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Monoclonal Anti-Acetylated Tubulin antibody produced in mouse

RRID:AB_609894

Antibody ID

AB_609894

Target Antigen

Acetylated Tubulin antibody produced in mouse human, mollusc, non-human primate, other invertebrate, plant, bacteria/archaea, c elegans/worm, rat, yeast/fungi, porcine, amoeba/protozoa, bovine, hamster, chicken/bird, drosophila/arthropod, mouse, xenopus/amphibian, monkey, protista, mouse, pig, human, bovine, invertebrates, rat, hamster, plant, frog, chicken

Proper Citation

(Sigma-Aldrich Cat# T7451, RRID:AB_609894)

Clonality

monoclonal antibody

Comments

Vendor recommendations: IgG2b Radioimmunoassay; Electron Microscopy; Other; Immunohistochemistry; Western Blot; electron microscopy: suitable, immunoblotting: 0.03-0.06 mug/mL

Host Organism

mouse

Vendor

Sigma-Aldrich

Post-Translational Tubulin Modifications in Human Astrocyte Cultures.

  • Knight VB
  • Neurochem. Res.
  • 2018 Jul 9

Literature context:


Abstract:

The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the 'EBImage' package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with βIII-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/βIII-tubulin was greater than the ratio for polyglutamylated tubulin/βIII-tubulin. The open source GEODATA set GSE819950, comprising RNA sequencing data for the NHA cell line, was mined for the expression of enzymes responsible for tubulin modifications. Our analysis uncovered greater expression at the mRNA level for enzymes reported to function in acetylation and deacetylation as compared to enzymes implicated in glutamylation and deglutamylation. Taken together, the results represent a step toward unraveling the tubulin isotypic expression profile and post-translational modification patterns in astrocytes during human brain development.

Funding information:
  • NIMHD NIH HHS - G12 MD007592()

Transmission electron microscopy of zebrafish spinal motor nerve roots.

  • Morris AD
  • Dev. Dyn.
  • 2018 Jul 3

Literature context:


Abstract:

BACKGROUND: Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing. RESULTS: Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging. CONCLUSIONS: We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.

Funding information:
  • NINDS NIH HHS - R01 NS072212()

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

  • Nakano Y
  • Cell
  • 2018 Jun 25

Literature context:


Abstract:

The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.

Funding information:
  • NIMH NIH HHS - 5 F32 MH064339-03(United States)

Brain Somatic Mutations in MTOR Disrupt Neuronal Ciliogenesis, Leading to Focal Cortical Dyslamination.

  • Park SM
  • Neuron
  • 2018 Jun 9

Literature context:


Abstract:

Focal malformations of cortical development (FMCDs), including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are major etiologies of pediatric intractable epilepsies exhibiting cortical dyslamination. Brain somatic mutations in MTOR have recently been identified as a major genetic cause of FMCDs. However, the molecular mechanism by which these mutations lead to cortical dyslamination remains poorly understood. Here, using patient tissue, genome-edited cells, and mouse models with brain somatic mutations in MTOR, we discovered that disruption of neuronal ciliogenesis by the mutations underlies cortical dyslamination in FMCDs. We found that abnormal accumulation of OFD1 at centriolar satellites due to perturbed autophagy was responsible for the defective neuronal ciliogenesis. Additionally, we found that disrupted neuronal ciliogenesis accounted for cortical dyslamination in FMCDs by compromising Wnt signals essential for neuronal polarization. Altogether, this study describes a molecular mechanism by which brain somatic mutations in MTOR contribute to the pathogenesis of cortical dyslamination in FMCDs.

Funding information:
  • NHLBI NIH HHS - HL073284(United States)

TRRAP is a central regulator of human multiciliated cell formation.

  • Wang Z
  • J. Cell Biol.
  • 2018 Jun 4

Literature context:


Abstract:

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.

Funding information:
  • NIDDK NIH HHS - R01DK-069884(United States)

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

  • Lynch TJ
  • Cell Stem Cell
  • 2018 May 3

Literature context:


Abstract:

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

Funding information:
  • Chief Scientist Office - CAF/10/15(United Kingdom)
  • NHLBI NIH HHS - P01 HL051670()
  • NIDDK NIH HHS - P30 DK054759()

Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling.

  • Klatt Shaw D
  • Dev. Cell
  • 2018 May 21

Literature context:


Abstract:

Graded Shh signaling across fields of precursor cells coordinates patterns of gene expression, differentiation, and morphogenetic behavior as precursors form complex structures, such as the nervous system, the limbs, and craniofacial skeleton. Here we discover that intracellular calcium mobilization, a process tightly controlled and readily modulated, regulates the level of Shh-dependent gene expression in responding cells and affects the development of all Shh-dependent cell types in the zebrafish embryo. Reduced expression or modified activity of ryanodine receptor (RyR) intracellular calcium release channels shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tube. Mosaic analysis revealed that RyR-mediated calcium mobilization is required specifically in Shh ligand-receiving cells. This work reveals that RyR channels participate in intercellular signal transduction events. As modulation of RyR activity modifies tissue patterning, we hypothesize that alterations in intracellular calcium mobilization contribute to both birth defects and evolutionary modifications of morphology.

Funding information:
  • NIAID NIH HHS - R01 AI043458(United States)

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

  • Best SA
  • Cell Metab.
  • 2018 Apr 3

Literature context:


Abstract:

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1f/f/Ptenf/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations.

Funding information:
  • NHGRI NIH HHS - U54 HG004592(United States)

Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules.

  • Martin M
  • Elife
  • 2018 Mar 16

Literature context:


Abstract:

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion.

Funding information:
  • China Scholarship Council - PhD fellowship()
  • European Research Council - Synergy 609822()
  • Fonds De La Recherche Scientifique - FNRS - FRIA fellowship()
  • H2020 Marie Skłodowska-Curie Actions - IEF fellowship()
  • Marie Sklodowska-Curie Actions - IEF fellowship()
  • NCI NIH HHS - F30 CA183497(United States)
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek - ALW Open Program grant 824.15.017()

Spatial-Temporal Lineage Restrictions of Embryonic p63+ Progenitors Establish Distinct Stem Cell Pools in Adult Airways.

  • Yang Y
  • Dev. Cell
  • 2018 Mar 26

Literature context:


Abstract:

Basal cells (BCs) are p63-expressing multipotent progenitors of skin, tracheoesophageal and urinary tracts. p63 is abundant in developing airways; however, it remains largely unclear how embryonic p63+ cells contribute to the developing and postnatal respiratory tract epithelium, and ultimately how they relate to adult BCs. Using lineage-tracing and functional approaches in vivo, we show that p63+ cells arising from the lung primordium are initially multipotent progenitors of airway and alveolar lineages but later become restricted proximally to generate the tracheal adult stem cell pool. In intrapulmonary airways, these cells are maintained immature to adulthood in bronchi, establishing a rare p63+Krt5- progenitor cell population that responds to H1N1 virus-induced severe injury. Intriguingly, this pool includes a CC10 lineage-labeled p63+Krt5- cell subpopulation required for a full H1N1-response. These data elucidate key aspects in the establishment of regionally distinct adult stem cell pools in the respiratory system, potentially with relevance to other organs.

Funding information:
  • Intramural NIH HHS - ZIA HL006151-02(United States)
  • NCI NIH HHS - R01 CA112403()
  • NCI NIH HHS - R01 CA193455()
  • NHLBI NIH HHS - R35 HL135834()
  • NIAID NIH HHS - HHSN272201400008C()

Spatially resolved RNA-sequencing of the embryonic heart identifies a role for Wnt/β-catenin signaling in autonomic control of heart rate.

  • Burkhard SB
  • Elife
  • 2018 Feb 5

Literature context:


Abstract:

Development of specialized cells and structures in the heart is regulated by spatially -restricted molecular pathways. Disruptions in these pathways can cause severe congenital cardiac malformations or functional defects. To better understand these pathways and how they regulate cardiac development we used tomo-seq, combining high-throughput RNA-sequencing with tissue-sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart. Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments. Pacemaker cells in the sinoatrial region induce heart contractions, but little is known about the mechanisms underlying their development. Using our transcriptome map, we identified spatially restricted Wnt/β-catenin signaling activity in pacemaker cells, which was controlled by Islet-1 activity. Moreover, Wnt/β-catenin signaling controls heart rate by regulating pacemaker cellular response to parasympathetic stimuli. Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially restricted molecular pathways critical for specific cardiac functions.

Funding information:
  • NIMH NIH HHS - K99 MH104259(United States)

Hyperinnervation improves Xenopus laevis limb regeneration.

  • Mitogawa K
  • Dev. Biol.
  • 2018 Jan 15

Literature context:


Abstract:

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.

Nestin expression and in vivo proliferative potential of tanycytes and ependymal cells lining the walls of the third ventricle in the adult rat brain.

  • Hendrickson ML
  • Eur. J. Neurosci.
  • 2018 Jan 24

Literature context:


Abstract:

There is a disagreement in the literature concerning the degree of proliferation of cells in the walls of the third ventricle (3rdV) under normal conditions in the adult mammalian brain. To address this issue, we mapped the cells expressing the neural stem/progenitor cell marker nestin along the entire rostrocaudal extent of the 3rdV in adult male rats and observed a complex distribution. Abundant nestin was present in tanycyte cell bodies and processes and also was observed in patches of ependymal cells as well as in isolated ependymal cells throughout the walls of the 3rdV. However, we observed very limited ependymal cell or tanycyte proliferation in normal adult rats as determined by bromodeoxyuridine (BrdU) incorporation or the expression of Ki-67. Moreover, fewer than 13% of the cells that were BrdU-positive (BrdU+) or Ki-67-positive (Ki-67+) expressed nestin. These observations stand in contrast to those made in the subventricular zone of the lateral ventricle (SVZ) and subgranular zone of the hippocampal formation (SGZ), where cell proliferation measured by BrdU incorporation or Ki-67 expression is observed frequently in cells that also express nestin. Thus, while ependymal cell or tanycyte cell proliferation can be promoted by the addition of mitogens, dietary modifications or other in vivo manipulations, the proliferation of ependymal cells and tanycytes in the walls of the 3rdV is very limited in the normal adult male rat brain.

Funding information:
  • NIGMS NIH HHS - 5R01 GM083084-03(United States)

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

  • Krey JF
  • J. Neurosci.
  • 2018 Jan 24

Literature context:


Abstract:

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout (rda)]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

Funding information:
  • NCI NIH HHS - CA077735(United States)
  • NEI NIH HHS - P30 EY010572()
  • NIDCD NIH HHS - P30 DC005983()
  • NIDCD NIH HHS - R01 DC002368()
  • NIDCD NIH HHS - R01 DC011034()
  • NINDS NIH HHS - P30 NS061800()

mTORC1 Activation during Repeated Regeneration Impairs Somatic Stem Cell Maintenance.

  • Haller S
  • Cell Stem Cell
  • 2017 Dec 7

Literature context:


Abstract:

The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCRR NIH HHS - UL1 RR024989(United States)
  • NHLBI NIH HHS - R01 HL132996()
  • NIA NIH HHS - K99 AG041764()
  • NIA NIH HHS - P01 AG036695()
  • NIA NIH HHS - R00 AG041764()
  • NIA NIH HHS - R01 AG047497()
  • NIA NIH HHS - R01 AG047820()
  • NIA NIH HHS - R37 AG023806()
  • NIDDK NIH HHS - R01 DK100342()
  • NIDDK NIH HHS - R01 DK113144()

Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport.

  • Tas RP
  • Neuron
  • 2017 Dec 20

Literature context:


Abstract:

Microtubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly oriented minus-end out, while dynamic and tyrosinated microtubules are oriented oppositely. In addition, microtubules with similar orientations and modifications form bundles that bias transport. Importantly, because the plus-end-directed Kinesin-1 selectively interacts with acetylated microtubules, this organization guides this motor out of dendrites and into axons. In contrast, Kinesin-3 prefers tyrosinated microtubules and can enter both axons and dendrites. This separation of distinct microtubule subsets into oppositely oriented bundles constitutes a key architectural principle of the neuronal microtubule cytoskeleton that enables polarized sorting by different motor proteins.

Funding information:
  • Medical Research Council - G0901533(United Kingdom)

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii.

  • Özpolat BD
  • Elife
  • 2017 Dec 12

Literature context:


Abstract:

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

Funding information:
  • Canadian Institutes of Health Research - P20 GM103440(Canada)

Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination.

  • Kasioulis I
  • Elife
  • 2017 Oct 23

Literature context:


Abstract:

Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment.

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.

  • Thorpe SD
  • J. Cell. Physiol.
  • 2017 Sep 3

Literature context:


Abstract:

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.

The CEP19-RABL2 GTPase Complex Binds IFT-B to Initiate Intraflagellar Transport at the Ciliary Base.

  • Kanie T
  • Dev. Cell
  • 2017 Jul 10

Literature context:


Abstract:

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT.

Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids.

  • Nikolić MZ
  • Elife
  • 2017 Jun 30

Literature context:


Abstract:

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood-Nerve Barrier.

  • Morris AD
  • J. Neurosci.
  • 2017 May 3

Literature context:


Abstract:

Precisely orchestrated interactions between spinal motor axons and their ensheathing glia are vital for forming and maintaining functional spinal motor nerves. Following perturbations to peripheral myelinating glial cells, centrally derived oligodendrocyte progenitor cells (OPCs) ectopically exit the spinal cord and myelinate peripheral nerves in myelin with CNS characteristics. However, whether remaining peripheral ensheathing glia, such as perineurial glia, properly encase the motor nerve despite this change in glial cell and myelin composition, remains unknown. Using zebrafish mutants in which OPCs migrate out of the spinal cord and myelinate peripheral motor axons, we assayed perineurial glial development, maturation, and response to injury. Surprisingly, in the presence of OPCs, perineurial glia exited the CNS normally. However, aspects of their development, response to injury, and function were altered compared with wildtype larvae. In an effort to better understand the plasticity of perineurial glia in response to myelin perturbations, we identified transforming growth factor-β1 as a partial mediator of perineurial glial development. Together, these results demonstrate the incredible plasticity of perineurial glia in the presence of myelin perturbations.SIGNIFICANCE STATEMENT Peripheral neuropathies can result from damage or dysregulation of the insulating myelin sheath surrounding spinal motor axons, causing pain, inefficient nerve conduction, and the ectopic migration of oligodendrocyte progenitor cells (OPCs), the resident myelinating glial cell of the CNS, into the periphery. How perineurial glia, the ensheathing cells that form the protective blood-nerve barrier, are impacted by this myelin composition change is unknown. Here, we report that certain aspects of perineurial glial development and injury responses are mostly unaffected in the presence of ectopic OPCs. However, perineurial glial function is disrupted along nerves containing centrally derived myelin, demonstrating that, although perineurial glial cells display plasticity despite myelin perturbations, the blood-nerve barrier is compromised in the presence of ectopic OPCs.

Funding information:
  • NINDS NIH HHS - F31 NS051140()
  • NINDS NIH HHS - R01 NS072212()

Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization.

  • Tortosa E
  • Neuron
  • 2017 May 17

Literature context:


Abstract:

Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/β Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.

Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ.

  • Coulthard LG
  • J. Neurosci.
  • 2017 May 31

Literature context:


Abstract:

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Planarian Epidermal Stem Cells Respond to Positional Cues to Promote Cell-Type Diversity.

  • Wurtzel O
  • Dev. Cell
  • 2017 Mar 13

Literature context:


Abstract:

Successful regeneration requires that progenitors of different lineages form the appropriate missing cell types. However, simply generating lineages is not enough. Cells produced by a particular lineage often have distinct functions depending on their position within the organism. How this occurs in regeneration is largely unexplored. In planarian regeneration, new cells arise from a proliferative cell population (neoblasts). We used the planarian epidermal lineage to study how the location of adult progenitor cells results in their acquisition of distinct functional identities. Single-cell RNA sequencing of epidermal progenitors revealed the emergence of distinct spatial identities as early in the lineage as the epidermal neoblasts, with further pre-patterning occurring in their post-mitotic migratory progeny. Establishment of dorsal-ventral epidermal identities and functions, in response to BMP signaling, required neoblasts. Our work identified positional signals that activate regionalized transcriptional programs in the stem cell population and subsequently promote cell-type diversity in the epidermis.

