The role of organizers in patterning the nervous system.

The foundation for the anatomical and functional complexity of the vertebrate central nervous system is laid during embryogenesis. After Spemann's organizer and its derivatives have endowed the neural plate with a coarse pattern along its anteroposterior and mediolateral axes, this basis is progressively refined by the activity of secondary organizers within the neuroepithelium that function by releasing diffusible signaling factors. Dorsoventral patterning is mediated by two organizer regions that extend along the dorsal and ventral midlines of the entire neuraxis, whereas anteroposterior patterning is controlled by several discrete organizers. Here we review how these secondary organizers are established and how they exert their signaling functions. Organizer signals come from a surprisingly limited set of signaling factor families, indicating that the competence of target cells to respond to those signals plays an important part in neural patterning.

Irx3 and Pax6 establish differential competence for Shh-mediated induction of GABAergic and glutamatergic neurons of the thalamus.

During embryonic development, the presumptive GABAergic rostral thalamus (rTh) and glutamatergic caudal thalamus (cTh) are induced by Sonic hedgehog (Shh) signaling from the zona limitans intrathalamica (ZLI) at the rostral border of the thalamic primordium. We found that these inductions are limited to the neuroepithelium between the ZLI and the forebrain-midbrain boundary, suggesting a prepattern that limits thalamic competence. We hypothesized that this prepattern is established by the overlapping expression of two transcription factors: Iroquois-related homeobox gene 3 (Irx3) posterior to the ZLI, and paired box gene 6 (Pax6) anterior to the forebrain-midbrain boundary. Consistent with this assumption, we show that misexpression of Irx3 in the prethalamus or telencephalon results in ectopic induction of thalamic markers in response to Shh, that it functions as a transcriptional repressor in this context, and that antagonizing its function in the diencephalon attenuates thalamic specification. Similarly, misexpression of Pax6 in the midbrain together with Shh pathway activation results in ectopic induction of cTh markers in clusters of cells that fail to integrate into tectal layers and of atypical long-range projections, whereas antagonizing Pax6 function in the thalamus disrupts cTh formation. However, rTh markers are negatively regulated by Pax6, which itself is down-regulated by Shh from the ZLI in this area. Our results demonstrate that the combinatorial expression of Irx3 and Pax6 endows cells with the competence for cTh formation, whereas Shh-mediated down-regulation of Pax6 is required for rTh formation. Thus, thalamus induction and patterning depends both on a prepattern of Irx3 and Pax6 expression that establishes differential cellular competence and on Shh signaling from the ZLI organizer.

Lhx2 and Lhx9 determine neuronal differentiation and compartition in the caudal forebrain by regulating Wnt signaling.

Initial axial patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs during gastrulation. After this patterning phase, further diversification within the brain is thought to proceed largely independently in the different primordia. However, mechanisms that maintain the demarcation of brain subdivisions at later stages are poorly understood. In the alar plate of the caudal forebrain there are two principal units, the thalamus and the pretectum, each of which is a developmental compartment. Here we show that proper neuronal differentiation of the thalamus requires Lhx2 and Lhx9 function. In Lhx2/Lhx9-deficient zebrafish embryos the differentiation process is blocked and the dorsally adjacent Wnt positive epithalamus expands into the thalamus. This leads to an upregulation of Wnt signaling in the caudal forebrain. Lack of Lhx2/Lhx9 function as well as increased Wnt signaling alter the expression of the thalamus specific cell adhesion factor pcdh10b and lead subsequently to a striking anterior-posterior disorganization of the caudal forebrain. We therefore suggest that after initial neural tube patterning, neurogenesis within a brain compartment influences the integrity of the neuronal progenitor pool and border formation of a neuromeric compartment.

Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick.

The midbrain-hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB--loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.

Her6 regulates the neurogenetic gradient and neuronal identity in the thalamus.

