The organizer and neural induction in birds and mammals.

In avian and mammalian embryos the "organizer" property associated with neural induction of competent ectoderm into a neural plate and its subsequent patterning into rostro-caudal domains resides at the tip of the primitive streak before neurulation begins, and before a morphological Hensen's node is discernible. The same region and its later derivatives (like the notochord) also have the ability to "dorsalize" the adjacent mesoderm, for example by converting lateral plate mesoderm into paraxial (pre-somitic) mesoderm. Both neural induction and dorsalization of the mesoderm involve inhibition of BMP, and the former also requires other signals. This review surveys the key experiments done to elucidate the functions of the organizer and the mechanisms of neural induction in amniotes. We conclude that the mechanisms of neural induction in amniotes and anamniotes are likely to be largely the same; apparent differences are likely to be due to differences in experimental approaches dictated by embryo topology and other practical constraints. We also discuss the relationships between "neural induction" assessed by grafts of the organizer and normal neural plate development, as well as how neural induction relates to the generation of neuronal cells from embryonic and other stem cells in vitro.

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin.

The nuclear lamina couples mechanical forces to cell fate in the preimplantation embryo via actin organization.

During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.

A gene regulatory network for neural induction.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

Sequential changes in cellular properties accompanying amniote somite formation.

Somites are transient structures derived from the pre-somitic mesoderm (PSM), involving mesenchyme-to-epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail-to-head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be 'trapped' within the somitocoele after cells at the surfaces of the PSM undergo MET. Three-dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N-cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation.

Exploring the roles of FGF/MAPK and cVG1/GDF signalling on mesendoderm induction and convergent extension during chick primitive streak formation.

During primitive streak formation in the chick embryo, cells undergo mesendoderm specification and convergent extension at the same time and in the same cells. Previous work has implicated cVG1 (GDF3) as a key factor for induction of primitive streak identity and positioning the primitive streak, whereas FGF signalling was implicated in regulating cell intercalation via regulation of components of the WNT-planar cell polarity (PCP) pathway. FGF has also been reported to be able to induce a primitive streak (but lacking the most axial derivatives such as notochord/prechordal mesendoderm). These signals emanate from different cell populations in the embryo, so how do they interact to ensure that the same cells undergo both cell intercalation and acquire primitive streak identity? Here we begin to address this question by examining in more detail the ability of the two classes of signals in regulating the two developmental events. Using misexpression of inducers and/or exposure to inhibitors and in situ hybridisation, we study how these two signals regulate expression of Brachyury (TBXT) and PRICKLE1 as markers for the primitive streak and the PCP, respectively. We find that both signals can induce both properties, but while FGF seems to be required for induction of the streak by cVG1, it is not necessary for induction of PRICKLE1. The results are consistent with cVG1 being a common regulator for both primitive streak identity and the initiation of convergent extension that leads to streak elongation.

Molecular characteristics of the edge cells responsible for expansion of the chick embryo on the vitelline membrane.

During early avian development, only a narrow band of cells (the edge cells, also called 'margin of overgrowth') at the rim of the embryo is responsible for blastoderm expansion by crawling over the vitelline membrane (VM) to cover the whole egg yolk in just 4 days (a process called epiboly). Surprisingly, this has not yet been studied in detail. Here we explore the edge cells of the chick embryo using in situ hybridization, immunohistochemistry and live imaging. Morphological and molecular properties reveal that the edge has a distinctive structure, being subdivided into sub-regions, including at least four distinct zones (which we name as leading, trailing, deep and stalk zones). This allows us to study reorganization of the edge region that accompanies reattachment of an explanted blastoderm to the VM. Immunohistochemistry uncovers distinct polarized cellular features resembling the process of collective cell migration described in other systems. Live imaging reveals dynamic lamellipodial and filopodial activity at the leading edge of the outermost cells. Our data provide evidence that edge cells are a distinct tissue. We propose that edge cells may be a useful model system for the study of wound healing and other closure events in epithelial cell sheets.

A niche for axial stem cells - A cellular perspective in amniotes.

The head-tail axis in birds and mammals develops from a growth zone in the tail-end, which contains the node. This growth zone then forms the tailbud. Labelling experiments have shown that while many cells leave the node and tailbud to contribute to axial (notochord, floorplate) and paraxial (somite) structures, some cells remain resident in the node and tailbud. Could these cells be resident axial stem cells? If so, do the node and tailbud represent an instructive stem cell niche that specifies and maintains these stem cells? Serial transplantation and single cell labelling studies support the existence of self-renewing stem cells and heterotopic transplantations suggest that the node can instruct such self-renewing behaviour. However, only single cell manipulations can reveal whether self-renewing behaviour occurs at the level of a cell population (asymmetric or symmetric cell divisions) or at the level of single cells (asymmetric divisions only). We combine data on resident cells in the node and tailbud and review it in the context of axial development in chick and mouse, summarising our current understanding of axial stem cells and their niche and highlighting future directions of interest.

The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo.

Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.

Reflections on the past, present and future of developmental biology.

