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.

Retinoic acid influences the timing and scaling of avian wing development.

A fundamental question in biology is how embryonic development is timed between different species. To address this problem, we compared wing development in the quail and the larger chick. We reveal that pattern formation is faster in the quail as determined by the earlier activation of 5'Hox genes, termination of developmental organizers (Shh and Fgf8), and the laying down of the skeleton (Sox9). Using interspecies tissue grafts, we show that developmental timing can be reset during a critical window of retinoic acid signaling. Accordingly, extending the duration of retinoic acid signaling switches developmental timing between the quail and the chick and the chick and the larger turkey. However, the incremental growth rate is comparable between all three species, suggesting that the pace of development primarily governs differences in the expansion of the skeletal pattern. The widespread distribution of retinoic acid could coordinate developmental timing throughout the embryo.

Cell geometry, signal dampening, and a bimodal transcriptional response underlie the spatial precision of an ERK-mediated embryonic induction.

Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.

[The saga of neural induction: almost a century of research]. / La saga de l'induction neurale : presque un siècle de recherche.

Neural induction is a developmental process that allows cells from the ectoderm (the target tissue) to acquire a neural fate in response to signals coming from a specific adjacent embryonic region, the dorsal mesoderm (the inducing tissue). This process described in 1924 in amphibian embryos has not received a molecular explanation until the mid-1990s. Most of the work on neural induction has been carried out in amphibians. At these times, although the role played by the membrane of the target tissue had been suggested, no definitive work had been performed on the transduction of the neuralizing signal. Between 1990 and 2019 our aim was to decipher this transduction. We have underlined the necessary and sufficient role played by calcium signaling to induce ectoderm cells towards a neural fate from the activation of calcium channels to the direct transcription of early neural genes by calcium.

TITLE: La saga de l'induction neurale : presque un siècle de recherche. ABSTRACT: La formation du système nerveux débute par l'induction neurale, un processus qui permet aux cellules de l'ectoderme (tissu cible) d'acquérir un destin neural en réponse à des signaux provenant du mésoderme dorsal (tissu inducteur). Ce processus, décrit en 1924 sur l'amphibien, n'a reçu une explication moléculaire qu'au milieu des années 1990. Pendant cette période, plusieurs auteurs se sont intéressés au rôle joué par la membrane du tissu cible mais peu de travaux décisifs ont décrit la transduction du signal neuralisant. Entre 1990 et 2019, nous avons disséqué la transduction du signal neuralisant, un sujet très peu abordé alors. Nous avons souligné le rôle nécessaire et suffisant du calcium pour orienter les cellules de l'ectoderme vers un destin neural et établi la cascade moléculaire allant de l'activation de canaux membranaires à la transcription de gènes.

E. E. Just's broad, yet hidden, influence on modern cell and developmental biology.

This year, 2019, marks the centennial of embryologist E. E. Just's discovery of what is known as the fast block to polyspermy. Just's observation of the subtle changes that occur at the egg's surface during fertilization (and in experimental parthenogenesis) led him to postulate that the egg, and indeed every cell, possesses a property he called independent irritability, which represents the cell's ability to respond in a physiologically-relevant way to a variety of signals or triggers. In this paper, I argue that Just's concept of independent irritability informed his contemporary Johannes Holtfreter as Holtfreter attempted to explain the phenomena of embryonic induction and competence and that Holtfreter, in turn, influenced Marc Kirschner and John Gerhart in their formulation of the theory of facilitated variation. Just's influence is especially evident in Gerhart and Kirschner's presentations of what they call weak linkage-a property of living systems that allows core processes and components to be mixed and matched in different ways to generate novel traits. Unfortunately, the connection between Holtfreter's work and Just's has remained hidden. This paper gives examples of phenomena that exhibit weak linkage, and it lays out the case that Just's concept of independent irritability, through Holtfreter, Gerhart, and Kirschner, has broadly infiltrated modern cell and developmental biology.

Three-dimensional induction of dorsal, intermediate and ventral spinal cord tissues from human pluripotent stem cells.

The spinal cord contains more than 20 distinct subclasses of neurons that form well-organized neural circuits capable of sensing the environment and generating motor behavior. Although recent studies have described the efficient in vitro generation of spinal motor neurons, the induction of the spinal cord as a whole tissue has not been achieved. In the present study, we demonstrate three-dimensional (3D) induction of dorsal spinal cord-like tissues from human pluripotent stem cells. Our 3D spinal cord induction (3-DiSC) condition recapitulates patterning of the developing dorsal spinal cord and enables the generation of four types of dorsal interneuron marker-positive cell populations. By activating Shh signaling, intermediate and ventral spinal cord-like tissues are successfully induced. After dissociation of these tissues, somatosensory neurons and spinal motor neurons are detected and express neurotransmitters in an in vivo manner. Our approach provides a useful experimental tool for the analysis of human spinal cord development and will contribute to research on the formation and organization of the spinal cord, and its application to regenerative medicine.

