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Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis., (© 2024. The Author(s).)

The intricate dynamics of Hes expression across diverse cell types in the developing vertebrate embryonic tail have remained elusive. To address this, we have developed an endogenously tagged Hes1-Achilles mouse line, enabling precise quantification of dynamics at the single-cell resolution across various tissues. Our findings reveal striking disparities in Hes1 dynamics between presomitic mesoderm (PSM) and preneural tube (pre-NT) cells. While pre-NT cells display variable, low-amplitude oscillations, PSM cells exhibit synchronized, high-amplitude oscillations. Upon the induction of differentiation, the oscillation amplitude increases in pre-NT cells. Additionally, our study of Notch inhibition on Hes1 oscillations unveils distinct responses in PSM and pre-NT cells, corresponding to differential Notch ligand expression dynamics. These findings suggest the involvement of separate mechanisms driving Hes1 oscillations. Thus, Hes1 demonstrates dynamic behaviour across adjacent tissues of the embryonic tail, yet the varying oscillation parameters imply differences in the information that can be transmitted by these dynamics., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Synchronization of coupled oscillators is a universal phenomenon encountered across different scales and contexts, e.g., chemical wave patterns, superconductors, and the unison applause we witness in concert halls. The existence of common underlying coupling rules defines universality classes, revealing a fundamental sameness between seemingly distinct systems. Identifying rules of synchronization in any particular setting is hence of paramount relevance. Here, we address the coupling rules within an embryonic oscillator ensemble linked to vertebrate embryo body axis segmentation. In vertebrates, the periodic segmentation of the body axis involves synchronized signaling oscillations in cells within the presomitic mesoderm (PSM), from which somites, the prevertebrae, form. At the molecular level, it is known that intact Notch-signaling and cell-to-cell contact are required for synchronization between PSM cells. However, an understanding of the coupling rules is still lacking. To identify these, we develop an experimental assay that enables direct quantification of synchronization dynamics within mixtures of oscillating cell ensembles, for which the initial input frequency and phase distribution are known. Our results reveal a "winner-takes-it-all" synchronization outcome, i.e., the emerging collective rhythm matches one of the input rhythms. Using a combination of theory and experimental validation, we develop a coupling model, the "Rectified Kuramoto" (ReKu) model, characterized by a phase-dependent, nonreciprocal interaction in the coupling of oscillatory cells. Such nonreciprocal synchronization rules reveal fundamental similarities between embryonic oscillators and a class of collective behaviors seen in neurons and fireflies, where higher-level computations are performed and linked to nonreciprocal synchronization., Competing Interests: Competing interests statement:The authors declare no competing interest.

Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Skeletal muscles of the head and trunk originate in distinct lineages with divergent regulatory programmes converging on activation of myogenic determination factors. Branchiomeric head and neck muscles share a common origin with cardiac progenitor cells in cardiopharyngeal mesoderm (CPM). The retinoic acid (RA) signalling pathway is required during a defined early time window for normal deployment of cells from posterior CPM to the heart. Here, we show that blocking RA signalling in the early mouse embryo also results in selective loss of the trapezius neck muscle, without affecting other skeletal muscles. RA signalling is required for robust expression of myogenic determination factors in posterior CPM and subsequent expansion of the trapezius primordium. Lineage-specific activation of a dominant-negative RA receptor reveals that trapezius development is not regulated by direct RA signalling to myogenic progenitor cells in CPM, or through neural crest cells, but indirectly through the somitic lineage, closely apposed with posterior CPM in the early embryo. These findings suggest that trapezius development is dependent on precise spatiotemporal interactions between cranial and somitic mesoderm at the head/trunk interface., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Segmentation is a fundamental feature of the vertebrate body plan. This metameric organization is first implemented by somitogenesis in the early embryo, when paired epithelial blocks called somites are rhythmically formed to flank the neural tube. Recent advances in in vitro models have offered new opportunities to elucidate the mechanisms that underlie somitogenesis. Notably, models derived from human pluripotent stem cells introduced an efficient proxy for studying this process during human development. In this Review, we summarize the current understanding of somitogenesis gained from both in vivo studies and in vitro studies. We deconstruct the spatiotemporal dynamics of somitogenesis into four distinct modules: dynamic events in the presomitic mesoderm, segmental determination, somite anteroposterior polarity patterning, and epithelial morphogenesis. We first focus on the segmentation clock, as well as signalling and metabolic gradients along the tissue, before discussing the clock and wavefront and other models that account for segmental determination. We then detail the molecular and cellular mechanisms of anteroposterior polarity patterning and somite epithelialization., (© 2024. Springer Nature Limited.)

