Coupling segmentation to axis formation

A characteristic feature of the vertebrate body is its segmentation along the anteroposterior axis, as illustrated by the repetition of vertebrae that form the vertebral column. The vertebrae and their associated muscles derive from metameric structures of mesodermal origin, the somites. The segmentation of the body is established by somitogenesis, during which somites form sequentially in a rhythmic fashion from the presomitic mesoderm. This review highlights recent findings that show how dynamic gradients of morphogens and retinoic acid, coupled to a molecular oscillator, drive the formation of somites and link somitogenesis to the elongation of the anteroposterior axis.     

Segmental relationship between somites and vertebral column in zebrafish

The segmental heritage of all vertebrates is evident in the character of the vertebral column. And yet, the extent to which direct translation of pattern from the somitic mesoderm and de novo cell and tissue interactions pattern the vertebral column remains a fundamental, unresolved issue. The elements of vertebral column pattern under debate include both segmental pattern and anteroposterior regional specificity. Understanding how vertebral segmentation and anteroposterior positional identity are patterned requires understanding vertebral column cellular and developmental biology. In this study, we characterized alignment of somites and vertebrae, distribution of individual sclerotome progeny along the anteroposterior axis and development of the axial skeleton in zebrafish. Our clonal analysis of zebrafish sclerotome shows that anterior and posterior somite domains are not lineage-restricted compartments with respect to distribution along the anteroposterior axis but support a 'leaky' resegmentation in development from somite to vertebral column. Alignment of somites with vertebrae suggests that the first two somites do not contribute to the vertebral column. Characterization of vertebral column development allowed examination of the relationship between vertebral formula and expression patterns of zebrafish Hox genes. Our results support co-localization of the anterior expression boundaries of zebrafish hoxc6 homologs with a cervical/thoracic transition and also suggest Hox-independent patterning of regionally specific posterior vertebrae.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation,patterning and differentiation of somites

The paraxial mesoderm of the somites of the vertebrate embryo contains the precursors of the axial skeleton, skeletal muscles and dermis. The Meox1 and Meox2 homeobox genes are expressed in the somites and their derivatives during embryogenesis. Mice homozygous for a null mutation in Meox1 display relatively mild defects in sclerotome derived vertebral and rib bones, whereas absence of Meox2 function leads to defective differentiation and morphogenesis of the limb muscles. By contrast, mice carrying null mutations for both Meox genes display a dramatic and wide-ranging synthetic phenotype associated with extremely disrupted somite morphogenesis, patterning and differentiation. Mutant animals lack an axial skeleton and skeletal muscles are severely deficient. Our results demonstrate that Meox1 and Meox2 genes function together and upstream of several genetic hierarchies that are required for the development of somites. In particular, our studies place Meox gene function upstream of Pax genes in the regulation of chondrogenic and myogenic differentiation of paraxial mesoderm.     

Zic2 and Zic3 synergistically control neurulation and segmentation of paraxial mesoderm in mouse embryo

Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1-5) have been reported. Despite the partly overlapping expression profiles of these genes, mouse mutants for each Zic show distinct phenotypes. To uncover possible redundant roles, we characterized Zic2/Zic3 compound mutant mice. Zic2 and Zic3 are both expressed in presomitic mesoderm, forming and newly generated somites with differential spatiotemporal accentuation. Mice heterozygous for the hypomorphic Zic2 allele together with null Zic3 allele generally showed severe malformations of the axial skeleton, including asymmetric or rostro-caudally bridged vertebrae, and reduction of the number of caudal vertebral bones, that are not obvious in single mutants. These defects were preceded by perturbed somitic marker expression, and reduced paraxial mesoderm progenitors in the primitive streak. These results suggest that Zic2 and Zic3 cooperatively control the segmentation of paraxial mesoderm at multiple stages. In addition to the segmentation abnormality, the compound mutant also showed neural tube defects that ran the entire rostro-caudal extent (craniorachischisis), suggesting that neurulation is another developmental process where Zic2 and Zic3 have redundant functions.     

Characterization of the Skeletal Fusion with Sterility (sks) Mouse Showing Axial Skeleton Abnormalities Caused by Defects of Embryonic Skeletal Development

The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs.     

