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.     

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.     

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.     

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.     

Hox gene colinear expression in the avian medulla oblongata is correlated with pseudorhombomeric domains

The medulla oblongata (or caudal hindbrain) is not overtly segmented, since it lacks observable interrhombomeric boundaries. However, quail-chick fate maps showed that it is formed by 5 pseudorhombomeres (r7-r11) which were empirically found to be delimited consistently at planes crossing through adjacent somites (Cambronero and Puelles, 2000). We aimed to reexamine the possible segmentation or rostrocaudal regionalisation of this brain region attending to molecular criteria. To this end, we studied the expression of Hox genes from groups 3 to 7 correlative to the differentiating nuclei of the medulla oblongata. Our results show that these genes are differentially expressed in the mature medulla oblongata, displaying instances of typical antero-posterior (3′ to 5′) Hox colinearity. The different sensory and motor columns, as well as the reticular formation, appear rostrocaudally regionalised according to spaced steps in their Hox expression pattern. The anterior limits of the respective expression domains largely fit boundaries defined between the experimental pseudorhombomeres. Therefore the medulla oblongata shows a Hox-related rostrocaudal molecular regionalisation comparable to that found among rhombomeres, and numerically consistent with the pseudorhombomere list. This suggests that medullary pseudorhombomeres share some AP patterning mechanisms with the rhombomeres present in the rostral, overtly-segmented hindbrain, irrespective of variant boundary properties.     

Multiple morphogens and rapid elongation promote segmental patterning during development

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.     

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.     

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.     

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.     

Signalling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis

During central nervous system development, neural progenitors are patterned to form discrete neurogenic and non-neurogenic zones. In the zebrafish hindbrain, neurogenesis is organised by Fgf20a emanating from neurons located at each segment centre that inhibits neuronal differentiation in adjacent progenitors. Here, we have identified a molecular mechanism that clusters fgf20a-expressing neurons in segment centres and uncovered a requirement for this positioning in the regulation of neurogenesis. Disruption of hindbrain boundary cell formation alters the organisation of fgf20a-expressing neurons, consistent with a role of chemorepulsion from boundaries. The semaphorins Sema3fb and Sema3gb, which are expressed by boundary cells, and their receptor Nrp2a are required for clustering of fgf20a-expressing neurons at segment centres. The dispersal of fgf20a-expressing neurons that occurs following the disruption of boundaries or of Sema3fb/Sema3gb signalling leads to reduced FGF target gene expression in progenitors and an increased number of differentiating neurons. Sema3 signalling from boundaries thus links hindbrain segmentation to the positioning of fgf20a-expressing neurons that regulates neurogenesis.     

An evaluation of myelomeres and segmentation of the chick embryo spinal cord.

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of K?llén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.     

Segmental development of reticulospinal and branchiomotor neurons in lamprey: insights into the evolution of the vertebrate hindbrain

During development, the vertebrate hindbrain is subdivided along its anteroposterior axis into a series of segmental bulges called rhombomeres. These segments in turn generate a repeated pattern of rhombomere-specific neurons, including reticular and branchiomotor neurons. In amphioxus (Cephalochordata), the sister group of the vertebrates, a bona fide segmented hindbrain is lacking, although the embryonic brain vesicle shows molecular anteroposterior regionalization. Therefore, evaluation of the segmental patterning of the central nervous system of agnathan embryos is relevant to our understanding of the origin of the developmental plan of the vertebrate hindbrain. To investigate the neuronal organization of the hindbrain of the Japanese lamprey, Lethenteron japonicum, we retrogradely labeled the reticulospinal and branchial motoneurons. By combining this analysis with a study of the expression patterns of genes identifying specific rhombomeric territories such as LjKrox20, LjPax6, LjEphC and LjHox3, we found that the reticular neurons in the lamprey hindbrain, including isthmic, bulbar and Mauthner cells, develop in conserved rhombomere-specific positions, similar to those in the zebrafish. By contrast, lamprey trigeminal and facial motor nuclei are not in register with rhombomere boundaries, unlike those of gnathostomes. The trigeminal-facial boundary corresponds to the rostral border of LjHox3 expression in the middle of rhombomere 4. Exogenous application of retinoic acid (RA) induced a rostral shift of both the LjHox3 expression domain and branchiomotor nuclei with no obvious repatterning of rhombomeric segmentation and reticular neurons. Therefore, whereas subtype variations of motoneuron identity along the anteroposterior axis may rely on Hox-dependent positional values, as in gnathostomes, such variations in the lamprey are not constrained by hindbrain segmentation. We hypothesize that the registering of hindbrain segmentation and neuronal patterning may have been acquired through successive and independent stepwise patterning changes during evolution.     

Cell mixing between the embryonic midbrain and hindbrain

Segmentation is a mechanism that controls spatial organization along the anteroposterior axis of the neural tube and is particularly well characterized for the hindbrain region [1]. The generation of distinct and regionally specific structures from each rhombomere is achieved with the almost complete absence of cell mixing between neighboring rhombomeres [2, 3]. Here, we have examined cell mingling at the isthmus, where Otx2-expressing midbrain cells abut Gbx2-expressing hindbrain cells [4]. The sharp line of demarcation between the two expression domains suggests that this interface would be a compartment boundary, with no intermixing of cells, but this has not been directly tested. We have used short-term reaggregation assays to compare the adhesive properties of cells derived from midbrain and anterior hindbrain and cell labeling in vivo directly to monitor cell behavior at the midbrain/hindbrain boundary. Interestingly, our data demonstrate that, in contrast to the rhombomeres, differential adhesion does not seem to operate between the midbrain and anterior hindbrain and that cells move between the two territories. We conclude that these two subdivisions are not maintained by cell lineage restriction but by cells maintaining labile fates.     

Asymmetric division in fucoid zygotes is positioned by telophase nuclei

The relative contributions of cell polarity and nuclear position in specifying the plane of asymmetric division in fucoid zygotes were investigated. In zygotes developing normally, telophase nuclei were positioned parallel to the polar growth axis, and the division plane bisected both axes. To assess division plane specification, the colinearity of the nuclear and growth axes was uncoupled by treatment with pharmacological agents. Spatial correlations between the growth axis, telophase nuclei, and the division plane were analyzed in the treated zygotes. In all cases, cytokinesis was oriented transverse to the telophase mitotic array and was less well aligned with the growth axis. Telophase nuclei also played a predominant role in positioning the division plane in polyspermic zygotes. Microtubules from the telophase nuclei interdigitated throughout the plane of subsequent cytokinesis, and we speculate that they specify the division plane. Morphological markers of the division plane were not observed before telophase; the earliest division marker detected was a plate of actin that assembled in the zone of microtubule overlap late in telophase. These findings are consistent with division plane specification at cytoplast boundaries.     

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division.
In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.     

Developmental biology: Notching the hindbrain

One of the best-characterized lineage restrictions in developing vertebrates occurs between adjacent -rhombomeres of the hindbrain. It was recently shown that cells at the boundaries of zebrafish rhombomeres also differ from non-boundary cells in their migratory abilities, a difference driven by Notch signaling.     

Retinoic acid synthesis and hindbrain patterning in the mouse embryo

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.