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Papers by Elke Stein
Neuron, 2002
dendrites of pyramidal cells require directional informa-1 Department of Neuroscience tion to gro... more dendrites of pyramidal cells require directional informa-1 Department of Neuroscience tion to grow toward the pia, in the direction opposite the Johns Hopkins University School of Medicine axons. After the initial polarity of a neuron is established, 725 N. Wolfe Street several primary dendrites emerge from the cell body, Baltimore, Maryland 21205 which subsequently undergo extensive growth and 2 Institut de la Santé et de le Recherche branching. At the same time, the apical dendrite sends Mé dicale U106 out collateral branches that serve as major synaptic Bâ timent de Pé diatrie sites. This process allows neurons to achieve their ma-Hô pital de la Salpê triè re ture morphology and to receive input from afferent 47 Boulevard de l'Hô pital axons. 75013 Paris In previous work directed at characterizing signals France that regulate patterning of axons and dendrites in the 3 Howard Hughes Medical Institute cortex, we demonstrated the presence of a soluble axon Department of Anatomy chemorepellant activity concentrated near the pial sur-Department of Biochemistry and Biophysics face of the developing cortical plate (Polleux et al., 1998).
The Journal of cell biology, Jan 25, 2016
Axons navigate long distances through complex 3D environments to interconnect the nervous system ... more Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon gui...
Cell, 2023
Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Delet... more Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.
Host-Pathogen Interactions, 2017
Bacterial cells use the quorum sensing system to communicate with each other. The gram-negative s... more Bacterial cells use the quorum sensing system to communicate with each other. The gram-negative species very often use N-acyl homoserine lactones for this purpose. One of the easiest ways to detect these molecules is the use of particular reporter strains, which possess different kinds of reporter genes under the control of AHL-responsive promoters. Here we present some of the possibilities available today, even for not specialized researchers.
F1000 - Post-publication peer review of the biomedical literature, 2008
Insulin receptor signaling has been postulated to play a role in synaptic plasticity, however the... more Insulin receptor signaling has been postulated to play a role in synaptic plasticity, however the function of the insulin receptor in CNS is not clear. To test whether insulin receptor signaling affects visual system function, we recorded light-evoked responses in optic tectal neurons in living Xenopus tadpoles. Tectal neurons transfected with dominant negative insulin receptor (dnIR) which reduces insulin receptor phosphorylation or morpholino against insulin receptor which reduces total insulin receptor protein level have significantly smaller light-evoked responses than controls. dnIR-expressing neurons have reduced synapse density assessed by EM, decreased AMPA mEPSC frequency and altered experience-dependent dendritic arbor structural plasticity, although synaptic vesicle release probability, assessed by paired pulse responses, synapse maturation, assessed by AMPA/NMDA ratio and ultrastructural criteria, is unaffected by dnIR expression. These data indicate that insulin receptor signaling regulates circuit function and plasticity by controlling synapse density.
Journal of Neuroscience, 2012
Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental pr... more Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental processes including axon guidance, dendrite arborization, and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM del17) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization, volume, and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness, but these reductions could not be attributed to changes in neuron production or cell death. DSCAM del17 mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM del17 dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42, mutant mice displayed a decrease in the percentage of large, stable spines and an increase in the percentage of small, immature spines. Together, our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development.
Science, 2001
Axonal growth cones that cross the nervous system midline change their responsiveness to midline ... more Axonal growth cones that cross the nervous system midline change their responsiveness to midline guidance cues: They become repelled by the repellent Slit and simultaneously lose responsiveness to the attractant netrin. These mutually reinforcing changes help to expel growth cones from the midline by making a once-attractive environment appear repulsive. Here, we provide evidence that these two changes are causally linked: In the growth cones of embryonic Xenopus spinal axons, activation of the Slit receptor Roundabout (Robo) silences the attractive effect of netrin-1, but not its growth-stimulatory effect, through direct binding of the cytoplasmic domain of Robo to that of the netrin receptor DCC. Biologically, this hierarchical silencing mechanism helps to prevent a tug-of-war between attractive and repulsive signals in the growth cone that might cause confusion. Molecularly, silencing is enabled by a modular and interlocking design of the cytoplasmic domains of these potentially ...