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.

Reciprocal signaling by Wnt and Notch specifies a muscle precursor in the C. elegans embryo.

The MS blastomere produces one-third of the body wall muscles (BWMs) in the C. elegans embryo. MS-derived BWMs require two distinct cell-cell interactions, the first inhibitory and the second, two cell cycles later, required to overcome this inhibition. The inductive interaction is not required if the inhibitory signal is absent. Although the Notch receptor GLP-1 was implicated in both interactions, the molecular nature of the two signals was unknown. We now show that zygotically expressed MOM-2 (Wnt) is responsible for both interactions. Both the inhibitory and the activating interactions require precise spatiotemporal expression of zygotic MOM-2, which is dependent upon two distinct Notch signals. In a Notch mutant defective only in the inductive interaction, MS-derived BWMs can be restored by preventing zygotic MOM-2 expression, which removes the inhibitory signal. Our results suggest that the inhibitory interaction ensures the differential lineage specification of MS and its sister blastomere, whereas the inductive interaction promotes the expression of muscle-specifying genes by modulating TCF and ß-catenin levels. These results highlight the complexity of cell fate specification by cell-cell interactions in a rapidly dividing embryo.

Patterning of brain precursors in ascidian embryos.

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. Here, we address the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Our functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp Delta-like/Notch signalling induces intermediate (Gsx) over medial (Meis) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. We conclude that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar.

Hedgehog-BMP signalling establishes dorsoventral patterning in lateral plate mesoderm to trigger gonadogenesis in chicken embryos.

The gonad appears in the early embryo after several events: cells at the lateral plate mesoderm (LPM) undergo ingression, begin gonadal differentiation and then retain primordial germ cells (PGCs). Here we show that in the chicken embryo, these events are triggered on the basis of dorsoventral patterning at the medial LPM. Gonadal progenitor cells (GPCs) at the ventromedial LPM initiate gonadogenesis by undergoing ingression, whereas mesonephric capsule progenitor cells (MCPCs) at the dorsomedial LPM do not. These contrasting behaviours are caused by Hedgehog signalling, which is activated in GPCs but not in MCPCs. Inhibiting Hedgehog signalling prevents GPCs from forming gonadal structures and collecting PGCs. When activated by Hedgehog signalling, MCPCs form an ectopic gonad. This Hedgehog signalling is mediated by BMP4. These findings provide insight into embryonic patterning and gonadal initiation in the chicken embryo.

Scaling Pattern to Variations in Size during Development of the Vertebrate Neural Tube.

Anatomical proportions are robustly maintained in individuals that vary enormously in size, both within a species and between members of related taxa. However, the mechanisms underlying scaling are still poorly understood. We have examined this phenomenon in the context of the patterning of the ventral neural tube in response to a gradient of the morphogen Sonic hedgehog (SHH) in the chick and zebra finch, two species that differ in size during the time of neural tube patterning. We find that scaling is achieved, at least in part, by altering the sensitivity of the target cells to SHH and appears to be achieved by modulating the ratio of the repressive and activating transcriptional regulators, GLI2 and GLI3. This mechanism contrasts with previous experimental and theoretical analyses of morphogenic scaling that have focused on compensatory changes in the morphogen gradient itself.

Physiologic Course of Female Reproductive Function: A Molecular Look into the Prologue of Life.

The genetic, endocrine, and metabolic mechanisms underlying female reproduction are numerous and sophisticated, displaying complex functional evolution throughout a woman's lifetime. This vital course may be systematized in three subsequent stages: prenatal development of ovaries and germ cells up until in utero arrest of follicular growth and the ensuing interim suspension of gonadal function; onset of reproductive maturity through puberty, with reinitiation of both gonadal and adrenal activity; and adult functionality of the ovarian cycle which permits ovulation, a key event in female fertility, and dictates concurrent modifications in the endometrium and other ovarian hormone-sensitive tissues. Indeed, the ultimate goal of this physiologic progression is to achieve ovulation and offer an adequate environment for the installation of gestation, the consummation of female fertility. Strict regulation of these processes is important, as disruptions at any point in this evolution may equate a myriad of endocrine-metabolic disturbances for women and adverse consequences on offspring both during pregnancy and postpartum. This review offers a summary of pivotal aspects concerning the physiologic course of female reproductive function.

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells.

