Local positional cues in the neuroepithelium guide retinal axons in embryonic Xenopus brain

Growing retinal axons home to their distant target, the tectum, even when they are displaced from their normal pathway. This argues for long-range guidance mechanisms in the embryonic brain. Growth cones may orientate to diffusible attractants released from the target, as proposed in other systems, or they may use a stable distribution of positional information in the neuroepithelium. To distinguish between these possibilities, small pieces of the presumptive optic tract, through which retinal axons will normally grow, were rotated by approximately 90 degrees either clockwise or counterclockwise. When the retinal axons later encountered the rotated neuroepithelium, they also turned clockwise or counterclockwise, in correspondence with the direction of rotation. This demonstrates that long-range navigation of retinal axons in the vertebrate brain is based partly on stable, local positional factors, rather than on remote diffusible factors.     

Retinotopic order in the absence of axon competition

The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.     

Homing behaviour of axons in the embryonic vertebrate brain

In embryonic nervous systems, growing axons must often travel long distances through diverse extracellular terrains to reach their postsynaptic partners. In most embryos, axons grow to their appropriate targets along particular tracts or nerves, as though they were following guidance cues confined to specific pathways. For example, in all vertebrates, axons from the retina invariably grow to the tectum along the well-defined optic tract. Yet, transplant experiments demonstrate that retinal axons make tectal projections even though they enter the brain at locations which are distinctly off the optic tract. Only recently has it become possible to label discreet growing projections in the embryonic vertebrate brain. Thus, it is not yet known whether displaced retinal axons grow directly towards the tectum or find it accidently, through random extension. To resolve this question, pioneering axons from normal and transplanted eyes in embryonic Xenopus were labelled using a short-survival horseradish peroxidase (HRP) method, and their orientation during growth was quantitatively assessed. The finding that the ectopic fibres head towards their distant targets implies that guidance cues are not restricted to specific pathways but are distributed throughout the embryonic brain. The significance of this result is discussed with respect to the ontogeny and evolution of the visual pathway.     

Intrinsic differences in the growth rate of early nerve fibres related to target distance

Target field innervation in the developing vertebrate nervous system coincides with the onset of important trophic interactions. Two factors that determine the timing of this event are the distance axons have to grow to reach their targets, which are known to vary, and the rate at which they grow. There have been few studies of axonal growth rate at this stage of development and no comparative study of the relationship between growth rate and target distance. Embryonic chick cranial sensory neurons are located in discrete ganglia and the distance axons have to grow to reach their targets is different for each ganglion, ranging from several hundred to several thousand microns. Here, I show that these neurons differ in their in vivo growth rates; neurons with more distant targets growing faster. In vitro, single isolated neurons from each of these populations grow at a similar rate to that observed in vivo, indicating that growth rate is an intrinsically determined property of neurons before they reach their targets.     

Notch signalling and the synchronization of the somite segmentation clock

In vertebrates with mutations in the Notch cell-cell communication pathway, segmentation fails: the boundaries demarcating somites, the segments of the embryonic body axis, are absent or irregular. This phenotype has prompted many investigations, but the role of Notch signalling in somitogenesis remains mysterious. Somite patterning is thought to be governed by a "clock-and-wavefront" mechanism: a biochemical oscillator (the segmentation clock) operates in the cells of the presomitic mesoderm, the immature tissue from which the somites are sequentially produced, and a wavefront of maturation sweeps back through this tissue, arresting oscillation and initiating somite differentiation. Cells arrested in different phases of their cycle express different genes, defining the spatially periodic pattern of somites and controlling the physical process of segmentation. Notch signalling, one might think, must be necessary for oscillation, or to organize subsequent events that create the somite boundaries. Here we analyse a set of zebrafish mutants and arrive at a different interpretation: the essential function of Notch signalling in somite segmentation is to keep the oscillations of neighbouring presomitic mesoderm cells synchronized.     

Migratory neural crest-like cells form body pigmentation in a urochordate embryo

The neural crest, a source of many different cell types in vertebrate embryos, has not been identified in other chordates. Current opinion therefore holds that neural crest cells were a vertebrate innovation. Here we describe a migratory cell population resembling neural crest cells in the ascidian urochordate Ecteinascidia turbinata. Labelling of embryos and larvae with the vital lipophilic dye DiI enabled us to detect cells that emerge from the neural tube, migrate into the body wall and siphon primordia, and subsequently differentiate as pigment cells. These cells express HNK-1 antigen and Zic gene markers of vertebrate neural crest cells. The results suggest that migratory cells with some of the features of neural crest cells are present in the urochordates. Thus, we propose a hypothesis for neural crest evolution beginning with the release of migratory cells from the CNS to produce body pigmentation in the common ancestor of the urochordates and vertebrates. These cells may have gained additional functions or were joined by other cell types to generate the variety of derivatives typical of the vertebrate neural crest.     

