Transcriptional Codes and the Control of Neuronal Identity

Annual Review of Neuroscience. 2002  |  Pubmed ID: 12052910

The topographic assembly of neural circuits is dependent upon the generation of specific neuronal subtypes, each subtype displaying unique properties that direct the formation of selective connections with appropriate target cells. Studies of motor neuron development in the spinal cord have begun to elucidate the molecular mechanisms involved in controlling motor projections. In this review, we first describe the actions of transcription factors within motor neuron progenitors, which initiate a cascade of transcriptional interactions that lead to motor neuron specification. We next highlight the contribution of the LIM homeodomain (LIM-HD) transcription factors in establishing motor neuron subtype identity. Importantly, it has recently been shown that the combinatorial expression of LIM-HD transcription factors, the LIM code, confers motor neuron subtypes with the ability to select specific axon pathways to reach their distinct muscle targets. Finally, the downstream targets of the LIM code are discussed, especially in the context of subtype-specific motor axon pathfinding.

LIM Factor Lhx3 Contributes to the Specification of Motor Neuron and Interneuron Identity Through Cell-type-specific Protein-protein Interactions

Cell. Jul, 2002  |  Pubmed ID: 12150931

LIM homeodomain codes regulate the development of many cell types, though it is poorly understood how these factors control gene expression in a cell-specific manner. Lhx3 is involved in the generation of two adjacent, but distinct, cell types for locomotion, motor neurons and V2 interneurons. Using in vivo function and protein interaction assays, we found that Lhx3 binds directly to the LIM cofactor NLI to trigger V2 interneuron differentiation. In motor neurons, however, Isl1 is available to compete for binding to NLI, displacing Lhx3 to a high-affinity binding site on the C-terminal region of Isl1 and thereby transforming Lhx3 from an interneuron-promoting factor to a motor neuron-promoting factor. This switching mechanism enables specific LIM complexes to form in each cell type and ensures that neuronal fates are tightly segregated.

Inhibition of Retroviral Pathogenesis by RNA Interference

Current Biology : CB. Aug, 2002  |  Pubmed ID: 12176358

RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates.

Fine-tuning Motor Neuron Properties: Signaling from the Periphery

Neuron. Aug, 2002  |  Pubmed ID: 12372278

Our understanding of motor neuron differentiation is rapidly evolving. New studies demonstrate that cells in the periphery of the embryo provide feedback signals for spinal cord motor neurons that are instrumental in the timing and regulation of their development. Two papers in this issue of Neuron identify a motor neuron survival factor, GDNF, and the ETS transcription factor, PEA3, as key components of a signal transduction pathway whose goals are 2-fold: to cluster motor pool-specific cell bodies and to promote axon arborization.

Synchronization of Neurogenesis and Motor Neuron Specification by Direct Coupling of BHLH and Homeodomain Transcription Factors

Neuron. Jun, 2003  |  Pubmed ID: 12797958

Inductive signaling leads to the coactivation of regulatory pathways for specifying general neuronal traits in parallel with instructions for neuronal subtype specification. Nevertheless, the mechanisms that ensure that these pathways are synchronized have not been defined. To address this, we examined how bHLH proteins Ngn2 and NeuroM controlling neurogenesis functionally converge with LIM-homeodomain (LIM-HD) factors Isl1 and Lhx3 involved in motor neuron subtype specification. We found that Ngn2 and NeuroM transcriptionally synergize with Isl1 and Lhx3 to specify motor neurons in the embryonic spinal cord and in P19 stem cells. The mechanism underlying this cooperativity is based on interactions that directly couple the activity of the bHLH and LIM-HD proteins, mediated by the adaptor protein NLI. This functional link acts to synchronize neuronal subtype specification with neurogenesis.

Isl1 Identifies a Cardiac Progenitor Population That Proliferates Prior to Differentiation and Contributes a Majority of Cells to the Heart

Developmental Cell. Dec, 2003  |  Pubmed ID: 14667410

Hearts of mice lacking Isl1, a LIM homeodomain transcription factor, are completely missing the outflow tract, right ventricle, and much of the atria. isl1 expression and lineage tracing of isl1-expressing progenitors demonstrate that Isl1 is a marker for a distinct population of undifferentiated cardiac progenitors that give rise to the cardiac segments missing in isl1 mutants. Isl1 function is required for these progenitors to contribute to the heart. In isl1 mutants, isl1-expressing progenitors are progressively reduced in number, and FGF and BMP growth factors are downregulated. Our studies define two sets of cardiogenic precursors, one of which expresses and requires Isl1 and the other of which does not. Our results have implications for the development of specific cardiac lineages, left-right asymmetry, cardiac evolution, and isolation of cardiac progenitor cells.

