Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.     

BMP-2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin-3 (NT-3) and also glial cell line- derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum-free conditions, none of the factors tested, including NT-3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein-2 (BMP-2), promoted the survival of tyrosine hydroxylase (TH)-immunoreactive neurons at 6 days in vitro. However, when BMP-2 was coadministered with any of these factors the number of LC TH-positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT-3, bFGF, or BMP-2. The strongest effect (a fourfold increase in the number of TH-positive cells) was induced by cotreatment with forskolin, BMP-2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors.     

BMP‐2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin‐3 (NT‐3) and also glial cell line‐ derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum‐free conditions, none of the factors tested, including NT‐3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein‐2 (BMP‐2), promoted the survival of tyrosine hydroxylase (TH)‐immunoreactive neurons at 6 days in vitro. However, when BMP‐2 was coadministered with any of these factors the number of LC TH‐positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT‐3, bFGF, or BMP‐2. The strongest effect (a fourfold increase in the number of TH‐positive cells) was induced by cotreatment with forskolin, BMP‐2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 291–304, 2002; DOI 10.1002/neu.10034     

Control of the maturation and the survival of central noradrenergic neurons in culture.

Central noradrenergic neurons from the locus coeruleus express unique plastic properties. The aim of this study was to identify factors that specifically regulate the development and the survival of the noradrenergic cells. Primary dissociated cultures of embryonic locus coeruleus (LC) neurons were established. Norepinephrine (NE) uptake was used as an index of maturation of the noradrenergic neurons. The noradrenergic cells were identified and quantified following immunocytochemical staining for tyrosine hydroxylase antibody. We have examined the effect of hippocampal target tissue and of cyclic-AMP (cAMP) on the development of these cells. Coculturing LC cells with a low density of hippocampal target cells, resulted in a significant increase in NE uptake. However, when the amount of hippocampal target cells was doubled an enormous decrease in NE uptake occurred. The target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to target effect, we also tested the effect of elevated intracellular cAMP level on NE uptake versus GABA uptake. GABA uptake served as a developmental index of the non noradrenergic cells. Increasing the intracellular cAMP level, by application of the membrane permeable analog dibutyryl cyclic AMP (DbcAMP), resulted in a selective stimulation of NE uptake, due to enhanced survival of noradrenergic neurons. GABA uptake and the number of non-noradrenergic cells were not changed in the presence of DbcAMP. DbcAMP could maintain the survival of LC neurons in the absence of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation

Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification

Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.     

Evidence for Involvement of Wnt Signalling in Body Polarities,Cell Proliferation,and the Neuro-Sensory System in an Adult Ctenophore

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.     

Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem cells

This study examines the role of Wnt signaling events in regulating the differential potential of mesenchymal stem cells (MSCs) from adult bone marrow (BM). Immunohistochemical analysis of BM revealed co-localization of Wnt5a protein, a non-canonical Wnt, with CD45(+) cells and CD45(-) STRO-1(+) cells, while Wnt3a expression, a canonical Wnt, was associated with the underlying stroma matrix, suggesting that Wnts may regulate MSCs in their niche in BM. To elucidate the role of Wnts in MSC development, adult human BM-derived mononuclear cells were maintained as suspension cultures to recapitulate the marrow cellular environment, in serum-free, with the addition of Wnt3a and Wnt5a protein. Results showed that Wnt3a increased cell numbers and expanded the pool of MSCs capable of colony forming unit -- fibroblast (CFU-F) and CFU -- osteoblast (O), while Wnt5a maintained cell numbers and CFU-F and CFU-O numbers. However, when cells were cultured directly onto tissue culture plastic, Wnt5a increased the number of CFU-O relative to control conditions. These findings suggest the potential dual role of Wnt5a in the maintenance of MSCs in BM and enhancing osteogenesis ex vivo. Our work provides evidence that Wnts can function as mesenchymal regulatory factors by providing instructive cues for the recruitment, maintenance, and differentiation of MSCs.     

Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons

Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c-ret, which binds ligands of the glial cell line-derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)-positive neurons in the LC express both GFRalpha1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down-regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH-positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6-hydroxydopamine (6-OHDA) lesion model, but induced the sprouting of TH-positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.     

Insulin and insulin-like growth factor-I can trigger the differentiation of catecholaminergic precursors in cultures of dorsal root ganglia

Avian sensory ganglia contain a population of normally latent autonomic-type precursors with noradrenergic potentialities. Their differentiation in vitro into cells expressing tyrosine hydroxylase immunoreactivity is acutely dependent on the presence of one or more substances found in chick embryo extract (CEE). We have used cultures of dissociated dorsal root ganglia from embryonic quail as a model system in which to assay factors promoting catecholaminergic differentiation, the latter being appreciated quantitatively in terms of the number of tyrosine hydroxylase-positive cells present after 6 days in vitro; over a large range of concentrations, the number of such cells is directly proportional to the amount of CEE in the medium. In the course of attempts to replace CEE by defined bioactive molecules, we found that epidermal growth factor, fibroblast growth factor or nerve growth factor possessed negligible, or only marginal, noradrenergic differentiation-promoting activity. In contrast, insulin, at nanomolar levels, triggered expression of the catecholaminergic phenotype as well as did CEE. Insulin-like growth factor-I, at similar concentrations, had an analogous effect. It is suggested that an insulin-like molecule may play a role in the normal differentiation of sympathoblast precursors in vivo.     

Neuronal expression of mutations affecting primarily glia in the mouse: studies in the dysmyelinated and convulsive mutant quaking

1. Various aspects of the noradrenergic system in the brain of the dysmyelinating convulsive mutant mice quaking have been examined. 2. Determination of the endogenous contents of noradrenaline and its metabolite 3-methoxy 4-hydroxyphenyl-ethyleneglycol (MOPEG), as well as measurement of the electrically-evoked release of (3H)-noradrenaline shows an increased noradrenergic activity in the brain of the mutants, when compared to non convulsive controls of the same strain. 3. Ontogenic development of alpha adrenergic receptors indicate that an increased density of alpha-2 sites precedes the appearance of the first convulsions by approximately one week. 4. Anatomical determination of the number of noradrenergic neuronal cell bodies in the locus coeruleus shows a hyperplasia of this nucleus in the mutants. 5. Electrolytic coagulation of the locus coeruleus inhibits the convulsions of the quaking mice. 6. These results suggest that an alteration of the embryonic differentiation of the locus coeruleus, which gives rise to the majority of brain noradrenergic neurons, provokes a hyperactivity of this neuronal system, thereby triggering the convulsions of the quaking mutant mice. 7. The possible involvement of other neurotransmitter systems in the convulsions of these mutants, together with the nature of the relationship between neuronal abnormalities and dysmyelination phenomenon, are discussed.     

Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers

Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Here we showed that the bone morphogenetic protein antagonist noggin is critical in preventing differentiation of hES cells in culture. Furthermore, we found that the combination of noggin and basic fibroblast growth factor (bFGF) was sufficient to maintain the prolonged growth of hES cells while retaining all hES cell features. Since both noggin and bFGF are expressed in MEF, our findings suggest that they may be important factors secreted by MEF for maintaining undifferentiated pluripotent hES cells. Our data provide new insight into the mechanism how hES cell self-renewal is regulated. The newly developed feeder-free culture system will provide a more reliable alternative for future therapeutic applications of hES cells.     

Multiple roles of Activin/Nodal,bone morphogenetic protein,fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.     

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.     

Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo.

The dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin in the early patterning of organisms other than Xenopus. We have studied the contribution of Noggin to the embryonic development of the zebrafish. While single-copy noggin genes have been characterized in several vertebrate species, we report that the zebrafish genome harbors three noggin homologues. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Finally we show that noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish.