Molecular control of neural crest formation, migration and differentiation.

Induction, migration and differentiation of the neural crest are crucial for the development of the vertebrate embryo, and elucidation of the underlying mechanisms remains an important challenge. In the past year, a novel signal regulating the formation of neural crest cells has been identified, and advances have been made in uncovering roles for bone morphogenetic protein signals and for a transcription factor in the onset of neural crest migration. There have been new insights into the migration and plasticity of branchial neural crest cells. Important progress has been made in dissecting the roles of bone morphogenetic protein, Wnt and Notch signalling systems and their associated downstream transcription factors in the control of neural crest cell differentiation.

Control of cell behavior during vertebrate development by Slug, a zinc finger gene.

Slug, a vertebrate gene encoding a zinc finger protein of the Snail family, is expressed in the neural crest and in mesodermal cells emigrating from the primitive streak. Early chick embryos were incubated with antisense oligonucleotides to chick Slug. These oligonucleotides specifically inhibit the normal change in cell behavior that occurs at the two sites in the emerging body plan in which the gene is expressed. This change, which is the transition from epithelial to mesenchymal character, occurs at the formation of mesoderm during gastrulation and on emigration of the neutral crest from the neural tube.

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

Topographic mapping: organising by repulsion and competition?

The establishment of topographic maps of neuronal connections is believed to involve graded repulsion mediated by EphA receptors and ephrin-A ligands. Gene knockouts show that ephrin-A ligands do indeed have a crucial role in mapping, and that mechanisms in addition to graded repulsion must also be at work.

The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells.

BACKGROUND: During vertebrate head development, neural crest cells migrate from hindbrain segments to specific branchial arches, where they differentiate into distinct patterns of skeletal structures. The rostrocaudal identity of branchial neural crest cells appears to be specified prior to migration, so it is important that they are targeted to the correct destination. In Xenopus embryos, branchial neural crest cells segregate into four streams that are adjacent during early stages of migration. It is not known what restricts the intermingling of these migrating cell populations and targets them to specific branchial arches. Here, we investigated the role of Eph receptors and ephrins-mediators of cell-contact-dependent interactions that have been implicated in neuronal pathfinding-in this targeted migration. RESULTS: Xenopus EphA4 and EphB1 are expressed in migrating neural crest cells and mesoderm of the third arch, and third plus fourth arches, respectively. The ephrin-B2 ligand, which interacts with these receptors, is expressed in the adjacent second arch neural crest and mesoderm. Using truncated receptors, we show that the inhibition of EphA4/EphB1 function leads to abnormal migration of third arch neural crest cells into second and fourth arch territories. Furthermore, ectopic activation of these receptors by overexpression of ephrin-B2 leads to scattering of third arch neural crest cells into adjacent regions. Similar disruptions occur when the expression of ephrin-B2 or truncated receptors is targeted to the neural crest. CONCLUSIONS: These data indicate that the complementary expression of EphA4/EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of third and second arch neural crest and in targeting third arch neural crest to the correct destination. Together with previous work showing that Eph receptors and ligands mediate neuronal growth cone repulsion, our findings suggest that similar mechanisms are used for neural crest and axon pathfinding.

Metallothionein genes MTa and MTb expressed under distinct quantitative and tissue-specific regulation in sea urchin embryos.

Sea urchin embryo metallothionein (MT) mRNAs MTa and MTb have distinct cDNA sequences and are transcripts of different genes of a multigene family. These MT mRNAs differ in size and in their 3'-untranslated sequences. They encode proteins that are unusual among MT isotypes in that the relative positions of their cysteine residues are partially out of register, suggesting potential differences in function. In pluteus larvae MTa mRNA is expressed abundantly and exclusively in the ectoderm, while MTb mRNA, which is restricted to the endomesoderm at a low endogenous level, can be induced to a high level by heavy metal ions (M2+). MT mRNA is present in the maternal reservoir of the egg and is predominantly (greater than 95%) MTa mRNA. Endogenous expression in the embryo, which is at a much higher level than in the egg, requires M2+ for gene transcription, is developmentally regulated, and is greater than 90% MTa mRNA. When induced by added M2+, however, MTa and MTb mRNAs accumulate to almost equal levels. The differences in the ratios of MTa/MTb expressed endogenously and inductively are not attributable to differences in the stabilities of these MT mRNAs, which were observed under conditions of M2+ depletion, or in their inducibilities, which were observed at moderate to high M2+ levels. We found, instead, that the MTa gene responds to M2+ at a lower threshold level than MTb, so that at very low M2+ concentrations the ratio of induced MTa/MTb mRNA is high and equivalent to the endogenous ratio. Thus, endogenous expression of the MTa gene is selectively enhanced in the ectoderm by determinants that are responsive at low M2+ threshold concentrations.

Molecular approaches to the segmentation of the hindbrain.

