Gastruloids - a minimalistic model to study complex developmental metabolism.

Metabolic networks are well placed to orchestrate the coordination of multiple cellular processes associated with embryonic development such as cell growth, proliferation, differentiation and cell movement. Here, we discuss the advantages that gastruloids, aggregates of mammalian embryonic stem cells that self-assemble a rudimentary body plan, have for uncovering the instructive role of metabolic pathways play in directing developmental processes. We emphasise the importance of using such reductionist systems to link specific pathways to defined events of early mammalian development and their utility for obtaining enough material for metabolomic studies. Finally, we review the ways in which the basic gastruloid protocol can be adapted to obtain specific models of embryonic cell types, tissues and regions. Together, we propose that gastruloids are an ideal system to rapidly uncover new mechanistic links between developmental signalling pathways and metabolic networks, which can then inform precise in vivo studies to confirm their function in the embryo.

A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration.

The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-ß inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/- HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/- HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/- NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro.

On the nature and function of organizers.

Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.

Evo-engineering and the cellular and molecular origins of the vertebrate spinal cord.

The formation of the spinal cord during early embryonic development in vertebrate embryos is a continuous process that begins at gastrulation and continues through to the completion of somitogenesis. Despite the conserved usage of patterning mechanisms and gene regulatory networks that act to generate specific spinal cord progenitors, there now exists two seemingly disparate models to account for their action. In the first, a posteriorly localized signalling source transforms previously anterior-specified neural plate into the spinal cord. In the second, a population of bipotent stem cells undergo continuous self-renewal and differentiation to progressively lay down the spinal cord and axial mesoderm by posterior growth. Whether this represents fundamental differences between the experimental model organisms utilised in the generation of these models remains to be addressed. Here we review lineage studies across four key vertebrate models: mouse, chicken, Xenopus and zebrafish and relate them to the underlying gene regulatory networks that are known to be required for spinal cord formation. We propose that by applying a dynamical systems approach to understanding how distinct neural and mesodermal fates arise from a bipotent progenitor pool, it is possible to begin to understand how differences in the dynamical cell behaviours such as proliferation rates and cell movements can map onto conserved regulatory networks to generate diversity in the timing of tissue generation and patterning during development.

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Directional cell movements downstream of Gbx2 and Otx2 control the assembly of sensory placodes.

Cranial placodes contribute to sensory structures including the inner ear, the lens and olfactory epithelium and the neurons of the cranial sensory ganglia. At neurula stages, placode precursors are interspersed in the ectoderm surrounding the anterior neural plate before segregating into distinct placodes by as yet unknown mechanisms. Here, we perform live imaging to follow placode progenitors as they aggregate to form the lens and otic placodes. We find that while placode progenitors move with the same speed as their non-placodal neighbours, they exhibit increased persistence and directionality and these properties are required to assemble morphological placodes. Furthermore, we demonstrate that these factors are components of the transcriptional networks that coordinate placode cell behaviour including their directional movements. Together with previous work, our results support a dual role for Otx and Gbx transcription factors in both the early patterning of the neural plate border and the later segregation of its derivatives into distinct placodes.

Species-specific contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.

Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.

Neural crest and placode interaction during the development of the cranial sensory system.

In the vertebrate head, the peripheral components of the sensory nervous system are derived from two embryonic cell populations, the neural crest and cranial sensory placodes. Both arise in close proximity to each other at the border of the neural plate: neural crest precursors abut the future central nervous system, while placodes originate in a common preplacodal region slightly more lateral. During head morphogenesis, complex events organise these precursors into functional sensory structures, raising the question of how their development is coordinated. Here we review the evidence that neural crest and placode cells remain in close proximity throughout their development and interact repeatedly in a reciprocal manner. We also review recent controversies about the relative contribution of the neural crest and placodes to the otic and olfactory systems. We propose that a sequence of mutual interactions between the neural crest and placodes drives the coordinated morphogenesis that generates functional sensory systems within the head.

Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning.

In the vertebrate head, central and peripheral components of the sensory nervous system have different embryonic origins, the neural plate and sensory placodes. This raises the question of how they develop in register to form functional sense organs and sensory circuits. Here we show that mutual repression between the homeobox transcription factors Gbx2 and Otx2 patterns the placode territory by influencing regional identity and by segregating inner ear and trigeminal progenitors. Activation of Otx2 targets is necessary for anterior olfactory, lens and trigeminal character, while Gbx2 function is required for the formation of the posterior otic placode. Thus, like in the neural plate antagonistic interaction between Otx2 and Gbx2 establishes positional information thus providing a general mechanism for rostro-caudal patterning of the ectoderm. Our findings support the idea that the Otx/Gbx boundary has an ancient evolutionary origin to which different modules were recruited to specify cells of different fates.

Early neural crest induction requires an initial inhibition of Wnt signals.

Neural crest (NC) induction is a long process that continues through gastrula and neurula stages. In order to reveal additional stages of NC induction we performed a series of explants where different known inducing tissues were taken along with the prospective NC. Interestingly the dorso-lateral marginal zone (DLMZ) is only able to promote the expression of a subset of neural plate border (NPB) makers without the presence of specific NC markers. We then analysed the temporal requirement for BMP and Wnt signals for the NPB genes Hairy2a and Dlx5, compared to the expression of neural plate (NP) and NC genes. Although the NP is sensitive to BMP levels at early gastrula stages, Hairy2a/Dlx5 expression is unaffected. Later, the NP becomes insensitive to BMP levels at late gastrulation when NC markers require an inhibition. The NP requires an inhibition of Wnt signals prior to gastrulation, but becomes insensitive during early gastrula stages when Hairy2a/Dlx5 requires an inhibition of Wnt signalling. An increase in Wnt signalling is then important for the switch from NPB to NC at late gastrula stages. In addition to revealing an additional distinct signalling event in NC induction, this work emphasizes the importance of integrating both timing and levels of signalling activity during the patterning of complex tissues such as the vertebrate ectoderm.

Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction.

The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm we show for the first time that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula stage dependent on signals arising from the dorsolateral mesoderm; and second, a maintenance step at the neurula stage dependent on signals from tissues adjacent to the neural crest. By performing tissue recombination experiments and using specific inhibitors of different inductive signals, we show that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula to BMP activation at neurula stages is further supported by the dynamic expression of BMP4 and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The differential requirements of BMP activity allow us to propose an explanation for apparently discrepant results between chick and frog experiments. The demonstration that Wnt signals are required for neural crest induction by mesoderm solves an additional long-standing controversy. Finally, our results emphasise the importance of considering the order of exposure to signals during an inductive event.

Genetic network during neural crest induction: from cell specification to cell survival.

The concerted action of extracellular signals such as BMP, Wnt, FGF, RA and Notch activate a genetic program required to transform a naïve ectodermal cell into a neural crest cell. In this review we will analyze the extracellular signals and the network of transcription factors that are required for this transformation. We will propose the division of this complex network of factors in two main steps: an initial cell specification step followed by a maintenance or cell survival step.