Control of segment number in vertebrate embryos

The vertebrate body axis is subdivided into repeated segments, best exemplified by the vertebrae that derive from embryonic somites. The number of somites is precisely defined for any given species but varies widely from one species to another. To determine the mechanism controlling somite number, we have compared somitogenesis in zebrafish, chicken, mouse and corn snake embryos. Here we present evidence that in all of these species a similar 'clock-and-wavefront' mechanism operates to control somitogenesis; in all of them, somitogenesis is brought to an end through a process in which the presomitic mesoderm, having first increased in size, gradually shrinks until it is exhausted, terminating somite formation. In snake embryos, however, the segmentation clock rate is much faster relative to developmental rate than in other amniotes, leading to a greatly increased number of smaller-sized somites.     

Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head

The comparison of Hox genes between vertebrates and their closest invertebrate relatives (amphioxus and ascidia) highlights two derived features of Hox genes in vertebrates: duplication of the Hox gene cluster, and an elaboration of Hox expression patterns and roles compared with non-vertebrate chordates. We have investigated how new expression domains and their associated developmental functions evolved, by testing the cis-regulatory activity of genomic DNA fragments from the cephalochordate amphioxus Hox cluster in transgenic mouse and chick embryos. Here we present evidence for the conservation of cis-regulatory mechanisms controlling gene expression in the neural tube for half a billion years of evolution, including a dependence on retinoic acid signalling. We also identify amphioxus Hox gene regulatory elements that drive spatially localized expression in vertebrate neural crest cells, in derivatives of neurogenic placodes and in branchial arches, despite the fact that cephalochordates lack both neural crest and neurogenic placodes. This implies an elaboration of cis-regulatory elements in the Hox gene cluster of vertebrate ancestors during the evolution of craniofacial patterning.     

Noise-resistant and synchronized oscillation of the segmentation clock

Periodic somite segmentation in vertebrate embryos is controlled by the 'segmentation clock', which consists of numerous cellular oscillators. Although the properties of a single oscillator, driven by a hairy negative-feedback loop, have been investigated, the system-level properties of the segmentation clock remain largely unknown. To explore these characteristics, we have examined the response of a normally oscillating clock in zebrafish to experimental stimuli using in vivo mosaic experiments and mathematical simulation. We demonstrate that the segmentation clock behaves as a coupled oscillator, by showing that Notch-dependent intercellular communication, the activity of which is regulated by the internal hairy oscillator, couples neighbouring cells to facilitate synchronized oscillation. Furthermore, the oscillation phase of individual oscillators fluctuates due to developmental noise such as stochastic gene expression and active cell proliferation. The intercellular coupling was found to have a crucial role in minimizing the effects of this noise to maintain coherent oscillation.     

Wnt-mediated axon guidance via the Drosophila Derailed receptor

In nervous systems with bilateral symmetry, many neurons project axons across the midline to the opposite side. In each segment of the Drosophila embryonic nervous system, axons that display this projection pattern choose one of two distinct tracts: the anterior or posterior commissure. Commissure choice is controlled by Derailed, an atypical receptor tyrosine kinase expressed on axons projecting in the anterior commissure. Here we show that Derailed keeps these axons out of the posterior commissure by acting as a receptor for Wnt5, a member of the Wnt family of secreted signalling molecules. Our results reveal an unexpected role in axon guidance for a Wnt family member, and show that the Derailed receptor is an essential component of Wnt signalling in these guidance events.     

Position-dependent properties of retinal axons and their growth cones

The formation of the very orderly neuronal projection from the retina to the optic tectum is not yet understood, but several mechanisms are thought to be involved in a coordinated fashion. These mechanisms may include mechanical or chemical guidance in channels, guidance by spatial gradients of positional markers, gradients of temporal (maturation) markers or specific inter-axon interactions (see ref. 1 for review). The last-mentioned mechanism could explain the fibre order found in optic nerve and tract. It requires that some or all growing retinal axons can distinguish between retinal axons of various origins and grow preferentially along retinal axons originating from the same area as themselves. The in vitro experiments described here show that growth cones from the temporal half of the chick retina grow preferentially along temporal axons, whereas growth cones from nasal retina do not distinguish between nasal and temporal axons.     

