Axial Identity Research Articles

The Medaka zic1/zic4 Mutant Provides Molecular Insights into Teleost Caudal Fin Evolution

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

FoxA transcription factors are essential for the development of dorsal axial structures

In vertebrates, embryonic structures present at the dorsal midline, prechordal plate, notochord, hypochord and floor plate share a common embryonic origin. In zebrafish, they derive from a pool of progenitors located within the embryonic shield at the onset of gastrulation. The molecular mechanisms responsible for the common development of these structures remain unknown. Based on their spatial and temporal expression, transcription factors of the Forkhead box A (FoxA) family appeared to be good candidates to play such a role. In agreement with this hypothesis, we found that simultaneous knockdown of FoxA2 and FoxA3 abolish the formation of all axial derivatives, while overexpression of these transcription factors strongly enlarges dorsal mesodermal territories. We establish that, in FoxA2–FoxA3 double morphants, precursors of axial tissues are correctly induced at early gastrula stage, but their dorsal midline identity is not maintained during development and we found that progenitors of these tissues are cell-autonomously re-specified to form muscle fibers as well as cells of the ventral neural tube. Our study provides the first example of a specific loss of all dorsal midline tissues and demonstrates that members of the FoxA family have redundant functions essential to maintain the axial identity of prechordal plate, notochord, floor plate and hypochord progenitors during gastrulation.

O27. Somite formation without a clock

It is been widely assumed that the “segmentation clock” controls the timing of somite formation, or the size of individual somites, or both. However it is possible experimentally to generate somites that form neither in orderly head–tail sequence nor in a neat row. This suggests that it is possible to separate somite formation from a somitogenesis clock. What appears to go wrong in these apparently clockless somites is the partition of each somite into rostral and caudal halves: markers for these are inappropriately expressed. It also appears that somites generated without the clock have very specific axial identities. These findings suggest that the major functions of the “segmentation clock” are to impart positional addresses along the axis and to pattern individual somites into rostral and caudal halves.

Segment Number and Axial Identity in a Segmentation Clock Period Mutant

A species-specific number of segments is a hallmark of the vertebrate body plan. The first segmental structures in the vertebrate embryo are the somites, which bud sequentially from the growing presomitic mesoderm (PSM). The Clock and Wavefront model for somitogenesis proposes that the total number of somites is determined by the period of an oscillator or clock operating in the PSM and the total duration of PSM growth. Furthermore, the number of oscillations of the segmentation clock has been suggested to regulate the regional identity of segments along the body axis. Here we test these two ideas in a zebrafish mutant in which the segmentation clock is specifically slowed. This reduces segment number as predicted, but hox gene expression and posterior anatomical markers align with lower segmental counts in mutants compared to the wild-type, arguing against an instructive role of the segmentation clock in determining axial identities. Our data therefore suggest that precise control of segmentation clock period in relation to axial growth ensures a species-specific segment number and that during evolution modulating the clock's period through genetic mutations may have been a relevant way to vary segment number independently of axial regionalization.

Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.

Comparative analysis of Hoxa5 allelic series

Analysis of the Hoxa5(-/-) mutants has revealed the critical role of Hoxa5 in survival, specification of axial identity, and ontogeny of organs, including the respiratory tract. The presence of the selection cassette in the original Hoxa5(-/-) mutation may interfere with the interpretation of the phenotypes. To circumvent this aspect and to bypass the lethality of the Hoxa5 mutation, we have designed a conditional approach and generated Hoxa5 allelic variants. The conditional allele (Hoxa5(floxed)) behaves as a wild-type allele. In contrast, both the Hoxa5(Delta) and the Hoxa5(floxneo) alleles are characterized by the loss of the functional transcript and protein, the lethality due to lung defects and the skeletal homeotic transformations similar to those of the Hoxa5(-/-) mutants. Analysis of neighboring Hox gene expression patterns in the Hoxa5 mutants produced further confirmed that the Hoxa5 allelic variants are true null alleles.

Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

An early and crucial event in vertebrate inner ear development is the acquisition of axial identities that in turn dictate the positions of all subsequent inner ear components. Here, we focus on the role of the hindbrain in establishment of inner ear axes and show that axial specification occurs well after otic placode formation in chicken. Anteroposterior (AP) rotation of the hindbrain prior to specification of this axis does not affect the normal AP orientation and morphogenesis of the inner ear. By contrast, reversing the dorsoventral (DV) axis of the hindbrain results in changing the DV axial identity of the inner ear. Expression patterns of several ventrally expressed otic genes such as NeuroD, Lunatic fringe (Lfng) and Six1 are shifted dorsally, whereas the expression pattern of a normally dorsal-specific gene, Gbx2, is abolished. Removing the source of Sonic Hedgehog (SHH) by ablating the floor plate and/or notochord, or inhibiting SHH function using an antibody that blocks SHH bioactivity results in loss of ventral inner ear structures. Our results indicate that SHH, together with other signals from the hindbrain, are important for patterning the ventral axis of the inner ear. Taken together, our studies suggest that tissue(s) other than the hindbrain confer AP axial information whereas signals from the hindbrain are necessary and sufficient for the DV axial patterning of the inner ear.

