Development, regeneration, and even day-to-day physiology require plant and animal cells to make decisions based on their locations. The principles by which cells may do this are deceptively straightforward. But when reliability needs to be high—as often occurs during development—successful strategies tend to be anything but simple. Increasingly, the challenge facing biologists is to relate the diverse diffusible molecules, control circuits, and gene regulatory networks that help cells know where they are to the varied, sometimes stringent, constraints imposed by the need for real-world precision and accuracy.
Borders are important as they demarcate developing tissue into distinct functional units. A key challenge is the discovery of mechanisms that can convert morphogen gradients into tissue borders. While mechanisms that produce ultrasensitive cellular responses provide a solution, how extracellular morphogens drive such mechanisms remains poorly understood. Here, we show how Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) pathways interact to generate ultrasensitivity and borders in the dorsal telencephalon. BMP and FGF signaling manipulations in explants produced border defects suggestive of cross inhibition within single cells, which was confirmed in dissociated cultures. Using mathematical modeling, we designed experiments that ruled out alternative cross inhibition mechanisms and identified a cross-inhibitory positive feedback (CIPF) mechanism, or “toggle switch”, which acts upstream of transcriptional targets in dorsal telencephalic cells. CIPF explained several cellular phenomena important for border formation such as threshold tuning, ultrasensitivity, and hysteresis. CIPF explicitly links graded morphogen signaling in the telencephalon to switch-like cellular responses and has the ability to form multiple borders and scale pattern to size. These benefits may apply to other developmental systems.
During development, morphogen gradients play a crucial role in transforming a uniform field of cells into regions with distinct cell identities (marked by the expression of specific genes). Finding mechanisms that convert morphogen gradients into sharp borders of gene expression, however, remains a challenge. Cellular ultrasensitivity mechanisms that convert a linear stimulus into an on-off target response offer a good solution for making such borders. In this paper, we show how a cross-inhibitory positive feedback or toggle switch mechanism driven by two extracellular morphogens – BMP and FGF - produces ultrasensitivity in forebrain cells. Experiments with cells and explanted brain tissue reveal that BMPs and FGFs cross inhibit each other's signaling pathway. Such cross inhibition could occur through four possible mechanisms. By an iterative combination of modeling and experiment, we show the toggle switch to be the mechanism underlying cross inhibition, the ultrasensitive expression of multiple genes, and hysteresis in forebrain cells. As the toggle switch explicitly links extracellular morphogens to cellular ultrasensitivity, it provides a mechanism for making multiple sharp borders that can also scale with tissue size – an important issue in pattern formation. This might explain the abundance of BMP-FGF cross inhibition during development.
Retinoic acid (RA) regulates many cellular behaviors during embryonic development and adult homeostasis. Like other morphogens, RA forms gradients through the use of localized sources and sinks, feedback, and interactions with other signals; this has been particularly well studied in the context of hindbrain segmentation in vertebrate embryos. Yet, as a small lipophilic molecule derived from a dietary source—vitamin A—RA differs markedly from better-studied polypeptide morphogens in its mechanisms of transport, signaling, and removal. Computational models suggest that the distinctive features of RA gradients make them particularly robust to large perturbations. Such features include combined positive and negative feedback effects via intracellular fatty acid binding proteins and RA-degrading enzymes. Here, we discuss how these features, together with feedback interactions among RA target genes, help enable RA to specify multiple, accurate pattern elements in the developing hindbrain, despite operating in an environment of high cellular and biochemical uncertainty and noise.
Quasi-stable gradients of signaling protein molecules (known as morphogens or ligands) bound to cell receptors are known to be responsible for differential cell signaling and gene expressions. From these follow different stable cell fates and visually patterned tissues in biological development. Recent studies have shown that the relevant basic biological processes yield gradients that are sensitive to small changes in system characteristics (such as expression level of morphogens or receptors) or environmental conditions (such as temperature changes). Additional biological activities must play an important role in the high level of robustness observed in embryonic patterning for example. It is natural to attribute observed robustness to various type of feedback control mechanisms. However, our own simulation studies have shown that feedback control is neither necessary nor sufficient for robustness of the morphogen decapentaplegic (Dpp) gradient in wing imaginal disc of Drosophilas. Furthermore, robustness can be achieved by substantial binding of the signaling morphogen Dpp with nonsignaling cell surface bound molecules (such as heparan sulfate proteoglygans) and degrading the resulting complexes at a sufficiently rapid rate. The present work provides a theoretical basis for the results of our numerical simulation studies.
