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.

Distinct molecular profile of the chick organizer as a stem zone during axial elongation.

The vertebrate organizer plays a crucial role in building the main (antero-posterior) axis of the embryo: it neuralizes the surrounding ectoderm, and is the site of emigration for cells making axial and paraxial mesendoderm during elongation. The chick organizer becomes a stem zone at the onset of elongation; it stops recruiting cells from the neighbouring ectoderm and generates all its derivatives from the small number of resident cells it contains at the end of gastrulation stages. Nothing is known about the molecular identity of this stem zone. Here, we specifically labelled long-term resident cells of the organizer and compared their RNA-seq profile to that of the neighbouring cell populations. Screening by reverse transcription-polymerase chain reaction and in situ hybridization identified four genes (WIF1, PTGDS, ThPO and UCKL1) that are upregulated only in the organizer region when it becomes a stem zone and remain expressed there during axial elongation. In experiments specifically labelling the resident cells of the mature organizer, we show that only these cells express these genes. These findings molecularly define the organizer as a stem zone and offer a key to understanding how this zone is set up, the molecular control of its cells' behaviour and the evolution of axial growth zones.

The amniote primitive streak is defined by epithelial cell intercalation before gastrulation.

During gastrulation, a single epithelial cell layer, the ectoderm, generates two others: the mesoderm and the endoderm. In amniotes (birds and mammals), mesendoderm formation occurs through an axial midline structure, the primitive streak, the formation of which is preceded by massive 'polonaise' movements of ectoderm cells. The mechanisms controlling these processes are unknown. Here, using multi-photon time-lapse microscopy of chick (Gallus gallus) embryos, we reveal a medio-lateral cell intercalation confined to the ectodermal subdomain where the streak will later form. This intercalation event differs from the convergent extension movements of the mesoderm described in fish and amphibians (anamniotes): it occurs before gastrulation and within a tight columnar epithelium. Fibroblast growth factor from the extraembryonic endoderm (hypoblast, a cell layer unique to amniotes) directs the expression of Wnt planar-cell-polarity pathway components to the intercalation domain. Disruption of this Wnt pathway causes the mesendoderm to form peripherally, as in anamniotes. We propose that the amniote primitive streak evolved from the ancestral blastopore by acquisition of an additional medio-lateral intercalation event, preceding gastrulation and acting independently of mesendoderm formation to position the primitive streak at the midline.

Movements of chick gastrulation.

In birds as in all amniotes, the site of gastrulation is a midline structure, the primitive streak. This appears as cells in the one cell-thick epiblast undergo epithelial-to-mesenchymal transition to ingress and form definitive mesoderm and endoderm. Global movements involving tens of thousands of cells in the embryonic epiblast precede gastrulation. They position the primitive streak precursors from a marginal position (equivalent to the situation in anamniotes) along the future antero-posterior axis (typical for amniotes). These epithelial movements continue in modified form during gastrulation, when they are accompanied by collective movements of different class in the forming mesoderm and endoderm. Here I discuss the nature of these collective cell movements shaping the embryo, their interplay with signaling events controlling fate specification and significance in an evolutionary perspective.

Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS.

Boundary cap (BC) cells are neural crest derivatives that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various knock-in alleles of the mouse gene Egr2 (also known as Krox20), the expression of which, in trunk regions, is initially restricted to BC cells, we were able to trace BC cell progeny during development and analyze their fate. Trunk BC-derived cells migrated along peripheral axons and colonized spinal nerve roots and dorsal root ganglia (DRG). All Schwann cell precursors occupying the dorsal roots were derived from BC cells. In the DRG, BC-derived cells were the progenitors of both neurons, mainly nociceptive afferents, and satellite cells. These data indicate that BC cells constitute a source of peripheral nervous system (PNS) components that, after the major neural crest ventrolateral migratory stream, feeds a secondary wave of migration to the PNS.

Manipulating Gene Expression in the Chick Embryo.

The chick embryo is a well-established representative of amniote embryos, which has been used to make many discoveries, including many major concepts, which have moved our knowledge of developmental biology in hugely important ways. The chick has a relatively compact genome and is easily amenable to embryological manipulations and in vivo imaging. Morpholino gene manipulations have been used in a variety of contexts, and constitute a quick and versatile molecular tool. Here we describe methods to deliver morpholinos to chick embryos, allowing targeting of specific cell populations at defined developmental stages, using two stages as examples: the epiblast of the embryo in the first day of incubation, when the primary germ layers of the embryo are specified, and in ovo electroporation of the neural tube as an example of a later stage. With slight modifications, these general methods can be used to target other embryonic tissues.