Developmental Biology embodies some of the most fundamental questions in Biology and can trace its roots back to several thousand years ago; the last 100 years have been particularly extraordinary. In part the advances have been fuelled by new technical advances and knowledge in many other areas, which have contributed to shaping the field as truly interdisciplinary. During those 100 years some of our predecessors identified some key questions and a few important principles especially by trying to find general rules that govern what cells are able to do and how they choose between different options, as well as principles of experimental design that can be used to uncover those rules even before we know their physicochemical underpinnings. But the field has been changing rapidly in the last two decades. Here I present a brief overview of some of the changes that have taken place over the last Century and a personal view of current directions. The picture that emerges is of some dark clouds on the horizon, so this is also a call to arms for our colleagues to try to regain what the field has been losing.

'Neighbourhood watch' model: embryonic epiblast cells assess positional information in relation to their neighbours.

In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.

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.

Identical twins carry a persistent epigenetic signature of early genome programming.

Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.

Cellular aspects of somite formation in vertebrates.

Vertebrate segmentation, the process that generates a regular arrangement of somites and thereby establishes the pattern of the adult body and of the musculoskeletal and peripheral nervous systems, was noticed many centuries ago. In the last few decades, there has been renewed interest in the process and especially in the molecular mechanisms that might account for its regularity and other spatial-temporal properties. Several models have been proposed but surprisingly, most of these do not provide clear links between the molecular mechanisms and the cell behaviours that generate the segmental pattern. Here we present a short survey of our current knowledge about the cellular aspects of vertebrate segmentation and the similarities and differences between different vertebrate groups in how they achieve their metameric pattern. Taking these variations into account should help to assess each of the models more appropriately.

A mechanical model of early somite segmentation.

Somitogenesis is often described using the clock-and-wavefront (CW) model, which does not explain how molecular signaling rearranges the pre-somitic mesoderm (PSM) cells into somites. Our scanning electron microscopy analysis of chicken embryos reveals a caudally-progressing epithelialization front in the dorsal PSM that precedes somite formation. Signs of apical constriction and tissue segmentation appear in this layer 3-4 somite lengths caudal to the last-formed somite. We propose a mechanical instability model in which a steady increase of apical contractility leads to periodic failure of adhesion junctions within the dorsal PSM and positions the future inter-somite boundaries. This model produces spatially periodic segments whose size depends on the speed of the activation front of contraction (F), and the buildup rate of contractility (Λ). The Λ/F ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment size increases with the front speed.

Molecular anatomy of the pre-primitive-streak chick embryo.

The early stages of development of the chick embryo, leading to primitive streak formation (the start of gastrulation), have received renewed attention recently, especially for studies of the mechanisms of large-scale cell movements and those that position the primitive streak in the radial blastodisc. Over the long history of chick embryology, the terminology used to define different regions has been changing, making it difficult to relate studies to each other. To resolve this objectively requires precise definitions of the regions based on anatomical and functional criteria, along with a systematic molecular map that can be compared directly to the functional anatomy. Here, we undertake these tasks. We describe the characteristic cell morphologies (using scanning electron microscopy and immunocytochemistry for cell polarity markers) in different regions and at successive stages. RNAseq was performed for 12 regions of the blastodisc, from which a set of putative regional markers was selected. These were studied in detail by in situ hybridization. Together this provides a comprehensive resource allowing the community to define the regions unambiguously and objectively. In addition to helping with future experimental design and interpretation, this resource will also be useful for evolutionary comparisons between different vertebrate species.

Signaling events regulating embryonic polarity and formation of the primitive streak in the chick embryo.

The avian embryo is a key experimental model system for early development of amniotes. One key difference with invertebrates and "lower" vertebrates like fish and amphibians is that amniotes do not rely so heavily on maternal messages because the zygotic genome is activated very early. Early development also involves considerable growth in volume and mass of the embryo, with cell cycles that include G1 and G2 phases from very early cleavage. The very early maternal to zygotic transition also allows the embryo to establish its own polarity without relying heavily on maternal determinants. In many amniotes including avians and non-rodent mammals, this enables an ability of the embryo to "regulate": a single multicellular embryo can give rise to more than one individual-monozygotic twins. Here we discuss the embryological, cellular, molecular and evolutionary underpinnings of gastrulation in avian embryos as a model amniote embryo. Many of these properties are shared by human embryos.

Transplantation of Neural Tissue: Quail-Chick Chimeras.

Tissue transplantation is an important approach in developmental neurobiology to determine cell fate, to uncover inductive interactions required for tissue specification and patterning as well as to establish tissue competence and commitment. Combined with state-of-the-art molecular approaches, transplantation assays have been instrumental for the discovery of gene regulatory networks controlling cell fate choices and how such networks change over time. Avian species are among the favorite model systems for these approaches because of their accessibility and relatively large size. Here we describe two culture techniques used to generate quail-chick chimeras at different embryonic stages and methods to distinguish graft and donor tissue.

A 3D molecular atlas of the chick embryonic heart.

We present a detailed analysis of gene expression in the 2-day (HH12) embryonic chick heart. RNA-seq of 13 micro-dissected regions reveals regionalised expression of 15,570 genes. Of these, 132 were studied by in situ hybridisation and a subset (38 genes) was mapped by Optical Projection Tomography or serial sectioning to build a detailed 3-dimensional atlas of expression. We display this with a novel interactive 3-D viewer and as stacks of sections, revealing the boundaries of expression domains and regions of overlap. Analysis of the expression domains also defines some sub-regions distinct from those normally recognised by anatomical criteria at this stage of development, such as a previously undescribed subdivision of the atria into two orthogonal sets of domains (dorsoventral and left-right). We also include a detailed comparison of expression in the chick with the mouse and other species.