Morphogen gradient; nonlinear boundary value problem; robustness; mathematical modeling
How morphogen gradients form has long been a subject of controversy. The strongest support for the view that morphogens do not simply spread by free diffusion has come from a variety of studies of the Decapentaplegic (Dpp) gradient of the Drosophila larval wing disc.
In the present study, we initially show how the failure, in such studies, to consider the coupling of transport to receptor-mediated uptake and degradation has led to estimates of transport rates that are orders of magnitude too low, lending unwarranted support to a variety of hypothetical mechanisms, such as “planar transcytosis” and “restricted extracellular diffusion”. Using several independent dynamic methods, we obtain data that are inconsistent with such models, and that show directly that Dpp transport occurs by simple, rapid diffusion in the extracellular space. We discuss the implications of these findings for other morphogen systems in which complex transport mechanisms have been proposed.
We believe that these findings resolve a major, longstanding question about morphogen gradient formation, and provide a solid framework for interpreting experimental observations of morphogen gradient dynamics.
Cell surface heparan sulfate (HS) potentiates the activities of various growth factors. Here we show that HS stimulates bone morphogenetic protein (BMP) activity by enhancing recruitment of type II receptor subunits to BMP-type I receptor complexes, suggesting a view of HS as a catalyst of the formation of signaling complexes.
Cell surface heparan sulfate (HS) not only binds several major classes of growth factors but also sometimes potentiates their activities—an effect usually termed “coreception.” A view that coreception is due to the stabilization of growth factor–receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here, we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and we provide evidence that HS–BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I receptor subunits but instead enhances the subsequent recruitment of type II receptor subunits to BMP-type I receptor complexes. This suggests a view of HS as a catalyst of the formation of signaling complexes, rather than as a stabilizer of growth factor binding.
Few mechanistic ideas from the pre-molecular era of biology have had as enduring an impact as the morphogen concept. In the classical view, cells in developing embryos obtain positional information by measuring morphogen concentrations and comparing them with fixed concentration thresholds; as a result, graded morphogen distributions map into discrete spatial arrangements of gene expression. Recent studies on Hedgehog and other morphogens suggest that establishing patterns of gene expression may be less a function of absolute morphogen concentrations, than of the dynamics of signal transduction, gene expression, and gradient formation. The data appoint away from any universal model of morphogen interpretation and suggest that organisms use multiple mechanisms for reading out developmental signals in order to accomplish specific patterning goals.
Colon crypts, a single sheet of epithelia cells, consist of a periodic pattern of stem cells, transit-amplifying cells, and terminally differentiated cells that constantly renew and turnover. Experimental evidence suggests that Wnt signaling promotes and regulates stem cell division, differentiation, and possible cell migrations while intestinal BMP signaling inhibits stem cell self-renewal and repression in crypt formation. As more molecular details on Wnt and BMP in crypts are being discovered, little is still known about how complex interactions among Wnt, BMP, and different types of cells, and surrounding environments may lead to de novo formation of multiple crypts or how such interactions affect regeneration and stability of crypts.
We present a mathematical model that contains Wnt and BMP, a cell lineage, and their feedback regulations to study formation, regeneration, and stability of multiple crypts. The computational explorations and linear stability analysis of the model suggest a reaction–diffusion mechanism, which exhibits a short-range activation of Wnt plus a long-range inhibition with modulation of BMP signals in a growing tissue of cell lineage, can account for spontaneous formation of multiple crypts with the spatial and temporal pattern observed in experiments. Through this mechanism, the model can recapitulate some distinctive and important experimental findings such as crypt regeneration and crypt multiplication. BMP is important in maintaining stability of crypts and loss of BMP usually leads to crypt multiplication with a fingering pattern.
The study provides a mechanism for de novo formation of multiple intestinal crypts and demonstrates a synergetic role of Wnt and BMP in regeneration and stability of intestinal crypts. The proposed model presents a robust framework for studying spatial and temporal dynamics of cell lineages in growing tissues driven by multiple signaling molecules.
The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican independent, whereas those that trigger syndecan-1 shedding make initial FGF2 responses glypican dependent. We further show that syndecan-1 shedding is mediated by matrix metalloproteinase-7 (MMP7), which, being anchored to cells by HSPGs, also causes its own release in a complex with syndecan-1 ectodomains. These results support a specific role for shed syndecan-1 or MMP7–syndecan-1 complexes in tumor progression and add to accumulating evidence that syndecans and glypicans have nonequivalent functions in vivo.