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility., (© 2024 The Author(s).)

Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis., (© 2024. The Author(s).)

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations., Competing Interests: The authors declare no conflicts of interests.

The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression that propagate from the posterior to the anterior end of the tissue. In a new study, Klepstad and Marcon propose a new theoretical framework that recapitulates the dynamics of mouse somitogenesis observed in vivo and in vitro. To learn more about the story behind the paper, we caught up with first author Julie Klepstad and corresponding author Luciano Marcon, Principal Investigator at the Andalusian Center for Developmental Biology., (© 2024. Published by The Company of Biologists Ltd.)

During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans 1,2 . Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing 3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data 4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb., (© 2024. The Author(s).)

The Segmentation Clock is a tissue-level patterning system that enables the segmentation of the vertebral column precursors into transient multicellular blocks called somites. This patterning system comprises a set of elements that are essential for correct segmentation. Under the so-called "Clock and Wavefront" model, the system consists of two elements, a genetic oscillator that manifests itself as traveling waves of gene expression, and a regressing wavefront that transforms the temporally periodic signal encoded in the oscillations into a permanent spatially periodic pattern of somite boundaries. Over the last twenty years, every new discovery about the Segmentation Clock has been tightly linked to the nomenclature of the "Clock and Wavefront" model. This constrained allocation of discoveries into these two elements has generated long-standing debates in the field as what defines molecularly the wavefront and how and where the interaction between the two elements establishes the future somite boundaries. In this review, we propose an expansion of the "Clock and Wavefront" model into three elements, "Clock", "Wavefront" and signaling gradients. We first provide a detailed description of the components and regulatory mechanisms of each element, and we then examine how the spatiotemporal integration of the three elements leads to the establishment of the presumptive somite boundaries. To be as exhaustive as possible, we focus on the Segmentation Clock in zebrafish. Furthermore, we show how this three-element expansion of the model provides a better understanding of the somite formation process and we emphasize where our current understanding of this patterning system remains obscure., (Copyright © 2024. Published by Elsevier Inc.)

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come., (Copyright © 2024. Published by Elsevier Inc.)

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system 1-4 . How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches 1-5 , models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Sequential segmentation creates modular body plans of diverse metazoan embryos 1-4 . Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation 4 . However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish 5 . Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species 3,4,6-8 . Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro 1-5 , but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells 6-11 . Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis., (© 2022. The Author(s).)

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3'UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3'UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized "trunk-like structures" (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020)., Competing Interests: C.S.Y. and A.D.P. are co-founders of and have financial interests in MyoGene Bio. The Regents of the University of California have licensed intellectual property invented by C.S.Y. and A.D.P. to MyoGene Bio. A.D.P. serves on the scientific advisory board of MyoGene Bio. C.S.Y. is currently CEO of MyoGene Bio. H.X. declares no competing interests.