A Postmitotic Role for Isl-class LIM Homeodomain Proteins in the Assignment of Visceral Spinal Motor Neuron Identity

Neuron. Feb, 2004  |  Pubmed ID: 14766174

LIM homeobox genes have a prominent role in the regulation of neuronal subtype identity and distinguish motor neuron subclasses in the embryonic spinal cord. We have investigated the role of Isl-class LIM homeodomain proteins in motor neuron diversification using mouse genetic methods. All spinal motor neuron subtypes initially express both Isl1 and Isl2, but Isl2 is rapidly downregulated by visceral motor neurons. Mouse embryos lacking Isl2 function exhibit defects in the migration and axonal projections of thoracic level motor neurons that appear to reflect a cell-autonomous switch from visceral to somatic motor neuron character. Additional genetic mutations that reduce or eliminate both Isl1 and Isl2 activity result in more pronounced defects in visceral motor neuron generation and erode somatic motor neuron character. Thus, an early phase of high Isl expression and activity in newly generated motor neurons permits the diversification of visceral and somatic motor neuron subtypes in the developing spinal cord.

Analysis of Embryonic Motoneuron Gene Regulation: Derepression of General Activators Function in Concert with Enhancer Factors

Development (Cambridge, England). Jul, 2004  |  Pubmed ID: 15201216

The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. We have characterized how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. We found that non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9 located from -7096 to -6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9 expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord.

Recognition and Accommodation at the Androgen Receptor Coactivator Binding Interface

PLoS Biology. Sep, 2004  |  Pubmed ID: 15328534

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Axon Fasciculation Defects and Retinal Dysplasias in Mice Lacking the Immunoglobulin Superfamily Adhesion Molecule BEN/ALCAM/SC1

Molecular and Cellular Neurosciences. Sep, 2004  |  Pubmed ID: 15345243

The immunoglobulin superfamily adhesion molecule BEN (other names include ALCAM, SC1, DM-GRASP, neurolin, and CD166) has been implicated in the control of numerous developmental and pathological processes, including the guidance of retinal and motor axons to their targets. To test hypotheses about BEN function, we disrupted its gene via homologous recombination and analyzed the resulting mutant mice. Mice lacking BEN are viable and fertile, and display no external morphological defects. Despite grossly normal trajectories, both motor and retinal ganglion cell axons fasciculated poorly and were occasionally misdirected. In addition, BEN mutant retinae exhibited evaginated or invaginated regions with photoreceptor ectopias that resembled the "retinal folds" observed in some human retinopathies. Together, these results demonstrate that BEN promotes fasciculation of multiple axonal populations and uncover an unexpected function for BEN in retinal histogenesis.

Beta1 Integrins in Muscle, but Not in Motor Neurons, Are Required for Skeletal Muscle Innervation

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2004  |  Pubmed ID: 15371519

In vitro studies have provided evidence that beta1 integrins in motor neurons promote neurite outgrowth, whereas beta1 integrins in myotubes regulate acetylcholine receptor (AChR) clustering. Surprisingly, using genetic studies in mice, we show here that motor axon outgrowth and neuromuscular junction (NMJ) formation in large part are unaffected when the integrin beta1 gene (Itgb1) is inactivated in motor neurons. In the absence of Itgb1 expression in skeletal muscle, interactions between motor neurons and muscle are defective, preventing normal presynaptic differentiation. Motor neurons fail to terminate their growth at the muscle midline, branch excessively, and develop abnormal nerve terminals. These defects resemble the phenotype of agrin-null mice, suggesting that signaling molecules such as agrin, which coordinate presynaptic and postsynaptic differentiation, are not presented properly to nerve terminals. We conclude that Itgb1 expression in muscle, but not in motor neurons, is critical for NMJ development.

Magnitude of Binocular Vision Controlled by Islet-2 Repression of a Genetic Program That Specifies Laterality of Retinal Axon Pathfinding

Cell. Nov, 2004  |  Pubmed ID: 15537545

Pathfinding of retinal ganglion cell (RGC) axons at the midline optic chiasm determines whether RGCs project to ipsilateral or contralateral brain visual centers, critical for binocular vision. Using Isl2tau-lacZ knockin mice, we show that the LIM-homeodomain transcription factor Isl2 marks only contralaterally projecting RGCs. The transcription factor Zic2 and guidance receptor EphB1, required by RGCs to project ipsilaterally, colocalize in RGCs distinct from Isl2 RGCs in the ventral-temporal crescent (VTC), the source of ipsilateral projections. Isl2 knockout mice have an increased ipsilateral projection originating from significantly more RGCs limited to the VTC. Isl2 knockouts also have increased Zic2 and EphB1 expression and significantly more Zic2 RGCs in the VTC. We conclude that Isl2 specifies RGC laterality by repressing an ipsilateral pathfinding program unique to VTC RGCs and involving Zic2 and EphB1. This genetic hierarchy controls binocular vision by regulating the magnitude and source of ipsilateral projections and reveals unique retinal domains.

