Anterior expansion and posterior addition to the notochord mechanically coordinate zebrafish embryo axis elongation.

How force generated by the morphogenesis of one tissue impacts the morphogenesis of other tissues to achieve an elongated embryo axis is not well understood. The notochord runs along the length of the somitic compartment and is flanked on either side by somites. Vacuolating notochord cells undergo a constrained expansion, increasing notochord internal pressure and driving its elongation and stiffening. Therefore, the notochord is appropriately positioned to play a role in mechanically elongating the somitic compartment. We used multi-photon cell ablation to remove specific regions of the zebrafish notochord and quantify the impact on axis elongation. We show that anterior expansion generates a force that displaces notochord cells posteriorly relative to adjacent axial tissues, contributing to the elongation of segmented tissue during post-tailbud stages. Unexpanded cells derived from progenitors at the posterior end of the notochord provide resistance to anterior notochord cell expansion, allowing for stress generation along the anterior-posterior axis. Therefore, notochord cell expansion beginning in the anterior, and addition of cells to the posterior notochord, act as temporally coordinated morphogenetic events that shape the zebrafish embryo anterior-posterior axis.

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.

A fishy tail: Insights into the cell and molecular biology of neuromesodermal cells from zebrafish embryos.

Vertebrate embryos establish their primary body axis in a conserved progressive fashion from the anterior to the posterior. During this process, a posteriorly localized neuromesodermal cell population called neuromesodermal progenitors (NMps) plays a critical role in contributing new cells to the spinal cord and mesoderm as the embryo elongates. Defects in neuromesodermal population development can cause severe disruptions to the formation of the body posterior to the head. Given their importance during development and their potential, some of which has already been realized, for revealing new methods of in vitro tissue generation, there is great interest in better understanding NMp biology. The zebrafish model system has been instrumental in advancing our understanding of the molecular and cellular attributes of the NM cell population and its derivatives. In this review, we focus on our current understanding of the zebrafish NM population and its contribution to body axis formation, with particular emphasis on the lineage potency, morphogenesis, and niche factors that promote or inhibit differentiation.

An epiblast stem cell-derived multipotent progenitor population for axial extension.

The caudal lateral epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the body axis elongation. These progenitors, called neural mesodermal progenitors (NMPs), are identified as cells that co-express Sox2 and T/brachyury, a criterion used to derive NMP-like cells from embryonic stem cells in vitro However, unlike embryonic NMPs, these progenitors do not self-renew. Here, we find that the protocols that yield NMP-like cells in vitro initially produce a multipotent population that, in addition to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that epiblast stem cells (EpiSCs) are an effective source of these multipotent progenitors, which are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs exhibit limited self-renewal in vitro and a gene expression signature like their embryonic counterparts.

Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics.

During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. Using a genetic clone-tracing method, we labelled early embryonic progenitors and found a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages using light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid-gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, the lineage of which varies in a species-specific manner due to vastly different rates of differentiation and growth.

Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids.

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/ß-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.

The Chick Caudolateral Epiblast Acts as a Permissive Niche for Generating Neuromesodermal Progenitor Behaviours.

Neuromesodermal progenitors (NMps) are a population of bipotent progenitors that maintain competence to generate both spinal cord and paraxial mesoderm throughout the elongation of the posterior body axis. Recent studies have generated populations of NMp-like cells in culture, which have been shown to differentiate to both neural and mesodermal cell fates when transplanted into either mouse or chick embryos. Here, we aim to compare the potential of mouse embryonic stem (ES) cell-derived progenitor populations to generate NMp behaviour against both undifferentiated and differentiated populations. We define NMp behaviour as the ability of cells to: (i) contribute to a significant proportion of the anterior-posterior body axis, (ii) enter into both posterior neural and somitic compartments, and (iii) retain a proportion of the progenitor population within the posterior growth zone. We compare previously identified ES cell-derived NMp-like populations to undifferentiated mouse ES cells and find that they all display similar potentials to generate NMp behaviour in vivo. To assess whether this competence is lost upon further differentiation, we generated anterior and posterior embryonic cell types through the generation of 3D gastruloids and show that NMp competence is lost within the anterior (Brachyury-negative) portion of the gastruloid. Together this suggests that in vitro-derived NMp-like cells maintain an ability to contribute to multiple germ layers that is also present within pluripotent ES cells, rather than acquiring a neuromesodermal competent state through differentiation.

