HTHSCI 1DT3 Lecture Notes - Lecture 9: Platelet-Derived Growth Factor, Mx1, Basic Helix-Loop-Helix
23 Jun 2018
School
Department
Course
Professor
dorsal
ventral
FD6:616GA6-.E609:1:;//6:919D>9124./25605-27896:1HCI1E292-106;-206:1<29D
56=642A1<815-.=.0/1E67D>0.:E:13-2E19D6102927D2-5J
K>996-01231F->0:7-.A9.201L>792-1MGA-6::.201.01C6;->41F;<6
Expression#of#two#sets#of#genes#occur
based#on#position#(i.e.#SHH
concentration#gradient):
Class#I#genes#are#expressed
predominantly#in#the#dorsal#half,#while
Class#II#genes#are#expressed#mainly
ventrally.
It#is#the#overlap#of#these
various#genes#that
determine#which#cell#types#are
generated#(e.g.#low#Pax#6,#high#Olig2
leads#to#pMN/Motor#Neurone
development).
Motor#neurones#are#produced#first#in#their#particular#domains#of#the#spinal#cord,#and#at#some#point
this#ceases#(E4.0#in#chicks),#then#oligodendrocytes#are#produced#shortly#after:
A#number#of#genes#called#proneural#genes,#are#important#in#neuronal#generation:#neurogenin
1##(Ngn1&2).
These#genes#are#downregulated#in#ventral#neuroepithelium#of#neural#tube,#once#motor
neurone#production#ceases.
Once#Ngn#is#downregulated,#glial#cell#production#occurs.#(Extinction#of#neurogenins#from#the
olig2#expressing#domain#precedes#oligodendrocyte#progenitor#formation).
04/10/2012
6
- shortly after MN production ceases, proneural genes Ngn1 & 2 are
downregulated in the ventral neuroepithelium
- extinction of neurogenins from the olig2 expressing domain precedes
oligodendrocyte progenitor formation
Chick spinal cord
- the domains of Nkx2.2 and olig2 expression begin to shift relative to
one another to create a region of overlap
- migratory olig2+Nkx2.2+ progenitors appear within the overlap and
disperse throughout the spinal cord
Chick spinal cord
Zhou et al (2001)
As#diagram#above#shows#(in#chick#spinal#cord),#red#shows#cells#expressing#Ngn2#(neurogenin
2),#and#green#shows#cell#expression#of#Olig2.#Overlapping#regions#show#where#motor#neurons
are#being#developed.
Around#E4.0/E5.0#–#separation#of#Olig2#(green)#and#Ngn2#(red)#expressing#domains.#As#this
complete#separation#occurs,#oligodendrocyte#cells#begin#to#be#produced.
Same#concept#applies#for#Ngn1#and#Olig2#(shown#below)#–#with#separation#required#for
oligodendrocyte#development.
04/10/2012
6
- shortly after MN production ceases, proneural genes Ngn1 & 2 are
downregulated in the ventral neuroepithelium
- extinction of neurogenins from the olig2 expressing domain precedes
oligodendrocyte progenitor formation
Chick spinal cord
- the domains of Nkx2.2 and olig2 expression begin to shift relative to
one another to create a region of overlap
- migratory olig2+Nkx2.2+ progenitors appear within the overlap and
disperse throughout the spinal cord
Chick spinal cord
Zhou et al (2001)
.J6J1:6A>->9.201231C/0!N#1>051,4./#16GA-6::.201-6/.20:1.:1-6O;.-65132-
24./25605-27896156=642AE6091P.019D61,4./#152E>.0:"J
Q20=6-:648R124./25605-27896156=642AE6091277;-:1SD6019D6-61.:1>012=6-4>A12319D6
CTG#J#1>05124./#1/606:J
,4./1U606:1>051,4./25605-278961I6=642AE609
The#olig#bHLH#group#of#transcription#factors#were#identified#as#essential#for#oligodendrocyte
development#by#three#groups.
If#we#use#retroviruses#to#insert#the#olig1#genes#in#rat#neuroepithelial#cells#(that#have#not#yet
differentiated)#in#vitro,#and#mouse#brain#in#vivo#–#it#can#drive#the#production#of#oligodendrocytes.
Essentially#olig#genes#can#turn#on#the#development#of#oligodendrocyte.#,4./1!1V1##act#in#conjunction
with#other#transcription#factors#and#homeodomain#proteins,#to#specify#oligodendrocytes#during
embryonic#development.
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04/10/2012
9
- OPCs generated from pMN domain starting at
E12.5
- No ventral OPCs in Nkx6 null mice
- PDGFaR+Olig2+ OPCs still produced in dorsal cord
of Nkx6 null mice
- Dorsal OPCs arise from Pax7 and Msx-3
expressing regions
- Small numbers of dorsal Pax7+ OPCs seen in
normal mice but are suppressed by ventral OPCs
- 85% of all spinal cord OPCs generated from ventral
domains
- 3% from Dbx1+ domains, 10-15% from Msx+
domains
Cai et al, 2005
Reporter mice
Cre-recombinase
Cell restricted Cre mice Nkx-2.1
Cre-lox fate mapping
X
loxP
loxP
stop EGFP or Lacz
Nkx2.1/EGFP mice Nkx-2.1 EGFP or Lacz
combine with immunohistochemistry to
fate map specific cell types
Kessaris et al 2006
embryonic#development.
