A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos

The notochord is one of the defining features of chordates. The ascidian notochord is a rod like structure consisting of a single row of 40 cells. The anterior 32 ;primary' notochord cells arise from the A-line (anterior vegetal) blastomeres of the eight-cell stage embryo, whereas the posterior 8 ;secondary' notochord cells arise from the B-line (posterior vegetal) blastomeres of the eight-cell stage embryo. Specification of notochord precursors within these two lineages occurs in a spatially and temporally distinct manner. We show that specification of the secondary but not the primary notochord in Ciona intestinalis requires a relay mechanism involving two signalling pathways. First, we show evidence that acquisition of secondary notochord fate is dependent upon lateral Nodal signalling sources, situated in the adjacent b-line animal cells. Expression of the notochord specific gene Ci-Brachyury in the secondary notochord precursor was downregulated following selective inhibition of Nodal signal reception in B-line derivatives and also, strikingly, following selective inhibition of Nodal signal reception in A-line cell derivatives. Within the A-line, Nodal signals are required for localised expression of Delta2, which encodes a divergent form of Delta ligand. Using four distinct reagents to inhibit Delta2/Notch signals, we showed that Delta2 signalling from A-line cells, which activates the Notch/Su(H) pathway in adjacent B-line cells, is required for specification of the secondary notochord precursor. We propose a model whereby laterally produced Nodal acts to specify the secondary notochord precursor both directly in the B-line cells and via Delta2 induction in adjacent A-line cells.     

Multiple roles of Activin/Nodal,bone morphogenetic protein,fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.     

Inhibition of Wnt/Axin/beta-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity

Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/beta-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.     

Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system

The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch–mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.     

Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.     

FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos

Fibroblast growth factor (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/16/20 has been implicated in both of these processes. However, in FGF9/16/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/16/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/16/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.     

Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. II. Neural plate morphogenesis and cell lineages during neurulation

We describe the lineage and morphogenesis of neural plate cells in the ascidian, Ciona intestinalis, from reconstructed cell maps of embryos at 12-min intervals during and after neurulation, between 31 and 61% of embryonic development. Neurulation commences in a posterior to anterior wave following in the wake of the ninth cleavage, when all cells, except possibly four, are in their 10th generation. The neural plate then comprises 76 cells, in up to four posterior rows each of eight vegetal-hemisphere cells, and eight anterior rows each of six animal-hemisphere cells. Two cells are lost from the neural plate to the muscle cell line during neurulation and four cells are gained from ectoderm outside the plate. All cells become wedge-shaped. Simple, stereotyped positional changes transform cells from lateral locations in the plate to posterior locations in the tube; bilateral partners shear their midline positions to form the keel, and ectodermal cells zipper up dorsally to form the capstone, of a tube which is four cells in cross section posteriorly, but more complex anteriorly. Neither cell death nor migration occur during neurulation. Divisions become asynchronous and the cell-cycle extends; 170 10th- to 12th-generation cells exist by the time the neural tube becomes completely internalized. Generally, only one further division is required to complete the lineage analysis, two at the most. Neural plate cell divisions were invariant using our observational methods, and their lineage is compared with that from recent studies of H. Nishida (1987, Dev. Biol. 121, 526-541).     

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate,the ascidian Ciona intestinalis

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.     

Distinct and cooperative roles for Nodal and Hedgehog signals during hypothalamic development

Despite its evolutionary conservation and functional importance, little is known of the signaling pathways that underlie development of the hypothalamus. Although mutations affecting Nodal and Hedgehog signaling disrupt hypothalamic development, the time and site of action and the exact roles of these pathways remain very poorly understood. Unexpectedly, we show here that cell-autonomous reception of Nodal signals is neither required for the migration of hypothalamic precursors within the neural plate, nor for further development of the anterior-dorsal hypothalamus. Nodal signaling is, however, cell-autonomously required for establishment of the posterior-ventral hypothalamus. Conversely, Hedgehog signaling antagonizes the development of posterior-ventral hypothalamus, while promoting anterior-dorsal hypothalamic fates. Besides their distinct roles in the regionalization of the diencephalon, we reveal cooperation between Nodal and Hedgehog pathways in the maintenance of the anterior-dorsal hypothalamus. Finally we show that it is the prechordal plate and not the head endoderm that provides the early signals essential for establishment of the hypothalamus.     

