Induction and axial patterning of the neural plate: Planar and vertical signals

In this review I summarize recent findings on the contributions of different cell groups to the formation of the basic plan of the nervous system of vertebrate embryos. Midline cells of the mesoderm—the organizer, notochord, and prechordal plate—and midline cells of the neural ectoderm—the notoplate and floor plate—appear to have a fundamental role in the induction and patterning of the neural plate. Vertical signals acting across tissue layers and planar signals acting through the neural epithelium have distinct roles and cooperate in induction and pattern formation. Whereas the prechordal plate and notochord have distinct vertical signaling properties, the initial anteroposterior (A-P) pattern of the neural plate may be induced by planar signals originating from the organizer region. Planar signals from the notoplate may also contribute to the mediolateral (M-L) patterning of the neural plate. These and other findings suggest a general view of neural induction and axial patterning. © 1993 John Wiley & Sons, Inc.     

Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium.     

Discontinuous organization and specification of the lateral floor plate in zebrafish

The floor plate is a signaling center in the ventral neural tube of vertebrates with important functions during neural patterning and axon guidance. It is composed of a centrally located medial floor plate (MFP) and a bilaterally positioned lateral floor plate (LFP). While the role of the MFP as source of signaling molecules like, e.g., Sonic Hedgehog (Shh) is well understood, the exact organization and function of the LFP are currently unclear. Based on expression analyses, the one cell wide LFP in zebrafish has been postulated to be a homogenous structure. We instead show that the zebrafish trunk LFP is discontinuously arranged. Single LFP cells alternate with p3 neuronal precursor cells, which develop V3 interneurons along the anteroposterior (AP) axis. Our mutant analyses indicate that both, formation of LFP and p3 cells require Delta-Notch signaling. Importantly, however, the two cell types are differentially regulated by Hedgehog (HH) and Nkx2.2 activities. This implicates a novel mechanism of neural tube patterning, in which distinct cell populations within one domain of the ventral neural tube are differently specified along the AP axis. We conclude that different levels of HH and Nkx2.2 activities are responsible for the alternating appearance of LFP and p3 neuronal progenitor cells in the zebrafish ventral neural tube.     

Cell fate determination in embryonic ectoderm

During gastrulation in vertebrates the cells of the embryonic ectoderm give rise to epidermal progenitors in the ventral side and neural progenitors in the dorsal side. Despite many years of scrutiny, the molecular basis of these important embryonic cell fate decisions have not been solved. Only recently have we witnessed swift progress in the quest for factors involved in neural and epidermal induction. Several of what seem to be bona fide in vivo neural and epidermal inducers have been cloned, and the mechanism of their functions in embryos is also beginning to be understood. These new molecular results have revolutionized our view on the patterning of embryonic ectoderm and suggest that while the induction of epidermis requires instructive inductive signals, the establishment of neural fate occurs by default when epidermal inducers are inhibited. In this review, we discuss recent advances of our knowledge on epidermal and neural induction in the context of the “default model”. We will then address the process of neurogenesis as well as recent findings on neural patterning. Emphasis is placed on, but not limited to, discoveries made in Xenopus, as most of our progress in understanding the ectodermal patterning is obtained from studies using this organism. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 128–151, 1998     

Induction and differentiation of the neural crest

The neural crest is a population of cells that forms at the junction between the epidermis and neural plate in vertebrate embryos. Recent progress has elucidated the identity and timing of molecular events responsible for the earliest steps in neural crest development, particularly those involving the induction and its migration. Concomitantly, advances have been made in the identification, purification and generation of neural crest stem cells at various developmental stages that deepens our understanding of the plasticity and restriction of neural crest differentiation.     

The mechanisms of dorsoventral patterning in the vertebrate neural tube

We describe the essential features of and the molecules involved in dorsoventral (DV) patterning in the neural tube. The neural tube is, from its very outset, patterned in this axis as there is a roof plate, floor plate, and differing numbers and types of neuroblasts. These neuroblasts develop into different types of neurons which express a different range of marker genes. Early embryological experiments identified the notochord and the somites as being responsible for the DV patterning of the neural tube and we now know that 4 signaling molecules are involved and are generated by these surrounding structures. Fibroblast growth factors (FGFs) are produced by the caudal mesoderm and must be down-regulated before neural differentiation can occur. Retinoic acid (RA) is produced by the paraxial mesoderm and is an inducer of neural differentiation and patterning and is responsible for down-regulating FGF. Sonic hedgehog (Shh) is produced by the notochord and floor plate and is responsible for inducing ventral neural cell types in a concentration-dependent manner. Bone morphogenetic proteins (BMPs) are produced by the roof plate and are responsible for inducing dorsal neural cell types in a concentration-dependent manner. Subsequently, RA is used twice more. Once from the somites for motor neuron differentiation and secondly RA is used to define the motor neuron subtypes, but in the latter case it is generated within the neural tube from differentiating motor neurons rather than from outside. These 4 signaling molecules also interact with each other, generally in a repressive fashion, and DV patterning shows how complex these interactions can be.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.     