Local cell interactions and self-amplifying individual cell ingression drive amniote gastrulation.

Gastrulation generates three layers of cells (ectoderm, mesoderm, endoderm) from a single sheet, while large scale cell movements occur across the entire embryo. In amniote (reptiles, birds, mammals) embryos, the deep layers arise by epithelial-to-mesenchymal transition (EMT) at a morphologically stable midline structure, the primitive streak (PS). We know very little about how these events are controlled or how the PS is maintained despite its continuously changing cellular composition. Using the chick, we show that isolated EMT events and ingression of individual cells start well before gastrulation. A Nodal-dependent 'community effect' then concentrates and amplifies EMT by positive feedback to form the PS as a zone of massive cell ingression. Computer simulations show that a combination of local cell interactions (EMT and cell intercalation) is sufficient to explain PS formation and the associated complex movements globally across a large epithelial sheet, without the need to invoke long-range signalling.DOI: http://dx.doi.org/10.7554/eLife.01817.001.

Assembly of imaging chambers and high-resolution imaging of early chick embryos.

The chick embryo is a classical model system for developmental biology. Its flat disk morphology exemplifies the amniote embryos from reptiles, birds, and most mammals (including humans). It is also particularly amenable to manipulation and culture both in vitro and in ovo. As a consequence, studies with this system have been important for promoting our understanding of cell and tissue movements, lineages, inductive processes, and cell fate decisions. In recent years, imaging techniques have been devised that allow these questions to be studied at the cellular level at late neurulation and segmentation stages. Modern improvements to imaging techniques now allow direct visualization of molecular interactions and cellular events and are likely to be instrumental in linking these processes to whole tissue and embryo morphogenesis. Here, we describe a method for high-resolution (confocal or multiphoton) time-lapse imaging of chick embryos at stages spanning pregastrulation to early neurulation. We give detailed instructions for assembling the specialized imaging chambers required for this procedure.

Dedifferentiation of foetal CNS stem cells to mesendoderm-like cells through an EMT process.

Tissue-specific stem cells are considered to have a limited differentiation potential. Recently, this notion was challenged by reports that showed a broader differentiation potential of neural stem cells, in vitro and in vivo, although the molecular mechanisms that regulate plasticity of neural stem cells are unknown. Here, we report that neural stem cells derived from mouse embryonic cortex respond to Lif and serum in vitro and undergo epithelial to mesenchymal transition (EMT)-mediated dedifferentiation process within 48 h, together with transient upregulation of pluripotency markers and, more notably, upregulation of mesendoderm genes, Brachyury (T) and Sox17. These induced putative mesendoderm cells were injected into early gastrulating chick embryos, which revealed that they integrated more efficiently into mesoderm and endoderm lineages compared to non-induced cells. We also found that TGFß and Jak/Stat pathways are necessary but not sufficient for the induction of mesendodermal phenotype in neural stem cells. These results provide insights into the regulation of plasticity of neural stem cells through EMT. Dissecting the regulatory pathways involved in these processes may help to gain control over cell fate decisions.

Spatially and temporally controlled electroporation of early chick embryos.

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.

Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice.

During embryonic development, restricted expression of the regulatory genes Krox20 and kreisler are involved in segmentation and antero-posterior patterning of the hindbrain neural tube. The analysis of transgenic mice in which specific rhombomeres (r) are eliminated points to an important role of segmentation in the generation of neuronal networks controlling vital rhythmic behaviours such as respiration. Thus, elimination of r3 and r5 in Krox20-/- mice suppresses a pontine antiapneic system (Jacquin et al., 1996). We now compare Krox20-/- to kreisler heterozygous (+/kr) and homozygous (kr/kr) mutant neonates. In +/kr mutant mice, we describe hyperactivity of the antiapneic system: analysis of rhythm generation in vitro revealed a pontine modification in keeping with abnormal cell specifications previously reported in r3 (Manzanares et al., 1999b). In kr/kr mice, elimination of r5 abolished all +/kr respiratory traits, suggesting that +/kr hyperactivity of the antiapneic system is mediated through r5-derived territories. Furthermore, collateral chemosensory pathways that normally mediate delayed responses to hypoxia and hyperoxia were not functional in kr/kr mice. We conclude that the pontine antiapneic system originates from r3r4, but not r5. A different rhythm-promoting system originates in r5 and kreisler controls the development of antiapneic and chemosensory signal transmission at this level.