Anterior-posterior Polarity Research Articles

Fgf-Dependent Etv4/5 Activity Is Required for Posterior Restriction of Sonic hedgehog and Promoting Outgrowth of the Vertebrate Limb

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

The membrane-anchored metalloproteinase regulator RECK stabilizes focal adhesions and anterior–posterior polarity in fibroblasts

Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anterior-posterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell-substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.

Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo

Genetic studies have identified the key signalling pathways and developmentally regulated transcription factors that govern cell lineage allocation and axis patterning in the early mammalian embryo. Recent advances have uncovered details of the molecular circuits that tightly control cell growth and differentiation in the mammalian embryo from the blastocyst stage, through the establishment of initial anterior-posterior polarity, to gastrulation, when the germ cells are set aside and the three primary germ layers are specified. Relevant studies in lower vertebrates indicate the conservation and divergence of regulatory mechanisms for cell lineage allocation and axis patterning.

PLK-1 asymmetry contributes to asynchronous cell division ofC. elegansembryos

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development.

Expression of the Drosophila melanogaster GADD45 Homolog (CG11086) Affects Egg Asymmetric Development That Is Mediated by the c-Jun N-Terminal Kinase Pathway

The mammalian GADD45 (growth arrest and DNA-damage inducible) gene family is composed of three highly homologous small, acidic, nuclear proteins: GADD45alpha, GADD45beta, and GADD45gamma. GADD45 proteins are involved in important processes such as regulation of DNA repair, cell cycle control, and apoptosis. Annotation of the Drosophila melanogaster genome revealed that it contains a single GADD45-like protein (CG11086; D-GADD45). We found that, as its mammalian homologs, D-GADD45 is a nuclear protein; however, D-GADD45 expression is not elevated following exposure to genotoxic and nongenotoxic agents in Schneider cells and in adult flies. We showed that the D-GADD45 transcript increased following immune response activation, consistent with previous microarray findings. Since upregulation of GADD45 proteins has been characterized as an important cellular response to genotoxic and nongenotoxic agents, we aimed to characterize the effect of D-GADD45 overexpression on D. melanogaster development. Overexpression of D-GADD45 in various tissues led to different phenotypic responses. Specifically, in the somatic follicle cells overexpression caused apoptosis, while overexpression in the germline affected the dorsal-ventral polarity of the eggshell and disrupted the localization of anterior-posterior polarity determinants. In this article we focused on the role of D-GADD45 overexpression in the germline and found that D-GADD45 caused dorsalization of the eggshell. Since mammalian GADD45 proteins are activators of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways, we tested for a genetic interaction in D. melanogaster. We found that eggshell polarity defects caused by D-GADD45 overexpression were dominantly suppressed by mutations in the JNK pathway, suggesting that the JNK pathway has a novel, D-GADD45-mediated, function in the Drosophila germline.

Wnt and TGF-beta expression in the sponge Amphimedon queenslandica and the origin of metazoan embryonic patterning.

BackgroundThe origin of metazoan development and differentiation was contingent upon the evolution of cell adhesion, communication and cooperation mechanisms. While components of many of the major cell signalling pathways have been identified in a range of sponges (phylum Porifera), their roles in development have not been investigated and remain largely unknown. Here, we take the first steps toward reconstructing the developmental signalling systems used in the last common ancestor to living sponges and eumetazoans by studying the expression of genes encoding Wnt and TGF-β signalling ligands during the embryonic development of a sponge.Methodology/Principal FindingsUsing resources generated in the recent sponge Amphimedon queenslandica (Demospongiae) genome project, we have recovered genes encoding Wnt and TGF-β signalling ligands that are critical in patterning metazoan embryos. Both genes are expressed from the earliest stages of Amphimedon embryonic development in highly dynamic patterns. At the time when the Amphimedon embryos begin to display anterior-posterior polarity, Wnt expression becomes localised to the posterior pole and this expression continues until the swimming larva stage. In contrast, TGF-β expression is highest at the anterior pole. As in complex animals, sponge Wnt and TGF-β expression patterns intersect later in development during the patterning of a sub-community of cells that form a simple tissue-like structure, the pigment ring. Throughout development, Wnt and TGF-β are expressed radially along the anterior-posterior axis.Conclusions/SignificanceWe infer from the expression of Wnt and TGF-β in Amphimedon that the ancestor that gave rise to sponges, cnidarians and bilaterians had already evolved the capacity to direct the formation of relatively sophisticated body plans, with axes and tissues. The radially symmetrical expression patterns of Wnt and TGF-β along the anterior-posterior axis of sponge embryos and larvae suggest that these signalling pathways contributed to establishing axial polarity in the very first metazoans.