Network motifs provided a “conceptual tool” for understanding the functional principles of biological networks, but such motifs have primarily been used to consider static network structures. Static networks, however, cannot be used to reveal time- and region-specific traits of biological systems. To overcome this limitation, we proposed the concept of a “spatiotemporal network motif,” a spatiotemporal sequence of network motifs of sub-networks which are active only at specific time points and body parts.
On the basis of this concept, we analyzed the developmental gene regulatory network of the Drosophila melanogaster embryo. We identified spatiotemporal network motifs and investigated their distribution pattern in time and space. As a result, we found how key developmental processes are temporally and spatially regulated by the gene network. In particular, we found that nested feedback loops appeared frequently throughout the entire developmental process. From mathematical simulations, we found that mutual inhibition in the nested feedback loops contributes to the formation of spatial expression patterns.
Taken together, the proposed concept and the simulations can be used to unravel the design principle of developmental gene regulatory networks.
The view of biology as goal-directed engineering has deep historical roots in developmental biology, a field currently benefitting from an influx of ideas and methods from systems biology. Systems biology draws on non-biological paradigms to explain developmental mechanisms of control, the specific type of regulation that achieves or maintains a desired end. This review highlights some of the current efforts designed to elucidate basic design principles underlying the engineering objectives of robustness, precision, and scaling that are required during developmental control of growth and pattern formation. Examples from vertebrate and invertebrate development are used to illustrate general principles including the value of integral feedback in achieving set-point control; the usefulness of self-organizing behavior; the importance of recognizing and appropriately handling noise; and the No Free Lunch theory. Through the examination of such principles, systems biology offers a functional framework to make sense of the mechanistic complexity of organismal development.
nipbl-deficient zebrafish provide evidence that heart and gut defects in Cornelia de Lange Syndrome are caused by combined effects of multiple gene expression changes that occur during early embryonic development.
Cornelia de Lange Syndrome (CdLS) is the founding member of a class of multi-organ system birth defect syndromes termed cohesinopathies, named for the chromatin-associated protein complex cohesin, which mediates sister chromatid cohesion. Most cases of CdLS are caused by haploinsufficiency for Nipped-B-like (Nipbl), a highly conserved protein that facilitates cohesin loading. Consistent with recent evidence implicating cohesin and Nipbl in transcriptional regulation, both CdLS cell lines and tissues of Nipbl-deficient mice show changes in the expression of hundreds of genes. Nearly all such changes are modest, however—usually less than 1.5-fold—raising the intriguing possibility that, in CdLS, severe developmental defects result from the collective action of many otherwise innocuous perturbations. As a step toward testing this hypothesis, we developed a model of nipbl-deficiency in zebrafish, an organism in which we can quantitatively investigate the combinatorial effects of gene expression changes. After characterizing the structure and embryonic expression of the two zebrafish nipbl genes, we showed that morpholino knockdown of these genes produces a spectrum of specific heart and gut/visceral organ defects with similarities to those in CdLS. Analysis of nipbl morphants further revealed that, as early as gastrulation, expression of genes involved in endodermal differentiation (sox32, sox17, foxa2, and gata5) and left-right patterning (spaw, lefty2, and dnah9) is altered. Experimental manipulation of the levels of several such genes—using RNA injection or morpholino knockdown—implicated both additive and synergistic interactions in causing observed developmental defects. These findings support the view that birth defects in CdLS arise from collective effects of quantitative changes in gene expression. Interestingly, both the phenotypes and gene expression changes in nipbl morphants differed from those in mutants or morphants for genes encoding cohesin subunits, suggesting that the transcriptional functions of Nipbl cannot be ascribed simply to its role in cohesin loading.
Although best known for its role in chromatid cohesion, cohesin is increasingly seen as a regulator of gene expression. In Cornelia de Lange Syndrome (CdLS), partial deficiency for NIPBL, which encodes a cohesin regulator, is associated with small changes in the expression of many genes (similar effects are seen in Nipbl-deficient mice and flies). Are such changes responsible for pervasive developmental defects in CdLS? To address this, we used morpholino oligonucleotides to quantitatively reduce levels of Nipbl protein and Nipbl target genes in zebrafish embryos. Combined knockdown of both zebrafish nipbl genes produced heart and gut defects with similarities to those observed in CdLS. Nipbl-deficient embryos showed quantitative changes in the expression of several genes involved in the specification of endoderm, which both gives rise to gut and provides a substrate for cardiac precursor migration, as well as genes that regulate left-right asymmetry. Functional studies of these putative targets suggest that changes in their expression collectively, and in some cases synergistically, contribute to the observed phenotypes. These findings suggest that birth defects in CdLS result from combinatorial, quantitative effects of NIPBL on gene expression, and suggest that cardiac and visceral organ defects in CdLS arise during early embryonic development.