Homeodomain Transcription Factors in the Development of Subsets of Hindbrain Reticulospinal Neurons

Molecular and Cellular Neurosciences. Jan, 2005  |  Pubmed ID: 15607939

Hindbrain reticulospinal neurons are involved in complex neural functions that are mediated by spinal elements, including posture control and modulation of respiration and cardiovascular function. Recent descriptive studies with chick, mouse, and rat embryos have provided anatomical insight into the development of the different reticulospinal nuclei and the establishment of their axonal projection pathways into the spinal cord. In this study, we have addressed the molecular control of this process. Retrograde labeling of reticulospinal neurons in chick and mouse embryos combined with immunostaining for the homeodomain factors Lhx1/Lhx5, Lhx3/Lhx4, and Chx10 have defined transcriptional codes that label subsets of neurons with different axon projection patterns. Gain of function and loss of function experiments using in ovo electroporation implicate these transcription factors in the determination of reticulospinal neuron identity. Furthermore, our studies reveal novel gene interactions between the transcription factors analyzed that may determine the final patterns of reticulospinal axon projection.

Olig2 and Ngn2 Function in Opposition to Modulate Gene Expression in Motor Neuron Progenitor Cells

Genes & Development. Jan, 2005  |  Pubmed ID: 15655114

Spinal motor neurons and oligodendrocytes are generated sequentially from a common pool of progenitors termed pMN cells. Olig2 is a bHLH-class transcription factor in pMN cells, but it has remained unclear how its transcriptional activity is modulated to first produce motor neurons and then oligodendrocytes. Previous studies have shown that Olig2 primes pMN cells to become motor neurons by triggering the expression of Ngn2 and Lhx3. Here we show that Olig2 also antagonizes the premature expression of post-mitotic motor neuron genes in pMN cells. This blockade is counteracted by Ngn2, which accumulates heterogeneously in pMN cells, thereby releasing a subset of the progenitors to differentiate and activate expression of post-mitotic motor neuron genes. The antagonistic relationship between Ngn2 and Olig2 is mediated by protein interactions that squelch activity as well as competition for shared DNA-binding sites. Our data support a model in which the Olig2/Ngn2 ratio in progenitor cells serves as a gate for timing proper gene expression during the development of pMN cells: Olig2(high) maintains the pMN state, thereby holding cells in reserve for oligodendrocyte generation, whereas Ngn2(high) favors the conversion of pMN cells into post-mitotic motor neurons.

Small CTD Phosphatases Function in Silencing Neuronal Gene Expression

Science (New York, N.Y.). Jan, 2005  |  Pubmed ID: 15681389

Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) complex. To understand how this silencing is achieved, we examined a family of class-C RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) phosphatases [small CTD phosphatases (SCPs) 1 to 3], whose expression is restricted to non-neuronal tissues. We show that REST/NRSF recruits SCPs to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. Phosphatase-inactive forms of SCP interfere with REST/NRSF function and promote neuronal differentiation of P19 stem cells. Likewise, small interfering RNA directed to the single Drosophila SCP unmasks neuronal gene expression in S2 cells. Thus, SCP activity is an evolutionarily conserved transcriptional regulator that acts globally to silence neuronal genes.

The LIM Domain-only Protein LMO4 is Required for Neural Tube Closure

Molecular and Cellular Neurosciences. Feb, 2005  |  Pubmed ID: 15691703

Nuclear LIM domain-only proteins (LMOs), which consist of two closely spaced 50 amino acid Zn2+-finger protein interaction modules mediate interactions between several classes of transcription factors important for development. LMO2 is necessary for development of the entire hematopoietic system and overexpression of LMO1 or LMO2 results in human acute T cell leukemia. LMO4 is the most widely expressed LMO but its normal function is unknown. During development, LMO4 is expressed in dividing neuroepithelial cells within the ventricular zone along the entire rostrocaudal axis of the nervous system. In telencephalic and spinal cord regions of the CNS, LMO4 is highly expressed in ventral but is low in dorsal proliferating neuroepithelial cells. To understand the role of LMO4 during mouse development, we generated a homozygous null mutation in the gene. We found that LMO4 is required for proper closure of the anterior neural tube. In the absence of LMO4, elevation, bending, and proliferation of the ventral neural epithelium and consequent fusion of the prospective dorsal ends of the neural tube do not occur. LMO4 mutant mice die embryonically and exhibit exencephaly, which is associated with abnormal patterns of cell proliferation and with high levels of apoptotic cell death within the neuroepithelium. LMO4 is thus essential for normal patterns of proliferation and for survival of neural epithelial cells in the rostral neural tube. LMO4 is also expressed in Schwann cell progenitors after these contact neurites, a process mediated in part by neuregulin (Nrg).

