2017 KRIEGSTEIN, Arnold, Cortical Development Cell Migration, UCSF, June 1, 2017
<p class="MsoNormal" >KRIEGSTEIN, Arnold, Cortical Development Radial Glial Cell Migration, UCSF, June 1, 2017</p>
<p>
Prof<b>. AK = Arnold Kriegstein </b>is Direc of Regenerative Med at UCSF ( a superstar in cortical embryology);
This is to a packed audience in Clark Auditorium at Stanford at noon in the SNI Thursday series.
full title for today is " genomic insights into human cortical development, evolution, and disease."</p>
<p>
his PhD thesis advisor was Eric Kandel. (AK is an MD, PhD (from NYU))...
has been at UCSF since 2004.
His abstract appears below: (note: <b>oRG = outer Radial Glia</b>)
<b>Radial glia</b>, the <b>neural stem cells of the neocortex</b>, are located in two niches:</p>
<p>
the ventricular zone and the outer subventricular zone. Although outer subventricular zone radial glia (oRG) appear to generate the majority of human cortical neurons, their molecular features remain elusive. </p>
<p>
We have begun to sequence mRNA from single human progenitor cells for unbiased classification of cell identity and for detection of activated signaling pathways. By analyzing gene expression across single cells, we find that oRG cells preferentially express genes related to extracellular matrix formation, <b>migration</b>, and <b>stemness</b>, and we relate these genes to the position, morphology, and behaviors previously used to classify these cells. </p>
<p>
Many of these genes are involved in growth factor signaling and self-renewal pathways,
suggesting that outer radial glia cells establish a self-sustaining proliferative niche in the OSVZ.</p>
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Using single cell clonal lineage analysis, we find that oRG cells can generate hundreds of daughter neurons of deep and upper layer identity, establishing the extensive proliferative and neurogenic capacity of this cell type. Finally, by using novel markers that reveal the morphology of oRG or ventricular radial glia cells selectively, we find that oRG cells form the primary scaffold for migration of neurons to the cortical plate during mid- and late-phases of cortical neurogenesis. More generally, we have expanded this approach to identify the genes and pathways distinguishing diverse cell types during cortical development.</p>
<p>
These molecular insights have already informed a novel model of primate corticogenesis, suggested a relationship between oRG cells and brain tumors, provided insights into the specific cell types affected by genetic forms of lissencephaly, and have helped identify the mechanism of Zika virus microcephaly.
* end of AK's abstract
(Note: a <b>similar recent lecture given by AK in Oct, 2016 at the Wellcome Genomics Campus</b>
appears on <b>YouTube</b>.)
* AK begins his lecture *
we've been working our way up the evolutionary ladder:
we started on aplysia (in Kandel Lab), then turtles, mice and humans..</p>
<p>
our focus today is on the development of human cortex.</p>
<p>
he is an MD, PhD (and is interested in human disease);</p>
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we will talk abt differences btwn human and mouse brain development...</p>
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first , size difference:</p>
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human brain is 1000 fold bigger than the mouse brain and that is mostly due to cortex.</p>
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but elephants and porpoise brains are even bigger than ours and are more highly folded.</p>
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but (despite that) they have 1/3 fewer neurons than human brains.
(shows slides from brainmuseum.org)</p>
<p>
NE cells (<b>neuroepithelial </b>cells transform into <b>RADIAL GLIAL CELLS </b>in the embryo.
RGCs are the cortical stem cells.</p>
<p>
we will not talk about adult neurogenesis today.
(BB = bob blum, note: the <b>ventricular zone </b>is the zone of cells immediately adjacent to the ventricles: hence,
those cells are the "lowest" in most diagrams... ie closest to the ground in a standing human.
