Esther Bell

Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.

Bone morphogenetic proteins are members of the transforming growth factor beta (TGF beta) superfamily which are involved in a range of developmental processes including modelling of the skeleton. We show here that Bmp-2 is expressed in mesenchyme surrounding early cartilage condensations in the developing chick limb, and that Bmp-4 is expressed in the perichondrium of developing cartilage elements. To investigate their roles during cartilage development, BMP-2 and BMP-4 were expressed ectopically in developing chick limbs using retroviral vectors. Over-expression of BMP-2 or BMP-4 led to a dramatic increase in the volume of cartilage elements, altered their shapes and led to joint fusions. This increase in volume appeared to result from an increase in the amount of matrix and in the number of chondrocytes. The latter did not appear to be due to increased proliferation of chondrocytes, suggesting that it may result from increased recruitment of precursors. BMP-2 and BMP-4 also delayed hypertrophy of chondrocytes and formation of the osteogenic periosteum. These data provide insights into how BMP-2 and BMP-4 may model and control the growth of skeletal elements during normal embryonic development, suggesting roles for both molecules in recruiting non-chondrogenic precursors to chondrogenic fate.

DRAGON: a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)β superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFβ ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFβ signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFβ ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.

Cell fate specification and competence by Coco, a maternal BMP, TGFbeta and Wnt inhibitor.

Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFβ/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFβ signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.

DRAGON: A Member of the Repulsive Guidance Molecule-Related Family of Neuronal- and Muscle-Expressed Membrane Proteins Is Regulated by DRG11 and Has Neuronal Adhesive Properties

DRG11, a transcription factor expressed in embryonic dorsal root ganglion (DRG) and dorsal horn neurons, has a role in the development of sensory circuits. We have used a genomic binding strategy to screen for the promoter region of genes regulated by DRG11. One gene with a promoter region binding to the DNA binding domain of DRG11 encodes a novel membrane-associated [glycosyl-phosphatidylinositol (GPI)-anchored] protein that we call DRAGON. DRAGON expression is transcriptionally regulated by DRG11, and it is coexpressed with DRG11 in embryonic DRG and spinal cord. DRAGON expression in these areas is reduced in DRG11 null mutants. DRAGON is expressed, however, in the neural tube before DRG11, and unlike DRG11 it is expressed in the brain and therefore must be regulated by other transcriptional regulatory elements. DRAGON shares high sequence homology with two other GPI-anchored membrane proteins: the mouse ortholog of chick repulsive guidance molecule (mRGM), which is expressed in the mouse nervous system in areas complementary to DRAGON, and DRAGON-like muscle (DL-M), the expression of which is restricted to skeletal and cardiac muscle. A comparative genomic analysis indicates that the family of RGM-related genes—mRGM, DRAGON, and DL-M—are highly conserved among mammals, zebrafish, chick, and Caenorhabditis elegans but not Drosophila. DRAGON, RGM, and DL-M mRNA expression in the zebrafish embryo is similar to that in the mouse. Neuronal cell adhesion assays indicate that DRAGON promotes and mRGM reduces adhesion of mouse DRG neurons. We show that DRAGON interacts with itself homophilically. The dynamic expression, ordered spatial localization, and adhesive properties of the RGM-related family of membrane-associated proteins are compatible with specific roles in development.

Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein

The earliest decision in vertebrate neural development is the acquisition of a neural identity by embryonic ectodermal cells. The default model for neural induction postulates that neural fate specification in the vertebrate embryo occurs by inhibition of epidermal inducing signals in the gastrula ectoderm. Bone morphogenetic proteins (BMPs) act as epidermal inducers, and all identified direct neural inducers block BMP signaling either intra- or extracellularly. Although the mechanism of action of the secreted neural inducers has been elucidated, the relevance of intracellular BMP inhibitors in neural induction is not clear. In order to address this issue and to identify downstream targets after BMP inhibition, we have monitored the transcriptional changes in ectodermal explants neuralized by Smad7 using a Xenopus laevis 5000-clone gastrula-stage cDNA microarray. We report the identification and initial characterization of 142 genes whose transcriptional profiles change in the neuralized explants. In order to address the potential involvement during neural induction of genes identified in the array, we performed gain-of-function studies in ectodermal explants. This approach lead to the identification of four genes that can function as neural inducers in Xenopus and three others that can synergize with known neural inducers in promoting neural fates. Based on these studies, we propose a role for post-transcriptional control of gene expression during neural induction in vertebrates and present a model whereby sustained BMP inhibition is promoted partly through the regulation of TGFβ activated kinase (TAK1) activity by a novel TAK1-binding protein (TAB3).

Fluorescent labeling of endothelial cells allows in vivo, continuous characterization of the vascular development of Xenopus laevis

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.

Neuronatin Promotes Neural Lineage in ESCs via Ca2+ Signaling

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underling neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca2+ ([Ca2+]i) by antagonizing Ca2+‐ATPase isoform 2 (sarco/endoplasmic reticulum Ca2+‐ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway. STEM CELLS 2010;28:1950–1960

Segmental identity and cerebellar granule cell induction in rhombomere 1

BackgroundCerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?ResultsWe show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2.ConclusionsDifferent overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.

PRDC regulates placode neurogenesis in chick by modulating BMP signalling

The epibranchial placodes generate the neurons of the geniculate, petrosal, and nodose cranial sensory ganglia. Previously, it has been shown that bone morphogenetic proteins (BMPs) are involved in the formation of these structures. However, it has been unclear as to whether BMP signalling has an ongoing function in directing the later development of the epibranchial placodes, and how this signalling is regulated. Here, we demonstrate that BMPs maintain placodal neurogenesis and that their activity is modulated by a member of the Cerberus/Dan family of BMP antagonists, Protein Related to Dan and Cerberus (PRDC). We find that Bmp4 is expressed in the epibranchial placodes while Bmp7 and PRDC are expressed in the pharyngeal pouches. The timing and regional expression of these three genes suggest that BMP7 is involved in inducing placode neurogenesis and BMP4 in maintaining it and that BMP activity is modulated by PRDC. To investigate this hypothesis, we have performed both gain- and loss- of-function experiments with PRDC and find that it can modulate the BMP signals that induce epibranchial neurogenesis: a gain of PRDC function results in a loss of Bmp4 and hence placode neurogenesis is inhibited; conversely, a loss of PRDC function induces ectopic Bmp4 and an expansion of placode neurogenesis. This modulation is therefore necessary for the number and positioning of the epibranchial neurons.

Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo

Replication-competent retroviral vectors based on Rous sarcoma virus (RSV) are becoming increasingly popular for expressing genes in both primary cell cultures and embryonic chick tissuesin ovo. In this article, we review the features of RSV and its life cycle that make it suitable for use as a vector. We describe the design and use of the RCAS and RCAS(BP) series of vectors, which are currently the most widely used RSV-based vectors, illustrating both their strengths and weaknesses. Finally, we outline laboratory protocols suitable for the handling of these retroviral vectors.