The Flightless I Homolog, fli-1, Regulates Anterior/Posterior Polarity, Asymmetric Cell Division and Ovulation During Caenorhabditis elegans Development

Flightless I (Fli I) is an evolutionarily conserved member of the gelsolin family, containing actin-binding and severing activity in vitro. The physiological function of Fli I during animal development remains largely undefined. In this study, we reveal a key role of the Caenorhabditis elegans Fli I homolog, fli-1, in specifying asymmetric cell division and in establishing anterior-posterior polarity in the zygote. The fli-1 gene also regulates the cytokinesis of somatic cells and the development of germline and interacts with the phosphoinositol-signaling pathway in the regulation of ovulation. The fli-1 reporter gene shows that the localization of FLI-1 coincides with actin-rich regions and that the actin cytoskeleton is impaired in many tissues in the fli-1 mutants. Furthermore, the function of fli-1 in C. elegans can be functionally substituted by the Drosophila Fli I. Our studies demonstrate that fli-1 plays an important role in regulating the actin-dependent events during C. elegans development.

Regionalisation of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder

BackgroundReciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue.ResultsAs a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5.ConclusionWe propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.

TheCaenorhabditis eleganscell‐cycle regulator ZYG‐11 defines a conserved family of CUL‐2 complex components

The cullin CUL-2 is a crucial component of a subclass of multisubunit cullin-RING ubiquitin-ligases. The specificity of CUL-2-based complexes is provided by variable substrate-recognition subunits that bind to specific substrates. In Caenorhabditis elegans, CUL-2 regulates several key processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity and mitotic chromatin condensation. However, the substrate recognition subunits that work in these CUL-2-dependent processes were unknown. Here, we present evidence that ZYG-11 is the substrate-recognition subunit for a CUL-2-based complex that regulates these functions. We show that ZYG-11 interacts with CUL-2 in vivo and binds to the complex adaptor protein Elongin C using a nematode variant of the VHL-box motif. We show that the ZYG11 gene family encompasses two main branches in metazoa, and provide evidence that members of the extended ZYG11 family in nematodes and humans are conserved components of CUL2-based ubiquitin-ligases.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.

Regulatory constraints in the evolution of the tetrapod limb anterior–posterior polarity

The anterior to posterior (A-P) polarity of the tetrapod limb is determined by the confined expression of Sonic hedgehog (Shh) at the posterior margin of developing early limb buds, under the control of HOX proteins encoded by gene members of both the HoxA and HoxD clusters. Here, we use a set of partial deletions to show that only the last four Hox paralogy groups can elicit this response: that is, precisely those genes whose expression is excluded from most anterior limb bud cells owing to their collinear transcriptional activation. We propose that the limb A-P polarity is produced as a collateral effect of Hox gene collinearity, a process highly constrained by its crucial importance during trunk development. In this view, the co-option of the trunk collinear mechanism, along with the emergence of limbs, imposed an A-P polarity to these structures as the most parsimonious solution. This in turn further contributed to stabilize the architecture and operational mode of this genetic system.

Dystroglycan down-regulation links EGF receptor signaling and anterior–posterior polarity formation in the Drosophila oocyte

Anterior-posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. Here, we show that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. Our data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior-posterior polarity formation in the oocyte.

Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos

During development, the establishment of cell polarity is important for cells to undergo asymmetric cell divisions that give rise to diverse cell types. In C. elegans embryos, cues from the centrosome trigger the cortical flow of an actomyosin network, leading to the formation of anterior-posterior polarity. However, its precise mechanism is poorly understood. Here, we show that small GTPases have sequential and crucial functions in this process. ECT-2, a potential guanine nucleotide-exchange factor (GEF) for RHO-1, was uniformly distributed at the cortex before polarization, but was excluded from the posterior cortex by the polarity cue from the centrosomes. This local exclusion of ECT-2 led to an asymmetric RHO-1 distribution, which generated a cortical flow of the actomyosin that translocated PAR proteins and CDC-42 (Refs 4, 5) to the anterior cortex. Polarized CDC-42 was, in turn, involved in maintaining the established anterior-cortical domains. Our results suggest that a local change in the function of ECT-2 and RHO-1 links the centrosomal polarity cue with the polarization of the cell cortex.

