Embryo-scale reverse genetics at single-cell resolution.

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1-4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

Hinge point emergence in mammalian spinal neurulation.

Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hinge point, then both the median hinge point and dorsolateral hinge points, followed by dorsolateral hinge points only. The biomechanical mechanism of hinge point formation in the mammalian neural tube is poorly understood. Here we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, nonneural ectoderm expansion, and neural plate adhesion to the notochord. Dorsolateral hinge points emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hinge point formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

The embryonic node behaves as an instructive stem cell niche for axial elongation.

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Single-cell morphometrics reveals ancestral principles of notochord development.

Embryonic tissues are shaped by the dynamic behaviours of their constituent cells. To understand such cell behaviours and how they evolved, new approaches are needed to map out morphogenesis across different organisms. Here, we apply a quantitative approach to learn how the notochord forms during the development of amphioxus: a basally branching chordate. Using a single-cell morphometrics pipeline, we quantify the geometries of thousands of amphioxus notochord cells, and project them into a common mathematical space, termed morphospace. In morphospace, notochord cells disperse into branching trajectories of cell shape change, revealing a dynamic interplay between cell shape change and growth that collectively contributes to tissue elongation. By spatially mapping these trajectories, we identify conspicuous regional variation, both in developmental timing and trajectory topology. Finally, we show experimentally that, unlike ascidians but like vertebrates, posterior cell division is required in amphioxus to generate full notochord length, thereby suggesting this might be an ancestral chordate trait that is secondarily lost in ascidians. Altogether, our novel approach reveals that an unexpectedly complex scheme of notochord morphogenesis might have been present in the first chordates. This article has an associated 'The people behind the papers' interview.

Patterns of brachyury expression in chordomas.

INTRODUCTION: Chordomas are rare malignant midline tumors, presumed to arise from notochordal remnants. This was further suggested by the discovery of the brachyury in chordomas pathogenesis. Its immunohistochemical expression has become the principal adjunct in the diagnosis of chordomas. However, studies about brachyury expression in chordomas are not fully comparable, mainly because they use different primary antibodies. Thus, the aim of this study is to investigate the expression of brachyury expression in a series of chordomas in conjunction to clinicopathological characteristics and to review the relevant literature providing all the details needed in the immunohistochemical study of brachyury. MATERIALS AND METHODS: This is a retrospective study of 62 chordomas, diagnosed over a 22-year period. No dedifferentiated or poorly differentiated cases were included. A monoclonal primary antibody (clone A-4) was used and brachyury expression was evaluated by the H-score. Clinicopathological parameters studied were age, sex, tumor localization, decalcification status and tissue age. Fetal notochords were used for comparison. RESULTS: Mean H-score of nuclear brachyury expression was 129.8. The tissue age significantly influenced brachyury expression, the older samples expressing less brachyury. Decalcification demonstrated a trend to weaken brachyury expression. Clinical characteristics were not correlated with the patterns of brachyury expression. Notochords were negative. Literature review reveals several polyclonal antibodies used and a positivity of 75%-100% in chordomas with even more variable results in notochords. CONCLUSION: In chordomas, as in other tumor types, an uniformization of studies about brachyury expression is needed, by considering the clone used, and the decalcification and the age of the sample, given the growing importance of brachyury in diagnosis and therapeutic steps.

Heads and tails: The notochord develops differently in the cranium and caudal fin of Atlantic Salmon (Salmo salar, L.).

While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it.

Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation.

Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Development of a straight vertebrate body axis.

The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis.

Functional and genetic analyses of ZYG11B provide evidences for its involvement in OAVS.

