Cellular and molecular control of vertebrate somitogenesis.

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.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. 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 indexing3 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 data4-8 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.

The people behind the papers - Julie Klepstad and Luciano Marcon.

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.

Oscillatory control of embryonic development.

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.

The Clock and Wavefront Self-Organizing model recreates the dynamics of mouse somitogenesis in vivo and in vitro.

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.

An emerging role for tissue plasticity in developmental precision.

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.

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

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.

Emergence of a left-right symmetric body plan in vertebrate embryos.

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.

PatternJ: an ImageJ toolset for the automated and quantitative analysis of regular spatial patterns found in sarcomeres, axons, somites, and more.

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.

Generation of patterns in the paraxial mesoderm.

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.

An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm.

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.

Notochord segmentation in zebrafish controlled by iterative mechanical signaling.

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.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies

ABSTRACT The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.