The body axis of vertebrates is subdivided into repetitive compartments called somites, which give rise primarily to the segmented architecture of the musculoskeletal system in the adult body. Somites form in a sequential and rhythmic manner in embryos and a physical boundary separates each somite from the rest of the unsegmented tissue and adjoining somites. Precise positioning of somite boundaries and determination of boundary cell fate in a select group of cells is thought to be driven by gene expression patterns and morphogen gradients. This pre-patterning step is followed by a mechanical process involving actomyosin activation in boundary cells and formation of an extracellular matrix that results in morphological boundary formation. While genes involved in somite boundary formation have been identified, there are many open questions about the underlying pre-patterning dynamics and mechanics and how these processes are coupled to generate a morphological boundary. Here, focusing on segmentation of zebrafish embryos as a model, we review pre-patterning processes critical for boundary formation and how cytoskeletal activity drives tissue separation. Our outlook is that this system holds exciting new avenues for unearthing general principles of boundary formation in developing embryos., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Trunk muscles in vertebrates are classified as either dorsal epaxial or ventral hypaxial muscles. Epaxial and hypaxial muscles are defined as muscles innervated by the dorsal and ventral rami of spinal nerves, respectively. Each cluster of spinal motor neurons passing through dorsal rami innervates epaxial muscles, whereas clusters traveling on the ventral rami innervate hypaxial muscles. Herein, we show that some motor neurons exhibiting molecular profiles for epaxial muscles follow a path in the ventral rami. Dorsal deep-shoulder muscles and some body wall muscles are defined as hypaxial due to innervation via the ventral rami, but a part of these ventral rami has the molecular profile of motor neurons that innervate epaxial muscles. Thus, the epaxial and hypaxial boundary cannot be determined simply by the ramification pattern of spinal nerves. We propose that, although muscle innervation occurs via the ventral rami, dorsal deep-shoulder muscles and some body wall muscles represent an intermediate group that lies between epaxial and hypaxial muscles., (© 2020 Anatomical Society.)

Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal-epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications.

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 axes 1 . 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 vivo 2 . 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 embryos 3 . Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 μm for C. bicolor and 627.6 ± 7.8 μm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization 1-3 . To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth., Competing Interests: GC, CS, JS, KW, PJ, AK, EW, CC, SP, PC, GR, MR, NS, AH, AA, SW, RK No competing interests declared, (© 2020, Cook et al.)

During vertebrate somitogenesis an inherent segmentation clock coordinates the spatiotemporal signaling to generate segmented structures that pattern the body axis. Using our experimental and quantitative approach, we study the cell movements and the genetic oscillations of her1 expression level at single-cell resolution simultaneously and scale up to the entire pre-somitic mesoderm (PSM) tissue. From the experimentally determined phases of PSM cellular oscillators, we deduced an in vivo frequency profile gradient along the anterior-posterior PSM axis and inferred precise mathematical relations between spatial cell-level period and tissue-level somitogenesis period. We also confirmed a gradient in the relative velocities of cellular oscillators along the axis. The phase order parameter within an ensemble of oscillators revealed the degree of synchronization in the tailbud and the posterior PSM being only partial, whereas synchronization can be almost complete in the presumptive somite region but with temporal oscillations. Collectively, the degree of synchronization itself, possibly regulated by cell movement and the synchronized temporal phase of the transiently expressed clock protein Her1, can be an additional control mechanism for making precise somite boundaries., (Copyright © 2019. Published by Elsevier Inc.)

Spatial patterning during embryonic development emerges from the differentiation of progenitor cells that share the same genetic program. One of the main challenges in systems biology is to understand the relationship between gene network and patterning, especially how the cells communicate to coordinate their differentiation. This review aims to describe the principles of pattern formation from local cell-cell interactions mediated by the Notch signalling pathway. Notch mediates signalling via direct cell-cell contact and regulates cell fate decisions in many tissues during embryonic development. Here, I will describe the patterning mechanisms via different Notch ligands and the critical role of Notch oscillations during the segmentation of the vertebrate body, brain development, and blood vessel formation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Vertebrate segments called somites are generated by periodic segmentation of the presomitic mesoderm (PSM). In the most accepted theoretical model for somite segmentation, the clock and wavefront (CW) model, a clock that ticks to determine particular timings and a wavefront that moves posteriorly are presented in the PSM, and somite positions are determined when the clock meets the posteriorly moving wavefront somewhere in the PSM. Over the last two decades, it has been revealed that the molecular mechanism of the clock and wavefront in vertebrates is based on clock genes including Hes family transcription factors and Notch effectors that oscillate within the PSM to determine particular timings and fibroblast growth factor (FGF) gradients, acting as the posteriorly moving wavefront to determine the position of somite segmentation. A clock-less condition in the CW model was predicted to form no somites; however, irregularly sized somites were still formed in mice and zebrafish, suggesting that this was one of the limitations of the CW model. Recently, we performed interdisciplinary research of experimental and theoretical biological studies and revealed the mechanisms of somite boundary determination in normal and clock-less conditions by characterization of the FGF/extracellular signal-regulated kinase (ERK) activity dynamics. Since features of the molecular clock have already been described in-depth in several reviews, we summarized recent findings regarding the role of FGF/ERK signaling in somite boundary formation and described our current understanding of how FGF/ERK signaling contributes to somitogenesis in normal and clock-less conditions in this review., (© 2020 Japanese Society of Developmental Biologists.)