The human brain is arguably the most complex structure among living organisms. However, the specific mechanisms leading to this complexity remain incompletely understood, primarily because of the poor experimental accessibility of the human embryonic brain. Over recent years, technologies based on pluripotent stem cells (PSCs) have been developed to generate neural cells of various types. While the translational potential of PSC technologies for disease modeling and/or cell replacement therapies is usually put forward as a rationale for their utility, they are also opening novel windows for direct observation and experimentation of the basic mechanisms of human brain development. PSC-based studies have revealed that a number of cardinal features of neural ontogenesis are remarkably conserved in human models, which can be studied in a reductionist fashion. They have also revealed species-specific features, which constitute attractive lines of investigation to elucidate the mechanisms underlying the development of the human brain, and its link with evolution.

Resolving time and space constraints during neural crest formation and delamination.

A striking feature of neural crest development in vertebrates is that all the specification, delamination, migration, and differentiation steps occur consecutively in distinct areas of the embryo and at different timings of development. The significance and consequences of this partition into clearly separated events are not fully understood yet, but it ought to be related to the necessity of controlling precisely and independently each step, given the wide array of cell types and tissues derived from the neural crest and the long duration of their development spanning almost the entire embryonic life. In this chapter, using the examples of early neural crest induction and delamination, we discuss how time and space constraints influence their development and describe the molecular and cellular responses that are employed by cells to adapt. In the first example, we analyze how cell sorting and cell movements cooperate to allow nascent neural crest cells, which are initially mingled with other neurectodermal progenitors after induction, to segregate from the neural tube and ectoderm populations and settle at the apex of the neural tube prior to migration. In the second example, we examine how cadherins drive the entire process of neural crest segregation from the rest of the neurectoderm by their dual role in mediating first cell sorting and cohesion during specification and later in promoting their delamination. In the third example, we describe how the expression and activity of the transcription factors known to drive epithelium-to-mesenchyme transition (EMT) are regulated timely and spatially by the cellular machinery so that they can alternatively and successively regulate neural crest specification and delamination. In the last example, we briefly tackle the problem of how factors triggering EMT may elicit different cell responses in neural tube and neural crest progenitors.

The use of human pluripotent stem cells for the in vitro derivation of cranial placodes and neural crest cells.

Due to their intrinsic differentiation potential, human pluripotent stem cells (hPSCs) hold remarkable promise for their use in cell-based therapies as well as an in vitro model for early human embryogenesis and for modeling disease. During the development of the human embryo, transient structures such as the neural crest (NC) and the cranial placodes (CPs) are specified in the first 3-4 weeks of gestation. Because of this early occurrence and a scarce availability of embryos for research purposes, these transient structures remain largely unexplored in humans. Hence, investigators are now exploiting in vitro differentiation of hPSC to unveil these early events and to generate NC and CP cells in vitro. Derivatives of the NC and CPs will contribute to the formation of very important organs, including most of the peripheral nervous system (NC) and the sensory organs of the head (CP). There are many diseases and conditions that affect NC and CP derivatives, thus a better knowledge of how these structures specialize, and the derivation of functional NC and CP cells for therapeutic applications will have an impact on the understanding and treatment of these disorders. Here, we discuss the current state of the art in directing hPSCs into NC or CP cells, which in spite of their importance is still in its infancy.

Wnt/ß-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells.

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/ß-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation.

Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.

Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.

The dynein-triggered ciliary motion in embryonic nodes: an exploratory study based on computational models.

The cilia, presenting a rotational movement in the embryonic nodes, play a crucial role in the left-right specification during embryogenesis. The characteristic architecture of these cilia is based on a cylindrical arrangement of 9 doublet microtubules and the motion of the cilia is triggered by the dynein motors located between adjacent doublets by converting the chemical energy into mechanical work. Restricted by the inherent difficulties of experiments, the dynein activation patterns in moving cilia cannot be directly observed. Thus, the mechanism of nodal ciliary movement is still unclear. In this study, we present computational models of the nodal ciliary ultrastructure based on tomographic images of the ciliary body. By employing time accurate three-dimensional solid mechanics analysis, we investigate the dynein-triggered sliding between adjacent doublet microtubules and simulate the induced ciliary bending. As an exploratory study, two dynein activation patterns are proposed and their rationality is discussed. The mathematical model presented by this paper provides a platform to investigate various assumptions of dynein activity, facilitating us to propose the most possible dynein activation pattern and therefore improving our understandings regarding the protein-beating problems of cilia.

Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1.

OBJECTIVE: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. METHODOLOGY: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. RESULTS: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver-specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. CONCLUSION: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds.

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ-line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.