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.     

Stability and plasticity of neural crest patterning and branchial arch Hox code after extensive cephalic crest rotation

The extent to which the spatial organisation of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within crest while contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. But in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings thus identify processes of relevance to the course of normal development, we have performed a series of neural tube and crest rotations producing a more extensive reorganisation of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonised in normal development, rather it simply populates the nearest available branchial arches. We also find that crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations not occurring in normal development. We find that after exchange of first and third arch crest by rotation of r1-7, crest alters its expression of hoxa-2 and hoxa-3 to match its new location within the embryo resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistence of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. We suggest that the intrinsic properties of crest have an effect on the spatial organisation of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or "community effects" within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation we find that there is a tight link between the resulting branchial arch Hox code and a particular skeletal morphology.     

The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells

BACKGROUND: During vertebrate head development, neural crest cells migrate from hindbrain segments to specific branchial arches, where they differentiate into distinct patterns of skeletal structures. The rostrocaudal identity of branchial neural crest cells appears to be specified prior to migration, so it is important that they are targeted to the correct destination. In Xenopus embryos, branchial neural crest cells segregate into four streams that are adjacent during early stages of migration. It is not known what restricts the intermingling of these migrating cell populations and targets them to specific branchial arches. Here, we investigated the role of Eph receptors and ephrins-mediators of cell-contact-dependent interactions that have been implicated in neuronal pathfinding-in this targeted migration. RESULTS: Xenopus EphA4 and EphB1 are expressed in migrating neural crest cells and mesoderm of the third arch, and third plus fourth arches, respectively. The ephrin-B2 ligand, which interacts with these receptors, is expressed in the adjacent second arch neural crest and mesoderm. Using truncated receptors, we show that the inhibition of EphA4/EphB1 function leads to abnormal migration of third arch neural crest cells into second and fourth arch territories. Furthermore, ectopic activation of these receptors by overexpression of ephrin-B2 leads to scattering of third arch neural crest cells into adjacent regions. Similar disruptions occur when the expression of ephrin-B2 or truncated receptors is targeted to the neural crest. CONCLUSIONS: These data indicate that the complementary expression of EphA4/EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of third and second arch neural crest and in targeting third arch neural crest to the correct destination. Together with previous work showing that Eph receptors and ligands mediate neuronal growth cone repulsion, our findings suggest that similar mechanisms are used for neural crest and axon pathfinding.     

HOXD4 and regulation of the group 4 paralog genes

From an evolutionary perspective, it is important to understand the degree of conservation of cis-regulatory mechanisms between paralogous Hox genes. In this study, we have used transgenic analysis of the human HOXD4 locus to identify one neural and two mesodermal 3' enhancers that are capable of mediating the proper anterior limits of expression in the hindbrain and paraxial mesoderm (somites), respectively. In addition to directing expression in the central nervous system (CNS) up to the correct rhombomere 6/7 boundary in the hindbrain, the neural enhancer also mediates a three rhombomere anterior shift from this boundary in response to retinoic acid (RA), mimicking the endogenous Hoxd4 response. We have extended the transgenic analysis to Hoxa4 identifying mesodermal, neural and retinoid responsive components in the 3' flanking region of that gene, which reflect aspects of endogenous Hoxa4 expression. Comparative analysis of the retinoid responses of Hoxd4, Hoxa4 and Hoxb4 reveals that, while they can be rapidly induced by RA, there is a window of competence for this response, which is different to that of more 3' Hox genes. Mesodermal regulation involves multiple regions with overlapping or related activity and is complex, but with respect to neural regulation and response to RA, Hoxb4 and Hoxd4 appear to be more closely related to each other than Hoxa4. These results illustrate that much of the general positioning of 5' and 3' flanking regulatory regions has been conserved between three of the group 4 paralogs during vertebrate evolution, which most likely reflects the original positioning of regulatory regions in the ancestral Hox complex.     

Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.     

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.     

Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch

The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3'RARE)/Hoxb1(3'RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3'RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3'RARE) and Hoxa1(null)/Hoxb1(null )double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

Tooth development in urodele amphibians occurs from a restricted region of anterior cranial neural crest. An in vitro culture system was used to test the odontogenic potential of more caudal regions of neural crest, including an "intermediate region" of neural folds which has never previously been tested for either fate or potential. Explants of different axial levels of neural crest with stomodaeal ectoderm and endoderm demonstrated that odontogenic potential extends not only further caudally than the axial level fated to produce teeth, but also beyond that with potential to produce cartilage. Our results show that chondrogenic potential is found only within the most rostral portion of the intermediate region, but that odontogenic potential extends to its most caudal limit. This separation of skeletogenic cell lineages in the neural crest necessitates a reevaluation of the designations of "cranial" and "trunk" and a reconsideration of the evolutionary implications of developmentally distinct crest-derived mesenchyme populations. The proposal that odontogenic potential extends into the trunk neural crest may be explained as conserved from a phylogenetically older, more extensive skeletogenic ability which produced the exoskeleton of more basal vertebrates.     

