High-throughput sequencing for deep coverage transcriptomics of brain-to-spinal cord projection neurons
Prosjekt
- Prosjektnummer
- 2013022
- Ansvarlig person
- Joel Glover
- Institusjon
- Oslo universitetssykehus HF
- Prosjektkategori
- Doktorgradsstipend
- Helsekategori
- Neurological
- Forskningsaktivitet
- 1. Underpinning
Rapporter
Gene expression profiling reveals the expression of specific transcription factors and axon growth regulatory proteins in neurons of the LVST (lateral vestibulospinal tract) group and the cMVST (contralateral medial vestibulospinal tract) group, the two main sources of projections from the brain to the spinal cord.
Synaptic connections from the brain to the spinal cord are mediated predominantly by descending projection neurons in the brainstem. One of the major sources lies in the vestibular nuclei, namely the 3 different vestibulspinal tracts, of which the two largest are the LVST (lateral vestibulospinal tract) neuron group, which is excitatory and projects the entire length of the spinal cord and innervates motoneurons and interneurons differentially at different spinal levels, and the cMVST (contralateral medial vestibulospinal tract) neuron group, which is inhibitory and projects to cervical levels and innervates motoneurons and interneurons that control neck musculature. The aim of this project is to identify genes that specify the identities of the LVST and cMVST neurons and direct the growth of their axons to their specific targets in the spinal cord. During 2016, we analyzed transcription factor expression at the protein level using immunohistochemistry and defined a 4-transcription factor signature that defined the LVST neurons and a small population of neurons in their immediate vicinity. We also identified transcription factors that distinguished the cMVST neuron group from the LVST neuron group. During 2017 we extended this analysis and discovered that we could define a group of neurons that include the LVST neuron group with a 3-transcription factor signature, and that we could distinguish two different components of the cMVST group by the expression of other specific transcription factor signatures. In 2018, we determined that a specific 4-transcription factor signature uniquely defines the LVST and a small associated neuron group, and that a specific 4-transcription factor signature uniquely defines one of the components of the cMVST group - no other neurons in the entire CNS express these gene expression signatures. This is the first time neuron groups of such functional specificity have been characterized by unique transcription factor signatures. Our results provide powerful insight into the molecular programs that define the vestibulospinal projections, and a springboard for similar analyses of other neuron groups that link the brain and spinal cord, which are already underway. These results have now been published - the article is in press at the journal eNeuro (no PMID number yet). Another manuscript will be submitted soon that describes the image analysis protocol that was developed to facilitate this study, and another manuscript is being written that provides a detailed anatomical description of the spatial relationships between the LVST and cMVST neurons as they develop. PhD student Anders Lunde will submit his doctoral dissertation during the spring of 2019.
There are no immediate consequences for health services. However, the study provides new and important information about how the neurons that connect the brain and spinal cord develop and become specialized. This information may be used to facilitate in-depth analyses of the precise synpatic connectivity of these neurons and how they function to regulate spinal cord circuits. This in turn may enable the development of regenerative treatments for spinal cord injury, or alternatively the development of neuroprosthetic devices that simulate the function of these projections.
Gene expression profiling reveals the expression of specific transcription factors and axon growth regulatory proteins in neurons of the LVST (lateral vestibulospinal tract) group and the cMVST (contralateral medial vestibulospinal tract) group, the two main sources of projections from the brain to the spinal cord.Synaptic connections from the brain to the spinal cord are mediated predominantly by descending projection neurons in the brainstem. One of the major sources lies in the vestibular nuclei, namely the 3 different vestibulspinal tracts, of which the two largest are the LVST (lateral vestibulospinal tract) neuron group, which is excitatory and projects the entire length of the spinal cord and innervates motoneurons and interneurons differentially at different spinal levels, and the cMVST (contralateral medial vestibulospinal tract) neuron group, which is inhibitory and projects to cervical levels and innervates motoneurons and interneurons that control neck musculature. The aim of this project is to identify genes that specify the identities of the LVST and cMVST neurons and direct the growth of their axons to their specific targets in the spinal cord. During 2016, we analyzed transcription factor expression at the protein level using immunohistochemistry and defined a 4-transcription factor signature that defined the LVST neurons and a small population of neurons in their immediate vicinity. We also identified transcription factors that distinguished the cMVST neuron group from the LVST neuron group. During 2017 we have extended this analysis and have discovered that we can actually uniquely define the LVST neuron group with a 3-transcription factor signature, and we can define two different components of the cMVST group with two different 3--transcription factor signatures. This is the first time neuron groups of such functional specificity have been characterized by unique transcription factor signatures. Our results provide powerful insight into the molecular programs that define the vestibulospinal projections, and a springboard for similar analyses of other neuron groups that link the brain and spinal cord. We are currently writing a manuscript based on these results, which we plan to submit for publication during the spring of 2018. It is also planned that PhD student Anders Lunde will submit his doctoral dissertation during 2018.