BACKGROUND: The Oculo-Auriculo-Vertebral Spectrum (OAVS) or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical heterogeneity of this spectrum and its incomplete penetrance limited the molecular diagnosis. In this study, we describe a novel causative gene, ZYG11B. METHODS: A sporadic case of OAVS was analyzed by whole exome sequencing in trio strategy. The identified candidate gene, ZYG11B, was screened in 143 patients by next generation sequencing. Overexpression and immunofluorescence of wild-type and mutated ZYG11B forms were performed in Hela cells. Moreover, morpholinos were used for transient knockdown of its homologue in zebrafish embryo. RESULTS: A nonsense de novo heterozygous variant in ZYG11B, (NM_024646, c.1609G>T, p.Glu537*) was identified in a single OAVS patient. This variant leads in vitro to a truncated protein whose subcellular localization is altered. Transient knockdown of the zebrafish homologue gene confirmed its role in craniofacial cartilages architecture and in notochord development. Moreover, ZYG11B expression regulates a cartilage master regulator, SOX6, and is regulated by Retinoic Acid, a known developmental toxic molecule leading to clinical features of OAVS. CONCLUSION: Based on genetic, cellular and animal model data, we proposed ZYG11B as a novel rare causative gene for OAVS.

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates.

The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs ß cell differentiation, compatible with the antagonistic role of Bmp in ß cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs ß cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.

Cell population growth regulates dorsalization and caudalization during chick morphogenesis and programmed cell death in lens fibres.

The chick embryo ectoblast was examined for a possible relationship between the state of neural competence and cell population growth. It was found that although ectoblast cells with doubling times ranging between 5 to 20 h exhibit neural competence, the extent of neutralization induced by the Hensen's node depends on the duration of the cell cycle; the longer the doubling time of the competent ectoblast, the stronger the induction and the greater the induced neural tissue. Neural induction in the competent ectoblast occurs in at least two steps: the first lasts for 1-2 h of direct contact with the inducing Hensen's node graft; a contact for another 2 h with even a non-inducing post-nodal fragment is essential to consolidate neutralization. Hensen's node graft induces mitotic activity in the competent ectoblast in contact. Teratogens which inhibit cell population growth, development and blastoderm expansion in chick embryo gastrula cause concomitant caudalization of the embryonic axis. We confirm Yamada's hypothesis that dorsalization is under positive mitogenic control, whereas caudalization is controlled by a negative cell cycle regulation. Reverse transcripts of chick gastrula mRNA were cloned in pBR322. Colony hybridization with cDNA made against chicken yolk RNA showed positive clones. Thus chicken yolk contains maternal mRNAs. cDNA made against mRNA extracted from stage 10 foreheads was hybridized with RNA from stage 1 to 13 embryos, 19 day lens and egg yolk. The hybridization signal, which was low between stages 1 to 7, increased between stages 10-13 and decreased thereafter. Forehead cDNA also hybridized to yolk RNA. Thus, maternal RNA sequences are present in the early chick embryo. During lens development, epithelial cells retain proliferative activity and their progeny reaching a stationary phase join the fibre area and contribute to the growth of fibre cells. The rate of transfer from epithelium to fibre regulates the rate of programmed cell death of the non-dividing differentiated lens fibre cells.

The notochord gene regulatory network in chordate evolution: Conservation and divergence from Ciona to vertebrates.

The notochord is a structure required for support and patterning of all chordate embryos, from sea squirts to humans. An increasing amount of information on notochord development and on the molecular strategies that ensure its proper morphogenesis has been gleaned through studies in the sea squirt Ciona. This invertebrate chordate offers a fortunate combination of experimental advantages, ranging from translucent, fast-developing embryos to a compact genome and impressive biomolecular resources. These assets have enabled the rapid identification of numerous notochord genes and cis-regulatory regions, and provide a rather unique opportunity to reconstruct the gene regulatory network that controls the formation of this developmental and evolutionary chordate landmark. This chapter summarizes the morphogenetic milestones that punctuate notochord formation in Ciona, their molecular effectors, and the current knowledge of the gene regulatory network that ensures the accurate spatial and temporal orchestration of these processes.

Development and regeneration dynamics of the Medaka notochord.

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon, a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon â€‹+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo, and provides new insights in its development and regeneration properties.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

Iterative and Complex Asymmetric Divisions Control Cell Volume Differences in Ciona Notochord Tapering.