During vertebrate brain development, the onset of neuronal differentiation is under strict temporal control. In the mammalian thalamus and other brain regions, neurogenesis is regulated also in a spatially progressive manner referred to as a neurogenetic gradient, the underlying mechanism of which is unknown. Here we describe the existence of a neurogenetic gradient in the zebrafish thalamus and show that the progression of neurogenesis is controlled by dynamic expression of the bHLH repressor her6. Members of the Hes/Her family are known to regulate proneural genes, such as Neurogenin and Ascl. Here we find that Her6 determines not only the onset of neurogenesis but also the identity of thalamic neurons, marked by proneural and neurotransmitter gene expression: loss of Her6 leads to premature Neurogenin1-mediated genesis of glutamatergic (excitatory) neurons, whereas maintenance of Her6 leads to Ascl1-mediated production of GABAergic (inhibitory) neurons. Thus, the presence or absence of a single upstream regulator of proneural gene expression, Her6, leads to the establishment of discrete neuronal domains in the thalamus.

PRDC regulates placode neurogenesis in chick by modulating BMP signalling.

The epibranchial placodes generate the neurons of the geniculate, petrosal, and nodose cranial sensory ganglia. Previously, it has been shown that bone morphogenetic proteins (BMPs) are involved in the formation of these structures. However, it has been unclear as to whether BMP signalling has an ongoing function in directing the later development of the epibranchial placodes, and how this signalling is regulated. Here, we demonstrate that BMPs maintain placodal neurogenesis and that their activity is modulated by a member of the Cerberus/Dan family of BMP antagonists, Protein Related to Dan and Cerberus (PRDC). We find that Bmp4 is expressed in the epibranchial placodes while Bmp7 and PRDC are expressed in the pharyngeal pouches. The timing and regional expression of these three genes suggest that BMP7 is involved in inducing placode neurogenesis and BMP4 in maintaining it and that BMP activity is modulated by PRDC. To investigate this hypothesis, we have performed both gain- and loss- of-function experiments with PRDC and find that it can modulate the BMP signals that induce epibranchial neurogenesis: a gain of PRDC function results in a loss of Bmp4 and hence placode neurogenesis is inhibited; conversely, a loss of PRDC function induces ectopic Bmp4 and an expansion of placode neurogenesis. This modulation is therefore necessary for the number and positioning of the epibranchial neurons.

Intrinsic, Hox-dependent cues determine the fate of skeletal muscle precursors.

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. We show that instead, somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb.

Hedgehog signaling from the ZLI regulates diencephalic regional identity.

The zona limitans intrathalamica (ZLI), a narrow compartment in the vertebrate forebrain that bisects the diencephalon transversely, expresses the secreted factor sonic hedgehog (Shh). Because genetic disruption of Shh in mouse causes severe early developmental defects, this strategy has not been useful in identifying a ZLI-specific role for this gene. To modulate Shh signaling in a spatiotemporally restricted manner, we carried out gain- and loss-of-function experiments in chick embryos using in ovo electroporation and found that Shh signaling is required for region-specific gene expression in thalamus and prethalamus, the major diencephalic brain areas flanking the ZLI. We further show that differential competence of thalamic and prethalamic primordia in responding to Shh signaling is regulated by the transcription factor Irx3. We show that, through the release of Shh, the ZLI functions as a local signaling center that regulates the acquisition of identity for these important diencephalic regions.

The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation.

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Here we report characterization of Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes, that is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. Our data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates.

Arcuate plan of chick midbrain development.

In spinal cord and hindbrain development, neurons are generated as longitudinal cell columns aligned with the ventral and dorsal midlines. For rostral brain, however, the fundamental structure of early neuronal patterning remains poorly understood. We report here that, in the chick embryo, the ventral midbrain is remarkably regular in its cellular and molecular organization; it is arranged as a reiterative series of arcuate territories arrayed bilateral to the ventral midline. In the mantle layer of the ventral midbrain, an arcuate series of neuronal cell columns (midbrain arcs) is demonstrated by acetylcholinesterase histochemistry and gene expression for class III beta-tubulin, homeodomain transcription factors, and neurotransmitter synthetic enzymes. In the ventricular layer of midbrain progenitor cells, WNT and NOTCH ligand gene expression displays arcuate periodicities that form a tight three-dimensional registration with the arcs of the underlying mantle layer. Ventral midbrain arcuate patterning is even macroscopically visible, forming ridges along the ventricular surface. These observations establish that a single plan of arcuate organization governs the morphogenesis and cell-type specification of the ventral midbrain. Arcs are not restricted to the midbrain tegmentum but extend through the subthalamic tegmentum of the forebrain. Thus, the chick rostral brain, which is classically divided into midbrain and forebrain, can also be partitioned into the following: (1) a neuraxial region of arcs and (2) an anterodorsal cap that includes midbrain tectum and nonsubthalamic forebrain. We show that this partition of brain tissue is supported by the expression patterns of homologs of Drosophila gap genes.