Recent studies have shown that the rhombomeric bulges of the developing vertebrate hindbrain reflect segmental mechanisms that generate pattern in this region of the CNS. Although little is known of the genetic basis of this segmentation, in situ hybridization studies have provided circumstantial evidence that certain 'zinc-finger' and homeobox genes have roles in the development of segments in the early mouse hindbrain. We discuss the implications of these findings for the function of these genes in hindbrain development.

Control of cell behaviour by signalling through Eph receptors and ephrins.

Eph receptor tyrosine kinases and ephrins mediate contact-dependent cell interactions that regulate the repulsion and adhesion mechanisms involved in the guidance and assembly of cells. Recent work has revealed a role of overlapping Eph receptor and ephrin expression in modulating neuronal growth cone repulsion, and has shown that bidirectional activation restricts intermingling and communication between cell populations. In addition, progress has been made in understanding how Eph receptors and ephrins control cell adhesion.

Eph receptors and ephrins in neural development.

Ephrins, ligands for the Eph family of receptor tyrosine kinases, are pivotal players in many developmental phenomena in both the central and peripheral nervous systems. Ephrins appear to act typically, but not exclusively, as repellents throughout development to influence axon pathfinding and topographic mapping, as well as restricting cell migration and intermingling. Recent findings are beginning to characterize the function and signaling of ephrins, as well as major roles for them in other tissues.

Elk-L3, a novel transmembrane ligand for the Eph family of receptor tyrosine kinases, expressed in embryonic floor plate, roof plate and hindbrain segments.

The Eph family of receptor tyrosine kinases has 13 distinct members and seven ligands for these receptors have been described to date. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and compartmentalization role outside of the nervous system as well. The ligands are all membrane-attached, and this attachment appears to be crucial for their normal function; five of the known ligands are linked to the membrane via a glycosyl phosphotidylinositol (GPI) linkage, while two of the ligands are transmembrane proteins. Despite the large number of Eph family receptors and ligands, they can be divided into just two major subclasses based on their binding specificities. All the GPI-anchored ligands bind and activate one subclass of the Eph receptors (that represented by Eck) while the two transmembrane ligands bind and activate the other major subclass of receptors (represented by Elk). Here we report the identification and characterization of the third, and most divergent, member of the transmembrane group of Eph ligands, which we term Elk-L3 (Elk-related receptor ligand number 3). Elk-L3 is notable for its remarkably restricted and prominent expression in the floor plate and roof plate of the developing neural tube and its rhombomere-specific expression in the developing hindbrain. The Elk-L3 gene has been localized to mouse chromosome 11 and human chromosome 17.

An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain.

In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.

Eph receptors and ephrins: regulators of guidance and assembly.

Recent advances have started to elucidate the developmental functions and biochemistry of Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins. Interactions between these molecules are promiscuous, but they largely fall into two groups: EphA receptors bind to GPI-anchored ephrin-A ligands, while EphB receptors bind to ephrin-B proteins that have a transmembrane and cytoplasmic domain. Remarkably, ephrin-B proteins transduce signals, such that bidirectional signaling can occur upon interaction with Eph receptor. In many tissues, specific Eph receptors and ephrins have complementary domains, whereas other family members may overlap in their expression. An important role of Eph receptors and ephrins is to mediate cell-contact-dependent repulsion. Complementary and overlapping gradients of expression underlie establishment of a topographic map of neuronal projections in the retinotectal system. Eph receptors and ephrins also act at boundaries to channel neuronal growth cones along specific pathways, restrict the migration of neural crest cells, and via bidirectional signaling prevent intermingling between hindbrain segments. Intriguingly, Eph receptors and ephrins can also trigger an adhesive response of endothelial cells and are required for the remodeling of blood vessels. Biochemical studies suggest that the extent of multimerization of Eph receptors modulates the cellular response and that the actin cytoskeleton is one major target of the intracellular pathways activated by Eph receptors. Eph receptors and ephrins have thus emerged as key regulators of the repulsion and adhesion of cells that underlie the establishment, maintenance, and remodeling of patterns of cellular organization.

The zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites.

Several vertebrate genes of the Hairy/Enhancer-of-split (HES) family are involved in paraxial mesoderm segmentation and intersomitic boundary establishment/maintenance. Here, we show that the zebrafish hairy-related gene, her6, highly homologous to the mammalian and chicken HES-1 genes, is expressed in the posterior part of each segmented somite and in stripes in the anterior presomitic mesoderm (PSM), and also in a dynamic, segmentally restricted pattern during hindbrain segmentation, with all rhombomeres expressing her6 at different time points and at different levels.

The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest.