Divergent mechanisms specify chordate motoneurons: evidence from ascidians

Ascidians are members of the vertebrate sister group Urochordata. Their larvae exhibit a chordate body plan, which forms by a highly accelerated embryonic strategy involving a fixed cell lineage and small cell numbers. We report a detailed analysis of the specification of three of the five pairs of motoneurons in the ascidian Ciona intestinalis and show that despite well-conserved gene expression patterns and embryological outcomes compared with vertebrates, key signalling molecules have adopted different roles. We employed a combination of cell ablation and gene manipulation to analyse the function of two signalling molecules with key roles in vertebrate motoneuron specification that are known to be expressed equivalently in ascidians: the inducer Sonic hedgehog, produced ventrally by the notochord and floorplate; and the inhibitory BMP2/4, produced on the lateral/dorsal side of the neural plate. Our surprising conclusion is that neither BMP2/4 signalling nor the ventral cell lineages expressing hedgehog play crucial roles in motoneuron formation in Ciona. Furthermore, BMP2/4 overexpression induced ectopic motoneurons, the opposite of its vertebrate role. We suggest that the specification of motoneurons has been modified during ascidian evolution, such that BMP2/4 has adopted a redundant inductive role rather than a repressive role and Nodal, expressed upstream of BMP2/4 in the dorsal neural tube precursors, acts as a motoneuron inducer during normal development. Thus, our results uncover significant differences in the mechanisms used for motoneuron specification within chordates and also highlight the dangers of interpreting equivalent expression patterns as indicative of conserved function in evo-devo studies.     

Recruitment of cell groups through Delta/Notch signalling during spider neurogenesis

Early neurogenesis in the spider is characterised by a stereotyped pattern of sequential recruitment of neural cells from the neuroectoderm, comparable with neuroblast formation in Drosophila: However, in contrast to Drosophila, where single cells delaminate from the neuroectoderm, groups of cells adopt the neural fate and invaginate into the spider embryo. This raises the question of whether Delta/Notch signalling is involved in this process, as this system normally leads to a singling out of individual cells through lateral inhibition. I have therefore cloned homologues of Delta and Notch from the spider Cupiennius salei and studied their expression and function. The genes are indeed expressed during the formation of neural cells in the ventral neuroectoderm. Loss of function of either gene leads to an upregulation of the proneural genes and an altered morphology of the neuroectoderm that is comparable with Delta and Notch mutant phenotypes in Drosophila: Thus, although Delta/Notch signalling appears to be used in the same way as in Drosophila, the lateral inhibition process produces clusters of invaginating cells, rather than single cells. Intriguingly, neuroectodermal cells that are not invaginating seem to become neural cells at a later stage, while the epidermal cells are derived from lateral regions that overgrow the neuroectoderm. In this respect, the neuroectodermal region of the spider is more similar to the neural plate of vertebrates, than to the neuroectoderm of Drosophila:     

Global and local mechanisms of forebrain and midbrain patterning

During the past years, major advances have been made in understanding the sequential events involved in neural plate patterning. Positional information is already conferred to cells of the neural plate at the time of its induction in the ectoderm. The interplay between the BMP- and the Fgf- signaling pathways leads to the induction of neural cell fates. Thus, neural induction and neural plate patterning are overlapping processes. Later, at the end of gastrulation, positional cell identities within the neural plate are refined and maintained by the action of several neural plate organizers. By locally emitting signaling molecules, they influence the fate of the developing nervous system with high regional specificity. Recent advances have been made both in understanding the mechanisms that dictate the relative position of these organizers and in how signaling molecules spread from them with high spatial and temporal resolution.     

Zebrafish wnt4b expression in the floor plate is altered in sonic hedgehog and gli-2 mutants

The floor plate of the neural tube serves an important function as a source of signals that pattern cell fates in the nervous system as well as directing proper axon pathfinding. We have cloned a novel zebrafish wnt family member, wnt4b, which is expressed exclusively in the floor plate. To place wnt4b in the context of known regulators of midline development, its expression was analyzed in the zebrafish mutants cyclops (cyc), floating head (flh), you-too (yot), and sonic you (syu). wnt4b expression in the medial and lateral floor plate are shown to be regulated independently: medial floor plate expression occurs in the absence of a notochord, while lateral floor plate expression requires a functional notochord, sonic hedgehog and gli-2.     

Bmp activity establishes a gradient of positional information throughout the entire neural plate.

Bone morphogenetic proteins (Bmps) are key regulators of dorsoventral (DV) patterning. Within the ectoderm, Bmp activity has been shown to inhibit neural development, promote epidermal differentiation and influence the specification of dorsal neurons and neural crest. In this study, we examine the patterning of neural tissue in mutant zebrafish embryos with compromised Bmp signalling activity. We find that although Bmp activity does not influence anteroposterior (AP) patterning, it does affect DV patterning at all AP levels of the neural plate. Thus, we show that Bmp activity is required for specification of cell fates around the margin of the entire neural plate, including forebrain regions that do not form neural crest. Surprisingly, we find that Bmp activity is also required for patterning neurons at all DV levels of the CNS. In swirl/bmp2b(-) (swr(-)) embryos, laterally positioned sensory neurons are absent whereas more medial interneuron populations are hugely expanded. However, in somitabun(-) (sbn(-)) embryos, which probably retain higher residual Bmp activity, it is the sensory neurons and not the interneurons that are expanded. Conversely, in severely Bmp depleted embryos, both interneurons and sensory neurons are absent and it is the most medial neurons that are expanded. These results are consistent with there being a gradient of Bmp-dependent positional information extending throughout the entire neural and non-neural ectoderm.