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.     

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.     

Paralog group 1 hox genes regulate rhombomere 5/6 expression of vhnf1, a repressor of rostral hindbrain fates, in a meis-dependent manner

The vertebrate hindbrain is segmented into an array of rhombomeres (r), but it remains to be fully understood how segmentation is achieved. Here we report that reducing meis function transforms the caudal hindbrain to an r4-like fate, and we exploit this experimental state to explore how r4 versus r5-r6 segments are set aside. We demonstrate that r4 transformation of the caudal hindbrain is mediated by paralog group 1 (PG1) hox genes and can be repressed by vhnf1, a gene expressed in r5-r6. We further find that vhnf1 expression is regulated by PG1 hox genes in a meis-dependent manner. This implies that PG1 hox genes not only induce r4 fates throughout the caudal hindbrain, but also induce expression of vhnf1, which then represses r4 fates in the future r5-r6. Our results further indicate that r4 transformation of the caudal hindbrain occurs at intermediate levels of meis function, while extensive removal of meis function produces a hindbrain completely devoid of segments, suggesting that different hox-dependent processes may have distinct meis requirements. Notably, reductions in the function of another Hox cofactor, pbx, have not been reported to transform the caudal hindbrain, suggesting that Meis and Pbx proteins may also function differently in their roles as Hox cofactors.     

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.     

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.     

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.     

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.     

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.     

Complex Regulation of cyp26a1 Creates a Robust Retinoic Acid Gradient in the Zebrafish Embryo

Positional identities along the anterior–posterior axis of the vertebrate nervous system are assigned during gastrulation by multiple posteriorizing signals, including retinoic acid (RA), fibroblast growth factors (Fgfs), and Wnts. Experimental evidence has suggested that RA, which is produced in paraxial mesoderm posterior to the hindbrain by aldehyde dehydrogenase 1a2 (aldh1a2/raldh2), forms a posterior-to-anterior gradient across the hindbrain field, and provides the positional information that specifies the locations and fates of rhombomeres. Recently, alternative models have been proposed in which RA plays only a permissive role, signaling wherever it is not degraded. Here we use a combination of experimental and modeling tools to address the role of RA in providing long-range positional cues in the zebrafish hindbrain. Using cell transplantation and implantation of RA-coated beads into RA-deficient zebrafish embryos, we demonstrate that RA can directly convey graded positional information over long distances. We also show that expression of Cyp26a1, the major RA-degrading enzyme during gastrulation, is under complex feedback and feedforward control by RA and Fgf signaling. The predicted consequence of such control is that RA gradients will be both robust to fluctuations in RA synthesis and adaptive to changes in embryo length during gastrulation. Such control also provides an explanation for the fact that loss of an endogenous RA gradient can be compensated for by RA that is provided in a spatially uniform manner.     

Hindbrain patterning involves graded responses to retinoic acid signalling

Several recent studies have shown that retinoic acid signalling is required for correct patterning of the hindbrain. However, the data from these studies are disparate and the precise role of retinoic acid signalling in patterning the anteroposterior axis of the neural tube remains uncertain. To help clarify this issue, we have cultured a staged series of chick embryos in the presence of an antagonist to the all three retinoic acid receptors. Our data indicate that retinoic acid is the transforming signal involved in the expansion of posterior hindbrain structures. We find that the hindbrain region of the neural tube down to the level of the sixth somite acquires the identity of rhombomere 4 when retinoic acid signalling is blocked. Specification of future rhombomere boundaries has a retinoic acid dependency between stage 5 and stage 10(+) that is lost progressively in an anterior-to-posterior sequence. Furthermore, the application of various concentrations of antagonist shows that successively more posterior rhombomere boundaries require progressively higher concentration of endogenous retinoic acid for their correct positioning, a result that strengthens the hypothesis that a complex retinoid gradient acts to pattern the posterior hindbrain. Our dissection of early retinoic acid functions allows us to re-interpret the wide disparity of hindbrain phenotypes previously observed in various models of retinoic acid deficiency.     

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.     

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.     

Cyp26C1 encodes a novel retinoic acid-metabolizing enzyme expressed in the hindbrain,inner ear,first branchial arch and tooth buds during murine development

Retinoic acid (RA), an active metabolite of vitamin A, is a crucial signaling molecule involved in tissue morphogenesis during embryonic development. RA distribution and concentration is precisely regulated during embryogenesis by balanced complementary activities of RA synthesizing (RALDH) and metabolizing (CYP26) enzymes. Here, we describe the identification of a novel murine p450 cytochrome belonging to the CYP26 family, mCYP26C1. Sequence alignment show that mCYP26C1 is more closely related to mCYP26B1 than mCYP26A1. At early developmental stages (E8.0-E8.5), mCyp26C1 is expressed in prospective rhombomeres 2 and 4, in the first branchial arch and along the lateral surface mesenchyme adjacent to the rostral hindbrain. At E9.5, mCyp26C1 expression persists in rhombomere 2 and in the maxillary and mandibular components of the first branchial arch, and is strongly induced in the lateral cervical mesenchyme. By mid-gestation, mCyp26C1 is weakly expressed in the cervical mesenchyme and in the maxillary component of the first branchial arch. At E11.5, mCyp26C1 can only be seen in a narrow band in the lateral cervical mesenchyme. During late gestation, mCyp26C1 exhibits region-specific expression in the inner ear epithelium and a persistent expression in the inner dental epithelium of the developing teeth. This pattern of expression suggests that mCYP26C1 may play an important role in protecting the hindbrain, first branchial arch, otocyst and tooth buds against RA exposure during embryonic development.     

Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo

Positional identities along the anterior–posterior axis of the vertebrate nervous system are assigned during gastrulation by multiple posteriorizing signals, including retinoic acid (RA), fibroblast growth factors (Fgfs), and Wnts. Experimental evidence has suggested that RA, which is produced in paraxial mesoderm posterior to the hindbrain by aldehyde dehydrogenase 1a2 (aldh1a2/raldh2), forms a posterior-to-anterior gradient across the hindbrain field, and provides the positional information that specifies the locations and fates of rhombomeres. Recently, alternative models have been proposed in which RA plays only a permissive role, signaling wherever it is not degraded. Here we use a combination of experimental and modeling tools to address the role of RA in providing long-range positional cues in the zebrafish hindbrain. Using cell transplantation and implantation of RA-coated beads into RA-deficient zebrafish embryos, we demonstrate that RA can directly convey graded positional information over long distances. We also show that expression of Cyp26a1, the major RA-degrading enzyme during gastrulation, is under complex feedback and feedforward control by RA and Fgf signaling. The predicted consequence of such control is that RA gradients will be both robust to fluctuations in RA synthesis and adaptive to changes in embryo length during gastrulation. Such control also provides an explanation for the fact that loss of an endogenous RA gradient can be compensated for by RA that is provided in a spatially uniform manner.