Genetic analysis of molecular oscillators in mammalian somitogenesis: clues for studies of human vertebral disorders

The repeating pattern of the human vertebral column is shaped early in development, by a process called somitogenesis. In this embryonic process, pairs of mesodermal segments called somites are serially laid down along the developing neural tube. Somitogenesis is an iterative process, repeating at regular time intervals until the last somite is formed. This process lays down the vertebrate body axis from head to tail, making for a progression of developmental steps along the rostral-caudal axis. In this review, the roles of the Notch, Wnt, fibroblast growth factor, retinoic acid and other pathways are described during the following key steps in somitogenesis: formation of the presomitic mesoderm (PSM) and establishment of molecular gradients; prepatterning of the PSM by molecular oscillators; patterning of rostral-caudal polarity within the somite; formation of somite borders; and maturation and resegmentation of somites to form musculoskeletal tissues. Disruption of somitogenesis can lead to severe vertebral birth defects such as spondylocostal dysostosis (SCD). Genetic studies in the mouse have been instrumental in finding mutations in this disorder, and ongoing mouse studies should provide functional insights and additional candidate genes to help in efforts to identify genes causing human spinal birth defects.     

The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain.

We describe a new zebrafish mutation, neckless, and present evidence that it inactivates retinaldehyde dehydrogenase type 2, an enzyme involved in retinoic acid biosynthesis. neckless embryos are characterised by a truncation of the anteroposterior axis anterior to the somites, defects in midline mesendodermal tissues and absence of pectoral fins. At a similar anteroposterior level within the nervous system, expression of the retinoic acid receptor a and hoxb4 genes is delayed and significantly reduced. Consistent with a primary defect in retinoic acid signalling, some of these defects in neckless mutants can be rescued by application of exogenous retinoic acid. We use mosaic analysis to show that the reduction in hoxb4 expression in the nervous system is a non-cell autonomous effect, reflecting a requirement for retinoic acid signalling from adjacent paraxial mesoderm. Together, our results demonstrate a conserved role for retinaldehyde dehydrogenase type 2 in patterning the posterior cranial mesoderm of the vertebrate embryo and provide definitive evidence for an involvement of endogenous retinoic acid in signalling between the paraxial mesoderm and neural tube.     

Teratogenic effects of retinoic acid and dimethylsulfoxide on embryonic chick wing and somite

In order to document the stage(s) at which the embryonic chick wing bud is sensitive to vitamin A teratogenesis and the kinds of defects produced by vitamin A insult to the embryonic chick wing, 1-microgram doses of retinoic acid (1 microliter RA in 90% DMSO at a concentration of 1 microgram/microliter) were locally applied to the right wing bud of chick embryos at stages 17-23 (Hamburger and Hamilton: J. Morphol., 88:49-92, '51), and the resulting limb skeleton anatomy was observed at 10 days of incubation. Local application of RA at stages 17-20 resulted in distal wing skeleton defects. There were significantly more wing skeleton defects among embryos treated at these stages with RA solution than among solvent (DMSO)-treated control embryos and than among untreated control embryos. Wings of embryos treated with RA at stages 21-23 were always normal. Scapular and vertebral defects were seen at 10 days of incubation among embryos which had been treated prior to stage 21 with both the RA solution and the solvent control. Statistical analysis and histological data suggest that scapular and vertebral defects were caused by DMSO-induced damage to somites.     

Plasticity of axial identity among somites: cranial somites can generate vertebrae without expressing Hox genes appropriate to the trunk

Classic studies have shown that the presomitic mesoderm is already committed to a specific morphological fate, for example, the ability to generate a rib. Hox gene expression in the paraxial mesoderm has also been shown to be fixed early and not susceptible to modulation by an ectopic environment. This is in contrast to the plasticity of Hox expression in neuroectodermal derivatives. We reexamine here the potential of somites for morphological plasticity by transplanting the cranial (occipital) somites 1-4, that normally produce small contributions to the skull, to the trunk of avian embryos. Surprisingly, the transposed cranial somites are able to form reasonably normal vertebral anlage. In addition, the cranial somitic mesoderm produces intervertebral disks, structures not normally found in the skull. These somites are however unable to generate some elements of the vertebrae, such as the costal process. In contrast to the morphogenetic plasticity of the occipital somites, their characteristic inability to support survival of dorsal root ganglia was not significantly modified by posterior transplantation. Dorsal root ganglia initially developed and then degenerated with the same morphological stages as normally observed. In striking contrast to the plasticity of morphology, we found that all four members of the of the fourth paralogous group of Hox genes that are expressed endogenously at the level of the graft are not upregulated in the caudad-transposed cranial mesoderm. It therefore appears that genes other than those of the Hox family normally expressed at this axial level control the position-specific morphogenesis of ectopic vertebrae formed from cranial somites. In evolutionary terms, the present results imply that occipital somites that were incorporated into the "New Head" retain the ability to develop according to their original morphogenetic fate, into vertebrae.     