Formation and differentiation of the avian sclerotome.

The avian sclerotome forms by epitheliomesenchymal transition of the ventral half-somite. Sclerotome development is characterized by a craniocaudal polarization, resegmentation, and axial identity. Its formation is controlled by signals from the notochord, the neural tube, the lateral plate mesoderm, and the myotome. These signals and crosstalk between somite cells lead to the separation of various subdomains, such as the central and ventral sclerotomes that express Pax1 under the control of Sonic hedgehog and Noggin, and the dorsal and lateral sclerotome that do not express Pax1 and are controlled by Bmp-4. Further subdomains that give rise to specific derivatives are the syndetome, neurotome, meningotome, and arthrotome. The molecular control of subdomain formation and cell type specification is discussed.

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.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

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.

FGF Signaling Controls Somite Boundary Position and Regulates Segmentation Clock Control of Spatiotemporal Hox Gene Activation

Vertebrate segmentation requires a molecular oscillator, the segmentation clock, acting in presomitic mesoderm (PSM) cells to set the pace at which segmental boundaries are laid down. However, the signals that position each boundary remain unclear. Here, we report that FGF8 which is expressed in the posterior PSM, generates a moving wavefront at which level both segment boundary position and axial identity become determined. Furthermore, by manipulating boundary position in the chick embryo, we show that Hox gene expression is maintained in the appropriately numbered somite rather than at an absolute axial position. These results implicate FGF8 in ensuring tight coordination of the segmentation process and spatiotemporal Hox gene activation.

Association of bronchopulmonary sequestration with expression of the homeobox protein Hoxb-5

Bronchopulmonary sequestration (BPS) is caused by the abnormal development of an accessory lung diverticulum from the foregut very early in embryogenesis. The developmental abnormalities seen with BPS suggest that this anomaly is caused by abnormal expression of homeobox genes, which control axial identity and organ-specific patterning during embryogenesis. The authors previously have shown that the homeobox gene Hoxb-5 is necessary for normal airway branching during lung development. The authors now report that BPS is associated with aberrant developmental expression of Hoxb-5 protein, suggesting that this Hox gene is involved in the development of BPS. J Pediatr Surg 35:1817-1819. Copyright © 2000 by W.B. Saunders Company.

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.

One loop renormalization for the axial Ward-Takahashi identity in domain-wall QCD

We calculate the one loop correction to the axial Ward-Takahashi identity given by Furman and Shamir in domain-wall QCD. It is shown perturbatively that the renormalized axial Ward-Takahashi identity is satisfied without fine-tuning and the ``conserved'' axial vector current receives no renormalization, giving ${Z}_{A}=1$. This fact will simplify the calculation of the pion decay constant in numerical simulations since the decay constant defined by this current needs no lattice renormalization factor.

Transcriptional interferences at the Hoxa4/Hoxa5 locus: importance of correct Hoxa5 expression for the proper specification of the axial skeleton.

We have previously described a Hoxa5 mutant mouse line in which specification of axial identity is perturbed and viability is markedly reduced. In the present study, we assay the Hoxa5 mutation in different genetic backgrounds and carry out a complete analysis of skeletal transformations. Although Hoxa5 is expressed over a large domain during embryogenesis, homeotic transformations of the axial skeleton are confined between cervical vertebra C3 and thoracic vertebra T2, which corresponds to the specific expression domain of the major Hoxa5 transcript. Loss of Hoxa5 function also affects the formation of the acromion in the appendicular skeleton. Disruption of the adjacent Hoxa4 gene leads to similar homeotic transformations of the cervicothoracic vertebrae. To discriminate the respective role of each gene, we generated transheterozygous animals carrying inactivated Hoxa4 and Hoxa5 alleles on different chromosomes. Compound heterozygous mutants exhibit homeotic transformations in the cervicothoracic transition region more reminiscent to those observed in Hoxa5 homozygous mutants. Although the Hoxa5 mutation does not significantly affect Hoxa4 expression, the pattern of Hoxa5 expression is impaired in cis by the Hoxa4 mutation, specifically in the cervicothoracic region of the prevertebral column. The expression of Hoxa5 in this particular domain is also perturbed by the Hoxa5 mutation itself, raising the possibility of regional autoregulation. Altogether, these results demonstrate the crucial role of Hoxa5 in the specification of the cervical and upper thoracic region of the skeleton and establish the importance of its correct expression for the proper patterning of the embryo.

Positional apoptosis during vertebrate CNS development in the absence of endogenous retinoids.

