Navigating the anterior-posterior axis with Wnts

Recent studies have begun to shed light on the molecular guidance cues controlling anterior-posterior axon guidance. Two recent studies in the current issue of Developmental Cell show that Wnts play critical roles in patterning processes and directing neuronal migration in C. elegans. Together with previous findings in vertebrates and flies, these new results establish conserved function of Wnts in A-P guidance.     

Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.     

Requirement for beta-catenin in anterior-posterior axis formation in mice

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.     

Sall4 isoforms act during proximal-distal and anterior-posterior axis formation in the mouse embryo

Reciprocal signals from embryonic and extra-embryonic tissues pattern the embryo in proximal-distal (PD) and anterior-posterior (AP) fashion. Here we have analyzed three gene trap mutations of Sall4, of which one (Sall4-1a) led to a hypomorphic and recessive phenotype, demonstrating that Sall4-1a has yet undescribed extra-embryonic and embryonic functions in regulating PD and AP axis formation. In Sall4-1a mutants the self-maintaining autoregulatory interaction between Bmp4, Nodal and Wnt, which determines the PD axis was disrupted because of defects in the extra-embryonic visceral endoderm. More severely, two distinct Sall4 gene-trap mutants (Sall4-1a,b), resembling null mutants, failed to initiate Bmp4 expression in the extra-embryonic ectoderm and Nodal in the epiblast and were therefore unable to initiate PD axis formation. Tetraploid rescue underlined the extra-embryonic nature of the Sall4-1a phenotype and revealed a further embryonic function in Wnt/beta-catenin signaling to elongate the AP axis during gastrulation. This observation was supported through genetic interaction with beta-catenin mutants, since compound heterozygous mutants recapitulated the defects of Wnt3a mutants in posterior development.     

MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis

Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA--miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs--in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.     

Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis

The Drosophila bithorax complex (BX-C) controls segmental development by selectively deploying three protein products, Ubx, abd-A and Abd-B, within specific segments along the body axis. Expression of these products within any one segment (or, more accurately, parasegment) is affected by mutations clustered in a particular region of the BX-C. The regulatory regions defined by this genetic analysis span 20-50 kb and there is one region for each segmental unit. Here we describe regulatory elements from several of these regions, identified by fusion to a Ubx-lacZ gene and analysis in germline transformants. A small DNA fragment from the abx region programs expression with an anterior boundary in the second thoracic segment (parasegment 5). This anterior limit is appropriate, since the abx region normally controls Ubx in parasegment 5. Other regulatory regions of the BX-C that control development of parasegments 6, 7 or 8 contain similar regulatory elements that program expression with anterior limits in parasegments 6, 7 or 8, respectively. These experiments define a class of BX-C regulatory elements that control expression along the anterior-posterior axis. The early appearance of the lacZ patterns in embryos suggests a role for these elements in the initial activation of expression from the BX-C.     

Microtubules are involved in anterior-posterior axis formation in C. elegans embryos

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O'Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.     

Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis

The Xenopus homeobox gene Xhox3 shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identities during pattern formation of the axial mesoderm in early embryonic development.     

Development of a chordate anterior-posterior axis without classical retinoic acid signaling

Developmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior-posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior-posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny.     

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C(2)H(2) zinc finger genes, which has been implicated in notum growth and patterning. Here we show that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. Our data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior-posterior (AP) axis using both autonomous and non-autonomous mechanisms.     

A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium

The Drosophila egg develops through closely coordinated activities of associated germline and somatic cells. An essential aspect of egg development is the differentiation of the somatic follicle cells into several distinct subpopulations with specific functions. Here we demonstrate that the graded activity of the Janus kinase (JAK) pathway, stimulated by the Unpaired ligand, patterns the anterior-posterior axis of the follicular epithelium. Different levels of JAK activity instruct adoption of distinct anterior cell fates. Further, the coordinated activities of the JAK/STAT and epidermal growth factor receptor (EGFR) pathways are required to specify the posterior terminal cell fate. We propose that Upd secreted from the polar cells may act as a morphogen to stimulate A/P-derived follicular fates through JAK pathway activation.     

The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation

Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. We have identified reduction-of-function anaphase-promoting complex (APC) mutations that eliminate a-p polarity. We also demonstrate that the APC activator cdc20 is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects. We propose that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation.     

Migration of neuronal cells along the anterior-posterior body axis of C. elegans: Wnts are in control

Migrating neuronal cells are directed to their final positions by an array of guidance cues. It has been shown that guidance molecules such as UNC-6/Netrin and SLT-1/Slit play a major role in controlling cell and axon migrations along the dorsal-ventral body axis. Much less is known, however, about the mechanisms that mediate migration along the anterior-posterior (AP) body axis. Recent research in Caenorhabditis elegans has uncovered an important role of the Wnt family of signalling molecules in controlling AP-directed neuronal cell migration and polarity. A common theme that emerges from these studies is that multiple Wnt proteins function in parallel as instructive cues or permissive signals to control neuronal patterning along this major body axis.     

Making heads from tails: Development of a reversed anterior-posterior axis during budding in an acoel

The anterior-posterior axis is a key feature of the bilaterian body plan. Although axis specification during embryogenesis has been studied extensively, virtually nothing is known about how this axis can be established post-embryonically, as occurs in budding animals. We investigated bud formation in the acoel Convolutriloba retrogemma, which reproduces by a remarkable process involving the formation of animals with linked but completely opposite body axes. Reverse axes are established anew during each round of budding and manifestations of the bud's new axis develop gradually, with regionalization of axial patterning genes (Hox and otx) and the establishment of organized musculature occurring secondarily, after bud initiation. A swath of tissue at the parent-bud boundary has no regenerative potential and appears devoid of inherent axial polarity. GSK-3 inhibitor trials suggest that Wnt/β-catenin or Hedgehog signalling may mediate the establishment of this unpolarized zone. Formation of unpolarized tissue may provide a buffer between opposing polarity cues and be a general mechanism by which budding animals establish and maintain linked body axes. In addition to elucidating the developmental basis of budding in a bilaterian, this study provides insight into convergence in animal budding mechanisms, redeployment of embryonic gene expression during budding, and Hox gene evolution.     

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.     

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.