The notochord of the invertebrate chordate Ciona forms a tapered rod at tailbud stages consisting of only 40 cylindrical cells in a single-file column. This tapered shape involves differences in notochord cell volume along the anterior-posterior axis. Here, we quantify sibling cell volume asymmetry throughout the developing notochord and find that there are distinctive patterns of unequal cleavage in all 4 bilateral pairs of A-line primary notochord founder cells and also in the B-line-derived secondary notochord founder cells. A quantitative model confirms that the observed patterns of unequal cleavage are sufficient to explain all the anterior-posterior variation in notochord cell volume. Many examples are known of cells that divide asymmetrically to give daughter cells of different size and fate. Here, by contrast, a series of subtle but iterative and finely patterned asymmetric divisions controls the shape of an entire organ. Quantitative 3D analysis of cell shape and spindle positioning allows us to infer multiple cellular mechanisms driving these unequal cleavages, including polarized displacements of the mitotic spindle, contributions from the shape of the mother cell, and late changes occurring between anaphase and abscission that potentially involve differential cortical contractility. We infer differential use of these mechanisms between different notochord blastomeres and also between different rounds of cell division. These results demonstrate a new role for asymmetric division in directly shaping a developing organ and point toward complex underlying mechanisms.

Comprehensive single-cell transcriptome lineages of a proto-vertebrate.

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development-from the onset of gastrulation to swimming tadpoles-in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

A unique role of thyroid hormone receptor ß in regulating notochord resorption during Xenopus metamorphosis.

Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that thyroid hormone (T3) receptor (TR) plays a necessary and sufficient role to mediate the causative effect of T3 on metamorphosis. Of the two known TR genes in vertebrates, TRα is highly expressed during both premetamorphosis and metamorphosis while TRß expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of T3 during metamorphosis, suggesting potentially different functions during metamorphosis. Indeed, gene knockout studies have shown that knocking out TRα and TRß has different effects on tadpole development. In particularly, homozygous TRß knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TRß-knockout tadpoles, an apparently normal notochord is present when the notochord in wild-type and TRα-knockout tadpoles disappears. Here, we have investigated how tail notochord resorption is regulated by TR. We show that TRß is selectively very highly expressed in the notochord compared to TRα. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by T3 and implicated to play a role in tissue resorption by degrading the extracellular matrix (ECM). In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of TRß expression in the notochord specifically upregulate these MMPs, which in turn degrades the ECM, leading to the collapse of the notochord and its subsequent resorption during metamorphosis.

A histocytological and radiological overview of the natural history of intervertebral disk: from embryonic formation to age-related degeneration.

PURPOSE: To elucidate the natural history of intervertebral disk (IVD) and characterize its embryonic beginnings and age-related degeneration. METHODS: Coronal sections of embryonic (E13.5-neonatal) and postnatal (4-60-week-old) Sprague-Dawley rat IVD were stained by a series of histological stainings (hematoxylin and eosin, Alcian blue, Picrosirius red, Masson, Periodic acid-Schiff). Growth kinetics within embryonic IVD were evaluated by immunohistochemical staining of Ki67 and proliferating cell nuclear antigen. Postnatal maturation and degeneration of IVD were visualized on radiology by X-ray, CT, and MR imaging. RESULTS: During the formation of rat IVD, inner annulus fibrosus (AF) and cartilaginous endplate (CEP) shared similar cell density, extracellular matrix, and potential of growth kinetics; notochord provided increased and enlarged cytoplasmic vacuoles to generate nucleus pulposus (NP), part of which was retained within CEP. Postnatally, vacuolated notochord cells were reduced by devacuolation, while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilaginous matrix accumulated and infiltrated outward. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP; only part of these histocytological changes could be reported on radiology. CONCLUSIONS: By showing that the natural history of IVD is orchestrated by a dynamic histocytological regulation, our study may facilitate better understanding of the developmental defects, cellular heterogeneity, age-related degenerative mechanisms, and biological regeneration of IVD. These slides can be retrieved under Electronic Supplementary Material.