Morphogen gradient interpretation.

A morphogen gradient is an important concept in developmental biology, because it describes a mechanism by which the emission of a signal from one part of an embryo can determine the location, differentiation and fate of many surrounding cells. The value of this idea has been clear for over half a century, but only recently have experimental systems and methods of analysis progressed to the point where we begin to understand how a cell can sense and respond to tiny changes in minute concentrations of extracellular signalling factors.     

Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds.

Outgrowth and patterning of the vertebrate limb are controlled by reciprocal interactions between the posterior mesenchyme (polarizing region) and a specialized ectodermal structure, the apical ectodermal ridge (AER). Sonic hedgehog (SHH) signalling by the polarizing region modulates fibroblast growth factor (FGF)4 signalling by the posterior AER, which in turn maintains the polarizing region (SHH/FGF4 feedback loop). Here we report that the secreted bone-morphogenetic-protein (BMP) antagonist Gremlin relays the SHH signal from the polarizing region to the AER. Mesenchymal Gremlin expression is lost in limb buds of mouse embryos homozygous for the limb deformity (Id) mutation, which disrupts establishment of the SHH/FGF4 feedback loop. Grafting Gremlin-expressing cells into ld mutant limb buds rescues Fgf4 expression and restores the SHH/FGF4 feedback loop. Analysis of Shh-null mutant embryos reveals that SHH signalling is required for maintenance of Gremlin and Formin (the gene disrupted by the ld mutations). In contrast, Formin, Gremlin and Fgf4 activation are independent of SHH signalling. This study uncovers the cascade by which the SHH signal is relayed from the posterior mesenchyme to the AER and establishes that Formin-dependent activation of the BMP antagonist Gremlin is sufficient to induce Fgf4 and establish the SHH/FGF4 feedback loop.     

Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning

In many developmental contexts, a locally produced morphogen specifies positional information by forming a concentration gradient over a field of cells. However, during embryonic dorsal-ventral patterning in Drosophila, two members of the bone morphogenetic protein (BMP) family, Decapentaplegic (Dpp) and Screw (Scw), are broadly transcribed but promote receptor-mediated signalling in a restricted subset of expressing cells. Here we use a novel immunostaining protocol to visualize receptor-bound BMPs and show that both proteins become localized to a sharp stripe of dorsal cells. We demonstrate that proper BMP localization involves two distinct processes. First, Dpp undergoes directed, long-range extracellular transport. Scw also undergoes long-range movement, but can do so independently of Dpp transport. Second, an intracellular positive feedback circuit promotes future ligand binding as a function of previous signalling strength. These data elicit a model in which extracellular Dpp transport initially creates a shallow gradient of BMP binding that is acted on by positive intracellular feedback to produce two stable states of BMP-receptor interactions, a spatial bistability in which BMP binding and signalling capabilities are high in dorsal-most cells and low in lateral cells.     

A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.     

Type IV collagens regulate BMP signalling in Drosophila

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. Here we show that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. We provide evidence that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of our findings that human type IV collagen binds BMP4, we predict that this role of type IV collagens will be conserved.     

Reading the Hedgehog morphogen gradient by measuring the ratio of bound to unbound Patched protein

Morphogens are 'form-generating' substances that spread from localized sites of production and specify distinct cellular outcomes at different concentrations. A cell's perception of morphogen concentration is thought to be determined by the number of active receptors, with inactive receptors making little if any contribution. Patched (Ptc), the receptor for the morphogen Hedgehog (Hh), is active in the absence of ligand and blocks the expression of target genes by inhibiting Smoothened (Smo), an essential transducer of the Hh signal. Hh binding to Ptc abrogates the ability of Ptc to inhibit Smo, thereby unleashing Smo activity and inducing target gene expression. Here, we show that a cell's measure of ambient Hh concentration is not determined solely by the number of active (unliganded) Ptc molecules. Instead, we find that Hh-bound Ptc can titrate the inhibitory action of unbound Ptc. Furthermore, we demonstrate that this effect is sufficient to allow normal reading of the Hh gradient in the presence of a form of Ptc that cannot bind the ligand but retains its ability to inhibit Smo. These results support a model in which the ratio of bound to unbound Ptc molecules determines the cellular response to Hh.     

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate

Signals elicited by binding of the T-cell antigen receptor and the CD4/CD8 co-receptor to major histocompatibility complex (MHC) molecules control the generation of CD4+ (helper) or CD8+ (cytotoxic) T cells from thymic precursors that initially express both co-receptor proteins. These precursors have unique, clonally distributed T-cell receptors with unpredictable specificity for the self-MHC molecules involved in this differentiation process. However, the mature T cells that emerge express only the CD4 (MHC class II-binding) or CD8 (MHC class I-binding) co-receptor that complements the MHC class-specificity of the T-cell receptor. How this matching of co-receptor-defined lineage and T-cell-receptor specificity is achieved remains unknown, as does whether signalling by the T-cell receptors, co-receptors and/or general cell-fate regulators such as Notch-1 contributes to initial lineage choice, to subsequent differentiation processes or to both. Here we show that the CD4 versus CD8 lineage fate of immature thymocytes is controlled by the co-receptor-influenced duration of initial T-cell receptor-dependent signalling. Notch-1 does not appear to be essential for this fate determination, but it is selectively required for CD8+ T-cell maturation after commitment directed by T-cell receptors. This indicates that the signals constraining CD4 versus CD8 lineage decisions are distinct from those that support subsequent differentiation events such as silencing of co-receptor loci.     

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.     

Polarizing activity and retinoid synthesis in the floor plate of the neural tube

In many developing organisms the establishment of axial polarity and the patterning of cells depend on local signals that derive from restricted regions of the embryo. In vertebrate embryos, the origins of tissue polarity have been examined extensively in the developing limb. The anteroposterior pattern of the chick limb seems to be controlled by a morphogen, possibly retinoic acid, that is enriched in a region of the limb known as the zone of polarizing activity (ZPA). Certain tissues other than the ZPA have also shown polarizing activity experimentally in the chick limb, raising the possibility that signalling molecules involved in pattern formation in different embryonic tissues are conserved. Here we provide evidence that a similar polarizing activity is also present in a restricted region of the developing central nervous system (CNS). We show that a specialized group of neural cells termed the floor plate, but not other regions of the CNS, mimics the ZPA in respecifying the digit pattern in the developing chick limb. In addition, using an in vitro biochemical assay, we show that the floor plate can synthesize retinoic acid and 3,4-didehydroretinol, the precursor of a second morphogenetically active retinoid, 3,4-didehydroretinoic acid. These results show that the floor plate is a local source of a ZPA-like polarizing signal, possibly a retinoid, which may regulate the pattern of cell differentiation in the developing CNS.     

The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium

Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.