Funding information:
  • NIGMS NIH HHS - R01 GM080639()

Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

  • Phua SC
  • Cell
  • 2017 Jan 12

Literature context:


Abstract:

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Funding information:
  • NIAMS NIH HHS - R01 AR054396()
  • NIDDK NIH HHS - R01 DK102910()
  • NIGMS NIH HHS - DP2 GM105448()
  • NIGMS NIH HHS - R01 GM095941()
  • NIGMS NIH HHS - R01 GM112988()

Simultaneous Loss of NCKX4 and CNG Channel Desensitization Impairs Olfactory Sensitivity.

  • Ferguson CH
  • J. Neurosci.
  • 2017 Jan 4

Literature context:


Abstract:

In vertebrate olfactory sensory neurons (OSNs), Ca2+ plays key roles in both mediating and regulating the olfactory response. Ca2+ enters OSN cilia during the response through the olfactory cyclic nucleotide-gated (CNG) channel and stimulates a depolarizing chloride current by opening the olfactory Ca2+-activated chloride channel to amplify the response. Ca2+ also exerts negative regulation on the olfactory transduction cascade, through mechanisms that include reducing the CNG current by desensitizing the CNG channel via Ca2+/calmodulin (CaM), to reduce the response. Ca2+ is removed from the cilia primarily by the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), and the removal of Ca2+ leads to closure of the chloride channel and response termination. In this study, we investigate how two mechanisms conventionally considered negative regulatory mechanisms of olfactory transduction, Ca2+ removal by NCKX4, and desensitization of the CNG channel by Ca2+/CaM, interact to regulate the olfactory response. We performed electro-olfactogram (EOG) recordings on the double-mutant mice, NCKX4-/-;CNGB1ΔCaM, which are simultaneously lacking NCKX4 (NCKX4-/-) and Ca2+/CaM-mediated CNG channel desensitization (CNGB1ΔCaM). Despite exhibiting alterations in various response attributes, including termination kinetics and adaption properties, OSNs in either NCKX4-/- mice or CNGB1ΔCaM mice show normal resting sensitivity, as determined by their unchanged EOG response amplitude. We found that OSNs in NCKX4-/-;CNGB1ΔCaM mice displayed markedly reduced EOG amplitude accompanied by alterations in other response attributes. This study suggests that what are conventionally considered negative regulatory mechanisms of olfactory transduction also play a role in setting the resting sensitivity in OSNs. SIGNIFICANCE STATEMENT: Sensory receptor cells maintain high sensitivity at rest. Although the mechanisms responsible for setting the resting sensitivity of sensory receptor cells are not well understood, it has generally been assumed that the sensitivity is set primarily by how effectively the components in the activation cascade of sensory transduction can be stimulated. Our findings in mouse olfactory sensory neurons suggest that mechanisms that are primarily responsible for terminating the olfactory response are also critical for proper resting sensitivity.

Funding information:
  • NIDCD NIH HHS - R01 DC007395()
  • NIGMS NIH HHS - T32 GM007231()

Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

  • Huang P
  • Cell
  • 2016 Aug 25

Literature context:


Abstract:

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia.

  • Bolar NA
  • Am. J. Hum. Genet.
  • 2016 Jul 7

Literature context:


Abstract:

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.

A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis.

  • Golding A
  • PLoS ONE
  • 2016 Jun 4

Literature context:


Abstract:

In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device's observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies.

Neomycin damage and regeneration of hair cells in both mechanoreceptor and electroreceptor lateral line organs of the larval Siberian sturgeon (Acipenser baerii).

  • Fan C
  • J. Comp. Neurol.
  • 2016 May 1

Literature context:


Abstract:

The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red-conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443-1456, 2016. © 2015 Wiley Periodicals, Inc.

Immunohistochemical Analysis of Human Vallate Taste Buds.

