Intrinsic transition of embryonic stem-cell differentiation into neural progenitors

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.     

Wnt signalling regulates adult hippocampal neurogenesis

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/beta-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.     

The molecular nature of the zebrafish tail organizer

Based on grafting experiments, Mangold and Spemann showed the dorsal blastopore lip of an amphibian gastrula to be able to induce a secondary body axis. The equivalent of this organizer region has been identified in different vertebrates including teleosts. However, whereas the graft can induce ectopic head and trunk, endogenous and ectopic axes fuse in the posterior part of the body, raising the question of whether a distinct organizer region is necessary for tail development. Here we reveal, by isochronic and heterochronic transplantation, the existence of a tail organizer deriving from the ventral margin of the zebrafish embryo, which is independent of the dorsal Spemann organizer. Loss-of-function experiments reveal that bone morphogenetic protein (BMP), Nodal and Wnt8 signalling pathways are required for tail development. Moreover, stimulation of naive cells by a combination of BMP, Nodal and Wnt8 mimics the tail-organizing activity of the ventral margin and induces surrounding tissues to become tail. In contrast to induction of the vertebrate head, known to result from the triple inhibition of BMP, Nodal and Wnt, here we show that induction of the tail results from the triple stimulation of BMP, Nodal and Wnt8 signalling pathways.     

The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.     

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.     

Twisted gastrulation is a conserved extracellular BMP antagonist

Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal-ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling.     

Homologues of Twisted gastrulation are extracellular cofactors in antagonism of BMP signalling

Twisted gastrulation (TSG) is involved in specifying the dorsal-most cell fate in Drosophila embryos, but its mechanism of action is poorly understood. TSG has been proposed to modify the action of Short gastrulation (SOG), thereby increasing signalling by the bone morphogenetic protein (BMP) Decapentaplegic. SOG, an inhibitor of BMP signalling, is in turn inactivated by the protease Tolloid. Here we identify Tsg gene products from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG orthologue, chordin. We show that Tsg binds both the vertebrate Decapentaplegic orthologue BMP4 and chordin, and that these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. We conclude that Tsg acts as a cofactor in chordin's antagonism of BMP signalling.     

Repressor activity of Headless/Tcf3 is essential for vertebrate head formation

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.     

Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds. The degree of receptor activation in response to these signals is a known determinant of cell fate, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signalling protein Sonic hedgehog (Shh). These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signalling. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity. However, Nkx2.2 expression expands dosally in Pax6 mutants, raising the possibility that Pax6 controls neuronal pattern indirectly. Here we provide evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity.     

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.     

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.     

Wnt signalling in stem cells and cancer

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.     

Casein kinase I transduces Wnt signals.

The Wnt signalling cascade is essential for the development of both invertebrates and vertebrates, and is altered during tumorigenesis. Although a general framework for Wnt signalling has been elucidated, not all of the components have been identified. Here we describe a serine kinase, casein kinase I (CKI), which was isolated by expression cloning in Xenopus embryos. CKI reproduces several properties of Wnt signals, including generation of complete dorsal axes, stabilization of beta-catenin and induction of genes that are direct targets of Wnt signals. Dominant-negative forms of CKI and a pharmacological blocker of CKI inhibited Wnt signals in Xenopus. Inhibiting CKI in Caenorhabditis elegans generated worms with a mom phenotype, indicative of a loss of Wnt signals. In addition, CKI bound to and increased the phosphorylation of dishevelled, a known component of the Wnt pathway. These data indicate that CKI may be a conserved component of the Wnt pathway.     

LDL-receptor-related proteins in Wnt signal transduction

The Wnt family of secreted signalling molecules are essential in embryo development and tumour formation. The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signalling is not understood. In Drosophila, arrow phenocopies the wingless (DWnt-1) phenotype, and encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6 (refs 12-15). Here we report that LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt-Fz signalling, and induced Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signalling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. Our results indicate that LRP6 may be a component of the Wnt receptor complex.     

Distalization of the Drosophila leg by graded EGF-receptor activity

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-beta (TGF-beta) and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. Here I revise our understanding of limb development in flies and show that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals.     

The Wnt/beta-catenin pathway regulates cardiac valve formation

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.