Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum

<p>Abstract</p> <p>Background</p> <p>Neural development consists of a series of steps, including neurogenesis, patterning, cell migration, axon guidance, and finally, synaptogenesis. Because all these steps proceed in a constantly changing environment, functional gene analyses during development have to take time into account. This is quite challenging, however, as loss-of-function approaches based on classic genetic tools do not allow for the precise temporal control that is required for developmental studies. Gene silencing by RNA interference (RNAi) in combination with the chicken embryo or with cultured embryos opens new possibilities for functional gene analysis <it>in vivo</it>. Axonin-1/TAG-1 is a cell adhesion molecule of the immunoglobulin superfamily with a well defined temporal and spatial expression pattern in the developing vertebrate nervous system. Axonin-1/TAG-1 was shown to promote neurite outgrowth <it>in vitro </it>and to be required for commissural and sensory axon pathfinding <it>in vivo</it>.</p> <p>Results</p> <p>To knock down axonin-1 in a temporally and spatially controlled manner during development of the nervous system, we have combined RNAi with the accessibility of the chicken embryo even at late stages of development. Using <it>ex ovo </it>RNAi, we analyzed the function of axonin-1/TAG-1 in cerebellar development. Axonin-1 is expressed in postmitotic granule cells while they extend their processes, the parallel fibers. In the absence of axonin-1 these processes still extend but no longer in a parallel manner to each other or to the pial surface of the cerebellum.</p> <p>Conclusion</p> <p>Axonin-1/TAG-1 is required for the navigation, but not for the elongation, of granule cell processes in the developing cerebellum <it>in vivo</it>.</p

Ex ovo RNAi for functional gene analysis during neural development

To study gene function in vivo during development of the central nervous system (CNS) efficient model systems that allow for temporal and spatial control of gene expression are required. In our lab, we have used the chicken embryo in combination with in ovo electroporation and RNA interference (RNAi) for gene silencing during early stages of nervous system development. In particular, we used dorsal commissural neurons to study axonal pathfinding. These neurons extend their axons toward the floor plate, the ventral midline of the spinal cord. Guidance cues derived from the floor plate, the intermediate target of these axons, are important for axon growth toward and across the midline. Using in ovo RNAi, we could show that interference with the function of the transmembrane glycoprotein Endoglycan (PODXL2) resulted in the failure to turn or in erroneous caudal turns after midline crossing. Furthermore, the morphology of the floor plate was severely disturbed in the absence of Endoglycan. During later stages of neural development, Endoglycan is expressed by Purkinje cells in the cerebellum. For the functional characterization of Endoglycan during cerebellar development, we extended the experimental accessibility of chicken embryos to much older stages. We established the procedure of ex ovo electroporation and RNAi to manipulate gene expression in the developing cerebellum. The cerebellum represents a well characterized neuronal structure, and therefore, an ideal system to study CNS development, including neurogenesis, differentiation, migration, axon guidance, as well as synapse formation. To demonstrate the applicability and the efficiency of ex ovo RNAi, we analyzed the function of the cell adhesion molecule Axonin-1 in cerebellar development. Axonin-1 is expressed by postmitotic granule cells at the time when they extend their axons, the parallel fibers. In the absence of Axonin-1 the arrangement of granule cell axons within the molecular layer was aberrant and fibers no longer extended parallel but towards the cerebellar surface. The effect of Axonin-1 was not on parallel fiber elongation but affected specifically parallel fiber navigation. The same effects on parallel fiber development observed after ex ovo RNAi were reproduced in embryos treated with function-blocking anti-Axonin-1 antibodies, indicating that ex ovo RNAi efficiently and reproducibly silenced axonin-1 in the developing cerebellum. Thus, we used ex ovo RNAi to study the function of Endoglycan during cerebellar development. Endoglycan is expressed by Purkinje cells at the time when they migrate towards the pial surface to establish the Purkinje cell monolayer. Interference with Endoglycan function caused a severe migration defect of Purkinje cells. Moreover, due to the aberrant formation of the Purkinje cell monolayer, the thickness of the external germinal layer and the proliferation rate of granule cells were significantly reduced causing disturbed foliation and reduced size of the cerebellum. Für die Analyse von Genfunktionen während der Entwicklung des zentralen Nervensystems (ZNS) werden effiziente Systeme benötigt, welche es ermöglichen, die Genexpression zeitlich und räumlich zu kontrollieren. In unserem Labor benutzen wir in ovo RNAi (RNA interference), um im Hühnerembryo während früher neuraler Entwicklungsstadien Gene gezielt auszuschalten. Anhand dorsaler Kommissuralneurone studieren wir Wegweisermoleküle, welche Axone zu ihren Zielzellen dirigieren. Axone der Kommissuralneurone wachsen gegen die ventrale Mittelline des Rückenmarks, die Bodenplatte, überqueren diese und wachsen anschliessend in rostraler Richtung weiter. Wegweisermoleküle, welche direkt auf der Bodenplatte lokalisiert sind oder von dieser sezerniert werden, sind wichtig für die korrekte Wegfindung dieser Axone. Mittels in ovo RNAi konnten wir zeigen, dass das Transmembran-Glykoprotein Endoglycan (PODXL2) nach dem Überqueren der Bodenplatte für die anschliessende rostrale Drehung von Kommissuralaxonen wichtig ist. Der Verlust von Endoglycan-Aktivität führte zu Stillstehen am Ausgang der Bodenplatte und fehlerhaftem caudalen Wachsen von Kommissuralaxonen. Das Blockieren von Endoglycan führte ausserdem zu einer Veränderung der Morphologie der Bodenplatte. Während späterer Entwicklungsstadien wird Endoglycan von migrierenden Purkinje- Zellen des Kleinhirns (Cerebellum) exprimiert. Um die Funktion von Endoglycan im Kleinhirn zu untersuchen, haben wir mit ex ovo RNAi eine Methode entwickelt, welche das gezielte Hemmen einzelner Gene während fortgeschrittener Stadien der Entwicklung des Nervensystems ermöglicht. Die neuronale Struktur des Kleinhirns ist gut charakterisiert und daher ein ideales System um verschiedene Entwicklungsschritte des Nervensystems zu studieren. Um die Anwendbarkeit und Effizienz von ex ovo RNAi auf unsere Fragestellung zu testen, haben wir zunächst die Funktion des Zelladhäsionsmoleküls Axonin-1 im Kleinhirn untersucht. Während des Auswachsens der Axone, den so genannten Parallelfasern, exprimieren Körnerzellen Axonin-1. Der Verlust von Axonin-1-Aktivität hat zur Folge, dass die Organisation der Parallelfasern in der Molekularschicht des Kleinhirns defekt ist. Dieser Defekt beruht auf der fehleraften Navigation von Parallelfasern. Den gleichen Effekt auf die Entwicklung der Parallelfasern konnten wir mittels Injektionen von funktionsblockierenden Antikörpern erzielen. Dies deutet darauf hin, dass ex ovo RNAi effizient und reproduzierbar Axonin-1-Aktivität im Kleinhirn hemmen kann. Daher verwendeten wir diese Methode für die Untersuchung von Endoglycan im Kleinhirn. Dieses Gen wird von Purkinje-Zellen während ihrer radialen Migration in Richtung Oberfläche des Kleinhirns exprimiert. Das Fehlen von Endoglycan verursachte einen Migrationsdefekt der Purkinje-Zellen und eine anormale Ausbildung der Purkinje-Zellschicht. Dies wiederum führte zu einer verminderten Dicke der Körnerzellschicht bedingt durch eine Reduktion der Zellteilungsrate