Extirpation experiments on embryonic rudiments of the carapace of Chelydra serpentina

Somites, along with adjacent neural tube and overlying ectoderm, were extirpated unilaterally from embryos of Chelydra serpentina. Mesoderm of three somites was removed from various levels. The operations included the last formed somite and were done on embryos with 12 to 22 pairs of somites. In practice it was found that ventromedial portions of the somites were not included in the extirpation. The animals were preserved before pigmentation became heavy. The cartilaginous skeleton was stained selectively. The extirpations resulted in depletions of ribs consonant with relating the second rib to the fourteenth somite. The somites behaved as mosaics; they did not reconstitute each other nor did they regenerate after partial extirpation. The rudiments for the ribs were separable from the rudiments of the vertebrae, the sclerotomes, and were found to arise from a more lateral portion of the somite. The scutes are ectodermal derivatives, which are held to be dependent upon underlying somitic mesoderm for their differentiation. The extirpations resulted in abnormalities and depletions of scutes.     

Specification of vertebral identity is coupled to Notch signalling and the segmentation clock

To further analyse requirements for Notch signalling in patterning the paraxial mesoderm, we generated transgenic mice that express in the paraxial mesoderm a dominant-negative version of Delta1. Transgenic mice with reduced Notch activity in the presomitic mesoderm as indicated by loss of Hes5 expression were viable and displayed defects in somites and vertebrae consistent with known roles of Notch signalling in somite compartmentalisation. In addition, these mice showed with variable expressivity and penetrance alterations of vertebral identities resembling homeotic transformations, and subtle changes of Hox gene expression in day 12.5 embryos. Mice that carried only one functional copy of the endogenous Delta1 gene also showed changes of vertebral identities in the lower cervical region, suggesting a previously unnoticed haploinsufficiency for Delta1. Likewise, in mice carrying a null allele of the oscillating Lfng gene, or in transgenic mice expressing Lfng constitutively in the presomitic mesoderm, vertebral identities were changed and numbers of segments in the cervical and thoracic regions were reduced, suggesting anterior shifts of axial identity. Together, these results provide genetic evidence that precisely regulated levels of Notch activity as well as cyclic Lfng activity are critical for positional specification of the anteroposterior body axis in the paraxial mesoderm.     

Hox patterning of the vertebrate rib cage

Unlike the rest of the axial skeleton, which develops solely from somitic mesoderm, patterning of the rib cage is complicated by its derivation from two distinct tissues. The thoracic skeleton is derived from both somitic mesoderm, which forms the vertebral bodies and ribs, and from lateral plate mesoderm, which forms the sternum. By generating mouse mutants in Hox5, Hox6 and Hox9 paralogous group genes, along with a dissection of the Hox10 and Hox11 group mutants, several important conclusions regarding the nature of the ;Hox code' in rib cage and axial skeleton development are revealed. First, axial patterning is consistently coded by the unique and redundant functions of Hox paralogous groups throughout the axial skeleton. Loss of paralogous function leads to anterior homeotic transformations of colinear regions throughout the somite-derived axial skeleton. In the thoracic region, Hox genes pattern the lateral plate-derived sternum in a non-colinear manner, independent from the patterning of the somite-derived vertebrae and vertebral ribs. Finally, between adjacent sets of paralogous mutants, the regions of vertebral phenotypes overlap considerably; however, each paralogous group imparts unique morphologies within these regions. In all cases examined, the next-most posterior Hox paralogous group does not prevent the function of the more-anterior Hox group in axial patterning. Thus, the ;Hox code' in somitic mesoderm is the result of the distinct, graded effects of two or more Hox paralogous groups functioning in any anteroposterior location.     

Effects of retinoic acid on the development of the facial skeleton in hamsters: early changes involving cranial neural crest cells

Treatment of gravid hamsters with 60/mg of retinoic acid on the 8th day of pregnancy resulted in facial skeleton defects in 100% of the survivors examined by alizarin staining at term. An investigation of the early stages in the development of these malformations indicated that the teratogen induced delayed and disorganized patterns of cranial neural crest cell migration as well as extensive death and damage of crest cells. The results demonstrate that retinoic acid provides a useful tool for studies in the pathogenesis of facial skeletal abnormalities in vivo. Moreover, the extensive defects seen in the teratogen-treated litters at term, together with the results of the microscopical analyses, support the hypothesis that cranial neural crest cells make an important contribution to the development of the mammalian facial skeleton.