Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific β-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs’ notochordal fate.     

Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral pathfinding of limb motor axons

The Eph family of tyrosine kinase receptors has recently been implicated in various processes involving the detection of environmental cues such as axonal guidance, targeted cell migration and boundary formation. We have inactivated the mouse EphA4 gene to investigate its functions during development. Homozygous EphA4 mutant animals show peroneal muscular atrophy correlating with the absence of the peroneal nerve, the main dorsal nerve of the hindlimb. This phenotype is also observed, although with a lower penetrance, in heterozygotes. During normal hindlimb innervation, motor axons converge towards the sciatic plexus region at the base of the limb bud, where they must choose between dorsal and ventral trajectories within the limb. Among the axons emerging from the sciatic plexus, dorsal projections show higher levels of EphA4 protein than ventral axons. In EphA4 mutant mice, presumptive dorsal motor axons fail to enter the dorsal compartment of the limb and join the ventral nerve. Our data therefore suggest that the level of EphA4 protein in growing limb motor axons is involved in the selection of dorsal versus ventral trajectories, thus contributing to the topographic organisation of motor projections.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia

Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.     

Regeneration of the tail bud in Xenopus embryos.

In an attempt to solve some aspect of the long-standing controversy about the regenerative ability of appendages in vertebrate embryos, the tail bud of Xenopus laevis embryos has beenamputated at stage sranging from St. 26 to St. 32 and its ability to regenerate duringa culture period of 2-3 days has been studied. At amputation stages 26-28,the tail bud consisted only undifferentialted mesoderm and ectoderm, but at stage 32 it had afully differentiated neural tube, a vaculotaed notochord and segmented somites. A total of 137amputations at differnt stages gace consistent results: a tail formed in all the operated larvacand it had normal, well-developed axial tissues in most cases. The relatively few cases with abnormal tail struture were stunted, oedematour larvae with defects in the trunk region as well. It is concluded from these experiments that cells near the original tail budare able to differentiate into tialbud tissues and to replace the amputated regoin, even at these late embryoic stages. The implications of these findings for comparative studies on regeneration in vertebrates are discussed.     

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.     

Two distinct sources for a population of maturing axial progenitors

In mammals, the primitive streak region and its descendant, the tail bud, are the source of nascent mesoderm and spinal cord throughout axial elongation. A localised population of long-term axial progenitors has been identified in a region of the tail bud, the chordoneural hinge, but the localisation of such progenitors at earlier stages is so far untested. By studying gene expression, we have shown that a specific topological arrangement of domains persists from the streak to the tail bud, and includes an area (the node-streak border) in which ectoderm that expresses primitive streak markers overlies the prospective notochord. This arrangement persists in the chordoneural hinge. Homotopic grafts show that, as in other vertebrates, cells in the streak and node predominantly produce mesoderm, whereas those in the node-streak border and lateral to the streak additionally produce neurectoderm. Node-streak border descendants populate not only neurectoderm, somites and notochord throughout the axis, but also the chordoneural hinge. Ectoderm lateral to the embryonic day (E)8.5 streak is later recruited to the midline, where it produces somites and chordoneural hinge cells, the position of which overlaps that of border-derived cells. Therefore, the E8.5 axial progenitors that will make the tail comprise cells from two distinct sources: the border and lateral ectoderm. Furthermore, heterotopic grafts of cells from outside the border to this region also populate the chordoneural hinge. Expression of several streak- and tail bud-specific genes declines well before elongation ends, even though this late population can be successfully transplanted into earlier embryos. Therefore, at least some aspects of progenitor status are conferred by the environment and are not an intrinsic property of the cells.     