Induction of a parafacial rhythm generator by rhombomere 3 in the chick embryo.

Observations of knock-out mice suggest that breathing at birth requires correct development of a specific hindbrain territory corresponding to rhombomeres (r) 3 and 4. Focusing on this territory, we examined the development of a neuronal rhythm generator in the chick embryo. We show that rhythmic activity in r4 is inducible after developmental stage 10 through interaction with r3. Although the nature of this interaction remains obscure, we find that the expression of Krox20, a segmentation gene responsible for specifying r3 and r5, is sufficient to endow other rhombomeres with the capacity to induce rhythmic activity in r4. Induction is robust, because it can be reproduced with r2 and r6 instead of r4 and with any hindbrain territory that normally expresses Krox20 (r3, r5) or can be forced to do so (r1, r4). Interestingly, the interaction between r4 and r3/r5 that results in rhythm production can only take place through the anterior border of r4, revealing a heretofore unsuspected polarity in individual rhombomeres. The r4 rhythm generator appears to be homologous to a murine respiratory parafacial neuronal system developing in r4 under the control of Krox20 and Hoxa1. These results identify a late role for Krox20 at the onset of neurogenesis.

Segmentation and compartition in the early avian hindbrain.

For the comparative embryologists of the early 20th century, the segment-like bulges that appear transiently during the early stages of vertebrate hindbrain development were both the object of fascination and the subject of vigorous dispute. Conflicting views were held as to the significance of these 'rhombomeres' to brain development and their more general relevance to head evolution. Whether rhombomeres are inconsequential bumps in the embryonic brain or true segments-iterative or metameric units-has only recently been resolved. A number of studies using more modern techniques (such as immunohistochemistry, in situ hybridisation, axonal tracing, single cell labelling, heterotopic and orthotopic grafting, and the manipulation of gene expression by electroporation) have shown that the hindbrain has a truly metameric cellular organisation. The avian embryo has played a particularly prominent role in such studies by virtue of its large size and accessibility, its amenability to microsurgery, and its well-described anatomy. Furthermore, electrophysiological studies, also on avian embryos, have shown that segmentation of the parent neuroepithelium into rhombomeres plays a crucial part in establishing the functional organization of the hindbrain. Segmentation suggests the early allocation of defined sets of precursor cells and is therefore presumed to allow a specific identity for each successive segment to emerge from a common ground plan. This short review will focus on the contribution the avian embryo has made to our understanding of this fly-like region of the vertebrate brain, in respect of its morphology and neuronal architecture, the cellular and molecular mechanisms involved in establishing and maintaining the segments, and the molecular controls of segmental identity.

Local inhibition guides the trajectory of early longitudinal tracts in the developing chick brain.

During development of the chick central nervous system, the trajectories of the descending medial and lateral longitudinal fascicles (MLF and LLF) are pioneered by axons originating from the interstitial nucleus of Cajal (INC) and the mesencephalic trigeminal nucleus (MTN), respectively. Both tracts cross rhombomere 1 at two specific locations in the basal plate. In this study, we have investigated the molecular properties of these crossing points and find that they are permissive regions situated in an otherwise inhibitory boundary region. We show that the dorsal part of rhombomere 1 is inhibitory for the growth of both MTN and INC axons. Ventrally, MLF and LLF axons are repelled from the midline by Slit proteins. Our results reveal the existence of a new repulsive/inhibitory mechanism for axons in the alar plate in addition to the ventral repulsion by Slit proteins. This suggests a model where MLF and LLF axons are channeled longitudinally within the neural tube by both dorsal and ventral constraints.

The paired-type homeobox gene Dmbx1 marks the midbrain and pretectum.