Neuron, 2001
tebrate plexins are divided into four subfamilies (A through D) based on structural similarity (T... more tebrate plexins are divided into four subfamilies (A through D) based on structural similarity (Tamagnone et al., 1999). All these proteins are transmembrane proteins with a semaphorin (sema) domain followed by a cysteinerich motif in their extracellular region and a conserved
Molecular and Cellular Neuroscience, 2005
Journal of Biological Chemistry, 1998
Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis;... more Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr 594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK. Eph family receptor tyrosine kinases transmit signals that direct cell migration, cell targeting, and cell-cell aggregation (1-5). These receptors are functionally subdivided into two subclasses (EphA or EphB) based on their overlapping affinities for either glycerolphosphatidylinositol-linked (ephrins A1-A5) or transmembrane (ephrins B1-B3) protein ligands (6, 7). Many Eph family receptor tyrosine kinases and their ligands are reciprocally compartmentalized during development, consistent with their roles in directing migration and organization of specialized cell-cell interactions (2, 8-10). For example, tectal gradients of the EphA3 (Mek4) ligand, ephrin A2 (ELF-1), direct developmental targeting of retinal axons expressing EphA3 receptor (11, 12). Similarly, axonal migratory paths of spinal motor neurons expressing Eph receptors, EphB4 and EphB3 (HTK/HEK2), are directed by segmental expression of the EphB4 ligand, ephrin B2 (5). In a reconstituted system, 32D cells transfected with EphB3 (Hek2) receptors aggregate with cells transfected with the EphB3 ligand, ephrin B1 (4). The intracellular mediators of these targeting responses are incompletely defined. EphA2 (Eck) and EphB1 (ELK) are pro
Genes & Development, 1998
Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons withi... more Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.
FEBS Letters, 1989
The muscular content of the mRNAs encoding the five subunits of the nicotinic acetylcholine recep... more The muscular content of the mRNAs encoding the five subunits of the nicotinic acetylcholine receptor was measured during postnatal development in the rat. Subunit specific mRNAs show differchtial regulation. The levels of the a-, yand &subunit specific mRNAs decrease steadily after birth, while the & and a-subunit mRNAs increase transiently and then decrease. The adult pattern of subunit specific mRNA levels is reached at 46 weeks postnatally. The content of y-and e-subunit mRNA changes in a reciprocal fashion during the first 2 postnatal weeks, supporting the view that differential regulation of y-and s-subunit mRNA during development is one mechanism mediating the appearance of the adult, s-subunit containing, subtype of end-plate channel. Denervation of neonatal muscle increases the levels of all subunitspecific mRNAs during further development. It prevents the postnatal decrease in y-subunit mRNA and enhances the initial increase in a-subunit mRNA. This makes it appear that the e-subunit gene is less sensitive to regulation by the nerve in the postnatal period than the y-subunit gene.
Current Opinion in Neurobiology, 2007
In the developing nervous system, nerve cells and axons respond to various attractive and repulsi... more In the developing nervous system, nerve cells and axons respond to various attractive and repulsive guidance cues while traveling to their final destination. Netrins are bifunctional guidance cues that attract several classes of axons but repel others. The response of an axon to netrins is dictated by the composition of netrin receptors on the cell surface and the internal state of the growth cone. Recent analyses have identified several signal transduction pathways that contribute to netrin-mediated guidance. A model emerges in which tyrosine phosphorylation, phosphatidylinositol signaling and regulation by Rho GTPases act in concert to trigger extension of axons and turning of growth cones in response to Netrin1.