Biological systems are so complex that we must ask: "What purpose does all this complexity serve?" Lander argues that computational biology may help provide answers
A culture's icons are a window onto its soul. Few would disagree that, in the culture of molecular biology that dominated much of the life sciences for the last third of the 20th century, the dominant icon was the double helix. In the present, post-modern, 'systems biology' era, however, it is, arguably, the hairball.
Developmental biology, regenerative medicine and cancer biology are increasingly occupied with the molecular characterization of stem cells. Yet recent work adds to a growing body of literature suggesting that 'stemness' cannot be reduced to the molecular features of cell types, and is instead an emergent property of cell lineages under feedback control.
Studies of developing and self-renewing tissues have shown that differentiated cell types are typically specified through the actions of multistage cell lineages. Such lineages commonly include a stem cell and multiple progenitor (transit amplifying; TA) cell stages, which ultimately give rise to terminally differentiated (TD) cells. In several cases, self-renewal and differentiation of stem and progenitor cells within such lineages have been shown to be under feedback regulation. Together, the existence of multiple cell stages within a lineage and complex feedback regulation are thought to confer upon a tissue the ability to autoregulate development and regeneration, in terms of both cell number (total tissue volume) and cell identity (the proportions of different cell types, especially TD cells, within the tissue). In this paper, we model neurogenesis in the olfactory epithelium (OE) of the mouse, a system in which the lineage stages and mediators of feedback regulation that govern the generation of terminally differentiated olfactory receptor neurons (ORNs) have been the subject of much experimental work. Here we report on the existence and uniqueness of steady states in this system, as well as local and global stability of these steady states. In particular, we identify parameter conditions for the stability of the system when negative feedback loops are represented either as Hill functions, or in more general terms. Our results suggest that two factors – autoregulation of the proliferation of transit amplifying (TA) progenitor cells, and a low death rate of TD cells – enhance the stability of this system.
cell lineage; olfactory epithelium; neurogenesis; feedback; stem cell; transit amplifying cell; terminally differentiated cell; neuronal progenitor; stability; modeling
Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75–80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only ∼30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/− mice and in individuals with CdLS.
Cornelia de Lange Syndrome (CdLS) is a genetic disease marked by growth retardation, cognitive and neurological problems, and structural defects in many organ systems. The majority of CdLS cases are due to mutation of one copy of the Nipped B-like (NIPBL) gene, the product of which regulates a complex of chromosomal proteins called cohesin. How reduction of NIPBL function gives rise to pervasive developmental defects in CdLS is not understood, so a model of CdLS was developed by generating mice that carry one null allele of Nipbl. Developmental defects in these mice show remarkable similarity to those observed in individuals with CdLS, including small stature, craniofacial abnormalities, reduced body fat, behavioral disturbances, and high perinatal mortality. Molecular analysis of tissues and cells from Nipbl mutant mice provide the first evidence that the major role of Nipbl in the etiology of CdLS is to exert modest, but significant, effects on the expression of diverse sets of genes, some of which are located in characteristic arrangements along the DNA. Among affected genes is a set involved in the development of adipocytes, the cells that make and accumulate body fat, potentially explaining reductions in body fat accumulation commonly observed in individuals with CdLS.
Cell surface heparan sulfate proteoglycans (HSPGs) act as co-receptors for multiple families of growth factors that regulate animal cell proliferation, differentiation and patterning. Elimination of heparan sulfate during brain development is known to produce severe structural abnormalities. Here we investigate the developmental role played by one particular HSPG, glypican-1 (Gpc1), which is especially abundant on neuronal cell membranes, and is the major HSPG of the adult rodent brain.