Transfer of Spectral Weight in Spectroscopies of Correlated Electron Systems

We study the transfer of spectral weight in the photoemission and optical spectra of strongly correlated electron systems. Within the LISA, that becomes exact in the limit of large lattice coordination, we consider and compare two models of correlated electrons, the Hubbard model and the periodic Anderson model. The results are discussed in regard of recent experiments. In the Hubbard model, we predict an anomalous enhancement optical spectral weight as a function of temperature in the correlated metallic state which is in qualitative agreement with optical measurements in $V_2O_3$. We argue that anomalies observed in the spectroscopy of the metal are connected to the proximity to a crossover region in the phase diagram of the model. In the insulating phase, we obtain an excellent agreement with the experimental data and present a detailed discussion on the role of magnetic frustration by studying the $k-$resolved single particle spectra. The results for the periodic Anderson model are discussed in connection to recent experimental data of the Kondo insulators $Ce_3Bi_4Pt_3$ and $FeSi$. The model can successfully explain the different energy scales that are associated to the thermal filling of the optical gap, which we also relate to corresponding changes in the density of states. The temperature dependence of the optical sum rule is obtained and its relevance for the interpretation of the experimental data discussed. Finally, we argue that the large scattering rate measured in Kondo insulators cannot be described by the periodic Anderson model.Comment: 19 pages + 29 figures. Submitted to PR

Advances in tenascin-C biology

Tenascin-C is an extracellular matrix glycoprotein that is specifically and transiently expressed upon tissue injury. Upon tissue damage, tenascin-C plays a multitude of different roles that mediate both inflammatory and fibrotic processes to enable effective tissue repair. In the last decade, emerging evidence has demonstrated a vital role for tenascin-C in cardiac and arterial injury, tumor angiogenesis and metastasis, as well as in modulating stem cell behavior. Here we highlight the molecular mechanisms by which tenascin-C mediates these effects and discuss the implications of mis-regulated tenascin-C expression in driving disease pathology

Perturbation of AX-1 expression did not interfere with granule cell migration from the EGL through the ML