Current Biology, 2004
glandular stage), the primordial buds give rise to the respiratory tree [1-3]. This process requi... more glandular stage), the primordial buds give rise to the respiratory tree [1-3]. This process requires FGFR2IIIb, a receptor isoform expressed in the endoderm, and FGFs made in the mesoderm. Expression of Fgf10 is highly localized to the distal mesoderm adjacent to the tips of
Cell, 2003
and subsequently across the floor plate, forming axon commissures (Figure 1A and see also Colamar... more and subsequently across the floor plate, forming axon commissures (Figure 1A and see also Colamarino and Tessier-Lavigne, 1995). These commissural axons project toward the midline in part because they are attracted by Netrin-1, a long-range chemoattractant secreted by
Cell, 2003
of the target regions to which it initially projected. Within each region, further branch formati... more of the target regions to which it initially projected. Within each region, further branch formation (terminal arborization) and retraction then shapes the final pattern of synaptic contacts. At the same time, naturally occurring neuronal cell death culls many of these neurons, estab
Cell, 2011
The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amylo... more The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloidb precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.
Proceedings of the National Academy of Sciences, 2009
Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical ... more Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8–16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb inte...
The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-... more The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-, delta- and epsilon-subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457-519 amino acids and reveal the structural characteristics common to subunits of ligand-gated ion channels. The pattern of subunit-specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the alpha-, beta- and delta-subunit mRNAs, suppresses the expression of the gamma-subunit mRNA, and induces expression of epsilon-subunit mRNA. Subunit-specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChR gamma, composed of the alpha-, beta-, gamma- and delta-subunits, has functional properties similar...
Neuron, 2002
dendrites of pyramidal cells require directional informa-1 Department of Neuroscience tion to gro... more dendrites of pyramidal cells require directional informa-1 Department of Neuroscience tion to grow toward the pia, in the direction opposite the Johns Hopkins University School of Medicine axons. After the initial polarity of a neuron is established, 725 N. Wolfe Street several primary dendrites emerge from the cell body, Baltimore, Maryland 21205 which subsequently undergo extensive growth and 2 Institut de la Santé et de le Recherche branching. At the same time, the apical dendrite sends Mé dicale U106 out collateral branches that serve as major synaptic Bâ timent de Pé diatrie sites. This process allows neurons to achieve their ma-Hô pital de la Salpê triè re ture morphology and to receive input from afferent 47 Boulevard de l'Hô pital axons. 75013 Paris In previous work directed at characterizing signals France that regulate patterning of axons and dendrites in the 3 Howard Hughes Medical Institute cortex, we demonstrated the presence of a soluble axon Department of Anatomy chemorepellant activity concentrated near the pial sur-Department of Biochemistry and Biophysics face of the developing cortical plate (Polleux et al., 1998).
The Journal of cell biology, Jan 25, 2016
Axons navigate long distances through complex 3D environments to interconnect the nervous system ... more Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon gui...
Cell, 2023
Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Delet... more Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.
Host-Pathogen Interactions, 2017
Bacterial cells use the quorum sensing system to communicate with each other. The gram-negative s... more Bacterial cells use the quorum sensing system to communicate with each other. The gram-negative species very often use N-acyl homoserine lactones for this purpose. One of the easiest ways to detect these molecules is the use of particular reporter strains, which possess different kinds of reporter genes under the control of AHL-responsive promoters. Here we present some of the possibilities available today, even for not specialized researchers.
F1000 - Post-publication peer review of the biomedical literature, 2008
Insulin receptor signaling has been postulated to play a role in synaptic plasticity, however the... more Insulin receptor signaling has been postulated to play a role in synaptic plasticity, however the function of the insulin receptor in CNS is not clear. To test whether insulin receptor signaling affects visual system function, we recorded light-evoked responses in optic tectal neurons in living Xenopus tadpoles. Tectal neurons transfected with dominant negative insulin receptor (dnIR) which reduces insulin receptor phosphorylation or morpholino against insulin receptor which reduces total insulin receptor protein level have significantly smaller light-evoked responses than controls. dnIR-expressing neurons have reduced synapse density assessed by EM, decreased AMPA mEPSC frequency and altered experience-dependent dendritic arbor structural plasticity, although synaptic vesicle release probability, assessed by paired pulse responses, synapse maturation, assessed by AMPA/NMDA ratio and ultrastructural criteria, is unaffected by dnIR expression. These data indicate that insulin receptor signaling regulates circuit function and plasticity by controlling synapse density.
