Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.     

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.     

FLRT3 as a key player on chick limb development

Limb outgrowth is maintained by a specialized group of cells, the apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip. It has been shown that fibroblast growth factor (FGF) activity and activation of the Erk pathway are crucial for AER function. Recently, FLRT3, a transmembrane protein able to interact with FGF receptors, has been implicated in the activation of ERK by FGFs. In this study, we show that flrt3 expression is restricted to the AER, co-localizing its expression with fgf8 and pERK activity. Loss-of-function studies have shown that silencing of flrt3 affects the integrity of the AER and, subsequently, its proper function during limb bud outgrowth. Our data also indicate that flrt3 expression is not regulated by FGF activity in the AER, whereas ectopic WNT3A is able to induce flrt3 expression. Overall, our findings show that flrt3 is a key player during chicken limb development, being necessary but not sufficient for proper AER formation and maintenance under the control of BMP and WNT signalling.     

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.     

PKCθ Regulates T Cell Motility via Ezrin-Radixin-Moesin Localization to the Uropod

Cell motility is a fundamental process crucial for function in many cell types, including T cells. T cell motility is critical for T cell-mediated immune responses, including initiation, activation, and effector function. While many extracellular receptors and cytoskeletal regulators have been shown to control T cell migration, relatively few signaling mediators have been identified that can modulate T cell motility. In this study, we find a previously unknown role for PKCθ in regulating T cell migration to lymph nodes. PKCθ localizes to the migrating T cell uropod and regulates localization of the MTOC, CD43 and ERM proteins to the uropod. Furthermore, PKCθ-deficient T cells are less responsive to chemokine induced migration and are defective in migration to lymph nodes. Our results reveal a novel role for PKCθ in regulating T cell migration and demonstrate that PKCθ signals downstream of CCR7 to regulate protein localization and uropod formation.     

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.     

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.     

Disabled-2 is an epithelial surface positioning gene

The formation of the primitive endoderm layer on the surface of the inner cell mass is one of the earliest epithelial morphogenesis in mammalian embryos. In mouse embryos deficient of Disabled-2 (Dab2), the primitive endoderm cells lose the ability to position on the surface, resulting in defective morphogenesis. Embryonic stem cells lacking Dab2 are also unable to position on the surface of cell aggregates and fail to form a primitive endoderm outer layer in the embryoid bodies. The cellular function of Dab2, a cargo-selective adaptor, in mediating endocytic trafficking of clathrin-coated vesicles is well established. We show here that Dab2 mediates directional trafficking and polarized distribution of cell surface proteins such as megalin and E-cadherin and propose that loss of polarity is the underlying mechanism for the loss of epithelial cell surface positioning in Dab2-deficient embryos and embryoid bodies. Thus, the findings indicate that Dab2 is a surface positioning gene and suggest a novel mechanism of epithelial cell surface targeting.     

Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo.

Apical ectodermal ridges (AERs) isolated from 3- to 4-day chick and quail embryos were prepared by means of trypsinization and microdissection and then were grafted to the dorsal or ventral side of a host chick wing bud. They induced supernumerary limb outgrowths from the host bud showing, respectively, a bidorsal or biventral organization, as determined by the patterns of feather germs. The grafted ridge cells persisted, as revealed by histological sections of supernumerary chick limb parts growing under the influence of quail AERs, whose cells are readily distinguished after application of the Feulgen reagent.These results show that the AER induces limb outgrowth regardless of whether it is associated with dorsal or ventral limb ectoderm and that its continued existence is not dependent on contributions of ectodermal cells from the opposed ectodermal faces of the limb bud. The AER is pictured as maintaining the subjacent mesoderm in a condition of developmental plasticity without specifying its differentiation with respect to the proximodistal axis. It remains uncertain whether the positional values of cells that develop under the influence of the AER arise within these cells themselves or appear in response to influences from proximal sources.     

A bidirectional kinesin motor in live Drosophila embryos

Spindle assembly and elongation involve poleward and away-from-the-pole forces produced by microtubule dynamics and spindle-associated motors. Here, we show that a bidirectional Drosophila Kinesin-14 motor that moves either to the microtubule plus or minus end in vitro unexpectedly causes only minor spindle defects in vivo. However, spindles of mutant embryos are longer than wild type, consistent with increased plus-end motor activity. Strikingly, suppressing spindle dynamics by depriving embryos of oxygen causes the bidirectional motor to show increased accumulation at distal or plus ends of astral microtubules relative to wild type, an effect not observed for a mutant motor defective in motility. Increased motor accumulation at microtubule plus ends may be due to increased slow plus-end movement of the bidirectional motor under hypoxia, caused by perturbation of microtubule dynamics or inactivation of the only other known Drosophila minus-end spindle motor, cytoplasmic dynein. Negative-stain electron microscopy images are consistent with highly cooperative motor binding to microtubules, and gliding assays show dependence on motor density for motility. Mutant effects of the bidirectional motor on spindle function may be suppressed under normal conditions by motor: motor interactions and minus-end movement induced by spindle dynamics. These forces may also bias wild-type motor movement toward microtubule minus ends in live cells. Our findings link motor : motor interactions to function in vivo by showing that motor density, together with cellular dynamics, may influence motor function in live cells.     

Membrane-bound steel factor maintains a high local concentration for mouse primordial germ cell motility, and defines the region of their migration

Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.     

Toxoplasma gondii: induction of egress by the potassium ionophore nigericin

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. Some of the devastating consequences of toxoplasmosis are in part due to the lysis of the host cell during parasite egress. The process of egress is poorly understood and since it is asynchronous in tissue culture its study has been limited to those conditions that induce it, such as artificial permeabilisation of the host cell and induction of calcium fluxes with ionophores. Given that permeabilisation leads to egress by the activation of motility upon a drop in host cell potassium concentration, we investigated whether the ionophore nigericin, which selectively causes efflux of potassium from the cell without the need for permeabilisation, would cause egress. Nigericin effectively causes intracellular parasites to exit their host cell within 30 min of treatment with the drug. Our results show that nigericin-induced egress depends on an efflux of potassium from the cell and requires phospholipase C function and parasite motility. This novel method of inducing and synchronising egress mimics the effect of artificial permeabilisation in all respects. Nevertheless, since the membrane remains intact during the treatment, in our nigericin-induced egress we are able to detect parasite-dependent permeabilisation of the host cell, a known step in induced egress. In addition, consistent with the model that loss of host cell potassium leads to egress through the activation of intraparasitic calcium fluxes, a previously isolated Toxoplasma mutant lacking a sodium hydrogen exchanger and defective in responding to calcium fluxes does not undergo nigericin-induced egress. Thus, the discovery that nigericin induces egress presents a novel assay that allows for the genetic and biochemical analysis of the signalling mechanisms that lead to the induction of motility and egress.