Induction of cranial and posterior trunk neural crest by exogenous retinoic acid in zebrafish

Retinoic acid (RA) plays an important role in development of vertebrate embryos. We demonstrate impacts of exogenous RA on the formation of neural crest cells in zebrafish using specific neural crest markers sox9b and crestin. Treatment with all -trans RA at 10₋₇ mmol/L at 50% epiboly induces sox9b expression in the forebrain and crestin expression in the forebrain and midbrain, resulting in significant increase of pigment cells in the head derived from the cranial neural crest. In addition, RA treatment induces expression of sox9b and crestin in the caudal marginal cells of the neuroectoderm during early segmentation. Earlier commitment of these cells to the neural crest fate in the posterior margins leads to abnormal development of the posterior body, probably by preventing mingling of ventral derived and dorsal-derived cells during the formation of the tailbud.     

The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity

The serially segmented (metameric) structures of vertebrates are based on somites that are periodically formed during embryogenesis. A 'clock and wavefront' model has been proposed to explain the underlying mechanism of somite formation, in which the periodicity is generated by oscillation of Notch components (the clock) in the posterior pre-somitic mesoderm (PSM). This temporal periodicity is then translated into the segmental units in the 'wavefront'. The wavefront is thought to exist in the anterior PSM and progress backwards at a constant rate; however, there has been no direct evidence as to whether the levels of Notch activity really oscillate and how such oscillation is translated into a segmental pattern in the anterior PSM. Here, we have visualized endogenous levels of Notch1 activity in mice, showing that it oscillates in the posterior PSM but is arrested in the anterior PSM. Somite boundaries formed at the interface between Notch1-activated and -repressed domains. Genetic and biochemical studies indicate that this interface is generated by suppression of Notch activity by mesoderm posterior 2 (Mesp2) through induction of the lunatic fringe gene (Lfng). We propose that the oscillation of Notch activity is arrested and translated in the wavefront by Mesp2.     

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. Here we show that abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. Our results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes.     

RGM is a repulsive guidance molecule for retinal axons

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.     

Differentiation of a bipotential glial progenitor cell in a single cell microculture

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.     

Neural crest origins of the neck and shoulder

The neck and shoulder region of vertebrates has undergone a complex evolutionary history. To identify its underlying mechanisms we map the destinations of embryonic neural crest and mesodermal stem cells using Cre-recombinase-mediated transgenesis. The single-cell resolution of this genetic labelling reveals cryptic cell boundaries traversing the seemingly homogeneous skeleton of the neck and shoulders. Within this assembly of bones and muscles we discern a precise code of connectivity that mesenchymal stem cells of both neural crest and mesodermal origin obey as they form muscle scaffolds. The neural crest anchors the head onto the anterior lining of the shoulder girdle, while a Hox-gene-controlled mesoderm links trunk muscles to the posterior neck and shoulder skeleton. The skeleton that we identify as neural crest-derived is specifically affected in human Klippel-Feil syndrome, Sprengel's deformity and Arnold-Chiari I/II malformation, providing insights into their likely aetiology. We identify genes involved in the cellular modularity of the neck and shoulder skeleton and propose a new method for determining skeletal homologies that is based on muscle attachments. This has allowed us to trace the whereabouts of the cleithrum, the major shoulder bone of extinct land vertebrate ancestors, which seems to survive as the scapular spine in living mammals.     

Chemotropic guidance of developing axons in the mammalian central nervous system

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.     

Drosophila nurse cells produce a posterior signal required for embryonic segmentation and polarity

The segmental pattern of insect embryos depends on influences from morphogenetic centres near each of the egg poles. In Drosophila, maternal effect mutations are known that impair the normal function of each centre. Injection of wild-type cytoplasm into mutant eggs has revealed that morphogenetic signals localized at the anterior and posterior pole of eggs can be transplanted. We show here that these activities can also be detected during oogenesis. Posterior activity can be recovered at an early stage (stage 10, ref. 5) from the oocyte-nurse cell complex, but anterior activity can only be detected in the mature oocytes (stage 14). We conclude that the bicoid-dependent anterior signal, although produced by the nurse cells, does not become active before it is localized to the anterior egg pole, whereas posterior activity can be detected in the nurse cells before, and therefore independently of, its localization to the posterior egg pole.