Mice with a null mutation in Gpc1 were generated and found to be viable and fertile. The major phenotype associated with Gpc1 loss is a highly significant reduction in brain size, with only subtle effects on brain patterning (confined to the anterior cerebellum). The brain size difference emerges very early during neurogenesis (between embryonic days 8.5 and 9.5), and remains roughly constant throughout development and adulthood. By examining markers of different signaling pathways, and the differentiation behaviors of cells in the early embryonic brain, we infer that Gpc1-/- phenotypes most likely result from a transient reduction in fibroblast growth factor (FGF) signaling. Through the analysis of compound mutants, we provide strong evidence that Fgf17 is the FGF family member through which Gpc1 controls brain size.
These data add to a growing literature that implicates the glypican family of HSPGs in organ size control. They also argue that, among heparan sulfate-dependent signaling molecules, FGFs are disproportionately sensitive to loss of HSPGs. Finally, because heterozygous Gpc1 mutant mice were found to have brain sizes half-way between homozygous and wild type, the data imply that endogenous HSPG levels quantitatively control growth factor signaling, a finding that is both novel and relevant to the general question of how the activities of co-receptors are exploited during development.
We have identified a unique heparan sulfate (HeS) proteoglycan synthesized by the neuronal-like cell line PC12. The proteoglycan, purified with monoclonal antibodies from medium conditioned by PC12 cells, has an apparent molecular weight of 350,000, and it contains a Mr 80,000 core protein and HeS side chains of Mr 15,000 each. The purified molecule has the same apparent size and density as it has in conditioned medium.
HeS proteoglycans that are indistinguishable antigenically but very difficult to solubilize are found on the external surface and in the interior of PC12 cells and neurons. Mild proteolysis converts the surface proteoglycan into a molecule closely resembling that found in the medium. The same surface antigens are also present on a subpopulation of T-cells and on a non-neuronal accessory cell found in dorsal root ganglion cultures.
The PC12 cell line and the non-neuronal dorsal root ganglion cells secrete a factor into medium that, after adsorption to polylysine-coated surfaces, induces rapid neurite outgrowth by primary sympathetic neurons. The monoclonal antibodies used to purify the neuronal HeS proteoglycan from PC12 cells are capable of depleting this conditioned medium of its neurite-promoting activity. These studies suggest that a HeS proteoglycan synthesized and secreted by neurons and certain accessory cells plays a role in regulating neurite outgrowth.
A large, diverse, and growing number of strategies have been proposed to explain how morphogen gradients achieve robustness and precision. We argue that, to be useful, the evaluation of such strategies must take into account the constraints imposed by competing objectives and performance tradeoffs. This point is illustrated through a mathematical and computational analysis of the strategy of self-enhanced morphogen clearance. The results suggest that the usefulness of this strategy comes less from its ability to increase robustness to morphogen source fluctuations per se, than from its ability to overcome specific kinds of noise, and to increase the fraction of a morphogen gradient within which robust threshold positions may be established. This work also provides new insights into the longstanding question of why morphogen gradients show a maximum range in vivo.
Mathematical “cost-benefit” analyses provide insight into the prices developing organisms pay for strategies that increase robustness in morphogen-mediated patterning.
It is widely accepted that the growth and regeneration of tissues and organs is tightly controlled. Although experimental studies are beginning to reveal molecular mechanisms underlying such control, there is still very little known about the control strategies themselves. Here, we consider how secreted negative feedback factors (“chalones”) may be used to control the output of multistage cell lineages, as exemplified by the actions of GDF11 and activin in a self-renewing neural tissue, the mammalian olfactory epithelium (OE). We begin by specifying performance objectives—what, precisely, is being controlled, and to what degree—and go on to calculate how well different types of feedback configurations, feedback sensitivities, and tissue architectures achieve control. Ultimately, we show that many features of the OE—the number of feedback loops, the cellular processes targeted by feedback, even the location of progenitor cells within the tissue—fit with expectations for the best possible control. In so doing, we also show that certain distinctions that are commonly drawn among cells and molecules—such as whether a cell is a stem cell or transit-amplifying cell, or whether a molecule is a growth inhibitor or stimulator—may be the consequences of control, and not a reflection of intrinsic differences in cellular or molecular character.