The Segmentation clock is a population of cellular genetic oscillators, located in the posterior of the elongating vertebrate embryo, that governs the rhythmic and sequential segmentation of the body axis into somites. Somites are blocks of cells that give rise to the segmented anatomy of the adult, including the backbone, muscles and skin. Malfunction of the segmentation clock results in malformations of these structures, a condition termed congenital scoliosis in the clinic. In all vertebrates, the oscillating cells of the segmentation clock are coordinated in a wave pattern, such that each new wave corresponds to a new segment. Maintenance of this wave pattern is important for precise segmentation and requires the local synchronization of the cellular oscillators. Existing models of the segmentation clock have explored the role of the Delta-Notch intercellular signaling pathway primarily as a coupling mechanism between neighboring autonomous oscillators. Recent work challenges several aspects of this simplification, suggesting that the mechanism of synchronization is more complex and may differ between species, and that Notch signaling may do more than synchronize cells. Here, we first examine evidence and models concerning the role of Notch signaling in driving, maintaining and synchronizing the mouse clock, highlighting results emerging from ex vivo culture systems of mouse segmentation clock cells. We then compare this to synchronization in the zebrafish, where accumulating evidence suggests that Notch signaling impacts the amplitude of the oscillating signal, and discuss whether the amplitude itself is meaningful for segmentation. Finally, we review work showing that multiple Delta ligands are active in segmentation, and consider how an interplay between these ligands could confer effective Notch functions in the segmentation clock. These lines of enquiry suggest that synchronization and Notch signaling are more complex than previously described, and reveal exciting new avenues for investigation into the coordination and precision of patterning the early embryo., (Copyright © 2020. Published by Elsevier Inc.)

During somite segmentation, the expression of clock genes such as Hes7 oscillates synchronously in the presomitic mesoderm (PSM). This synchronous oscillation slows down in the anterior PSM, leading to wave-like propagating patterns from the posterior to anterior PSM. Such dynamic expression depends on Notch signaling and is critical for somite formation. However, it remains to be determined how slowing oscillations in the anterior PSM are controlled, and whether the expression of the Notch ligand Delta-like1 (Dll1) oscillates on the surface of individual PSM cells, as postulated to be responsible for synchronous oscillation. Here, by using Dll1 fluorescent reporter mice, we performed live-imaging of Dll1 expression in PSM cells and found the oscillatory expression of Dll1 protein on the cell surface regions. Furthermore, a comparison of live-imaging of Dll1 and Hes7 oscillations revealed that the delay from Dll1 peaks to Hes7 peaks increased in the anterior PSM, suggesting that the Hes7 response to Dll1 becomes slower in the anterior PSM. These results raise the possibility that Dll1 protein oscillations on the cell surface regulate synchronous Hes7 oscillations, and that the slower response of Hes7 to Dll1 leads to slower oscillations in the anterior PSM., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Using a stochastic individual-based modelling approach, we examine the role that Delta-Notch signalling plays in the regulation of a robust and reliable somite segmentation clock. We find that not only can Delta-Notch signalling synchronize noisy cycles of gene expression in adjacent cells in the presomitic mesoderm (as is known), but it can also amplify and increase the coherence of these cycles. We examine some of the shortcomings of deterministic approaches to modelling these cycles and demonstrate how intrinsic noise can play an active role in promoting sustained oscillations, giving rise to noise-induced quasi-cycles. Finally, we explore how translational/transcriptional delays can result in the cycles in neighbouring cells oscillating in anti-phase and we study how this effect relates to the propagation of noise-induced stochastic waves.