Molecular mechanisms of neural crest induction

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.     

Roles of Wnt proteins in neural development and maintenance

Many constituents of Wnt signaling pathways are expressed in the developing and mature nervous systems. Recent work has shown that Wnt signaling controls initial formation of the neural plate and many subsequent patterning decisions in the embryonic nervous system, including formation of the neural crest. Wnt signaling continues to be important at later stages of development. Wnts have been shown to regulate the anatomy of the neuronal cytoskeleton and the differentiation of synapses in the cerebellum. Wnt signaling has been demonstrated to regulate apoptosis and may participate in degenerative processes leading to cell death in the aging brain.     

Sonic hedgehog in the nervous system: functions, modifications and mechanisms

Signaling by Sonic hedgehog (Shh) controls important developmental processes, including dorsoventral neural tube patterning, neural stem cell proliferation, and neuronal and glial cell survival. Shh signaling involves lipid modifications to Shh itself, as well as changes in protein subcellular localization. Recent advances have revealed the importance of palmitoylation and acylation of Shh on its potency and migration capacity. Subsequent trafficking and organelle sorting in the Shh signaling pathway have been observed; these observations offer a new dimension to our understanding of downstream signal transduction events.     

Fibroblast growth factors as regulators of central nervous system development and function

Fibroblast growth factors (FGFs) are multifunctional signaling proteins that regulate developmental processes and adult physiology. Over the last few years, important progress has been made in understanding the function of FGFs in the embryonic and adult central nervous system. In this review, I will first discuss studies showing that FGF signaling is already required during formation of the neural plate. Next, I will describe how FGF signaling centers control growth and patterning of specific brain structures. Finally, I will focus on the function of FGF signaling in the adult brain and in regulating maintenance and repair of damaged neural tissues.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Neural induction and patterning in the mouse in the absence of the node and its derivatives

The signals which induce vertebrate neural tissue and pattern it along the anterior-posterior (A-P) axis have been proposed to emanate from Spemann's organizer, which in mammals is a structure termed the node. However, mouse embryos mutant for HNF3 beta lack a morphological node and node derivatives yet undergo neural induction. Gene expression domains occur at their normal A-P axial positions along the mutant neural tubes in an apparently normal temporal manner, including the most anterior and posterior markers. This neural patterning occurs in the absence of expression of known organizer genes, including the neural inducers chordin and noggin. Other potential signaling centers in gastrulating mutant embryos appear to express their normal constellation of putative secreted factors, consistent with the possibility that neural-inducing and -patterning signals emanate from elsewhere or at an earlier time. Nevertheless, we find that the node and the anterior primitive streak, from which the node derives, are direct sources of neural-inducing signals, as judged by expression of the early midbrain marker Engrailed, in explant-recombination experiments. Similar experiments showed the neural-inducing activity in HNF3 beta mutants to be diffusely distributed. Our results indicate that the mammalian organizer is capable of neural induction and patterning of the neural plate, but that maintenance of an organizer-like signaling center is not necessary for either process.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.     

The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures

During vertebrate development, an organizing signaling center, the isthmic organizer, forms at the boundary between the midbrain and hindbrain. This organizer locally controls growth and patterning along the anteroposterior axis of the neural tube. On the basis of transplantation and ablation experiments in avian embryos, we show here that, in the caudal midbrain, a restricted dorsal domain of the isthmic organizer, that we call the isthmic node, is both necessary and sufficient for the formation and positioning of the roof plate, a signaling structure that marks the dorsal midline of the neural tube and that is involved in its dorsoventral patterning. This is unexpected because in other regions of the neural tube, the roof plate has been shown to form at the site of neural fold fusion, which is under the influence of epidermal ectoderm derived signals. In addition, the isthmic node contributes cells to both the midbrain and hindbrain roof plates, which are separated by a boundary that limits cell movements. We also provide evidence that mid/hindbrain roof plate formation involves homeogenetic mechanisms. Our observations indicate that the isthmic organizer orchestrates patterning along the anteroposterior and the dorsoventral axis.