Development of Circuits That Generate Simple Rhythmic Behaviors in Vertebrates

Current Opinion in Neurobiology. Feb, 2005  |  Pubmed ID: 15721739

Neurobiologists have long sought to understand how circuits in the nervous system are organized to generate the precise neural outputs that underlie particular behaviors. Given the complexity of the nervous system in higher vertebrates this is a daunting task. Nevertheless, recent advances in developmental genetics hold out hope that studies of locomotor and respiratory circuits will provide general insight for understanding how ensembles of neurons are wired to control specific behaviors.

Coexpressed EphA Receptors and Ephrin-A Ligands Mediate Opposing Actions on Growth Cone Navigation from Distinct Membrane Domains

Cell. Apr, 2005  |  Pubmed ID: 15820684

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.

Cholinergic Input is Required During Embryonic Development to Mediate Proper Assembly of Spinal Locomotor Circuits

Neuron. Apr, 2005  |  Pubmed ID: 15820692

Rhythmic limb movements are controlled by pattern-generating neurons within the ventral spinal cord, but little is known about how these locomotor circuits are assembled during development. At early stages of embryogenesis, motor neurons are spontaneously active, releasing acetylcholine that triggers the depolarization of adjacent cells in the spinal cord. To investigate whether acetylcholine-driven activity is required for assembly of the central pattern-generating (CPG) circuit, we studied mice lacking the choline acetyltransferase (ChAT) enzyme. Our studies show that a rhythmically active spinal circuit forms in ChAT mutants, but the duration of each cycle period is elongated, and right-left and flexor-extensor coordination are abnormal. In contrast, blocking acetylcholine receptors after the locomotor network is wired does not affect right-left or flexor-extensor coordination. These findings suggest that the cholinergic neurotransmitter pathway is involved in configuring the CPG during a transient period of development.

Hox Genes: the Instructors Working at Motor Pools

Cell. Nov, 2005  |  Pubmed ID: 16269326

Motor neurons are assigned unique subidentities preceding their axon navigation. This ensures proper innervation of muscle targets and is accompanied by a stereotypical clustering of motor neuron cell bodies into "motor pools" within the spinal cord. However, the mechanisms that drive motor neuron diversification have been poorly understood. A new study by Dasen et al. (2005) in this issue of Cell shows that a network of Hox genes is responsible for instructing motor pool development.

FGF As a Target-derived Chemoattractant for Developing Motor Axons Genetically Programmed by the LIM Code

Neuron. Jun, 2006  |  Pubmed ID: 16772167

LIM transcription factors confer developing axons with specific navigational properties, but the downstream guidance receptors and ligands are not well defined. The dermomyotome, a transient structure from which axial muscles arise, is the source of a secreted long-range chemoattractant specific for medial-class spinal motor neuron axons (MMCm axons). We show that fibroblast growth factors (FGFs) produced by the dermomyotome selectively attract MMCm axons in vitro. FGF receptor 1 (FGFR1) expression is restricted to MMCm neurons, and conditional deletion of FGFR1 causes motor axon guidance defects. Furthermore, reprogramming the identity of limb-innervating motor neurons to that of dermomyotome-innervating MMCm cells using the LIM factor Lhx3 induces FGFR1 expression and shifts an increased number of motor axons to an FGF-responsive state. These results point to a role for FGF signaling in axon guidance and further unravel how downstream effectors of LIM codes direct wiring of the developing nervous system.

Reduced Expression of the LIM-homeobox Gene Lhx3 Impairs Growth and Differentiation of Rathke's Pouch and Increases Cell Apoptosis During Mouse Pituitary Development

Mechanisms of Development. Aug, 2006  |  Pubmed ID: 16859901

The formation of the anterior and intermediate lobes of the pituitary gland is a multi-step process regulated by cell-cell interactions involving a number of signaling pathways and by cascades of cell-intrinsic transcription factors. The LIM-homeodoamin protein Lhx3 has previously been shown to play an essential role in the growth of Rathke's pouch, a primordium of the anterior and intermediate lobes of the pituitary. However, the mechanisms underlying the function and regulation of Lhx3 remain to be elucidated. Here we report that a targeted insertion of a DNA fragment in the 3'-untranslated region of the Lhx3 gene reduces the expression of both Lhx3 mRNA and protein in Rathke's pouch. Mutant mice homozygous for this Lhx3 allele show severe hypoplasia of the pouch, a defect identical to that observed in Lhx3-null mutants. To gain insights into the mechanism of Lhx3 function in pituitary development, we further analyzed the Lhx3 deficient mutants by examination of early pituitary marker expression, cell proliferation, and cell apoptosis. Our results revealed an increase in cell apoptosis and a loss of Islet1 and Calbindin marker expression in Rathke's pouch of these mutants. Recently, increased cell apoptosis in Rathke's pouch has been described in mutant mice impaired in the function of the bicoid-like homeodomain proteins Pitx1 and Pitx2. In those mutants, the expression of Lhx3 is absent. Our results thus underscore the view that Lhx3 functions downstream of the Pitx factors in the same transcriptional cascade that controls growth and early cell differentiation of the developing pituitary gland.

Calcium Activation of the LMO4 Transcription Complex and Its Role in the Patterning of Thalamocortical Connections

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2006  |  Pubmed ID: 16899735

Lasting changes in neuronal connectivity require calcium-dependent gene expression. Here we report the identification of LIM domain-only 4 (LMO4) as a mediator of calcium-dependent transcription in cortical neurons. Calcium influx via voltage-sensitive calcium channels and NMDA receptors contributes to synaptically induced LMO4-mediated transactivation. LMO4-mediated transcription is dependent on signaling via calcium/calmodulin-dependent protein (CaM) kinase IV and microtubule-associated protein (MAP) kinase downstream of synaptic stimulation. Coimmunoprecipitation experiments indicate that LMO4 can form a complex with cAMP response element-binding protein (CREB) and can interact with cofactor of LIM homeodomain protein 1 (CLIM1) and CLIM2. To evaluate the role of LMO4 in vivo, we examined the consequences of conditional loss of lmo4 in the forebrain, using the Cre-Lox gene-targeting strategy. The organization of the barrel field in somatosensory cortex is disrupted in mice in which lmo4 is deleted conditionally in the cortex. Specifically, in contrast to controls, thalamocortical afferents in conditional lmo4 null mice fail to segregate into distinct barrel-specific domains. These observations identify LMO4 as a calcium-dependent transactivator that plays a key role in patterning thalamocortical connections during development.

T-Box Transcription Factor Tbx20 Regulates a Genetic Program for Cranial Motor Neuron Cell Body Migration

Development (Cambridge, England). Dec, 2006  |  Pubmed ID: 17119020

Members of the T-box transcription factor family (Tbx) are associated with several human syndromes during embryogenesis. Nevertheless, their functions within the developing CNS remain poorly characterized. Tbx20 is expressed by migrating branchiomotor/visceromotor (BM/VM) neurons within the hindbrain during neuronal circuit formation. We examined Tbx20 function in BM/VM cells using conditional Tbx20-null mutant mice to delete the gene in neurons. Hindbrain rhombomere patterning and the initial generation of post-mitotic BM/VM neurons were normal in Tbx20 mutants. However, Tbx20 was required for the tangential (caudal) migration of facial neurons, the lateral migration of trigeminal cells and the trans-median movement of vestibuloacoustic neurons. Facial cell soma migration defects were associated with the coordinate downregulation of multiple components of the planar cell polarity pathway including Fzd7, Wnt11, Prickle1, Vang1 and Vang2. Our study suggests that Tbx20 programs a variety of hindbrain motor neurons for migration, independent of directionality, and in facial neurons is a positive regulator of the non-canonical Wnt signaling pathway.

Determinants for Dephosphorylation of the RNA Polymerase II C-terminal Domain by Scp1

Molecular Cell. Dec, 2006  |  Pubmed ID: 17157258

Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.

Cloning and Developmental Expression of a Chick G-protein-coupled Receptor SCGPR1

Gene Expression Patterns : GEP. Feb, 2007  |  Pubmed ID: 17251065

To identify spinal motor neuron subtype-specific transcripts, we employed a single cell subtractive screen of mRNAs in chick embryos. We cloned a differentially expressed gene that termed spinal cord G-protein-coupled receptor 1 (SCGPR1) from its expression pattern that change dynamically in the developing spinal cord. The vertebrate orthologue of SCGPR1 is termed Gpr37 (GPCR/CNS1, ET(B)R-LP-1, Pael-R), however the specific ligand of this receptor has not been identified. Recent studies indicate that Pael-R can associate with parkin, a ubiquitin ligase which accumulates in Lewy bodies in dopaminergic neurons and is associated with Parkinson's disease. Although SCGPR1 (Gpr37) expression has been examined in adult tissues, the embryonic expression has not reported. Here, we have defined the expression pattern of SCGPR1 by in situ hybridization during chick development. SCGPR1 was first detected at HH stage 7 in the neural tube and notochord. As development progressed, SCGPR1 expression became restricted to the ventral neural tube. SCGPR1 expression was also present in the developing telencephalon, mesencephalon, retina, visceral-class motor neurons, myotome and thyroid invagination.

A Functional Study of MiR-124 in the Developing Neural Tube

Genes & Development. Mar, 2007  |  Pubmed ID: 17344415

Neural development is a highly orchestrated process that entails precise control of gene expression. Although microRNAs (miRNAs) have been implicated in fine-tuning gene networks, the roles of individual miRNAs in vertebrate neural development have not been studied in vivo. We investigated the function of the most abundant neuronal miRNA, miR-124, during spinal cord development. Neither inhibition nor overexpression of miR-124 significantly altered the acquisition of neuronal fate, suggesting that miR-124 is unlikely to act as a primary determinant of neuronal differentiation. Two endogenous targets of miR-124, laminin gamma 1 and integrin beta1, were identified, both of which are highly expressed by neural progenitors but repressed upon neuronal differentiation. Thus miR-124 appears to ensure that progenitor genes are post-transcriptionally inhibited in neurons.

Notch and MAML Signaling Drives Scl-dependent Interneuron Diversity in the Spinal Cord

Neuron. Mar, 2007  |  Pubmed ID: 17359917

The ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Here we show that cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, our study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification.

Distinct Target-derived Signals Organize Formation, Maturation, and Maintenance of Motor Nerve Terminals

Cell. Apr, 2007  |  Pubmed ID: 17418794

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

The Ubiquitin Ligase Phr1 Regulates Axon Outgrowth Through Modulation of Microtubule Dynamics

Neuron. Nov, 2007  |  Pubmed ID: 18031680

To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.

Instructive Role of APKCzeta Subcellular Localization in the Assembly of Adherens Junctions in Neural Progenitors

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2008  |  Pubmed ID: 18162555

In the neurogenic phase of CNS development, the proliferating progenitors are found medially within the neuroepithelium. The adherens junctions on the apical membrane of proliferating neural progenitors allow for cell-cell adhesion and medial stratification. In contrast, differentiating neuronal precursors delaminate and migrate laterally, establishing the laminar layers. Apical adherens junctions also establish the apical-basal polarity in neural progenitors, which in turn is postulated to lead to asymmetric inheritance of cell fate determinants during neurogenic divisions. The signaling pathways and cellular mechanisms that regulate the assembly and asymmetric localization of adherens junctions in neural progenitors remain elusive. Here we show that atypical PKCzeta/lambda (aPKCzeta/lambda) localizes at the apical membrane of proliferating neural stem cells, but not postmitotic neuronal precursors, in the developing chicken neural tube. This precise subcellular compartmentalization of the kinase activity provides an instructive signal for apical assembly of adherens junctions in a PI3K, Rac/Cdc42 signaling-dependent pathway. Apical aPKCzeta coordinates neural stem cell proliferation and the overall stratification of cell types within the neural tube.

Regulation of Motor Neuron Specification by Phosphorylation of Neurogenin 2

Neuron. Apr, 2008  |  Pubmed ID: 18400164

The mechanisms by which proneural basic helix-loop-helix (bHLH) factors control neurogenesis have been characterized, but it is not known how they specify neuronal cell-type identity. Here, we provide evidence that two conserved serine residues on the bHLH factor neurogenin 2 (Ngn2), S231 and S234, are phosphorylated during motor neuron differentiation. In knockin mice in which S231 and S234 of Ngn2 were mutated to alanines, neurogenesis occurs normally, but motor neuron specification is impaired. The phosphorylation of Ngn2 at S231 and S234 facilitates the interaction of Ngn2 with LIM homeodomain transcription factors to specify motor neuron identity. The phosphorylation-dependent cooperativity between Ngn2 and homeodomain transcription factors may be a general mechanism by which the activities of bHLH and homeodomain proteins are temporally and spatially integrated to generate the wide diversity of cell types that are a hallmark of the nervous system.

Segregation of Axial Motor and Sensory Pathways Via Heterotypic Trans-axonal Signaling

Science (New York, N.Y.). Apr, 2008  |  Pubmed ID: 18403711

Execution of motor behaviors relies on circuitries effectively integrating immediate sensory feedback to efferent pathways controlling muscle activity. It remains unclear how, during neuromuscular circuit assembly, sensory and motor projections become incorporated into tightly coordinated, yet functionally separate pathways. We report that, within axial nerves, establishment of discrete afferent and efferent pathways depends on coordinate signaling between coextending sensory and motor projections. These heterotypic axon-axon interactions require motor axonal EphA3/EphA4 receptor tyrosine kinases activated by cognate sensory axonal ephrin-A ligands. Genetic elimination of trans-axonal ephrin-A --> EphA signaling in mice triggers drastic motor-sensory miswiring, culminating in functional efferents within proximal afferent pathways. Effective assembly of a key circuit underlying motor behaviors thus critically depends on trans-axonal signaling interactions resolving motor and sensory projections into discrete pathways.

A Regulatory Network to Segregate the Identity of Neuronal Subtypes

Developmental Cell. Jun, 2008  |  Pubmed ID: 18539116

Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. Here we show how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, our study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops.

Developmental Neuroscience: Hox and Fox

Nature. Sep, 2008  |  Pubmed ID: 18800121

YAP Regulates Neural Progenitor Cell Number Via the TEA Domain Transcription Factor

Genes & Development. Dec, 2008  |  Pubmed ID: 19015275

Tight control of cell proliferation is essential for proper growth during development and for tissue homeostasis in mature animals. The evolutionarily conserved Hippo pathway restrains proliferation through a kinase cascade that culminates in the inhibition of the transcriptional coactivator YAP. Unphosphorylated YAP activates genes involved in cell proliferation and survival by interacting with a DNA-binding factor. Here we show that during vertebrate neural tube development, the TEA domain transcription factor (TEAD) is the cognate DNA-binding partner of YAP. YAP and TEAD gain of function causes marked expansion of the neural progenitor population, partly owing to their ability to promote cell cycle progression by inducing cyclin D1 and to inhibit differentiation by suppressing NeuroM. Their loss of function results in increased apoptosis, whereas repressing their target genes leads to premature neuronal differentiation. Inhibiting the upstream kinases of the Hippo pathway also causes neural progenitor overproliferation. Thus, the Hippo pathway plays critical roles in regulating neural progenitor cell number by affecting proliferation, fate choice, and cell survival.

Islet-to-LMO Stoichiometries Control the Function of Transcription Complexes That Specify Motor Neuron and V2a Interneuron Identity

Development (Cambridge, England). Sep, 2009  |  Pubmed ID: 19666821

LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision.

Structural and Functional Analysis of the Phosphoryl Transfer Reaction Mediated by the Human Small C-terminal Domain Phosphatase, Scp1

Protein Science : a Publication of the Protein Society. May, 2010  |  Pubmed ID: 20222012

Human small C-terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C-terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg(2+) ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl-aspartate intermediate. This high-energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl-aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para-nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady-state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg(2+) coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1-mediated catalysis. Through structural-based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.

Motor Axon Pathfinding

Cold Spring Harbor Perspectives in Biology. Mar, 2010  |  Pubmed ID: 20300210

Motor neurons are functionally related, but represent a diverse collection of cells that show strict preferences for specific axon pathways during embryonic development. In this article, we describe the ligands and receptors that guide motor axons as they extend toward their peripheral muscle targets. Motor neurons share similar guidance molecules with many other neuronal types, thus one challenge in the field of axon guidance has been to understand how the vast complexity of brain connections can be established with a relatively small number of factors. In the context of motor guidance, we highlight some of the temporal and spatial mechanisms used to optimize the fidelity of pathfinding and increase the functional diversity of the signaling proteins.

Hormone Binding and Co-regulator Binding to the Glucocorticoid Receptor Are Allosterically Coupled

The Journal of Biological Chemistry. May, 2010  |  Pubmed ID: 20335180

The glucocorticoid receptor initiates the cellular response to glucocorticoid steroid hormones in vertebrates. Co-regulator proteins dock to the receptor in response to hormone binding and potentiate the transcriptional activity of the receptor by modifying DNA and recruiting essential transcription factors like RNA polymerase II. Hormones and co-regulators bind at distinct sites in the ligand binding domain yet function cooperatively to mediate transcriptional control. This study reveals and quantifies energetic coupling between two binding sites using purified components. Using a library of peptides taken from co-regulator proteins, we determine the pattern of co-regulator binding to the glucocorticoid receptor ligand binding domain. We show that peptides from co-regulators differ in their effects on hormone binding and kinetics. Peptides from DAX1 and SRC1 bind with similar affinity, but DAX1 binding is coupled to hormone binding, and SRC1 is not. Mechanistic details of co-regulator binding and coupling to the hormone binding pocket are uncovered by analysis of properties endowed by mutation of a key residue in the allosteric network connecting the sites.

Defining Rhythmic Locomotor Burst Patterns Using a Continuous Wavelet Transform

Annals of the New York Academy of Sciences. Jun, 2010  |  Pubmed ID: 20536927

We review an objective and automated method for analyzing locomotor electrophysiology data with improved speed and accuracy. Manipulating central pattern generator (CPG) organization via mouse genetics has been a critical advance in the study of this circuit. Better quantitative measures of the locomotor data will further enhance our understanding of CPG development and function. Current analysis methods aim to measure locomotor cycle period, rhythmicity, and left-right and flexor-extensor phase; however, these methods have not been optimized to detect or quantify subtle changes in locomotor output. Because multiple experiments suggest that development of the CPG is robust and that the circuit is able to achieve organized behavior by several means, we sought to find a more objective and sensitive method for quantifying locomotor output. Recently, a continuous wavelet transform (CWT) has been applied to spinal cord ventral root recordings with promising results. The CWT provides greater resolution of cycle period, phase, and rhythmicity, and is proving to be a superior technique in assessing subtle changes in locomotion due to genetic perturbations of the underlying circuitry.

Atypical Cadherins Celsr1-3 Differentially Regulate Migration of Facial Branchiomotor Neurons in Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2010  |  Pubmed ID: 20631168

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate.