The Mouse Embryo Autonomously Acquires Anterior-Posterior Polarity at Implantation

The earliest recognizable sign of patterning of the mouse embryo along the anteroposterior (A-P) axis is the migration of the distal visceral endoderm (DVE) toward the future anterior side. Here we report an asymmetry in the mouse embryo at an unexpectedly early stage. The gene for Lefty1, a Nodal antagonist that influences the direction of DVE migration, was found to be asymmetrically expressed in the primitive endoderm of the implanting blastocyst. Lefty1 expression begins randomly in the inner cell mass (ICM) of the blastocyst but is regionalized to one side of the tilted ICM shortly after implantation. Asymmetric expression of Lefty1 can be established by in vitro culture, indicating that it does not require interaction with the uterus. The asymmetric Lefty1 expression is induced by Nodal signaling, although Nodal and genes for its effectors are expressed symmetrically. This asymmetry in molecular patterning of the mouse embryo pushes back the origin of the A-P body axis to the peri-implantation stage.

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.

Wnt Signals and Frizzled Activity Orient Anterior-Posterior Axon Outgrowth in C. elegans

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.

Molecular characterization of the rostral-most somites in early somitic stages of the chick embryo

Segmentation consists on the progressive formation of repetitive embryonic structures, named somites, which are formed from the most rostral part of the presomitic mesoderm. Somites are subdivided into anterior and posterior compartments and several genes are differentially expressed in either compartment. This has provided evidence for the importance of establishing the anterior–posterior polarity within each somite, which is critical for the correct segmented pattern of the adult vertebrate body. Although all somites appear morphologically similar, fate map studies have shown that the first 4 somites do not give rise to segmented structures, in contrast to more posterior ones. Moreover, in several somitogenesis-related mutants the anterior somites are not affected while posterior somites present clear defects or do not form at all. Altogether these data suggest relevant differences between rostral and caudal somites. In order to check for molecular differences between anterior and posterior somites, we have performed a detailed expression pattern analysis of several Notch signalling related genes. For the first time, we show that the somitic expression pattern profile is not the same along the anterior–posterior axis and that the differences are not observed always at the same somite level.

Embryogenesis and larval differentiation in sponges

Having descended from the first multicellular animals on earth, sponges are a key group in which to seek innovations that form the basis of the metazoan body plan, but sponges themselves have a body plan that is extremely difficult to reconcile with that of other animals. Adult sponges lack overt anterior–posterior polarity and sensory organs, and whether they possess true tissues is even debated. Nevertheless, sexual reproduction occurs as in other metazoans, with the development of embryos through a structured series of cellular divisions and organized rearrangements of cellular material, using both mesenchymal and epithelial movements to form a multicellular embryo. In most cases, the embryo undergoes morphogenesis into a spatially organized larva that has several cell layers, anterior–posterior polarity, and sensory capabilities. Here we review original data on the mode of cleavage, timing of cellular differentiation, and the mechanisms involved in the organization of differentiated cells to form the highly structured sponge larva. Our ultimate goal is to develop interpretations of the phylogenetic importance of these data within the Porifera and among basal Metazoa.

153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS

In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 �m) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.

Egg development in parthenogenetic nematodes: variations in meiosis and axis formation

In the well studied model nematode Caenorhabditis elegans entrance of the sperm induces an anterior-posterior polarity in the egg and determines the orientation of the primary embryonic axis. Subsequently, fusion of two haploid gamete nuclei results in a diploid zygote as a prerequisite for normal embryogenesis. Here we analyze the establishment of embryonic polarity and diploidy in the absence of sperm in three parthenogenetic nematode species from three different families, Diploscapter coronatus (Diploscapteridae), Acrobeloides nanus (Cephalobidae) and Plectus sp. (Plectidae). We find that they not only differ from C. elegans in these two aspects but also from each other, indicating variant solutions for the same developmental challenges and supporting the view that the parthenogenetic mode of reproduction has been acquired multiple times independently.