A striking example of coupling between growth and form occurs during the segmentation of the vertebrate embryo. During segmentation, pairs of segments, one on either side of the anterior-posterior axis, bud off from the presomitic mesoderm (PSM) at regular intervals in time. In the clock and wavefront model, a multicellular oscillator regulates the time at which the next pair of segments form whilst a wavefront regulates their spatial location. In most mathematical models of segmentation, it is assumed that cells in the PSM are oscillators that have a constant natural frequency. Based on recent experimental findings, here we propose a model in which the natural oscillation frequency of each PSM cell is a function of its position in the cell cycle. Given adequate oscillator coupling and that cells in the PSM are randomly distributed in the cell cycle, we find that the emergent oscillator frequency is a weighted average of the constituent oscillator frequencies with the weightings dependent on the fraction of cells in a given cell cycle state. Here, we show that such a model can allow for coupling between pattern formation and growth rate in PSM tissue., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Skeletal muscle in the trunk derives from the somites, paired segments of paraxial mesoderm. Whereas axial musculature develops within the somite, appendicular muscle develops following migration of muscle precursors into lateral plate mesoderm. The development of muscles bridging axial and appendicular systems appears mixed., Results: We examine development of three migratory muscle precursor-derived muscles in zebrafish: the sternohyoideus (SH), pectoral fin (PF), and posterior hypaxial (PHM) muscles. We show there is an anterior to posterior gradient to the developmental gene expression and maturation of these three muscles. SH muscle precursors exhibit a long delay between migration and differentiation, PF muscle precursors exhibit a moderate delay in differentiation, and PHM muscle precursors show virtually no delay between migration and differentiation. Using lineage tracing, we show that lateral plate contribution to the PHM muscle is minor, unlike its known extensive contribution to the PF muscle and absence in the ventral extension of axial musculature., Conclusions: We propose that PHM development is intermediate between a migratory muscle mode and an axial muscle mode of development, wherein the PHM differentiates after a very short migration of its precursors and becomes more anterior primarily by elongation of differentiated muscle fibers., (© 2019 Wiley Periodicals, Inc.)

Somites are epithelial segments of the paraxial mesoderm. Shortly after their formation, the epithelial somites undergo extensive cellular rearrangements and form specific somite compartments, including the sclerotome and the myotome, which give rise to the axial skeleton and to striated musculature, respectively. The dynamics of somite development varies along the body axis, but most research has focused on somite development at thoracolumbar levels. The development of tail somites has not yet been thoroughly characterized, even though vertebrate tail development has been intensely studied recently with respect to the termination of segmentation and the limitation of body length in evolution. Here, we provide a detailed description of the somites in the avian tail from the beginning of tail formation at HH-stage 20 to the onset of degeneration of tail segments at HH-stage 27. We characterize the formation of somite compartment formation in the tail region with respect to morphology and the expression patterns of the sclerotomal marker gene paired-box gene 1 (Pax1) and the myotomal marker genes MyoD and myogenic factor 5 (Myf5). Our study gives insight into the development of the very last segments formed in the avian embryo, and provides a basis for further research on the development of tail somite derivatives such as tail vertebrae, pygostyle and tail musculature., (© 2019 Anatomical Society.)