Gene expression profiling reveals the expression of specific transcription factors and axon growth regulatory proteins in neurons of the LVST (lateral vestibulospinal tract) group and the cMVST (contralateral medial vestibulospinal tract) group, the two main sources of projections from the brain to the spinal cord.Synaptic connections from the brain to the spinal cord are mediated predominantly by descending projection neurons in the brainstem. One of the major sources lies in the vestibular nuclei, namely the 3 different vestibulspinal tracts, of which the two largest are the LVST (lateral vestibulospinal tract) neuron group, which is excitatory and projects the entire length of the spinal cord and innervates motoneurons and interneurons differentially at different spinal levels, and the cMVST (contralateral medial vestibulospinal tract) neuron group, which is inhibitory and projects to cervical levels and innervates motoneurons and interneurons that control neck musculature. The aim of this project is to identify genes that specify the identities of the LVST and cMVST neurons and direct the growth of their axons to their specific targets in the spinal cord.
During 2016, we have extended our analysis of transcription factor expression at the protein level using immunohistochemistry and defined a 4-transcription factor signature that appears to uniquely define the LVST neurons. This is the first time a neuron group of such functional specificity has been characterized by a unique transcription factor signature. In addition, we have identified several transcription factors which in combination distinguish the cMVST from the LVST. However, we have not yet identified a unique transcription factor signature for the cMVST group. We are currently writing a manuscript based on these results, which we plan to submit for publication during the spring of 2017.
Kartlegging av genekspresjonsprofiler avslører uttrykket av bestemte proteiner som regulerer genekspresjon og nervefibervekst i nerveceller som hører til vestibulospinalbanen, en av hovedfiberbanene fra hjernen til ryggmargen.Signaler fra hjernen til ryggmargen formidles hovedsaklig av nedadstigende fiberbaner fra hjernestammen. En av de viktigste springer ut fra de såkalte vestibulariskjernene, som formidler informasjon om balanse. Denne banen, den såkalte vestibulospinalbanen, rekker ned til enden av ryggmargen og forsyner motoriske nerveceller (som driver musklene) med ulike typer impulser avhengig av ryggmargsnivå.
Målet for dette prosjektet er å forstå hvordan ulike typer nerveceller i vestibulariskjernene som innegår i denne fiberbanen blir bestemt utviklingsmessig til å danne kontakter med ulike nivåer og ulike typer motoriske nerveceller i ryggmargen. Dette gjør vi ved å isolere nervecellene og kartlegge genene de utrykker på forskjellige utviklingsstadier. Når vi har identifisert gener som uttrykkes selektivt, bruker vi tekniker til å synliggjøre genuttrykket i snitt av hjernestammen slik at vi kan se nøyaktig hvilke nerveceller uttrykker hvilke gener. Vi kan da undersøke om genuttrykket er korrelert med bestemte funksjoner.