The Subventricular zone is next UP, ie above (or outward from) the ventricular zone: and it is divided into the
inner subventricular zone , which is below the OUTER ventricular zone (which eventually turns into the outer neocortex.)</p>
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<b>Outer Subventricular Zone </b>is an expanded progenitor zone in the developing human cortex.</p>
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he compares a mouse at E13 to human embryo at gestational week 11.5 = GW 11.5</p>
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it is the oSVZ = <b>outer subventric zone </b>that is the layer that hugely expands (to make up the neocortex)</p>
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we use new markers to follow this...described in Hansen in Nature 2010...</p>
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the radial glia cell has a huge apical process ...that goes all the way up to the pia..
oRG = outer Radial Glia cells... same as ventric radial glia cells.</p>
<p>
he shows a great video !!! it shows 24 hours of migrating oSVZ cells in human...</p>
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look earlier at NE cells (at the stage of neural tube)</p>
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they just expand the founder population...</p>
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but then its basal and apical fibers retract .</p>
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but in oRG cell = outer radial glia cell... here the apical fiber never retracts
(unlike the NE cells which have symmetric retraction of fibers)</p>
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oRG outer radial glia...</p>
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<b>oRG the nucleus jumps up the apical fiber</b>!!!</p>
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microtubules move the nucleus around...</p>
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eg in INM (earlier cells) nucleus is pulled down by microtubules in neuroepi cells.</p>
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we thought oRGs probably just flipped this around 180 degrees... but totally wrong!!!
(that is, we previously thought that oRGs were just like NE cells only upside down...
in that nucleus migrated up instead of down)</p>
<p>
but if you block MTs with nodazole then the oRG nuclei still jumps up.. what's going on?</p>
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can cell division be disassociated from migration of the nucleus?.... yes!</p>
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this mechanism employs myosin...(rather than MTs)</p>
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oRG jumps up and then divides...</p>
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the centrosome tranlocates with the nucleus.</p>
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mitotic translocation is really different in these cells.</p>
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and oRGs play a role in cortical malformations.</p>
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see (Alex) Pollen in Cell in 2015...</p>
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put a single oRG into a multiwell plate...</p>
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it puts out a process, then jumps and divides... v characteristic of oRGs</p>
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a single oRG cell can produce over 700 upper and lower layer neurons...</p>
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starting with one oRG can get almost a1000 cells.</p>
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(in a mouse only get 20 cells from a single founder cell)</p>
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these cells can also produce astroglia.</p>
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a single oRG cell can produce 700 upper and lower neural cells.</p>
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ferrets also have oRGs.</p>
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the apical fibers can also support cell migration (of other cells)</p>
<p>
OSVZ oRGs migrate up into OSVZ and they produce daughters that are identical
but also migrate and differentiate into other cell types.</p>
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MOLECULAR MARKERS:</p>
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we do single cell RNAseq to disentangle cellular composition.</p>
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we dissected different zones.</p>
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see Pollen in Nat Biotech in 2014...</p>
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layers (bottom layer is the VZ; then the SubVZ then the IZ = intermed zone then the SP then CP the MZ.)</p>
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RGs may transform into IPCs (intermediate progenitors) and then into Neurons</p>
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he shows a gene plot which shows the top 2 dozen genes.</p>
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how abt novel cell markers?</p>
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want to distinguish ventricular RGs vs outer RGs...</p>
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yes... using in situ hybridization... there are differences in expressed genes.</p>
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And markers suggest specialized functions of the oRGs...</p>
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oRGs have genes that potentiate growth factor signaling...</p>
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and also LFR pathway for self-renewal...</p>
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LIFR/STAT3... helps self-renewal when expanding in number.</p>
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EVOLUTION:</p>
<p>
in rodent... there are CSF growth factors (ie growth factors that are in the CSF that bathe the cells)...</p>
<p>
but when the cells (in primates) move away from the ventricles then they must secrete their own
growth factors... that allows them to expand in # despite being away from the ventricle.</p>
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now we do RNAseq/ dissection at the 8,000 cell level...</p>
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we can look at 8,000 cells</p>
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IPC = intermediate progenitors...</p>
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compare pseudo-age with pseudo differentiation</p>
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RGCs turn into IPs and then into neurons...</p>
<p>
LIX1 an epithelial marker... only expressed v early in NE cells.</p>
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compare Occip cortex and PFC , although similar ... look at</p>
<p>
pyrams... see distinct diffs btwn PFC and V1 pyramidals.</p>
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HCRTR2 is expressed only in PFC pyrams... but not at all in V1...