We have isolated a paired-type homeobox gene Dmbx1, previously known as Atx (Development 128 (2001) 4789), from chick and mouse. Sequence similarity reveals that this gene is highly related to the Otx genes. Expression of Dmbx1 commences during gastrulation, when transcripts are detected in a crescent around the anterior neural plate. As development progresses, Dmbx1 marks the prospective midbrain and pretectum. Dmbx1 shares its caudal border of expression with Otx2, while expression is sharply delimited rostrally by the synencephalic-parencephalic boundary, later becoming restricted to the posterior synencephalon. At later stages, Dmbx1 is expressed in dynamic domains of the hindbrain and spinal cord. Additional sites of expression comprise stomodeal ectoderm and foregut endoderm, presomitic mesoderm, and the nasal pit.

Wnt6 marks sites of epithelial transformations in the chick embryo.

In a screen for Wnt genes executing the patterning function of the vertebrate surface ectoderm, we have isolated a novel chick Wnt gene, chick Wnt6. This gene encodes the first pan-epidermal Wnt signalling molecule. Further sites of expression are the boundary of the early neural plate and surface ectoderm, the roof of mesencephalon, pretectum and dorsal thalamus, the differentiating heart, and the otic vesicle. The precise sites of Wnt6 expression coincide with crucial changes in tissue architecture, namely epithelial remodelling and epithelial-mesenchymal transformation (EMT). Moreover, the expression of Wnt6 is closely associated with areas of Bmp signalling.

What can vertebrates tell us about segmentation?

Segmentation is a feature of the body plans of a number of diverse animal groupings, including the annelids, arthropods and chordates. However, it has been unclear whether or not these different manifestations of segmentation are independently derived or have a common origin. Central to this issue is whether or not there are common developmental mechanisms that establish segmentation and the evolutionary origins of these processes. A fruitful way to address this issue is to consider how segmentation in vertebrates is directed. During vertebrate development three different segmental systems are established: the somites, the rhombomeres and the pharyngeal arches. In each an iteration of parts along the long axis is established. However, it is clear that the formation of the somites, rhombomeres or pharyngeal arches have little in common, and as such there is no single segmentation process. These different segmental systems also have distinct evolutionary histories, thus highlighting the fact that segmentation can and does evolve independently at multiple points. We conclude that the term segmentation indicates nothing more than a morphological description and that it implies no mechanistic similarity. Thus it is probable that segmentation has arisen repeatedly during animal evolution.

Transcriptional control of GABAergic neuronal subtype identity in the thalamus.

BACKGROUND: The thalamus is often defined as the 'gateway to consciousness', a feature that is supported by the specific connectivity and electrophysiological properties of its neurons. Inhibitory GABAergic neurons are required for the dynamic gating of information passing through the thalamus. The high degree of heterogeneity among thalamic GABA neurons suggests that, during embryonic development, alternative differentiation programmes exist to guide the acquisition of inhibitory neuron subtype identity. RESULTS: Taking advantage of the accessibility of the developing chick embryo, we have used in ovo manipulations of gene expression to test the role of candidate transcription factors in controlling GABAergic neuronal subtype identity in the developing thalamus. CONCLUSIONS: In this study, we describe two alternative differentiation programmes for GABAergic neurogenesis in the thalamus and identify Helt and Dlx2 as key transcription factors that are sufficient to direct neuronal progenitors along a specific differentiation pathway at the expense of alternative lineage choices. Furthermore, we identify Calb2, a gene encoding for the GABA subtype marker calretinin as a target of the transcription factor Sox14. This work is a step forward in our understanding of how GABA neuron diversity in the thalamus is achieved during development and will help future investigation of the molecular mechanisms that lead up to the acquisition of different synaptic targets and electrophysiological features of mature thalamic inhibitory neurons.

Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia.

In neurodegenerative conditions and following brain trauma it is not understood why neurons die while astrocytes and microglia survive and adopt pro-inflammatory phenotypes. We show here that the damaged adult brain releases diffusible factors that can kill cortical neurons and we have identified histone H1 as a major extracellular candidate that causes neurotoxicity and activation of the innate immune system. Extracellular core histones H2A, H2B H3 and H4 were not neurotoxic. Innate immunity in the central nervous system is mediated through microglial cells and we show here for the first time that histone H1 promotes their survival, up-regulates MHC class II antigen expression and is a powerful microglial chemoattractant. We propose that when the central nervous system is degenerating, histone H1 drives a positive feedback loop that drives further degeneration and activation of immune defences which can themselves be damaging. We suggest that histone H1 acts as an antimicrobial peptide and kills neurons through mitochondrial damage and apoptosis.