EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish

The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.     

EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.     

Bidirectional signals establish boundaries.

Recent studies have shown that the formation of boundaries between the segments - rhombomeres - of the vertebrate hindbrain depends on bidirectional signalling between neighbouring cells. This signalling is mediated by Eph receptors and their ligands, which has been found to restrict cell intermingling in vitro.     

Formation and regeneration of rhombomere boundaries in the developing chick hindbrain.

Development in the chick hindbrain is founded on a segmented pattern. Groups of cells are allocated to particular segmental levels early in development, the cells of each segment (rhombomere) mixing freely with each other, but not with those of adjacent segments. After rhombomere formation, cells in the boundary regions become increasingly specialised. Rhombomeres are thus separate territories that will ultimately pursue different developmental fates. We are investigating the mechanisms that establish and maintain the pattern of rhombomeres and their boundaries. Donor-to-host transplantation experiments were used to confront tissue from different axial levels within the hindbrain. The frequency of boundary regeneration and patterning in the hindbrain was then assessed, based on gross morphology, arrangement of motor neurons and immunohistochemistry. We found that when rhombomeres from adjacent positions or positions three rhombomeres distant from one another were confronted, a normal boundary was invariably reconstructed. Juxtaposition of rhombomere 5 with 7 also yielded a new boundary. By contrast, donor and host tissue of the same positional origin combined without forming a boundary. The same result was obtained in combinations of rhombomeres 3 and 5. Confrontation of tissue from even-numbered rhombomeres 4 with 6 or 2 with 4 also failed to regenerate a boundary in the majority of cases. These results suggest that cell surface properties vary according to rhombomeric level in the hindbrain, and may support the idea of a two-segment periodicity.     

Compartments and their boundaries in vertebrate brain development

Fifteen years ago, cell lineage restriction boundaries were discovered in the embryonic vertebrate hindbrain, subdividing it into a series of cell-tight compartments (known as rhombomeres). Compartition, together with segmentally reiterative neuronal architecture and the nested expression of Hox genes, indicates that the hindbrain has a truly metameric organization. This finding initiated a search for compartments in other regions of the developing brain. The results of recent studies have clarified where compartment boundaries exist, have shed light on molecular mechanisms that underlie their formation and have revealed an important function of these boundaries: the positioning and stabilization of local signalling centres.     

Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain.

Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.     

Differential adhesion in morphogenesis: a modern view

The spreading of one embryonic tissue over another, the sorting out of their cells when intermixed and the formation of intertissue boundaries respected by the motile border cells all have counterparts in the behavior of immiscible liquids. The 'differential adhesion hypothesis' (DAH) explains these liquid-like tissue behaviors as consequences of the generation of tissue surface and interfacial tensions arising from the adhesion energies between motile cells. The experimental verification of the DAH, the recent computational models simulating adhesion-mediated morphogenesis, and the evidence concerning the role of differential adhesion in a number of morphodynamic events, including teleost epiboly, the specification of boundaries between rhombomeres in the developing vertebrate hindbrain, epithelial-mesenchymal transitions in embryos, and malignant invasion are reviewed here.     

Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. Physical mechanisms shaping compartment boundaries were recently explored in Drosophila, where actomyosin‐based barriers were revealed to be important for keeping cells apart. In vertebrates, interhombomeric boundaries are straight interfaces, which often serve as signaling centers that pattern the surrounding tissue. Here, we demonstrate that in the hindbrain of zebrafish embryos cell sorting sharpens the molecular boundaries and, once borders are straight, actomyosin barriers are key to keeping rhombomeric cells segregated. Actomyosin cytoskeletal components are enriched at interhombomeric boundaries, forming cable‐like structures in the apical side of the neuroepithelial cells by the time morphological boundaries are visible. When myosin II function is inhibited, cable structures do not form, leading to rhombomeric cell mixing. Downregulation of EphA4a compromises actomyosin cables and cells with different rhombomeric identity intermingle, and the phenotype is rescued enhancing myosin II activity. Moreover, enrichment of actomyosin structures is obtained when EphA4 is ectopically expressed in even‐numbered rhombomeres. These findings suggest that mechanical barriers act downstream of EphA/ephrin signaling to segregate cells from different rhombomeres.     

Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain

Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.     

Differential Activity of Drosophila Hox Genes Induces Myosin Expression and Can Maintain Compartment Boundaries

Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. We show here that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. We also show a similar role in cell segregation for the Abdominal-B Hox gene. Our results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells.     