Journal of Neuroscience, 2012
Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental pr... more Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental processes including axon guidance, dendrite arborization, and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM del17) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization, volume, and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness, but these reductions could not be attributed to changes in neuron production or cell death. DSCAM del17 mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM del17 dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42, mutant mice displayed a decrease in the percentage of large, stable spines and an increase in the percentage of small, immature spines. Together, our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development.
Science, 2001
Axonal growth cones that cross the nervous system midline change their responsiveness to midline ... more Axonal growth cones that cross the nervous system midline change their responsiveness to midline guidance cues: They become repelled by the repellent Slit and simultaneously lose responsiveness to the attractant netrin. These mutually reinforcing changes help to expel growth cones from the midline by making a once-attractive environment appear repulsive. Here, we provide evidence that these two changes are causally linked: In the growth cones of embryonic Xenopus spinal axons, activation of the Slit receptor Roundabout (Robo) silences the attractive effect of netrin-1, but not its growth-stimulatory effect, through direct binding of the cytoplasmic domain of Robo to that of the netrin receptor DCC. Biologically, this hierarchical silencing mechanism helps to prevent a tug-of-war between attractive and repulsive signals in the growth cone that might cause confusion. Molecularly, silencing is enabled by a modular and interlocking design of the cytoplasmic domains of these potentially ...
Neuron, 2001
tebrate plexins are divided into four subfamilies (A through D) based on structural similarity (T... more tebrate plexins are divided into four subfamilies (A through D) based on structural similarity (Tamagnone et al., 1999). All these proteins are transmembrane proteins with a semaphorin (sema) domain followed by a cysteinerich motif in their extracellular region and a conserved
Molecular and Cellular Neuroscience, 2005
Journal of Biological Chemistry, 1998
Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis;... more Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr 594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK. Eph family receptor tyrosine kinases transmit signals that direct cell migration, cell targeting, and cell-cell aggregation (1-5). These receptors are functionally subdivided into two subclasses (EphA or EphB) based on their overlapping affinities for either glycerolphosphatidylinositol-linked (ephrins A1-A5) or transmembrane (ephrins B1-B3) protein ligands (6, 7). Many Eph family receptor tyrosine kinases and their ligands are reciprocally compartmentalized during development, consistent with their roles in directing migration and organization of specialized cell-cell interactions (2, 8-10). For example, tectal gradients of the EphA3 (Mek4) ligand, ephrin A2 (ELF-1), direct developmental targeting of retinal axons expressing EphA3 receptor (11, 12). Similarly, axonal migratory paths of spinal motor neurons expressing Eph receptors, EphB4 and EphB3 (HTK/HEK2), are directed by segmental expression of the EphB4 ligand, ephrin B2 (5). In a reconstituted system, 32D cells transfected with EphB3 (Hek2) receptors aggregate with cells transfected with the EphB3 ligand, ephrin B1 (4). The intracellular mediators of these targeting responses are incompletely defined. EphA2 (Eck) and EphB1 (ELK) are pro
Genes & Development, 1998
Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons withi... more Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.