The axial skeleton is divided into different regions based on its morphological features. In particular, in birds and mammals, ribs are present only in the thoracic region. The axial skeleton is derived from a series of somites. In the thoracic region of the axial skeleton, descendants of somites coherently penetrate into the somatic mesoderm to form ribs. In regions other than the thoracic, descendants of somites do not penetrate the somatic lateral plate mesoderm. We performed live-cell time-lapse imaging to investigate the difference in the migration of a somite cell after contact with the somatic lateral plate mesoderm obtained from different regions of anterior-posterior axis in vitro on cytophilic narrow paths. We found that a thoracic somite cell continues to migrate after contact with the thoracic somatic lateral plate mesoderm, whereas it ceases migration after contact with the lumbar somatic lateral plate mesoderm. This suggests that cell-cell interaction works as an important guidance cue that regulates migration of somite cells. We surmise that the thoracic somatic lateral plate mesoderm exhibits region-specific competence to allow penetration of somite cells, whereas the lumbosacral somatic lateral plate mesoderm repels somite cells by contact inhibition of locomotion. The differences in the behavior of the somatic lateral plate mesoderm toward somite cells may confirm the distinction between different regions of the axial skeleton., (Copyright © 2019. Published by Elsevier B.V.)

Segment formation in vertebrate embryos is a stunning example of biological self-organization. Here, we present an idealized framework, in which we treat the presomitic mesoderm (PSM) as a one-dimensional line of oscillators. We use the framework to derive constraints that connect the size of somites, and the timing of their formation, to the growth of the PSM and the gradient of the somitogenesis clock period across the PSM. Our analysis recapitulates the observations made recently in ex vivo cultures of mouse PSM cells, and makes predictions for how perturbations, such as increased Wnt levels, would alter somite widths. Finally, our analysis makes testable predictions for the shape of the phase profile and somite widths at different stages of PSM growth. In particular, we show that the phase profile is robustly concave when the PSM length is steady and slightly convex in an important special case when it is decreasing exponentially. In both cases, the phase profile scales with the PSM length; in the latter case, it scales dynamically. This has important consequences for the velocity of the waves that traverse the PSM and trigger somite formation, as well as the effect of errors in phase measurement on somite widths.

The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis -regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Defects in somitogenesis result in vertebral malformations at birth known as spondylocostal dysostosis (SCDO). Somites are formed with a species-specific periodicity controlled by the "segmentation clock," which comprises a group of oscillatory genes in the presomitic mesoderm. Here, we report that a segmentation clock model derived from human embryonic stem cells shows many hallmarks of the mammalian segmentation clock in vivo, including a dependence on the NOTCH and WNT signaling pathways. The gene expression oscillations are highly synchronized, displaying a periodicity specific to the human clock. Introduction of a point of mutation into HES7, a specific mutation previously associated with clinical SCDO, eliminated clock gene oscillations, successfully reproducing the defects in the segmentation clock. Thus, we provide a model for studying the previously inaccessible human segmentation clock to better understand the mechanisms contributing to congenital skeletal defects., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development., (Copyright © 2019. Published by Elsevier B.V.)

Consistent asymmetries between the left and right sides of animal bodies are common. For example, the internal organs of vertebrates are left-right (L-R) asymmetric in a stereotyped fashion. Other structures, such as the skeleton and muscles, are largely symmetric. This Review considers how symmetries and asymmetries form alongside each other within the embryo, and how they are then maintained during growth. I describe how asymmetric signals are generated in the embryo. Using the limbs and somites as major examples, I then address mechanisms for protecting symmetrically forming tissues from asymmetrically acting signals. These examples reveal that symmetry should not be considered as an inherent background state, but instead must be actively maintained throughout multiple phases of embryonic patterning and organismal growth., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The intermediate mesoderm is located between the somites and the lateral plate mesoderm and gives rise to renal progenitors that contribute to the three mammalian kidney types (pronephros, mesonephros and metanephros). In this review, focusing largely on murine kidney development, we examine how the intermediate mesoderm forms during gastrulation/axis elongation and how it progressively gives rise to distinct renal progenitors along the rostro-caudal axis. We highlight some of the potential signalling cues and core transcription factor circuits that direct these processes, up to the point of early metanephric kidney formation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)