We have previously shown that quail embryos that develop in the absence of vitamin A have severe defects in their central nervous system. One defect is a completely missing posterior hindbrain. Here we have studied how this comes about by examining cell death using a wholemount technique. In these A- embryos we observe two narrow bands of ectopic apoptosis. One is in the mesenchyme in the region of the first somite and occurs at the 4-6 somite stage, before neural tube closure. The second band follows immediately afterwards and occurs in the neuroepithelium of the presumptive posterior hindbrain at the 6-8 somite stage. Electron microscopy shows that the dying neuroepithelial cells exhibit the characteristics of apoptosis. Rescuing the embryos by injecting retinol before gastrulation completely prevents these apoptotic events. In an effort to identify some of the genes that may be involved in the apoptotic pathway we show that Msx-2 is upregulated in the apoptotic neuroepithelium and thus may be involved, whereas Bmp-4 is not altered and thus presumably not involved. Since these apoptotic event take place at the time of specification of axial identity and segmentation in the mesenchyme and neuroepithelium we conclude that these cells die because they are wrongly specified in terms of their rostrocaudal position, a novel phenomenon which we refer to as positional apoptosis.

Amphibian limb regeneration: rebuilding a complex structure.

The ability to regenerate complex structures is widespread in metazoan phylogeny, but among vertebrates the urodele amphibians are exceptional. Adult urodeles can regenerate their limbs by local formation of a mesenchymal growth zone or blastema. The generation of blastemal cells depends not only on the local extracellular environment after amputation or wounding but also on the ability to reenter the cell cycle from the differentiated state. The blastema replaces structures appropriate to its proximodistal position. Axial identity is probably encoded as a graded property that controls cellular growth and movement through local cell interactions. The molecular basis is not understood, but proximodistal identity in newt blastemal cells may be respecified by signaling through a retinoic acid receptor isoform. The possibility of inducing a blastema on a mammalian limb cannot be discounted, although the molecular constraints are becoming clearer as we understand more about the mechanisms of urodele regeneration.

Axial current conservation in the Bethe-Salpeter approach to the nuclear two-body problem.

We investigate the structure of the one- and two-nucleon axial-current operators necessary for the partial conservation of the nuclear axial-current elastic matrix element in the one-boson-exchange approximation to the two-nucleon Bethe-Salpeter equation. We use three models for this purpose: (a) the linear sigma model, (b) the nonlinear sigma model, (c) a hybrid model, which is, roughly speaking, a linear combination of (a) and (b). We construct a partially conserved nuclear axial-current elastic matrix element in models (a) and (c) provided that the associated nuclear wave functions are solutions to the Bethe-Salpeter equation with a potential made of one-boson-exchange diagrams. In the nonlinear sigma model the nuclear one-body axial current is partially conserved by itself, without reference to the nuclear wave function, whereas the two-body axial current partial conservation is violated by terms of order 1/${\mathit{f}}_{\mathrm{\ensuremath{\pi}}}^{2}$. The complete axial current in models (a) and (c) and the one-body axial current in model (b) are applicable to the construction of the deuteron electroweak process amplitudes, for example. The divergence of the axial-current matrix element is proportional to the pion absorption nuclear matrix element, which leads to another potential application in the foundation of chiral perturbation theory for pion-two-nucleon processes. Consistency between the nuclear axial-currents and the underlying nuclear dynamics in models (a) and (c) is a new condition imposed by the partial conservation of the nuclear axial-current matrix element. We also examine conditions imposed on the form of the nucleon self-energy by the nucleon and meson axial Ward-Takahashi identities, as well as the approximations that satisfy the said conditions. We show that besides the first Born approximation, the so-called Hartree+random-phase approximation satisfies chiral Ward-Takahashi idenitities in models (a) and (c). \textcopyright{} 1996 The American Physical Society.

Activation of a single retinoic acid receptor isoform mediates proximodistal respecification

Background: The regenerating limb of urodele amphibians is an important system for evaluating the effects of retinoic acid (RA) on pattern formation. Regeneration proceeds by local formation of the blastema, a mesenchymal growth zone which normally only gives rise to structures distal to its level of origin. RA can respecify proximodistal identity in amphibian limb regeneration, and this activity of RA on the blastema is observed in two contexts. First, exposure to RA proximalizes a distal blastema resulting in duplication of structures proximal to the level of amputation. Second, after transplantation of a distal blastema to a proximal stump, the transplanted cells normally make only a minor contribution to the intercalary regenerate, but if transplanted cells are exposed to RA they occupy positions proximal to their level of origin and contribute to the regeneration of the intermediate tissue. Multiple isoforms of RA receptors (RARs) are expressed in the newt limb and are thought to mediate the respecification of positional identity.Results To identify which receptor(s) mediates proximodistal respecification, we have used the biolistics (particle bombardment) technique to transfect the blastemal mesenchyme with plasmids encoding chimeric proteins containing partial amino-acid sequences of the various newt RAR isoforms fused to a partial sequence of the thyroid hormone (3,5,3′-triiodothyronine; T3) receptor. We then used T3 treatment to selectively activate individual RAR isoforms in vivo. By analyzing the distributions of transfected cells in regenerates derived from distal-to-proximal transplantation we find that activation of a single RAR isoform, δ 2, specifically mediates proximalization.Conclusion These results demonstrate that the ability of RA to respecify proximodistal identity is mediated by a specific RAR isoform, δ 2. Activation of the RA pathway in individual cells indicates that positional respecification can be cell-autonomous. RA can respecify axial identity in several contexts in vertebrate development, but this is the first case where the RAR mediating respecification has been identified.