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.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.     

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)     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

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.     

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.     

From hindbrain segmentation to breathing after birth

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated within central pattern-generating neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the most understood part with respect to the cellular and molecular mechanisms that underlie its development. Hox paralogs and Hox-regulating genes kreisler/mafB and Krox20 are required for the normal formation of rhombomeres in vertebrate embryos. From studies of rhombomeres r3 and r4, the authors review mechanisms whereby these developmental genes may govern the early embryonic development of para-facial neuronal networks and specify patterns of motor activities operating throughout life. A model whereby the regional identity of progenitor cells can be abnormally specified in r3 and r4 after a mutation of these genes is proposed. Novel neuronal circuits may develop from some of these misspecified progenitors while others are eliminated, eventually affecting respiration and survival after birth.     

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.     

From hindbrain segmentation to breathing after birth: developmental patterning in rhombomeres 3 and 4

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated within central pattern-generating neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the most understood part with respect to the cellular and molecular mechanisms that underlie its development. Hox paralogs and Hox-regulating genes kreisler/mafB and Krox20 are required for the normal formation of rhombomeres in vertebrate embryos. From studies of rhombomeres r3 and r4, the authors review mechanisms whereby these developmental genes may govern the early embryonic development of para-facial neuronal networks and specify patterns of motor activities operating throughout life. A model whereby the regional identity of progenitor cells can be abnormally specified in r3 and r4 after a mutation of these genes is proposed. Novel neuronal circuits may develop from some of these misspecified progenitors while others are eliminated, eventually affecting respiration and survival after birth.     

Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

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.     

Effects of 13-cis-retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis)

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid-exposed Macaca fascicularis embryos. 13-cis-retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb-1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre- and post-otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.     

DBHR, a gene with homology to dopamine beta-hydroxylase, is expressed in the neural crest throughout early development

In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine beta-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling.     

Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos.

Retinoic acid is a very potent teratogen and has also been implicated as an endogenous developmental signalling molecule in vertebrate embryos. One of the regions of the embryo reliably affected by exogenously applied RA is the hindbrain. In this paper, we describe in detail the hindbrain of Xenopus laevis embryos briefly treated with various levels of RA at gastrula stages. Such treatments lead to development of embryos with loss of anterior structures. In addition, RA has a general effect on rhombomere morphology and specific effects on the development of the anterior rhombomeres. This effect is demonstrated using neurofilament antibodies, HRP staining and in situ hybridisation using a probe for expression of the Xenopus Krox-20 gene. Anatomically it is evident that the development of the hindbrain normally anterior to the otocyst (rhombomeres 1-4) is abnormal following RA treatment. Sensory and motor axons of cranial nerves V and VII form a single root and the peripheral paths of V and VII and IX and X are also abnormal, as is the more anterior location of the otocyst. These anatomical changes are accompanied by changes in the pattern of expression for the gene XKrox-20, which normally expresses in rhombomeres 3 and 5, but is found in a single band in the anterior hindbrain of treated embryos which standardly fail to generate the normal external segmental appearance. The results are discussed in terms of both the teratogenic and possible endogenous roles of RA during normal development of the central nervous system. We conclude that low doses of RA applied during gastrulation have specific effects on the anterior Xenopus hindbrain which appear to be evolutionarily conserved in the light of similar recent findings in zebrafish.