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.     

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.     

FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain

The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. We show that rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, our findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate.     

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.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified 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.     

Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.     

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.     

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.     

Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries

In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib ^ta52b allele than in mib ^tfi91 allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib ^ta52b mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.     

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.     

Patterning of chick brain vesicles as revealed by peanut agglutinin and cholinesterases.

Differentiation of individual rhombomeres of the chicken hindbrain directly follows the emergence of primary brain vesicles. Immediately after the constriction of the prosencephalon at HH9, a series of vesicles of decreasing size is established almost simultaneously between HH9 and HH10, including mesencephalon, four preotic (R2-R5) and one postotic (R6/R7) rhombomeres. Thereby, the cranial neural tube is ventrally embedded in a mesodermal PNA-binding matrix that particularly accumulates underneath vesicular constriction sites, as demonstrated for the segregation of the prosencephalon at HH9 and the cerebellar rhombomere R1 from R2 at HH13. The subsequent period of hindbrain differentiation is analyzed by cholinesterase (AChE, BChE) and peanut lectin histochemistry, by the BrdU and the neurite-specific G4 antibodies. Preotically, differentiation of two pairs of rhombomeres (R4 + R5, R2 + R3) starts in R4, immediately followed by R2. The caudal rhombomeres of both pairs are delayed (R5, R3). Then the postotic rhombomere is subdivided, whereby R7 differentiates before R6. Thus, the development in the direct vicinity of the otic vesicle is delayed (R5, R6). R7 is the last rhombomere that is demarcated caudally. Based on these findings, we postulate two processes that may regulate rhombomere formation in the chicken embryo: (a) an early rostrocaudal wave establishing the major brain vesicles, (b) a superimposed pairwise segmentation emanating rostrally and caudally from the otic vesicle. The segregation of the cerebellar rhombomere is a late step.     

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.     

Key roles of retinoic acid receptors alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches and otocyst in the mouse.

Mouse fetuses carrying targeted inactivations of both the RAR(&agr;) and the RARbeta genes display a variety of malformations in structures known to be partially derived from the mesenchymal neural crest originating from post-otic rhombomeres (e.g. thymus and great cephalic arteries) (Ghyselinck, N., Dupé, V., Dierich, A., Messaddeq, N., Garnier, J.M., Rochette-Egly, C., Chambon, P. and Mark M. (1997). Int. J. Dev. Biol. 41, 425-447). In a search for neural crest defects, we have analysed the rhombomeres, cranial nerves and pharyngeal arches of these double null mutants at early embryonic stages. The mutant post-otic cranial nerves are disorganized, indicating that RARs are involved in the patterning of structures derived from neurogenic neural crest, even though the lack of RARalpha and RARbeta has no detectable effect on the number and migration path of neural crest cells. Interestingly, the double null mutation impairs early developmental processes known to be independent of the neural crest e.g., the initial formation of the 3rd and 4th branchial pouches and of the 3rd, 4th and 6th arch arteries. The double mutation also results in an enlargement of rhombomere 5, which is likely to be responsible for the induction of supernumerary otic vesicles, in a disappearance of the rhombomere 5/6 boundary, and in profound alterations of rhombomere identities. In the mutant hindbrain, the expression domain of kreisler is twice its normal size and the caudal stripe of Krox-20 extends into the presumptive rhombomeres 6 and 7 region. In this region, Hoxb-1 is ectopically expressed, Hoxb-3 is ectopically up-regulated and Hoxd-4 expression is abolished. These data, which indicate that retinoic acid signaling through RARalpha and/or RARbeta is essential for the specification of rhombomere identities and for the control of caudal hindbrain segmentation by restricting the expression domains of kreisler and of Krox-20, also strongly suggest that this signaling plays a crucial role in the posteriorization of the hindbrain neurectoderm.     

Distinct contributions from the hindbrain and mesenchyme to inner ear morphogenesis

A mature inner ear is a complex structure consisting of vestibular and auditory components. Microsurgical ablations, rotations, and translocations were performed in ovo to identify the tissues that control inner ear morphogenesis. We show that mesenchyme/ectoderm adjacent to the developing ear specifically governs the shape of vestibular components - the semicircular canals and ampullae - by conferring anteroposterior axial information to these structures. In contrast, removal of individual hindbrain rhombomeres adjacent to the developing ear preferentially affects the growth and morphogenesis of the auditory subdivision, the cochlear duct, or basilar papilla. Removal of rhombomere 5 affects cochlear duct growth, while rhombomere 6 removal affects cochlear growth and morphogenesis. Rotating rhombomeres 5 and 6 along the anteroposterior axis also impacts cochlear duct morphogenesis but has little effect on the vestibular components. Our studies indicate that discrete tissues, acting at a distance, control the morphogenesis of distinct elements of the inner ear. These results provide a basis for identifying factors that are essential to vestibular and auditory development in vertebrates.