Recent studies in the chick have indicated that rhombomeres (r) are segments that underlie the patterning of hindbrain nerves. These segments may also be important for the specification of branchial arch structures since alternating rhombomeres, r2, r4 and r6, each contribute crest to a specific arch. Krox-20 has been implicated in the segmental patterning of the hindbrain in the mouse by its expression prior to segment formation in alternating domains, which later correspond to r3 and r5. Here, we describe the sequence and developmental expression of the Xenopus Krox-20 gene, XKrox-20. Alternating domains of XKrox-20 expression appear in the early neurula, later correspond to r3 and r5, and persist until late tadpole stages. In contrast to this conserved spatial expression in rhombomeres, we find a pattern in the neural crest of Xenopus that appears different from that found in the mouse: expression occurs in crest that migrates from r5 into the third visceral arch. We speculate that this may reflect a distinct route of neural crest migration due to anatomical differences between these systems, rather than a difference in the site of origin of Krox-20-expressing crest.

Krox-20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain.

The morphogenesis of the vertebrate hindbrain involves a transient segmentation process leading to the formation of reiterated organisation units called rhombomeres (r). A number of regulatory genes expressed with a rhombomere-specific pattern have been identified, including the gene encoding the transcription factor Krox-20, which is restricted to r3 and r5. We have previously demonstrated that in r3 and r5 Krox-20 directly controls the transcription of Hoxa-2 and Hoxb-2. In the present study, we provide evidence that Krox-20 is required for the expression of another Hox gene, Hoxb-3, in r5 specifically. Furthermore, the regulatory role of Krox-20 is not restricted to the control of Hox gene expression, since it is also involved in the activation of a receptor tyrosine kinase gene, Sek-1, in r3 and r5 and in the repression of the follistatin gene in r3 but not in r5. In conclusion, at least five regulatory genes belonging to different families are under the direct or indirect control of Krox-20 in r3 and/or r5 and this transcription factor therefore appears as a key regulator of gene expression in the developing hindbrain.

Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development.

The vertebrate hindbrain is segmented into a series of transient structures called rhombomeres. Despite knowing several factors that are responsible for the segmentation and maintenance of the rhombomeres, there are still large gaps in understanding the genetic pathways that govern their development. To find previously unknown genes that are expressed within the embryonic hindbrain, a subtracted chick hindbrain cDNA library has been made and 445 randomly picked clones from this library have been analysed using whole mount in situ hybridisation. Thirty-six of these clones (8%) display restricted expression patterns within the hindbrain, midbrain or cranial neural crest and of these, twenty-two are novel and eleven encode peptides that correspond to or are highly related to proteins with previously uncharacterised roles during early neural development. The large proportion of genes with restricted expression patterns and previously unknown functions in the embryonic brain identified during this screen provides insights into the different types of molecules that have spatially regulated expression patterns in cranial neural tissue.

Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain.

Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, we have screened for protein kinase genes with restricted expression patterns in this region of the CNS. We present the cloning of three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases (RTKs)), the identification of their chromosomal locations, and the analysis of their expression between 7.5 and 10.5 days of development. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signalling between neuroepithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with our previous finding that Sek (Sek-1) is expressed in r3 and r5 (Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992), these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain.

Murine Wnt-11 and Wnt-12 have temporally and spatially restricted expression patterns during embryonic development.

The Wnt gene family encodes a set of signalling molecules implicated in the development of a wide range of organisms. We have recently cloned partial cDNA sequences of murine Wnt-11 and Wnt-12. Here, we describe the spatio-temporal expression patterns of both genes during mouse embryogenesis. Wnt-11 expression is first detected within the truncus arteriosus from 8.25 dpc. By 9.5 dpc, Wnt-11 expression is detected in the somites at the medial junction of the dermatome and the myotome. Wnt-11 transcripts are also detected in limb bud mesenchyme from the time the bud is first visible. Wnt-12 is detected in the apical ectodermal ridge from 10.5 dpc. The implications of these expression patterns are discussed.

RNA detection using non-radioactive in situ hybridization.

Methods of non-radioactive in situ hybridization to RNA now enable the simultaneous detection of two RNAs in the same tissue. Sensitivity has been increased by several modifications, including the use of the polymerase chain reaction. Recently, in situ hybridization has been combined with lineage tracing to determine the origin of cells expressing a specific gene.

Eph-related receptors and their ligands: mediators of contact dependent cell interactions.

Recent studies of the large families of Eph-related receptor tyrosine kinases and their ligands suggest that they have key roles in embryonic development. These receptors mediate cell contact dependent signalling by binding to membrane-bound ligands, and certain ligands may themselves transduce signals. Functional studies indicate that in a number of tissues the receptors and ligands are expressed in complementary domains and mediate repulsive interactions that restrict cell and axon migration. In addition, they can also stimulate cell migration. Eph-related receptors and their ligands are therefore mediators of cell interactions required for tissue patterning and neuronal pathfinding during development. The potential clinical implications of these findings are discussed.