FEBS Letters, 1989
The muscular content of the mRNAs encoding the five subunits of the nicotinic acetylcholine recep... more The muscular content of the mRNAs encoding the five subunits of the nicotinic acetylcholine receptor was measured during postnatal development in the rat. Subunit specific mRNAs show differchtial regulation. The levels of the a-, yand &subunit specific mRNAs decrease steadily after birth, while the & and a-subunit mRNAs increase transiently and then decrease. The adult pattern of subunit specific mRNA levels is reached at 46 weeks postnatally. The content of y-and e-subunit mRNA changes in a reciprocal fashion during the first 2 postnatal weeks, supporting the view that differential regulation of y-and s-subunit mRNA during development is one mechanism mediating the appearance of the adult, s-subunit containing, subtype of end-plate channel. Denervation of neonatal muscle increases the levels of all subunitspecific mRNAs during further development. It prevents the postnatal decrease in y-subunit mRNA and enhances the initial increase in a-subunit mRNA. This makes it appear that the e-subunit gene is less sensitive to regulation by the nerve in the postnatal period than the y-subunit gene.
Current Opinion in Neurobiology, 2007
In the developing nervous system, nerve cells and axons respond to various attractive and repulsi... more In the developing nervous system, nerve cells and axons respond to various attractive and repulsive guidance cues while traveling to their final destination. Netrins are bifunctional guidance cues that attract several classes of axons but repel others. The response of an axon to netrins is dictated by the composition of netrin receptors on the cell surface and the internal state of the growth cone. Recent analyses have identified several signal transduction pathways that contribute to netrin-mediated guidance. A model emerges in which tyrosine phosphorylation, phosphatidylinositol signaling and regulation by Rho GTPases act in concert to trigger extension of axons and turning of growth cones in response to Netrin1.
Current Biology, 2004
glandular stage), the primordial buds give rise to the respiratory tree [1-3]. This process requi... more glandular stage), the primordial buds give rise to the respiratory tree [1-3]. This process requires FGFR2IIIb, a receptor isoform expressed in the endoderm, and FGFs made in the mesoderm. Expression of Fgf10 is highly localized to the distal mesoderm adjacent to the tips of
Cell, 2003
and subsequently across the floor plate, forming axon commissures (Figure 1A and see also Colamar... more and subsequently across the floor plate, forming axon commissures (Figure 1A and see also Colamarino and Tessier-Lavigne, 1995). These commissural axons project toward the midline in part because they are attracted by Netrin-1, a long-range chemoattractant secreted by
Cell, 2003
of the target regions to which it initially projected. Within each region, further branch formati... more of the target regions to which it initially projected. Within each region, further branch formation (terminal arborization) and retraction then shapes the final pattern of synaptic contacts. At the same time, naturally occurring neuronal cell death culls many of these neurons, estab
Cell, 2011
The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amylo... more The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloidb precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.
Proceedings of the National Academy of Sciences, 2009
Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical ... more Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8–16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb inte...
The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-... more The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-, delta- and epsilon-subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457-519 amino acids and reveal the structural characteristics common to subunits of ligand-gated ion channels. The pattern of subunit-specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the alpha-, beta- and delta-subunit mRNAs, suppresses the expression of the gamma-subunit mRNA, and induces expression of epsilon-subunit mRNA. Subunit-specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChR gamma, composed of the alpha-, beta-, gamma- and delta-subunits, has functional properties similar...
Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical ... more Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8 –16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease , duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS—i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease. copy number variants genomic structural variation human genome congenital heart disease leukemia
of the target regions to which it initially projected. Within each region, further branch formati... more of the target regions to which it initially projected. Within each region, further branch formation (terminal arboriza-tion) and retraction then shapes the final pattern of syn-aptic contacts. At the same time, naturally occurring neuronal cell death culls many of these neurons, estab-Program in Neuroscience lishing the final numbers of each neuronal population. University of California, San Francisco Much headway has been made in understanding the San Francisco, California 94143 mechanisms underlying four of these five events. The 2 Department of Biological Sciences first two, growth cone guidance and branch formation, Howard Hughes Medical Institute are controlled by positive and negative guidance and Stanford University branching cues, which include members of the netrin, Stanford, California 94305 semaphorin, slit, and ephrin families (Tessier-Lavigne and Goodman, 1996). The last two, terminal arborization and cell death, are also controlled by specific molecular Summary regulators which include members of the neurotrophin family, as well as by competitive interactions among Like naturally occurring neuronal cell death, stereo-axons regulated by specific patterns of electrical activity typed pruning of long axon branches to temporary (Herrmann and Shatz, 1995). targets is a widespread regressive phenomenon in In contrast, our understanding of the mechanisms the developing mammalian brain that helps sculpt the controlling the fifth type of event, the stereotyped prun-pattern of neuronal connections. The mechanisms con-ing of projections to temporary target regions, is much trolling stereotyped pruning are, however, poorly un-more fragmentary. (In this paper, we will refer to this derstood. Here, we provide evidence that semapho-pruning as " stereotyped pruning " , to distinguish it from rins, activating the Plexin-A3 receptor, function as the local pruning of subsets of branches that occurs retraction inducers to trigger-stereotyped pruning of within target regions to regulate terminal arborization.) specific hippocampal mossy fiber and pyramidal axon Stereotyped pruning is extensive and specific, as is well branches. Both pruning events are defective in Plexin-illustrated by the studies of O'Leary and colleagues on A3 mutants, reflecting a cell-autonomous requirement layer 5 neurons in the visual and motor cortex (Stanfield for Plexin-A3. The distribution of mRNAs for Sema3F and O'Leary, 1985; O'Leary and Stanfield, 1986; O'Leary and Sema3A makes them candidates for triggering the et al., 1990). In the adult, these two populations of neu-pruning. In vitro, hippocampal neurons respond to rons connect to overlapping but distinct sets of target semaphorins by retracting axon branches. These re-regions in a functionally appropriate way: those in visual sults implicate semaphorins as retraction inducers but not motor cortex connect to the superior colliculus controlling stereotyped pruning in the mammalian brain. (a visual processing center), those in motor but not visual cortex connect to the spinal cord (a motor control cen-Introduction ter), and both connect to the pons (which is involved in both sensory and motor control). Remarkably, these Neurons in vertebrates usually make connections with divergent adult patterns arise from differential sculpting multiple target cells. In the mammalian brain, for exam-of initially concordant projection patterns: both classes ple, each neuron contacts on average over a hundred of neurons initially project to the spinal cord, then sprout synaptic partners, often in many different target areas. branches that invade all the targets of both sets of neu-The pattern of connections is reproducible from animal rons, including the superior colliculus and the pons. to animal and is established during embryonic develop-Later, the neurons in each class selectively prune just ment through five types of progressive and regressive those projections that are functionally inappropriate. Several aspects of long-range stereotyped branch events (Tessier-Lavigne and Goodman, 1996; Albright pruning mark it out as a cellular phenomenon that is et al., 2000). Initially, each neuron sends out a single distinct from other aspects of neuronal morphogenesis. axon, whose motile tip (or growth cone) is guided to an First, the branches that are retracted are indeed stereo-initial target. To connect to multiple targets, additional typed, i.e., they are identifiable prior to the onset of axon branches must form from the initial axon shaft by pruning. This is in sharp contrast to the pruning of short interstitial axon branching (or more rarely by splitting of terminal arbors, which occurs seemingly stochas-the initial growth cone), and be guided to the additional tically—perhaps reflecting competitive forces (Bern-target areas. Many of the projections are, however, tran-stein and Lichtman, 1999). Second, the length of the sient, and are later pruned in a stereotyped fashion, axon branches that is retracted is often enormous— leaving each neuron with connections only to a subset many millimeters in the case of visual cortical efferents that project to the spinal cord. Again, this is in sharp *Correspondence: marctl@stanford.edu (M.T.-L.); samuelp@itsa. contrast to terminal arbor pruning, which usually in-ucsf.edu (S.J.P) volves a few micrometers of branches—a difference of 3 These authors contributed equally to this work. three orders of magnitude in the amount of axon that