Intrinsic and extrinsic cues time somite progenitor contribution to the vertebrate primary body axis.

During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

Mannose controls mesoderm specification and symmetry breaking in mouse gastruloids.

Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem-cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. We found that the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids. However, glucose removal did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis demonstrated that mannose reversed glycosylation of the Wnt pathway regulator, secreted frizzled receptor Frzb. Our study showed how mannose controls mesoderm specification in mouse gastruloids.

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice.

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.

The unappreciated generative role of cell movements in pattern formation.

The mechanisms underpinning the formation of patterned cellular landscapes has been the subject of extensive study as a fundamental problem of developmental biology. In most cases, attention has been given to situations in which cell movements are negligible, allowing researchers to focus on the cell-extrinsic signalling mechanisms, and intrinsic gene regulatory interactions that lead to pattern emergence at the tissue level. However, in many scenarios during development, cells rapidly change their neighbour relationships in order to drive tissue morphogenesis, while also undergoing patterning. To draw attention to the ubiquity of this problem and propose methodologies that will accommodate morphogenesis into the study of pattern formation, we review the current approaches to studying pattern formation in both static and motile cellular environments. We then consider how the cell movements themselves may contribute to the generation of pattern, rather than hinder it, with both a species specific and evolutionary viewpoint.

Zebrafish neuromesodermal progenitors undergo a critical state transition in vivo.

The transition state model of cell differentiation proposes that a transient window of gene expression stochasticity precedes entry into a differentiated state. Here, we assess this theoretical model in zebrafish neuromesodermal progenitors (NMps) in vivo during late somitogenesis stages. We observed an increase in gene expression variability at the 24 somite stage (24ss) before their differentiation into spinal cord and paraxial mesoderm. Analysis of a published 18ss scRNA-seq dataset showed that the NMp population is noisier than its derivatives. By building in silico composite gene expression maps from image data, we assigned an 'NM index' to in silico NMps based on the expression of neural and mesodermal markers and demonstrated that cell population heterogeneity peaked at 24ss. Further examination revealed cells with gene expression profiles incongruent with their prospective fate. Taken together, our work supports the transition state model within an endogenous cell fate decision making event.

Quantitative Experimental Embryology: A Modern Classical Approach.

Experimental Embryology is often referred to as a classical approach of developmental biology that has been to some extent replaced by the introduction of molecular biology and genetic techniques to the field. Inspired by the combination of this approach with advanced techniques to uncover core principles of neural crest development by the laboratory of Roberto Mayor, we review key quantitative examples of experimental embryology from recent work in a broad range of developmental biology questions. We propose that quantitative experimental embryology offers essential ways to explore the reaction of cells and tissues to targeted cell addition, removal, and confinement. In doing so, it is an essential methodology to uncover principles of development that remain elusive such as pattern regulation, scaling, and self-organisation.

Tissue tectonics and the multi-scale regulation of developmental timing.

Development encompasses processes that occur at multiple length scales, including gene-regulatory interactions, cell movements and reorganization, cell signalling and growth. It is essential that the timing of events in all of these different processes is coordinated to generate well-patterned tissues and organs. However, how the timing of intrinsic cell state changes is coordinated with events occurring at the multi-tissue and whole-organism level is unknown. Here, we argue that an important mechanism that accounts for the integration of timing across levels of organization is provided by tissue tectonics, i.e. how morphogenetic events driving tissue shape changes result in the relative displacement of signalling and responding tissues and coordinate developmental timing across scales. In doing so, tissue tectonics provides a mechanism by which the cell specification events intrinsic to cells can be modulated by the temporal exposure to extracellular signals. This exposure is in turn regulated by higher-order properties of the embryo, such as their physical properties, rates of growth and the combination of dynamic cell behaviours, impacting tissue morphogenesis. Tissue tectonics creates a downward flow of information from higher to lower levels of biological organization, providing an instance of downward causation in development.

The zebrafish presomitic mesoderm elongates through compaction-extension.

In vertebrate embryos the presomitic mesoderm becomes progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase, and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease and an increase in cell density. We also find that individual cells decrease in volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Taken together, we propose a compaction-extension mechanism of tissue elongation that highlights the need to better understand the role tissue intrinsic and extrinsic forces in regulating morphogenesis.

Establishment of the vertebrate body plan: Rethinking gastrulation through stem cell models of early embryogenesis.

A striking property of vertebrate embryos is the emergence of a conserved body plan across a wide range of organisms through the process of gastrulation. As the body plan unfolds, gene regulatory networks (GRNs) and multicellular interactions (cell regulatory networks, CRNs) combine to generate a conserved set of morphogenetic events that lead to the phylotypic stage. Interrogation of these multilevel interactions requires manipulation of the mechanical environment, which is difficult in vivo. We review recent studies of stem cell models of early embryogenesis from different species showing that, independent of species origin, cells in culture form similar structures. The main difference between embryos and in vitro models is the boundary conditions of the multicellular ensembles. We discuss these observations and suggest that the mechanical and geometric boundary conditions of different embryos before gastrulation hide a morphogenetic ground state that is revealed in the stem-cell-based models of embryo development.

A deep learning approach for staging embryonic tissue isolates with small data.

Machine learning approaches are becoming increasingly widespread and are now present in most areas of research. Their recent surge can be explained in part due to our ability to generate and store enormous amounts of data with which to train these models. The requirement for large training sets is also responsible for limiting further potential applications of machine learning, particularly in fields where data tend to be scarce such as developmental biology. However, recent research seems to indicate that machine learning and Big Data can sometimes be decoupled to train models with modest amounts of data. In this work we set out to train a CNN-based classifier to stage zebrafish tail buds at four different stages of development using small information-rich data sets. Our results show that two and three dimensional convolutional neural networks can be trained to stage developing zebrafish tail buds based on both morphological and gene expression confocal microscopy images, achieving in each case up to 100% test accuracy scores. Importantly, we show that high accuracy can be achieved with data set sizes of under 100 images, much smaller than the typical training set size for a convolutional neural net. Furthermore, our classifier shows that it is possible to stage isolated embryonic structures without the need to refer to classic developmental landmarks in the whole embryo, which will be particularly useful to stage 3D culture in vitro systems such as organoids. We hope that this work will provide a proof of principle that will help dispel the myth that large data set sizes are always required to train CNNs, and encourage researchers in fields where data are scarce to also apply ML approaches.

Cell-to-cell heterogeneity in Sox2 and Bra expression guides progenitor motility and destiny.

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its biological functions. By studying progenitors of the posterior region of bird embryos, we found that expression levels of transcription factors Sox2 and Bra, respectively involved in neural tube (NT) and mesoderm specification, display a high degree of cell-to-cell heterogeneity. By combining forced expression and downregulation approaches with time-lapse imaging, we demonstrate that Sox2-to-Bra ratio guides progenitor's motility and their ability to stay in or exit the progenitor zone to integrate neural or mesodermal tissues. Indeed, high Bra levels confer high motility that pushes cells to join the paraxial mesoderm, while high levels of Sox2 tend to inhibit cell movement forcing cells to integrate the NT. Mathematical modeling captures the importance of cell motility regulation in this process and further suggests that randomness in Sox2/Bra cell-to-cell distribution favors cell rearrangements and tissue shape conservation.