Presenilin-dependent Receptor Processing is Required for Axon Guidance

Cell. Jan, 2011  |  Pubmed ID: 21215373

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β 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.

Endogenous Retroviruses and Neighboring Genes Are Coordinately Repressed by LSD1/KDM1A

Genes & Development. Mar, 2011  |  Pubmed ID: 21357675

Endogenous retroviruses (ERVs) constitute a substantial portion of mammalian genomes, and their retrotransposition activity helped to drive genetic variation, yet their expression is tightly regulated to prevent unchecked amplification. We generated a series of mouse mutants and embryonic stem (ES) cell lines carrying "deletable" and "rescuable" alleles of the lysine-specific demethylase LSD1/KDM1A. In the absence of KDM1A, the murine endogenous retrovirus MuERV-L/MERVL becomes overexpressed and embryonic development arrests at gastrulation. A number of cellular genes normally restricted to the zygotic genome activation (ZGA) period also become up-regulated in Kdm1a mutants. Strikingly, many of these cellular genes are flanked by MERVL sequences or have cryptic LTRs as promoters that are targets of KDM1A repression. Using genome-wide epigenetic profiling of Kdm1a mutant ES cells, we demonstrate that this subset of ZGA genes and MERVL elements displays increased methylation of histone H3K4, increased acetylation of H3K27, and decreased methylation of H3K9. As a consequence, Kdm1a mutant ES cells exhibit an unusual propensity to generate extraembryonic tissues. Our findings suggest that ancient retroviral insertions were used to co-opt regulatory sequences targeted by KDM1A for epigenetic silencing of cell fate genes during early mammalian embryonic development.

Isl1 is Required for Multiple Aspects of Motor Neuron Development

Molecular and Cellular Neurosciences. Jul, 2011  |  Pubmed ID: 21569850

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.

Identification of a MicroRNA That Activates Gene Expression by Repressing Nonsense-mediated RNA Decay

Molecular Cell. May, 2011  |  Pubmed ID: 21596314

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

SnapShot: Spinal Cord Development

Cell. Jul, 2011  |  Pubmed ID: 21729788

Rapid and Efficient Generation of Functional Motor Neurons from Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Molecular Therapy : the Journal of the American Society of Gene Therapy. Oct, 2011  |  Pubmed ID: 21772256

Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60-70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.

Islet1-mediated Activation of the β-catenin Pathway is Necessary for Hindlimb Initiation in Mice

Development (Cambridge, England). Oct, 2011  |  Pubmed ID: 21937598

The transcriptional basis of vertebrate limb initiation, which is a well-studied system for the initiation of organogenesis, remains elusive. Specifically, involvement of the β-catenin pathway in limb initiation, as well as its role in hindlimb-specific transcriptional regulation, are under debate. Here, we show that the β-catenin pathway is active in the limb-forming area in mouse embryos. Furthermore, conditional inactivation of β-catenin as well as Islet1, a hindlimb-specific factor, in the lateral plate mesoderm results in a failure to induce hindlimb outgrowth. We further show that Islet1 is required for the nuclear accumulation of β-catenin and hence for activation of the β-catenin pathway, and that the β-catenin pathway maintains Islet1 expression. These two factors influence each other and function upstream of active proliferation of hindlimb progenitors in the lateral plate mesoderm and the expression of a common factor, Fgf10. Our data demonstrate that Islet1 and β-catenin regulate outgrowth and Fgf10-Fgf8 feedback loop formation during vertebrate hindlimb initiation. Our study identifies Islet1 as a hindlimb-specific transcriptional regulator of initiation, and clarifies the controversy regarding the requirement of β-catenin for limb initiation.

Protease Regulation: the Yin and Yang of Neural Development and Disease

Neuron. Oct, 2011  |  Pubmed ID: 21982365

The formation, maintenance, and plasticity of neural circuits rely upon a complex interplay between progressive and regressive events. Increasingly, new functions are being identified for axon guidance molecules in the dynamic processes that occur within the embryonic and adult nervous system. The magnitude, duration, and spatial activity of axon guidance molecule signaling are precisely regulated by a variety of molecular mechanisms. Here we focus on recent progress in understanding the role of protease-mediated cleavage of guidance factors required for directional axon growth, with a particular emphasis on the role of metalloprotease and γ-secretase. Since axon guidance molecules have also been linked to neural degeneration and regeneration in adults, studies of guidance receptor proteolysis are beginning to define new relationships between neurodevelopment and neurodegeneration. These findings raise the possibility that the signaling checkpoints controlled by proteases could be useful targets to enhance regeneration.

Ret Is a Multifunctional Coreceptor That Integrates Diffusible- and Contact-Axon Guidance Signals

Cell. Feb, 2012  |  Pubmed ID: 22304922

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.