The#genes#are#now#known#to#govern#multiple#aspects#of#progenitor#cell#function#in#the#ventral#neural
tube,#including#specification#of#motor#neurons.
Olig#1#and#2#expression#is#-6/;4>9651<81@WW,#but#indirectly#by#Nkx6.1#and#6.2.
(All#malignant#gliomas#express#24./#"
X027T2;91MGA6-.E609:Y1,4./!
Homozygous#olig1#null#mice#(knockout)#are#viable#but#show#a#564>81.0124./25605-27896
E>9;->9.20.#Heterozygous#(olig1#null,#olig1#present)#mice#show#generally#normal#development.
#Delay#in#oligodendrocyte#maturation#means#these#mice#are#still#viable.
At#P14#stage,#in#wild#type#(normal#olig1)#and#heterozygotes#(olig1#null,#olig1#normal)#–#35%#of#optic
nerve#axons#are#myelinated.#In#0;441E;9>09:R1021E864.01.:1:660#(i.e.#olig1#plays#a#role#in#maturation
ZHFM[1,C#in#myelination;#P14#is#a#later#stage#of#development).
Oligodendrocytes#make#contact#with#axon,#but#do#not#myelinate#them#in#the#brain.#Myelinogenesis
and#axonal#recognition#are#;072;A4651A-276::6:.
There#is#a#severe#reduction#of#myelination#in#the#spinal#cord.#At#3#weeks,#mice#develop#severe
neurological#deficits#and#die.
X027T2;91MGA6-.E609:Y1,4./#
Olig2#null#mice#609.-64814>7T124./25605-2789614.06>/617644:1.019D61:A.0>4172-51P<;910291.019D6
<->.01:96E"1(i.e.#they#are#not#the#only#controlling#factor,#except#in#the#spinal#cord).
Olig1&2#double#null#mice#have#02124./25605-27896:1>08SD6-61.019D61QC@.
,4./#1.:10676::>-8132-124./25605-2/4.2/606:.:.
Motor#neurons#also#lacking#in#the#spinal#cord#of#olig2#null#mice.
I;>412-./.0132-124./25605-27896:1PQ>.1691>41#++&"
Oligodendrocyte#precursor#cells#(OPCs)#are#produced#from#the
pMN#domains#around#E12.5.
Nkx6#knockout#(null)#mice#4>7T1\MCF[HZ1,KQ:#(red),#but#OPCs
are#still#identified#to#be#produced#in#the#dorsal#cord#(green)
(PDGFaR+Olig2+#OPCs)
Dorsal#OPCs#arise#from#K>G(1>051B:G]$16GA-6::.0/1-6/.20:
In#normal#mice,#generally#small#number#of#dorsal#Pax7+#OPCs#are
seen,#but#are#typically#supressed#by#ventral#OPCs.
85%#of#all#spinal#cord#OPCs#are#generated#in#ventral#domains
(red).
3%#of#OPCs#arise#from#DBx1+#domains,#10<15%#from#Msx+#domains#(n.b.#Pax7#is#expressed#in#both
Msx#and#Dbx#regions)
04/10/2012
9
- OPCs generated from pMN domain starting at
E12.5
- No ventral OPCs in Nkx6 null mice
- PDGFaR+Olig2+ OPCs still produced in dorsal cord
of Nkx6 null mice
- Dorsal OPCs arise from Pax7 and Msx-3
expressing regions
- Small numbers of dorsal Pax7+ OPCs seen in
normal mice but are suppressed by ventral OPCs
- 85% of all spinal cord OPCs generated from ventral
domains
- 3% from Dbx1+ domains, 10-15% from Msx+
domains
Cai et al, 2005
Reporter mice
Cre-recombinase
Cell restricted Cre mice Nkx-2.1
Cre-lox fate mapping
X
loxP
loxP
stop EGFP or Lacz
Nkx2.1/EGFP mice Nkx-2.1 EGFP or Lacz
combine with immunohistochemistry to
fate map specific cell types
Kessaris et al 2006
Q-6]Z2G1L>961B>AA.0/1^1Used(to(show(this(idea(of(oligodendrocyte(domains
Reporter#mice#contain#reporter#genes#(markers).#Many#reporter#genes#can#be#used#(large#family#of
genes),#a#common#gene#is#EGFP#(Enhanced#Green#Fluorescent#Protein#from#jellyfish).
In#front#of#the#reporter#gene#is#a#STOP#codon#(which#normally#stops#the#expression#of#the#EGFP
protein).#The#STOP#codon#is#flagged#by#the#two#sites#loxP.
The#importance#of#these#sites#is#that#they#can#60?8E>9.7>44817;913-2E19D61/602E61<819D6160?8E6
Q-61PQ-61[672E<.0>:6".#
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