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.     

Expression of the mouse labial-like homeobox-containing genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain.

The sequence of a mouse Hox 2.9 cDNA clone is presented. The predicted homeodomain is similar to that of the Drosophila gene labial showing 80% identity. The equivalent gene in the Hox 1 cluster is Hox 1.6 which shows extensive similarity to Hox 2.9 both within and outside the homeodomain. Hox 2.9 and Hox 1.6 are the only two mouse members of the labial-like family of homeobox-containing genes as yet identified. Hox 2.9 has previously been shown to be expressed in a single segmental unit of the developing hindbrain (rhombomere) and has been predicted to be involved in conferring rhombomere identity. To analyse further the function of Hox 2.9 during development and to determine if the other mouse labial-like gene Hox 1.6, displays similar properties, we have investigated the expression patterns of these two genes and an additional rhombomere-specific gene, Krox 20, on consecutive embryonic sections at closely staged intervals. This detailed analysis has enabled us to draw the following conclusions: (1) There are extensive similarities in the temporal and spatial expression of Hox 2.9 and Hox 1.6, throughout the period that both genes are expressed in the embryo (7 1/2 to 10 days). At 8 days the genes occupy identical domains in the neuroectoderm and mesoderm with the same sharp anterior boundary in the presumptive hindbrain. These similarities indicate a functional relationship between the genes and further suggest that the labial-like genes are responding to similar signals in the embryo. (2) By 9 days the neuroectoderm expression of both genes retreats posteriorly along the anteroposterior (AP) axis. The difference at this stage between the expression patterns is the persistence of Hox 2.9 in a specific region of the hindbrain, illustrating the capacity of Hox 2.9 to respond to additional positional regulatory signals and indicating a unique function for this gene in the hindbrain. (3) The restriction of Hox 2.9 expression in the hindbrain occurs at 8 1/2 days, approximately the same time as Krox 20 is first detected in the posterior adjoining domain. The mutually exclusive expression of Hox 2.9 and Krox 20 demarcated by sharp expression boundaries suggest that compartmentalisation of cells within the hindbrain has occurred up to 6 h before rhombomeres (morphological segments) are clearly visible. (4) Hox 2.9 expression is confined to the region of rhombomere 4 that shows cell lineage restriction and, unlike Krox 20, is expressed throughout the period that rhombomeres are visible (to 11 1/2 days).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. Here, we show that during neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize in controlling the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain.     

Knockdown of the complete Hox paralogous group 1 leads to dramatic hindbrain and neural crest defects

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.     

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.     

Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression

Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the rl territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.     

Hox Genes and Segmental Patterning of the Vertebrate Hindbrain

SYNOPSIS. Pattern formation in the developing hindbrain andcranio-facial region has been studied in a range of vertebrateorganisms. The developing hindbrain is transiently segmentedinto units termed rhombomeres which correspond with domainsof gene expression, lineage restriction and neuronal organizationand serve to coordinate the migration of cranial neural crestinto the adjacent branchial arches. In this paper I review thecellular and molecular events underlying both hindbrain segmentationand the acquisition of segmental identity, consolidating recentresults from different model systems. Data suggesting that thevertebrate Hox genes play an important role in specifying positionalvalue to the rhombomeres and cranial neural crest are also examined.I compare expression patterns of the Hox genes between speciesand consider the mechanisms involved in controlling their appropriatespatial regulation. In addition I describe a recently characterizedzebraflsh hindbrain segmentation mutant, Valentino; morphological,cellular and gene expression data for this mutant are helpingto further our understanding of hindbrain patterning.     

Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins

Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. We have previously shown that in ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Here, we show that Phox2b is a direct target of Hoxb1 and Hoxb2. We found a highly conserved Phox2b proximal enhancer that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. We further show that both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2b enhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression mechanism. Finally, we show that induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context.     

Hindbrain respecification in the retinoid-deficient quail

We report here the development and rescue of the truncated hindbrain of retinoid-deprived quail embryos. The embryo is completely rescued by an injection of retinol into the egg; this confirms retinol, or a related retinoid, as a required molecule in hindbrain development. Staging the retinoid replacement enabled us to determine that the 3-4 somite stage is the period when retinoids are required for normal development. Analysis of the development of the retinoid-deprived hindbrain phenotype through somitogenesis has revealed a pathway of retinoid action in early hindbrain regionalization. The hindbrain of the retinoid-deprived embryo is normal in size, during early somitogenesis, but has a respecified pattern of Krox-20 expression. From the earliest expression of Krox-20, at the 5 somite stage, the rhombomere 3 stripe fills the caudal third of the developing hindbrain to the level of the first somite. Morphologically only 2, instead of the normal 5, rhombomere bulges form. These 2 bulges express genes and, later, develop morphology characteristic of rhombomeres 1 and 2 and rhombomere 3. Posterior hindbrain specific genes, Hoxb-1, Fgf3, MafB, and the rhombomere 5 stripe of Krox-20 are never expressed in the head neuroepithelium of these embryos. From the initial formation of the neural plate, there is no evidence of rhombomere 4-7 specific characteristics. These results indicate the specification of the posterior hindbrain is lost and its cells participate in the formation of an enlarged anterior hindbrain. In our previous study, we reported the absence of the posterior hindbrain in retinoid-deprived quails (Maden, M., Gale, E., Kostetskii, I., Zile, M., 1996. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417-426). Here, we show this phenotype to be the result of respecification of the hindbrain cells. This provides evidence for a region specific response to a single stimulus, retinol, which suggests a pre-rhombomeric regionalization of the hindbrain.     

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.     

Specification of distinct motor neuron identities by the singular activities of individual Hox genes.

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. We have employed a direct gain-of-function approach using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though it is normally devoid of this cell type. These ectopic neurons have the specific identity of branchiomotor neurons and, in the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. Next, we have attempted to identify the precise branchiomotor subtypes that are generated after misexpression and our results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. Our data demonstrate, therefore, that at least to a certain extent and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, as these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, our data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance.     

spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.