Many tissues and organs grow to precise sizes and, when injured, regenerate accurately and rapidly. Here, we ask whether the organization of cells into lineages, and the feedback interactions that occur within lineages, are necessary elements of control strategies that make such behavior possible. Drawing on mathematical modeling and the results of experimental manipulation of the mouse olfactory epithelium, we show that performance objectives, such as robust size specification, fast regeneration from a variety of initial conditions, and maintenance of high ratios of differentiated to undifferentiated cells, can be simultaneously achieved through a combination of lineage structures, signaling mechanisms, and spatial distributions of cell types that correspond well with what is observed in many growing and regenerating tissues. Key to successful control is an integral-feedback mechanism that is implemented when terminally differentiated cells secrete molecules that lower the probability that progenitor cells replicate versus differentiate. Interestingly, this mechanism also explains how the distinctive proliferative behaviors of stem cell and “transit-amplifying” cell populations can emerge as a consequence of feedback effects, rather than intrinsic programming of cell types.
Are common, generic strategies used in the quantitative control of tissue growth and regeneration? An investigation of feedback effects in multistage lineages suggests they are.
Cells isolated from many types of human cancers express heparin-binding growth factors (HBGFs) that drive tumor growth, metastasis, and angiogenesis. The heparan sulfate proteoglycan glypican-1 (GPC1) is a coreceptor for HBGFs. Here we show that both cancer cell–derived and host-derived GPC1 are crucial for efficient growth, metastasis, and angiogenesis of human and mouse cancer cells. Thus downregulation of GPC1 in the human pancreatic cancer cell line PANC-1, using antisense approaches, resulted in prolonged doubling times and decreased anchorage-independent growth in vitro as well as attenuated tumor growth, angiogenesis, and metastasis when these cells were transplanted into athymic mice. Moreover, athymic mice that lacked GPC1 exhibited decreased tumor angiogenesis and metastasis following intrapancreatic implantation with either PANC-1 or T3M4 human pancreatic cancer cells and fewer pulmonary metastases following intravenous injection of murine B16-F10 melanoma cells. In addition, hepatic endothelial cells isolated from these mice exhibited an attenuated mitogenic response to VEGF-A. These data indicate that cancer cell– and host-derived GPC1 are crucial for full mitogenic, angiogenic, and metastatic potential of cancer cells. Thus targeting GPC1 might provide new avenues for cancer therapy and for the prevention of cancer metastasis.
Positional identities along the anterior–posterior axis of the vertebrate nervous system are assigned during gastrulation by multiple posteriorizing signals, including retinoic acid (RA), fibroblast growth factors (Fgfs), and Wnts. Experimental evidence has suggested that RA, which is produced in paraxial mesoderm posterior to the hindbrain by aldehyde dehydrogenase 1a2 (aldh1a2/raldh2), forms a posterior-to-anterior gradient across the hindbrain field, and provides the positional information that specifies the locations and fates of rhombomeres. Recently, alternative models have been proposed in which RA plays only a permissive role, signaling wherever it is not degraded. Here we use a combination of experimental and modeling tools to address the role of RA in providing long-range positional cues in the zebrafish hindbrain. Using cell transplantation and implantation of RA-coated beads into RA-deficient zebrafish embryos, we demonstrate that RA can directly convey graded positional information over long distances. We also show that expression of Cyp26a1, the major RA-degrading enzyme during gastrulation, is under complex feedback and feedforward control by RA and Fgf signaling. The predicted consequence of such control is that RA gradients will be both robust to fluctuations in RA synthesis and adaptive to changes in embryo length during gastrulation. Such control also provides an explanation for the fact that loss of an endogenous RA gradient can be compensated for by RA that is provided in a spatially uniform manner.
The formation of gradients of morphogens, signaling molecules that determine cell fates in a concentration-dependent manner, is a fundamental process in developmental biology. Several morphogens pattern the anterior–posterior (head to tail) axis of the vertebrate nervous system, including the vitamin A derivative, retinoic acid (RA) and fibroblast growth factors (Fgfs). However, it remains unclear how the activities of such morphogen gradients are coordinated. We have addressed this question by combining genetic experiments in zebrafish and computational analyses. We show that RA acts as a graded signal over long distances and that its gradient is shaped, to a large extent, by local control of RA degradation. In particular, RA promotes and Fgf suppresses RA degradation, thereby linking the shapes of RA and Fgf gradients. Computational models suggest that this linkage helps make RA-mediated patterning robust to changes in the rate at which RA is synthesized (which may vary with levels of dietary vitamin A) as well as in the size and shape of the embryo during development. Analogous regulatory loops may be used for similar purposes in other tissues in which RA and Fgfs interact, as well as in other morphogen systems.
Experimental and computational studies in zebrafish reveal a complex system regulating degradation of the vitamin A derivative retinoic acid along the anterior-posterior axis, which helps explain how morphogen gradients are established and maintained.