No change in the distribution of Pax6-positive granule cells was detectable between untreated control embryos at HH35 (a) or HH38 (c) and embryos injected and electroporated with dsRNA derived from AX-1 at HH35 (b) or HH38 (d). As expected, downregulation of AX-1 by RNAi at HH34 did not interfere with proliferation of granule cell precursors, as AX-1 is expressed only after granule cells become postmitotic. Sagittal sections labeled with BrdU and counterstained with DAPI taken from control embryos (e) and embryos lacking AX-1 (f) at HH38 did not differ. Inserts in (b,d,f) show adjacent sections stained for EGFP to demonstrate that sections were taken from the electroporated area of the cerebellum. Bar: 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum"</p><p>http://www.neuraldevelopment.com/content/3/1/7</p><p>Neural Development 2008;3():7-7.</p><p>Published online 17 Mar 2008</p><p>PMCID:PMC2322981.</p><p></p

The cerebellar anlage is positioned right under the bifurcation of the middorsal sinus and the middle cerebral vein

The injection site is indicated by +. The head of the embryo was turned and stabilized with a hook prepared from a spatula (arrowhead). Tweezer electrodes were placed parallel to the head. It was important to avoid contact between embryo and electrode (indicated by white brackets) to prevent tissue damage. One half of the cerebellum was successfully transfected by electroporation. The untransfected half of the cerebellum could serve as an internal control for the analysis of phenotypes. (d) Successful transfection of one half of the cerebellum after electroporation with platelet electrodes is shown in a 250-μm-thick coronal slice. Depending on the depth of the injection, different cerebellar layers could be targeted: deep injections into the ventricle resulted predominantly in transfection of the ventricular zone labeling all cell types proliferating and migrating from there, such as Purkinje cells, interneurons, and glia cells (e). With superficial injections the developing ML (f,g) and granule cells of the external germinal layer (h,i) were transfected. Parallel fibers in the developing ML were visualized using AX-1 as a marker (g). Granule cells were identified by Pax6 (i). EGL, external germinal layer; ML, molecular layer; PS, pial surface; V, ventricle. Bar: 2 mm in (a,b), 1 mm in (c), 500 μm in (d), 100 μm in (e-g), 50 μm in (h,i).<p><b>Copyright information:</b></p><p>Taken from "Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum"</p><p>http://www.neuraldevelopment.com/content/3/1/7</p><p>Neural Development 2008;3():7-7.</p><p>Published online 17 Mar 2008</p><p>PMCID:PMC2322981.</p><p></p

The efficiency of AX-1 downregulation was demonstrated in caudal sections taken from HH35 cerebella one day after injection and electroporation of dsRNA derived from AX-1 mixed with a plasmid encoding EGFP (50:1)

EGFP expression was used to identify the electroporated area of the cerebellum. Expression of AX-1 was lost from many cells after RNAi, resulting in a patchy appearance of the granule cell layer (b; c, open arrows). In corresponding sections of a control embryo AX-1 staining appeared homogenous. (c) The number of EGFP-expressing cells (white arrows) was much smaller than the number of cells that failed to express AX-1 due to the molecular ratio of 50:1 for dsRNA and EGFP plasmid. Very few cells were yellow, indicating both EGFP and AX-1 expression (c, arrowhead). For quantitative analysis, 30-μm-thick sagittal sections were stained for AX-1. EGFP expression was used to identify the electroporated area of the cerebellum. Staining intensities were compared between experimental (e,f) and control (EGFP) embryos (h) at HH35, one day after electroporation. On average, the injection and electroporation of AX-1 dsRNA reduced the AX-1 protein level in the transfected area by 61.3 ± 9.4% (n = 4 embryos; ***< 0.0001 for comparison with both control groups). As expected, there was no change in AX-1 protein levels in the untransfected part of the cerebellum of embryos treated with dsRNA. AX-1 expression in transfected areas of embryos treated with the EGFP plasmid alone did not differ from untreated control embryos (not shown; i). The ratio of AX-1 staining was 3.4 ± 2.2% (n = 5) for EGFP-control embryos and 2.3 ± 0.9% (n = 4) in untreated control embryos. The insert in (e) shows a low magnification image of the section. The box corresponds to the high magnification image shown in (e). PS, pial surface. Bar: 50 μm in (a-d), 200 μm in (e-h).<p><b>Copyright information:</b></p><p>Taken from "Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum"</p><p>http://www.neuraldevelopment.com/content/3/1/7</p><p>Neural Development 2008;3():7-7.</p><p>Published online 17 Mar 2008</p><p>PMCID:PMC2322981.</p><p></p

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals

We compared expression of related immunoglobulin superfamily cell adhesion molecules between embryos lacking AX-1 and age-matched untreated control embryos at HH35, one day after RNAi

We found no changes in NgCAM (a,b), NrCAM (c,d), and Contactin/F11 expression (e,f). Inserts in (a,c,e) show adjacent sections stained for EGFP to demonstrate that sections were taken from the electroporated area of the cerebellum. Bar: 100 μm in (a-d), 50 μm in (e,f).<p><b>Copyright information:</b></p><p>Taken from "Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum"</p><p>http://www.neuraldevelopment.com/content/3/1/7</p><p>Neural Development 2008;3():7-7.</p><p>Published online 17 Mar 2008</p><p>PMCID:PMC2322981.</p><p></p