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.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

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.     

Axial patterning in the developing vertebrate inner ear

Axial patterning in the vertebrate inner ear has been studied for over eighty years, and recent work has made great progress towards an understanding of the molecular mechanisms responsible for establishing asymmetries about the otic axes. Tissues extrinsic to the ear provide sources of signalling molecules that are active early in development, at or before otic placode stages, while intrinsic factors interpret these signals to establish and maintain axial pattern. Key features of dorsoventral otic patterning in amniote embryos involve Wnt and Fgf signalling from the hindbrain and Hh signalling from midline tissues (notochord and floorplate). Mutual antagonism between these pathways and their downstream targets within the otic epithelium help to refine and maintain dorsoventral axial patterning in the ear. In the zebrafish ear, the same tissues and signals are implicated, but appear to play a role in anteroposterior, rather than dorsoventral, otic patterning. Despite this paradox, conservation of mechanisms may be higher than is at first apparent.     

Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate

The ascidian neural plate has a grid-like organisation, with six rows and eight columns of aligned cells, generated by a series of stereotypical cell divisions. We have defined unique molecular signatures for each of the eight cells in the posterior-most two rows of the neural plate - rows I and II. Using a combination of morpholino gene knockdown, dominant-negative forms and pharmacological inhibitors, we tested the role of three signalling pathways in defining these distinct cell identities. Nodal signalling at the 64-cell stage was found to be required to define two different neural plate domains - medial and lateral - with Nodal inducing lateral and repressing medial identities. Delta2, an early Nodal target, was found to then subdivide each of the lateral and medial domains to generate four columns. Finally, a separate signalling system along the anteroposterior axis, involving restricted ERK1/2 activation, was found to promote row I fates and repress row II fates. Our results reveal how the sequential integration of three signalling pathways - Nodal, Delta2/Notch and FGF/MEK/ERK - defines eight different sub-domains that characterise the ascidian caudal neural plate. Most remarkably, the distinct fates of the eight neural precursors are each determined by a unique combination of inputs from these three signalling pathways.     

The role of prechordal mesendoderm in neural patterning

Prechordal mesendoderm is formed in response to Nodal and maternal beta-Catenin signaling and is regulated by signals from anterior endoderm and chordamesoderm. Prechordal mesendodermal cells are involved in neural induction and in anteroposterior and dorsoventral neural patterning. Inhibitors of Wnt and BMP growth factors secreted by prechordal mesendoderm mediate neural induction and anteroposterior and dorsoventral patterning, whereas SHH and TGF betas mediate dorsoventral patterning.     

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/beta-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Different activities of the frizzled-related proteins frzb2 and sizzled2 during Xenopus anteroposterior patterning

In a search for factors that regulate patterning of the Xenopus anteroposterior (A/P) axis, particularly the anterior ectoderm, we isolated two members of the Frizzled-related protein (FRP) gene family that are thought to encode antagonists of Wnt signaling. frzb2 is expressed in head mesoderm while sizzled2 is expressed in ventral ectoderm and mesoderm, tissues that modulate anterior fates. Consistent with a role for these genes in A/P patterning, ectopically expressed frzb2 inhibited head formation, while sizzled2 dorsalized embryos, causing expansion of the head. The different activities of frzb2 and sizzled2 may be explained by their interaction with distinct proteins since frzb2 is an inhibitor of Xwnt8 activity, while sizzled2 is unable to inhibit the activity of Xwnt8 or any other Xwnt tested. The data suggest that anteroposterior patterning is modulated by multiple components of the Wnt signaling pathway.     

FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development

The infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. Here we describe that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. We show that collar cells are composed of Fgf3(+) SOX3(+) proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10(+) anterior-dorsal infundibular cells. Together, our study shows that a subset of anterior floor plate-like cells gives rise to Fgf3(+) SOX3(+) progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth.     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.     

Mechanisms of Gurken-dependent pipe regulation and the robustness of dorsoventral patterning in Drosophila

The restriction of Pipe, a potential glycosaminoglycan-modifying enzyme, to ventral follicle cells of the egg chamber is essential for dorsoventral axis formation in the Drosophila embryo. pipe repression depends on the TGFalpha-like ligand Gurken, which activates the Drosophila EGF receptor in dorsal follicle cells. An analysis of Raf mutant clones shows that EGF signalling is required cell-autonomously in all dorsal follicle cells along the anteroposterior axis of the egg chamber to repress pipe. However, the autoactivation of EGF signalling important for dorsal follicle cell patterning has no influence on pipe expression. Clonal analysis shows that also the mirror-fringe cassette suggested to establish a secondary signalling centre in the follicular epithelium is not involved in pipe regulation. These findings support the view that the pipe domain is directly delimited by a long-range Gurken gradient. Pipe induces ventral cell fates in the embryo via activation of the Sp?tzle/Toll pathway. However, large dorsal patches of ectopic pipe expression induced by Raf clones rarely affect embryonic patterning if they are separated from the endogenous pipe domain. This indicates that potent inhibitory processes prevent pipe dependent Toll activation at the dorsal side of the egg.     

The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embryo

The anterior midline tissue (AML) of the late gastrula mouse embryo comprises the axial mesendoderm and the ventral neuroectoderm of the prospective forebrain, midbrain and rostral hindbrain. In this study, we have investigated the morphogenetic role of defined segments of the AML by testing their inductive and patterning activity and by assessing the impact of their ablation on the patterning of the neural tube at the early-somite-stage. Both rostral and caudal segments of the AML were found to induce neural gene activity in the host tissue; however, the de novo gene activity did not show any regional characteristic that might be correlated with the segmental origin of the AML. Removal of the rostral AML that contains the prechordal plate resulted in a truncation of the head accompanied by the loss of several forebrain markers. However, the remaining tissues reconstituted Gsc and Shh activity and expressed the ventral forebrain marker Nkx2.1. Furthermore, analysis of Gsc-deficient embryos reveals that the morphogenetic function of the rostral AML requires Gsc activity. Removal of the caudal AML led to a complete loss of midline molecular markers anterior to the 4th somite. In addition, Nkx2.1 expression was not detected in the ventral neural tube. The maintenance and function of the rostral AML therefore require inductive signals emanating from the caudal AML. Our results point to a role for AML in the refinement of the anteroposterior patterning and morphogenesis of the brain.