areal specific...</p>
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radial unit hypothesis from Pasco Rakic in Science in 1988.</p>
<p>
he shows prolif in VZ then form ontogenetic column... with an RG vertical scaffold...
yes.. this works in rodents but not so much in humans...</p>
<p>
use HPOX marker for oRGs and shows it at GW18... gestational week 18.</p>
<p>
the early fibers end in the subventricular zone as seen with CRYAB marker.</p>
<p>
put D-ide xtals on pia and see what gets labelled...
big diff btwn GW 15 and GW18...</p>
<p>
VZ fibers retract and never again contact the upper cortical layers
which are produced by outer RGs in the OSVZ...</p>
<p>
upper cortical layers contain the dense connex => human assoc cortex.</p>
<p>
versus the ventriz zone cells... which are more... the plumbing... eg the CC = corpus callosum..
and other long tracts.</p>
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insights into disease *</p>
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GBM = glioblastome multiforme...</p>
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many of the cell surface proteins that are enriched in oRG cells are highly expressed in glioblastoma.</p>
<p>
they are assoc with stem cell maintenance and</p>
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we obtained slices of tumor from our clinical colleagues...</p>
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he shows a video of tumor GBM... cells also have jumping and dividing (just as in embryo)</p>
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oRGs might be the cells of origin of GBM....</p>
<p>
2nd example : lissencephaly = ILS. smooth cortex (no folds) a migration disorder.</p>
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Miller-Dieker syndrome MDS.. is even more severe...</p>
<p>
it's a disorder of chromo 17 in humans that leads to loss of LIS1.</p>
<p>
we can generate organoids from these: minibrains...(as in Sergui Pasca Lab)</p>
<p>
compare normal development to MDS (Miller Dieker)</p>
<p>
note that laminations in 10 week organoids (WT vs MDS) are v similar BUT
at 15 weeks... differences emerge.</p>
<p>
we see oRGs in both.</p>
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human ESC and iPSC cells pass thru an oRG stage...</p>
<p>
we looked at vRGs (ventric RGs) and Lissencephaly patients divide normally in that layer....</p>
<p>
BUT, difft in oRGs in MDS.</p>
<p>
cells jump further and divide later (this is a problem with MTs = microtubules)
problem is only in the oRGs... oRGs may be involved expansion and folding.</p>
<p>
* Zika and Microcephaly </p>
<p>
from Tang in 2016.</p>
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neural stem cells are readily infected with Zika...</p>
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AXL is THE receptor that allows entry into cells (eg in skin and in astrocytes and in radial glia cells.</p>
<p>
AXL is the flavavirus receptor.</p>
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this predicts tropism of ZIKV for radial glia in first trimester.</p>
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Zika is highly infective in human astrocytes...</p>
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they all label with Sox2... Zika infects human astrocytes (not the neurons during the 2nd trimester)</p>
<p>
yes AXL mediates viral entry.</p>
<p>
blocking the AXL receptor blocks Zika entry into human brain cells..
from DeRisi...</p>
<p>
Azithromycin inhibits ZIKA virus infection (and is safe during pregnancy)
cheap and readily avaialalbe</p>
<p>
Joe deRisi is using this in Brazil...</p>
<p>
Summary:</p>
<p>
single cell mRNAseq has given us novel markers (that we've used to elucidate basic mechanisms of
development as well as pathophysiology of cerebral diseases.</p>
<p>
<<send notes to Tom S and to John H and and David P. and Sergiu P.>>
</p>