Evidence that Hoxa expression domains are evolutionarily transposed in spinal ganglia, and are established by forward spreading in paraxial mesoderm.

Transposition of anatomical structures along the anteroposterior axis has been a commonly used mechanism for changing body proportions during the course of evolutionary time. Earlier work (Gaunt, S.J., 1994. Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38, 549-552; Burke, A.C., Nelson, C.E., Morgan, B.A., Tabin, C., 1995. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333-346) showed how transposition in mesodermal derivatives (vertebrae) could be attributed to transposition in the expression of Hox genes along the axial series of somites. We now show how transposition in the segmental arrangement of the spinal nerves can also be correlated with shifts in the expression domains of Hox genes. Specifically, we show how the expression domains of Hoxa-7, a-9 and a-10 in spinal ganglia correspond similarly in both mouse and chick with the positions of the brachial and lumbosacral plexuses, and that this is true even though the brachial plexus of chick is shifted posteriorly, relative to mouse, by seven segmental units. In spite of these marked species differences in the boundaries of Hoxa-7 expression, cis regulatory elements located up to 5 kb upstream of the chick Hoxa-7 gene showed much functional and structural conservation with those described in the mouse (Puschel, A.W., Balling, R., Gruss, P., 1991. Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression. Development 112, 279-287; Knittel, T., Kessel, M., Kim, M.H., Gruss, P., 1995. A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. Development 121, 1077-1088). We also show that chick Hoxa-7 and a-10 expression domains spread forward into regions of somites that are initially negative for the expression of these genes. We discuss this as evidence that Hox expression in paraxial mesoderm spreads forward, as earlier found for neurectoderm and lateral plate mesoderm, in a process that occurs independently of cell movement.     

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.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

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.     

Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid.

Exposure of murine embryos to teratogenic doses of retinoic acid (RA) induced homeotic transformations of vertebrae. Posterior transformations occurred along the complete body axis after RA administration on day 7 of gestation and were accompanied by anterior shifts of Hox gene expression domains in embryos. Anterior transformations of vertebrae in the caudal half of the vertebral column were induced on day 8.5. We suggest that the identity of a vertebral segment is specified by a combination of functionally active Hox genes, a "Hox code." In this concept the sequential activation of Hox genes defines sequentially more posterior axial levels, while mesodermal cells leave the primitive streak. Exogenous RA interferes with the normal establishment of Hox codes and thus with axial specification.     

Axial skeletal and Hox expression domain alterations induced by retinoic acid, valproic acid, and bromoxynil during murine development

Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.     

Intrinsic,Hox-dependent cues determine the fate of skeletal muscle precursors

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. We show that instead, somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb.     

Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

The period of the somite segmentation clock is sensitive to Notch activity

The number of vertebrae is defined strictly for a given species and depends on the number of somites, which are the earliest metameric structures that form in development. Somites are formed by sequential segmentation. The periodicity of somite segmentation is orchestrated by the synchronous oscillation of gene expression in the presomitic mesoderm (PSM), termed the "somite segmentation clock," in which Notch signaling plays a crucial role. Here we show that the clock period is sensitive to Notch activity, which is fine-tuned by its feedback regulator, Notch-regulated ankyrin repeat protein (Nrarp), and that Nrarp is essential for forming the proper number and morphology of axial skeleton components. Null-mutant mice for Nrarp have fewer vertebrae and have defective morphologies. Notch activity is enhanced in the PSM of the Nrarp(-/-) embryo, where the ~2-h segmentation period is extended by 5 min, thereby forming fewer somites and their resultant vertebrae. Reduced Notch activity partially rescues the Nrarp(-/-) phenotype in the number of somites, but not in morphology. Therefore we propose that the period of the somite segmentation clock is sensitive to Notch activity and that Nrarp plays essential roles in the morphology of vertebrae and ribs.     

A clock-work somite

Somites are transient structures which represent the most overt segmental feature of the vertebrate embryo. The strict temporal regulation of somitogenesis is of critical developmental importance since many segmental structures adopt a periodicity based on that of the somites. Until recently, the mechanisms underlying the periodicity of somitogenesis were largely unknown. Based on the oscillations of c-hairy1 and lunatic fringe RNA, we now have evidence for an intrinsic segmentation clock in presomitic cells. Translation of this temporal periodicity into a spatial periodicity, through somite formation, requires Notch signaling. While the Hox genes are certainly involved, it remains unknown how the metameric vertebrate axis becomes regionalized along the antero-posterior (AP) dimension into the occipital, cervical, thoracic, lumbar, and sacral domains. We discuss the implications of cell division as a clock mechanism underlying the regionalization of somites and their derivatives along the AP axis. Possible links between the segmentation clock and axial regionalization are also discussed. BioEssays 22:72-83, 2000.     

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.     

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.     

The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation.

The aim of this work was to investigate the role played by the axial organs, neural tube and notochord, on the differentiation of muscle cells from the somites in the avian embryo. Two of us have previously shown that neuralectomy and notochordectomy is followed by necrosis of the somites and consecutive absence of vertebrae and of most muscle cells derived from the myotomes while the limbs develop normally with muscles. Here we have focused our attention on muscle cell differentiation by using the 13F4 mAb that recognizes a cytoplasmic antigen specific of all types of muscle cells. We show that differentiation of muscle cells of myotomes can occur in the absence of notochord and neural tube provided that the somites from which they are derived have been in contact with the axial organs for a defined period of time, about 10 hours for the first somites formed at the cervical level, a duration that progressively reduces caudalward (i.e. for thoracic and lumbar somites). Either one or the other of the two axial organs, the neural tube or the notochord can prevent somitic cell death and fulfill the requirements for myotomal muscle cell differentiation. Separation of the neural tube/notochord complex from the somites by a surgical slit on one side of the embryo gave the same results as extirpation of these organs and provided a perfect control on the non-operated side. A striking finding was that limb and body wall muscles, although derived from the somites, differentiated in the absence of the axial organs. However, limb muscles that develop after excision of the neural tube started to degenerate from E10 onward due to lack of innervation. In vitro explantation of somites from different axial levels confirmed and defined precisely the chronology of muscle cell commitment in the myotomes as revealed by the in vivo experiments.     

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.     

Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation

Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogues, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. We have analysed the genetic interactions between Cdx1 and Cdx2 in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. We conclude that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminding of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice.     

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.