma and neuroblastoma map to different stages of SCP/Schwann cell development. Overall, SCPs resemble migrating neural crest cells that maintain multipotency and become transcriptionally primed towards distinct lineages.