  • Tizzano M
  • Chem. Senses
  • 2015 Nov 17

Literature context:


Abstract:

The morphology of the vallate papillae from postmortem human samples was investigated with immunohistochemistry. Microscopically, taste buds were present along the inner wall of the papilla, and in some cases in the outer wall as well. The typical taste cell markers PLCβ2, GNAT3 (gustducin) and the T1R3 receptor stain elongated cells in human taste buds consistent with the Type II cells in rodents. In the human tissue, taste bud cells that stain with Type II cell markers, PLCβ2 and GNAT3, also stain with villin antibody. Two typical immunochemical markers for Type III taste cells in rodents, PGP9.5 and SNAP25, fail to stain any taste bud cells in the human postmortem tissue, although these antibodies do stain numerous nerve fibers throughout the specimen. Car4, another Type III cell marker, reacted with only a few taste cells in our samples. Finally, human vallate papillae have a general network of innervation similar to rodents and antibodies directed against SNAP25, PGP9.5, acetylated tubulin and P2X3 all stain free perigemmal nerve endings as well as intragemmal taste fibers. We conclude that with the exception of certain molecular features of Type III cells, human vallate papillae share the structural, morphological, and molecular features observed in rodents.

Funding information:
  • NEI NIH HHS - EY012114(United States)

Exorhodopsin and melanopsin systems in the pineal complex and brain at early developmental stages of Atlantic halibut (Hippoglossus hippoglossus).

  • Eilertsen M
  • J. Comp. Neurol.
  • 2014 Dec 15

Literature context:


Abstract:

The complexity of the nonvisual photoreception systems in teleosts has just started to be appreciated, with colocalization of multiple photoreceptor types with unresolved functions. Here we describe an intricate expression pattern of melanopsins in early life stages of the marine flat fish Atlantic halibut (Hippoglossus hippoglossus), a period when the unpigmented brain is directly exposed to environmental photons. We show a refined and extensive expression of melanopsins in the halibut brain already at the time of hatching, long before the eyes are functional. We detect melanopsin in the habenula, suprachiasmatic nucleus, dorsal thalamus, and lateral tubular nucleus of first feeding larvae, suggesting conserved functions of the melanopsins in marine teleosts. The complex expression of melanopsins already at larval stages indicates the importance of nonvisual photoreception early in development. Most strikingly, we detect expression of both exorhodopsin and melanopsin in the pineal complex of halibut larvae. Double-fluorescence labeling showed that two clusters of melanopsin-positive cells are located lateral to the central rosette of exorhodopsin-positive cells. The localization of different photopigments in the pineal complex suggests that two parallel photoreceptor systems may be active. Furthermore, the dispersed melanopsin-positive cells in the spinal cord of halibut larvae at the time of hatching may be primary sensory cells or interneurons representing the first example of dispersed high-order photoreceptor cells. The appearance of nonvisual opsins early in the development of halibut provides an alternative model for studying the evolution and functional significance of nonvisual opsins.

Maturation of peptide-positive synaptic arbors in the medicinal leech requires rhythmic target activity.

  • Kueh D
  • J. Comp. Neurol.
  • 2013 Aug 15

Literature context:


Abstract:

The formation and refinement of synaptic connections are dependent on the activity that emerges from nascent synaptic connections. Such activity has the effect of regulating the production and release of specific neurotransmitters. To determine the role of activity in regulating the production of peptide-positive synapses, we used antibodies against Phe-Met-Arg-Phe-NH₂ and acetylated α-tubulin as well as intracellular injections of Neurobiotin to examine varicosities belonging to heart excitor (HE) neurons on the heart tubes of medicinal leeches, Hirudo spp. We found that the production of peptide-positive varicosities increased considerably during the last week of embryogenesis, which coincided with the emergence of rhythmic activity of the heart tube. When we compromised central input to HE neurons with bicuculline or by surgical ablation of the central pattern generator during early embryogenesis, we found that activity in the heart tubes and its rhythmicity were greatly diminished. Furthermore, the activity of HE neurons had also lost its rhythmicity and appeared tonic, and production of peptide-positive varicosities was substantially reduced as well. Partial surgical ablations that preserved rhythmic activity in the heart tube while disrupting heart tube innervation by some HE neurons still resulted in peptide-positive varicosity production. Taken together, our results suggest that postsynaptic rhythmic activity of the heart tube is necessary and sufficient for the development and maturation of peptide-positive synapses.