Segmental identity can change independently in the hindbrain and rhombencephalic neural crest

In this study we tested whether the segmental identities of the hindbrain and its derived neural crest are necessarily linked or, instead, if they can be altered independently. Using morphological criteria, we show that the hindbrains of Hoxa-2 mutant mice, in which the second arch skeletal derivatives assume first arch characteristics (Gendron-Maguire et al. [1993] Cell 75:1317-1331; Rijli et al. [1993] Cell 75:1333-1349), retain normal segmental identities. Also, by phenotypic analysis, we show that, with retinoic acid, changes can be induced in the identity of the preotic hindbrain without effects in its derived neural crest. Our data thus indicate that identity changes in the hindbrain and branchial arch neural crest can occur independently. Moreover, if Hoxa-2 is concomitantly induced by retinoic acid in the first branchial arch, the proximal derivatives of this arch are also affected. We propose a model for the patterning of the branchial region, according to which the segmental identity in this area is provided mainly by the branchial arches.     

Angiotensinogen genotype, sodium reduction, weight loss, and prevention of hypertension: trials of hypertension prevention, phase II

Hox genes are segmentally expressed in the developing vertebrate hindbrain, neural crest cells and pharyngeal arches suggesting an important role in patterning these structures. Here we discuss the cellular and molecular mechanisms controlling segmentation and specification in the branchial region of the head. In addition, based on the recent phenotypical and molecular analysis of loss-of-function mutants in the mouse, we speculate that Hox genes may act like Drosophila selector genes in this system.     

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.     

Modulation of cytokine gene expression by thymic lympho-stromal cell to cell interaction: effect of retinoic acid

We have examined the expression of a panel of cytokines in thymic epithelial cells and CD4-CD8- (DN) thymocytes following cell to cell lymphostromal interaction, in an experimental model which enhances in vitro thymocyte maturation. Since retinoic acid (RA) has been previously shown to be an inhibitor of thymocyte maturation process in this model, we wanted to analyse cytokine expression in DN thymocytes and thymic epithelial cells following the RA-induced impairment of in vitro thymocyte maturation. Cell to cell lymphostromal interaction results in increased IL2 and decreased IL7 expression in thymocytes while the expression of IL1 beta and IL7 increased and decreased, respectively, in thymic epithelial cells. Addition of RA to lympho-stromal cell co-culture results in the enhancement of IL4 and IL7 expression in thymocytes while in thymic epithelial cells IL1 alpha decreased and IL6 and IL7 increased. These data indicate that discrete patterns of cytokine expression are present in thymocyte precursors and in thymic epithelial cells during in vitro T-cell development. They furthermore suggest that specific cytokine modulation might contribute to the RA-induced impairment of thymocyte differentiation.     

Improved synthesis of an aldobiouronic acid related to hardwood xylans, and preparation of a derivative thereof suitable for linking to proteins

We investigated the expression pattern of the endothelin-A receptor and endothelin 1 genes, the mutations of which affect the development of the mesectodermal derivatives of the neural crest. We show here that endothelin 1 is expressed by the environment of the cephalic neural crest cells invading branchial arches. Later on, while the neural crest-derived tissues of the head continue to express endothelin-A receptor, endothelin 1 is no longer expressed in their environment.     

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.     

Modelling the prevalence of Echinococcus and Taenia species in small ruminants of different ages in northern Jordan

The effect of all-trans retinoic acid on embryogenesis was studied in a cyclostome, Lampetra japonica. Treatment with 0.05-0.5 microM retinoic acid on early gastrula and early neurula resulted in loss of the pharynx and in the rostral truncation of the neural tube. The mouth, pharynx, esophagus, heart, endostyle, and rostral brain were missing with graded severity. In the severest case, the embryo consisted only of trunk segments, especially myotomes that extended to the rostral end of the axis. The effect appeared to be dose- and stage-dependent: Rostral pharyngeal arches were more vulnerable to a lower amount of retinoic acid, and earlier treatment resulted in severer defects. The initial protrusion of the anterior axis started equally in control and retinoic acid-treated embryos, implying that the head morphogenesis is omitted in treated embryos. By identifying the number of myotomes based on the differentiation of hypobranchial muscles, there seemed to be no myotomes lost by retinoic acid-induced truncation. The rostral truncation, therefore, was not simply a limitation of the anterior axis but was restricted to the ventral portion; only the branchial arches disappeared with normally developing myotomes dorsally. The absent region can be defined as the vertebrate head in a morphological sense, including the branchiomeric and preotic paraxial regions as well as the heart. The results suggest the presence of distinct programs between somitomeric and branchiomeric portions of the body, providing a developmental basis for the dual-metamerical body plan of vertebrates.