Notch signalling stabilises boundary formation at the midbrain-hindbrain organiser

The midbrain-hindbrain interface gives rise to a boundary of particular importance in CNS development as it forms a local signalling centre, the proper functioning of which is essential for the formation of tectum and cerebellum. Positioning of the mid-hindbrain boundary (MHB) within the neuroepithelium is dependent on the interface of Otx2 and Gbx2 expression domains, yet in the absence of either or both of these genes, organiser genes are still expressed, suggesting that other, as yet unknown mechanisms are also involved in MHB establishment. Here, we present evidence for a role for Notch signalling in stabilising cell lineage restriction and regulating organiser gene expression at the MHB. Experimental interference with Notch signalling in the chick embryo disrupts MHB formation, including downregulation of the organiser signal Fgf8. Ectopic activation of Notch signalling in cells of the anterior hindbrain results in an exclusion of those cells from rhombomeres 1 and 2, and in a simultaneous clustering along the anterior and posterior boundaries of this area, suggesting that Notch signalling influences cell sorting. These cells ectopically express the boundary marker Fgf3. In agreement with a role for Notch signalling in cell sorting, anterior hindbrain cells with activated Notch signalling segregate from normal cells in an aggregation assay. Finally, misexpression of the Notch modulator Lfng or the Notch ligand Ser1 across the MHB leads to a shift in boundary position and loss of restriction of Fgf8 to the MHB. We propose that differential Notch signalling stabilises the MHB through regulating cell sorting and specifying boundary cell fate.     

Motor neuron pathfinding following rhombomere reversals in the chick embryo hindbrain.

Motor neurons are segmentally organised in the developing chick hindbrain, with groups of neurons occupying pairs of hindbrain segments or rhombomeres. The branchiomotor nucleus of the trigeminal nerve occupies rhombomeres 2 and 3 (r2 and r3), that of the facial nerve r4 and r5, and that of the glossopharyngeal nerve r6 and r7. Branchiomotor neuron cell bodies lie within the basal plate, forming columns on either side of the ventral midline floor plate. Axons originating in rhombomeres 2, 4 and 6 grow laterally (dorsally) towards the exit points located in the alar plates of these rhombomeres, while axons originating in odd-numbered rhombomeres 3 and 5 grow laterally and then rostrally, crossing a rhombomere boundary to reach their exit point. Examination of the trajectories of motor axons in odd-numbered segments at late stages of development (19-25) showed stereotyped pathways, in which axons grew laterally before making a sharp turn rostrally. During the initial phase of outgrowth (stage 14-15), however, axons had meandering courses and did not grow in a directed fashion towards their exit point. When r3 or r5 was transplanted with reversed rostrocaudal polarity prior to motor axon outgrowth, the majority of axons grew to their appropriate, rostral exit point, despite the inverted neuroepithelial polarity. In r3 reversals, however, there was a considerable increase in the normally small number of axons that grew out via the caudal, r4 exit point. These findings are discussed with relevance to the factors involved in motor neuron specification and axon outgrowth in the developing hindbrain.     

Independent roles for retinoic acid in segmentation and neuronal differentiation in the zebrafish hindbrain

Segmentation of the vertebrate hindbrain into rhombomeres is essential for the anterior-posterior patterning of cranial motor nuclei and their associated nerves. The vitamin A derivative, retinoic acid (RA), is an early embryonic signal that specifies rhombomeres, but its roles in neuronal differentiation within the hindbrain remain unclear. Here we have analyzed the formation of primary and secondary hindbrain neurons in the zebrafish mutant neckless (nls), which disrupts retinaldehyde dehydrogenase 2 (raldh2), and in embryos treated with retinoid receptor (RAR) antagonists. Mutation of nls disrupts secondary, branchiomotor neurons of the facial and vagal nerves, but not the segmental pattern of primary, reticulospinal neurons, suggesting that RA acts on branchiomotor neurons independent of its role in hindbrain segmentation. Very few vagal motor neurons form in nls mutants and many facial motor neurons do not migrate out of rhombomere 4 into more posterior segments. When embryos are treated with RAR antagonists during gastrulation, we observe more severe patterning defects than seen in nls. These include duplicated reticulospinal neurons and posterior expansions of rhombomere 4, as well as defects in branchiomotor neurons. However, later antagonist treatments after rhombomeres are established still disrupt branchiomotor development, suggesting that requirements for RARs in these neurons occur later and independent of segmental patterning. We also show that RA produced by the paraxial mesoderm controls branchiomotor differentiation, since we can rescue the entire motor innervation pattern by transplanting wild-type cells into the somites of nls mutants. Thus, in addition to its role in determining rhombomere identities, RA plays a more direct role in the differentiation of subsets of branchiomotor neurons within the hindbrain.     