I 2015 har vi kommet langt med kartlegging av genuttrykk i snitt av hjernestammen, slik at vi har klart å identifisere subpopulasjoner av nerveceller i vestibulariskjernene som gir opphav til ulike deler av den vestibulospinale banen. Dette gir oss økt innsikt i hvordan denne banen er satt sammen, og hvordan de ulike målcellene i ryggmargen styres funksjonelt. Denne kunnskapen gir oss i tillegg mulighet til å bruke genetikk til å manipulere målrettet de ulike nervecellesubpopulasjoner i levende dyr for å få enda bedre innsikt i hvordan de fungerer. Denne kunnskapen belyser viktige detaljer om ryggmargsfunksjon, som blant annet kan anvendes i forbindelse med behandling av ryggmargsskader.
Gene expression profiling reveals the expression of specific transcription factors and axon growth regulatory proteins in neurons of the LVST (lateral vestibulospinal tract) group, a major source of projections from the brain to the spinal cord.Synaptic connections from the brain to the spinal cord are mediated predominantly by descending projection neurons in the brainstem. One of the major sources lies in the vestibular nuclei, namely the LVST (lateral vestibulospinal tract) neuron group, which projects the entire length of the spinal cord and innervates motoneurons and interneurons differentially at different spinal levels. The aim of this project is to identify genes that specify the identity of the LVST neurons and directs the growth of their axons to specific targets in the spinal cord.
During 2014, we have used selective retrograde tracing followed by cell dissociation and separation techniques to identify and isolate LVST neurons from the brains of mouse embryos at a stage early in the development of the LVST projection to the spinal cord. mRNA was then extracted from these isolated LVST neurons and subjected to RNA sequencing. Two rounds of RNA sequencing have been carried out, one at the University of Oslo and the other at Harvard University. In each case, a list of highly expressed genes and a list of differentially expressed genes (relative to control neurons in a neighboring region) were generated, and comparison of these lists identified a number of coincident genes specific to the LVST neurons. Such genes include transcription factors and axon growth regulatory proteins that are thereby implicated in specifying the identity and synaptic connectivity of the LVST neurons. Screening of expression at the protein level using immunohistochemistry has been carried out, and has confirmed the expression of several transcription factors and shown that some of these are expressed by subpopulations of the LVST neurons. This indicates that functional and connectional differences among LVST neurons may be specified by these identified transcription factors. Current work is focused on further characterisation of the expression of highly expressed genes and on utilising transgenic mouse models to investigate experimentally how these genes influence the differentiation of the LVST neurons. In addition, we have started to isolate LVST neurons from chicken embryos and subject them to RNA sequencing and immunohistochemistry, working on the assumption that the most important genes for LVST neuron development are likely to be conserved among vertebrates. We have already discovered that some transcription factors are indeed expressed in a conserved fashion on comparing mammals to birds, strengthening the notion that they are also important in the development of the LVST in humans.
Et overordnet spørsmål når det gjelder hvordan nervecellekretser i hjernen utvikler seg er hvilke gener styrer differensieringen av bestemte nervecelletyper og hvordan disse kobler seg sammen for å sikre riktig hjernefunksjon.I dette prosjektet bruker vi kraftig sekvenseringsmetoder til å kartlegge alle gentranskripter som leses av i identifisierte nervecellepopulasjoner i en bestemt motorisk krets i hjernestammen til mus. Siden prosjektets start har stipendiaten Anders Lunde fått opplæring i metodene som kreves til å merke og isolere de relevante nerveceller slik at gentranskriptene kan ekstraheres og sekvenseres. De første sekvenseringene har identifisert en rekke gener som er uttrykt spesielt mye i disse nervecellene i forhold til andre nervecellene. Immunhistokjemiske metoder er også tatt i bruk til å bekrefte ekspresjon av disse genene på proteinnivå.
Vitenskapelige artikler
Lunde, A, Okaty B, Dymecki S, Glover JC
Molecular profiling defines evolutionarily conserved transcription factor signatures of major vestibulospinal neuron groups
eNeuro, 2019
Deltagere
- Anders Lunde Doktorgradsstipendiat (finansiert av denne bevilgning)
- Joel Glover Prosjektleder
- Anders Lunde Doktorgradsstipendiat
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
Alle henvendelser rettes til eRapport