Funding information:
  • NIGMS NIH HHS - R01 GM071966(United States)

A quantitative analysis of branching, growth cone turning, and directed growth in zebrafish retinotectal axon guidance.

  • Simpson HD
  • J. Comp. Neurol.
  • 2013 Apr 15

Literature context:


Abstract:

The topographic projection from the eye to the tectum (amphibians and fish)/superior colliculus (birds and mammals) is a paradigm model system for studying mechanisms of neural wiring development. It has previously been proposed that retinal ganglion cell axons use distinct guidance strategies in fish vs. mammals, with direct guidance to the tectal target zone in the former and overshoot followed by biased branching toward the target zone in the latter. Here we visualized individual retinal ganglion cell axons as they grew over the tectum in zebrafish for periods of 10-21 hours and analyzed these results using an array of quantitative measures. We found that, although axons were generally guided directly toward their targets, this occurred without growth cone turning. Instead, axons branched dynamically and profusely throughout pathfinding, and successive branches oriented growth cone extension toward a target zone in a stepwise manner. These data suggest that the guidance strategies used between fish and mammals may be less distinct than previously thought.

Funding information:
  • NEI NIH HHS - P30 EY008126(United States)
  • NEI NIH HHS - R01 EY12118(United States)

Peripheral axons of the adult zebrafish maxillary barbel extensively remyelinate during sensory appendage regeneration.

  • Moore AC
  • J. Comp. Neurol.
  • 2012 Dec 15

Literature context:


Abstract:

Myelination is a cellular adaptation allowing rapid conduction along axons. We have investigated peripheral axons of the zebrafish maxillary barbel (ZMB), an optically clear sensory appendage. Each barbel carries taste buds, solitary chemosensory cells, and epithelial nerve endings, all of which regenerate after amputation (LeClair and Topczewski [2010] PLoS One 5:e8737). The ZMB contains axons from the facial nerve; however, myelination within the barbel itself has not been established. Transcripts of myelin basic protein (mbp) are expressed in normal and regenerating adult barbels, indicating activity in both maintenance and repair. Myelin was confirmed in situ by using toluidine blue, an anti-MBP antibody, and transmission electron microscopy (TEM). The adult ZMB contains ∼180 small-diameter axons (<2 μm), approximately 60% of which are myelinated. Developmental myelination was observed via whole-mount immunohistochemistry 4-6 weeks postfertilization, showing myelin sheaths lagging behind growing axons. Early-regenerating axons (10 days postsurgery), having no or few myelin layers, were disorganized within a fibroblast-rich collagenous scar. Twenty-eight days postsurgery, barbel axons had grown out several millimeters and were organized with compact myelin sheaths. Fiber types and axon areas were similar between normal and regenerated tissue; within 4 weeks, regenerating axons restored ∼85% of normal myelin thickness. Regenerating barbels express multiple promyelinating transcription factors (sox10, oct6 = pou3f1; krox20a/b = egr2a/b) typical of Schwann cells. These observations extend our understanding of the zebrafish peripheral nervous system within a little-studied sensory appendage. The accessible ZMB provides a novel context for studying axon regeneration, Schwann cell migration, and remyelination in a model vertebrate.

Funding information:
  • NIH HHS - U42 OD011140(United States)

Localization of Cadm2a and Cadm3 proteins during development of the zebrafish nervous system.

  • Hunter PR
  • J. Comp. Neurol.
  • 2011 Aug 1

Literature context:


Abstract:

Members of the Cadm/SynCAM/Necl/IGSF/TSLC family of cell adhesion molecules are known to have diverse functions during development of the nervous system, but information regarding their role during central nervous system (CNS) development in vivo is scarce. The rapid development of a relatively simple nervous system in larval zebrafish makes them a highly tractable model organism for studying gene function during nervous system development. An essential prerequisite for functional studies is a description of protein localization. To address this we have generated subtype-specific antibodies to two members of the zebrafish cell adhesion molecule family: cadm2a and cadm3. Using these novel antibodies we show that cadm3 and cadm2a are expressed throughout the nervous system of larval stage zebrafish. Particularly striking, and largely nonoverlapping expression of cadm2a and cadm3 is observed in the developing retina and spinal cord. Using in vitro binding assays we show that cadm2a and cadm3 bind heterophilically and preferentially to cadm1 and cadm4, respectively. These binding preferences are very similar to those seen for tetrapod Cadms but our study of protein localization suggests novel and diverse functions of cadms during nervous system development.