How to build a vertebrate hindlbrain. lessons from genetics

During vertebrate embryogenesis, the hindbrain is the site of a segmentation process which leads to the formation, along the anterior-posterior axis, of 7–8 metameres called rhombomeres. This phenomenon plays an essential role in early hindbrain regionalisation and in the specification of the pattern of developing structures in this region of the brain. Data accumulated during the last 10 years have also shown that rhombomeres are units of gene expression and of cell lineage. Hence, a number of regulatory genes are expressed according to segment-specific patterns in the hindbrain and have been implicated in the pattern formation process. In this review, we focus on the analysis of the function and regulation of these genes along the different steps of hindbrain segmentation, from segment delimitation to acquisition of positional identity. On this basis, we propose a model for the control of early hindbrain development.     

Expression patterns of Slit and Robo family members during vertebrate limb development

Cell interactions involving Notch signaling are required for the demarcation of tissue boundaries in both invertebrate and vertebrate development. Members of the Fringe gene family encode beta-1,3 N-acetyl-glucosaminyltransferases that function to refine the spatial localization of Notch-receptor signaling to tissue boundaries. In this paper we describe the isolation and characterization of the zebrafish (Danio rerio) homologue of the lunatic fringe gene (lfng). Zebrafish lfng is generally expressed in equivalent structures to those reported for the homologous chick and mouse genes. These sites include expression along the A-P axis of the neural tube, within the lateral plate mesoderm, in the presomitic mesoderm and the somites and in specific rhombomeres of the hindbrain; however, within these general expression domains species-specific differences in lfng expression exist. In mouse, Lfng is expressed in odd-numbered rhombomeres, whereas in zebrafish, expression occurs in even-numbered rhombomeres. In contrast to reports in both mouse and chicken embryos showing a kinematic cyclical expression of Lfng mRNA in the presomitic paraxial mesoderm, we find no evidence for a cyclic pattern of expression for the zebrafish lfng gene; instead, the zebrafish lfng is expressed in two static stripes within the presomitic mesoderm. Nevertheless, in zebrafish mutants affecting the correct formation of segment boundaries in the hindbrain and somites, lfng expression is aberrant or lost.     

Rhombomere formation and hind-brain crest cell migration from prorhombomeric origins in mouse embryos

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind-brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind-brain, suggesting that prorhombomeres primarily underlie mammalian hind-brain segmentation.     

The role of kreisler in segmentation during hindbrain development.

The mouse kreisler gene is expressed in rhombomeres (r) 5 and 6 during neural development and kreisler mutants have patterning defects in the hindbrain that are not fully understood. Here we analyzed this phenotype with a combination of genetic, molecular, and cellular marking techniques. Using Hox/lacZ transgenic mice as reporter lines and by analyzing Eph/ephrin expression, we have found that while r5 fails to form in these mice, r6 is present. This shows that kreisler has an early role in the formation of r5. We also observed patterning defects in r3 and r4 that are outside the normal domain of kreisler expression. In both heterozygous and homozygous kreisler embryos some r5 markers are induced in r3, suggesting that there is a partial change in r3 identity that is not dependent upon the loss of r5. To investigate the cellular character of r6 in kreisler embryos we performed heterotopic grafting experiments in the mouse hindbrain to monitor its mixing properties. Control experiments revealed that cells from even- or odd-numbered segments only mixed freely with themselves, but not with cells of opposite character. Transposition of cells from the r6 territory of kreisler mutants reveals that they adopt mature r6 characteristics, as they freely mix only with cells from even-numbered rhombomeres. Analysis of Phox2b expression shows that some aspects of later neurogenesis in r6 are altered, which may be associated with the additional roles of kreisler in regulating segmental identity. Together these results suggest that the formation of r6 has not been affected in kreisler mutants. This analysis has revealed phenotypic and mechanistic differences between kreisler and its zebrafish equivalent valentino. While valentino is believed to subdivide preexisting segmental units, in the mouse kreisler specifies a particular segment. The formation of r6 independent of r5 argues against a role of kreisler in prorhombomeric segmentation of the mouse hindbrain. We conclude that the mouse kreisler gene regulates multiple steps in segmental patterning involving both the formation of segments and their A-P identity.     

Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals,retinoic acid and Hoxa3

We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.