Axial progenitors with extensive potency are localised to the mouse chordoneural hinge

Elongation of the mouse anteroposterior axis depends on a small population of progenitors initially located in the primitive streak and later in the tail bud. Gene expression and lineage tracing have shown that there are many features common to these progenitor tissues throughout axial elongation. However, the identity and location of the progenitors is unclear. We show by lineage tracing that the descendants of 8.5 d.p.c. node and anterior primitive streak which remain in the tail bud are located in distinct territories: (1) ventral node descendants are located in the widened posterior end of the notochord; and (2) descendants of anterior streak are located in both the tail bud mesoderm, and in the posterior end of the neurectoderm. We show that cells from the posterior neurectoderm are fated to give rise to mesoderm even after posterior neuropore closure. The posterior end of the notochord, together with the ventral neurectoderm above it, is thus topologically equivalent to the chordoneural hinge region defined in Xenopus and chick. A stem cell model has been proposed for progenitors of two of the axial tissues, the myotome and spinal cord. Because it was possible that labelled cells in the tail bud represented stem cells, tail bud mesoderm and chordoneural hinge were grafted to 8.5 d.p.c. primitive streak to compare their developmental potency. This revealed that cells from the bulk of the tail bud mesoderm are disadvantaged in such heterochronic grafts from incorporating into the axis and even when they do so, they tend to contribute to short stretches of somites suggesting that tail bud mesoderm is restricted in potency. By contrast, cells from the chordoneural hinge of up to 12.5 d.p.c. embryos contribute efficiently to regions of the axis formed after grafting to 8.5 d.p.c. embryos, and also repopulate the tail bud. These cells were additionally capable of serial passage through three successive generations of embryos in culture without apparent loss of potency. This potential for self-renewal in chordoneural hinge cells strongly suggests that stem cells are located in this region.     

Morphogenetic features in the tail region of the rat embryo.

The secondary (direct) body formation is a mechanism of development in which morphogenesis of various organs occurs directly from a mass of undifferentiated mesenchymal cells (blastema) without previous formation of germ layers. It is characteristic of the posterior end of the embryonic body, i.e. of the tail bud of tailless and the tail of tailed mammals. Development of the neural tube occurring by this mechanism (secondary neurulation) has been previously explained. We investigated the morphogenetic mechanism by which two other axial structures in the rat tail develop: the tail gut and the notochord. Both structures develop from an axial condensation of undifferentiated mesenchymal cells (tail cord) of tail bud origin. The tail gut forms in a similar way to the secondary neural tube: cells in the ventral part of the tail cord elongate, acquire an apicobasal polarity and form a rosette-like structure around a lumen in the centre. The notochord forms by detachment of a group of cells of the tail cord dorsally to the developing tail gut. The peculiarities of this morphogenetic mechanism in comparison with those in other parts of the embryo are discussed. Causal (including evolutionary) explanations of this mechanism are ruled out.     

Formation of the chordamesoderm in the amphioxus embryo: Analysis with Brachyury and fork head/HNF-3 genes

 The embryonic development of amphioxus (cephalochordates) has much in common with that of vertebrates, suggesting a close phylogenetic relationship between the two chordate groups. To gain insight into alterations in the genetic cascade that accompanied the evolution of vertebrate embryogenesis, we investigated the formation of the chordamesoderm in amphioxus embryos using the genes Brachyury and fork head/HNF-3 as probes. Am(Bb)Bra1 and Am(Bb)Bra2 are homologues of the mouse Brachyury gene isolated from Branchiostoma belcheri. Molecular phylogenetic analysis suggests that the genes are independently duplicated in the amphioxus lineage. Both genes are initially expressed in the involuting mesoderm of the gastrula, then in the differentiating somites of neurulae, followed by the differentiating notochord and finally in the tail bud of ten-somite stage embryos. On the other hand, Am(Bb)fkh/HNF3-1, an amphioxus (B. belcheri) homologue of the fork head/HNF-3 gene, is initially expressed in the invaginating endoderm and mesoderm, then later in the differentiating notochord and in the tail bud. With respect to these two types of genes, the formation of the notochord and tail bud in amphioxus embryos shows similarity and dissimilarity with that of the notochord and tail bud in vertebrate embryos. Received: 21 November 1996 / Accepted: 24 January 1997     

Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development

Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.     