Funding information:
  • NIBIB NIH HHS - EB006733(United States)

Patterns of expression of Bardet-Biedl syndrome proteins in the mammalian cochlea suggest noncentrosomal functions.

  • May-Simera HL
  • J. Comp. Neurol.
  • 2009 May 10

Literature context:


Abstract:

Bardet-Biedl syndrome is a heterogeneous disorder causing a spectrum of symptoms, including visual impairment, kidney disease, and hearing impairment. Evidence suggests that BBS gene mutations cause defective ciliogenesis and/or cilium dysfunction. Cochlear development is affected by BBS gene deletion, and adult Bbs6(-/-) and Bbs4(-/-) mice are hearing impaired. This study addresses BBS protein expression in the rodent cochlea, to gain a better understanding of its function in vivo. As predicted by in vitro studies, Bbs6 immunofluorescence was localized to the basal bodies of supporting cells and sensory hair cells prior to the onset of hearing. In adult tissue, Bbs6 expression persisted in afferent neurons, including within the dendrites that innervate hair cells, implicating Bbs6 in a sensory neuronal function. Bbs2, which interacts with Bbs6, was also localized to hair cell basal bodies and stereociliary bundles. Additionally, Bbs2 was expressed in supporting cells at their intercellular boundaries, in a spatiotemporal pattern mirroring the development of the microtubule network. Bbs4 localized to cilia and developing cytoplasmic microtubule arrays. Pcm-1, a microtubular protein that interacts with Bbs4 in vitro, showed a comparable expression. Depolymerization of microtubules in slice preparations of the living cochlea resulted in Bbs4 and Pcm-1 mislocalization. Pcm-1 was also mislocalized in Bbs4(-/-) mice. This suggests that Bbs4/Pcm-1 interactions may be important in microtubule-dependent cytoplasmic trafficking in vivo. In summary, our findings indicate that BBS proteins adopt a range of cellular distributions in vivo, not restricted to the centrosome or cilium, and so broaden the possible underlying pathomechanisms of the disease.

Embryonic differentiation of serotonin-containing neurons in the enteric nervous system of the locust (Locusta migratoria).

  • Stern M
  • J. Comp. Neurol.
  • 2007 Mar 1

Literature context:


Abstract:

The enteric nervous system (ENS) of the locust consists of four ganglia (frontal and hypocerebral ganglion, and the paired ingluvial ganglia) located on the foregut, and nerve plexus innervating fore- and midgut. One of the major neurotransmitters of the ENS, serotonin, is known to play a vital role in gut motility and feeding. We followed the anatomy of the serotonergic system throughout embryonic development. Serotonergic neurons are generated in the anterior neurogenic zones of the foregut and migrate rostrally along the developing recurrent nerve to contribute to the frontal ganglion. They grow descending neurites, which arborize in all enteric ganglia and both nerve plexus. On the midgut, the neurites closely follow the leading migrating midgut neurons. The onset of serotonin synthesis occurs around halfway through development-the time of the beginning of midgut closure. Cells developing to serotonergic phenotype express the serotonin uptake transporter (SERT) significantly earlier, beginning at 40% of development. The neurons begin SERT expression during migration along the recurrent nerve, indicating that they are committed to a serotonergic phenotype before reaching their final destination. After completion of the layout of the enteric ganglia (at 60%) a maturational phase follows, during which serotonin-immunoreactive cell bodies increase in size and the fine arborizations in the nerve plexus develop varicosities, putative sites of serotonin release (at 80%). This study provides the initial step for future investigation of potential morphoregulatory functions of serotonin during ENS development.

Funding information:
  • NEI NIH HHS - P30 EY003790(United States)
  • NIGMS NIH HHS - P20 GM103554(United States)