Expression of T Protein in the Primitive Streak Is Necessary and Sufficient for Posterior Mesoderm Movement and Somite Differentiation

A characteristic abnormality of chimeras composed of wildtype andT/T(Brachyury) mutant embryonic stem cells is the aggregation and accumulation of mutant cells in the primitive streak and its descendant, the tail bud (V. Wilson, L. Manson, W. C. Skarnes, and R. S. P. Beddington (1995).Development121, 877–886). To demonstrate that this aberrant behaviour of mutant cells in the streak is due only to the absence of wild-type T protein and to investigate dosage effects of T function on cell deployment during gastrulation, a vector expressingTunder the control of its own promoter (which results inTexpression in the primitive streak but not in the notochord) was introduced intoT/Tmutant ES cells carrying a ubiquitouslacZlineage marker. Four clones (TR clones) that express T appropriately in the streak and rescue abnormal chimeric morphology were recovered. In chimeras, these four clones fall into two distinct categories with respect to their ability to exit from the primitive streak and their subsequent tissue colonisation profile. TR1 and TR4 descendants no longer accumulated in the tail bud and gave rise to all types of mesoderm as well as colonising ventral neurectoderm. Interestingly, TR2 and TR5 cells (which express higher levels of T protein than TR1 and TR4in vitro) tended to exit the streak prematurely, showed a marked reduction in posterior mesoderm colonisation, and were virtually excluded from ventral neurectoderm. However, while descendants of all four TR clones can colonise dermomyotome at all axial levels, the parentT/Tmutant cells only contribute to this tissue rostral to the forelimb bud and are completely excluded from more caudal dermomyotome. These results show that the abnormal aggregation of mutant cells homozygous for theBrachyurydeletion (∼200 kb) can be ascribed solely to the lack of wild-type T protein, as can the failure ofT/Tcells to colonise caudal dermomyotome. They also suggest that patterns of cell recruitment from the streak can be influenced by the level of T expression.     

Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln¹² and Glu¹⁴ and ephrin-B2. Mutation of Gln¹² and Glu¹⁴ does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ∼10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln¹² and Glu¹⁴ with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.     

EphA4, RhoB and the molecular development of feather buds are maintained by the integrity of the actin cytoskeleton

The development of feather buds is a highly ordered process involving epithelial-mesenchymal signalling. Cellular morphology is determined by the actin cytoskeleton, which is controlled by networks of regulators such as the GTPases. EphA4 belongs to a receptor tyrosine kinase family that has been consistently shown to regulate the cytoskeleton via Rho family GTPases in neural development and is expressed in early stages of feather bud development though its role has not been defined. We therefore used an in vitro skin culture system to interfere with EphA4 levels in feather buds using anti-sense oligonucleotides, demonstrating a severe effect on both their number and morphological form. Analysis of the Rho family of GTPases revealed that this effect was mediated by the GTPase RhoB, the expression of which was altered in response to altered levels of EphA4. In addition, the inhibition of RhoB mimicked the effects of reduced EphA4 levels on feather development. Significantly, manipulation of cytoskeletal dynamics revealed that those cells undergoing morphogenetic change regulate the patterning signals responsible for initiating feather development. We propose that this molecular maintenance mechanism between EphA4-RhoB and the actin cytoskeleton converges or coordinates with other morphogenic signalling systems to control feather bud development.     

Ephrin‐A5 regulates the formation of the ascending midbrain dopaminergic pathways

Dopaminergic neurons from the substantia nigra and the ventral tegmental area of the midbrain project to the caudate/putamen and nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. However, the mechanisms underlying the development of these pathways are not well understood. In the current study, the EphA5 receptor and its corresponding ligand, ephrin‐A5, were shown to regulate dopaminergic axon outgrowth and influence the formation of the midbrain dopaminergic pathways. Using a strain of mutant mice in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, EphA5 protein expression was detected in both the ventral tegmental area and the substantia nigra of the midbrain. Ephrin‐A5 was found in both the dorsolateral and the ventromedial regions of the striatum, suggesting a role in mediating dopaminergic axon‐target interactions. In the presence of ephrin‐A5, dopaminergic neurons extended longer neurites in in vitro coculture assays. Furthermore, in mice lacking ephrin‐A5, retrograde tracing studies revealed that fewer neurons sent axons to the striatum. These observations indicate that the interactions between ephrin‐A ligands and EphA receptors promote growth and targeting of the midbrain dopaminergic axons to the striatum. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009