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Suppression of interneuron programs andmaintenance of selected spinal motor neuronfates by the transcription factor AML1/Runx1
Nicolas Stifani*, Adriana R. O. Freitas*, Anna Liakhovitskaia
†
, Alexander Medvinsky
†
, Artur Kania
द
,and Stefano Stifani*
*Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, QC, Canada H3A 2B4;
†
Institute for Stem Cell Research/MedicalResearch Council Centre for Stem Cell Biology, University of Edinburgh, Edinburgh EH9 3JQ, United Kingdom;
‡
Department of Anatomy and Cell Biology,Division of Experimental Medicine, McGill University, Montreal, QC, Canada H3A 2B2;
§
De´partement de Me´decine, Universite´ de Montreal, Montreal, QC,Canada H3C 3JY; and
¶
Neural Circuit Development Laboratory, Institut de recherches cliniques de Montreal, Montreal, QC, Canada H2W 1R7Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved February 29, 2008 (received for review November 29, 2007)
Individualspinalmotorneuronidentitiesarespecifiedinlargepartby the intrinsic repertoire of transcription factors expressed byundifferentiated progenitors and maturing neurons. It is shownherethatthetranscriptionfactorAML1/Runx1(Runx1)isexpressedin selected spinal motor neuron subtypes after the onset ofdifferentiation and is both necessary and sufficient to suppressinterneuron-specific developmental programs and promote main-tenance of motor neuron characteristics. These findings show animportant role for Runx1 during the consolidation of selectedspinal motor neuron identities. Moreover, they suggest a require-ment for a persistent suppression of interneuron genes withinmaturing motor neurons.
lateral motor column
median motor column
runt
spinal cordspinal accessory column
S
pecific transcription factor codes within exclusive ventralprogenitor domains regulate motor neuron and interneurondifferentiation in the developing spinal cord (1, 2). Some deter-minants of both lineages are coexpressed in mitotic progenitors(3), raising the questions of what molecules control the diver-gence and maintenance of motor neuron and interneuron dif-ferentiation programs. Genetic studies suggest that the gene
Hb9
is required to suppress interneuron programs actively in matur-ing motor neurons (3, 4), but other effectors of the mechanismsthat promote the divergence of motor and interneuron fatesremain to be determined.In both invertebrates and vertebrates, the
runt
/
Runx
genefamily encodes DNA-binding transcription factors that mediatetransactivation or repression depending on specific contexts (5).Members of this transcription factor family regulate neuronsubtype specification and axon target connectivity in
Drosophila
(6–8), chick (9, 10), and mice (11–16). The
runt
/
Runx
familymember
AML1
/
Runx1
(
Runx1
) is expressed in selected popula-tions of motor neurons in the murine and avian spinal cord,suggesting that it is involved in motor neuron development (13,17). Here, we show that mouse
Runx1
is expressed in restrictedgroups of ventrally exiting cervical motor neurons during theirpostmitotic development. Loss of
Runx1
function does not affectthe survival of those motor neurons but results in a loss of expression of motor neuron-specific genes and a concomitantactivation of expression of interneuron-specific genes. Con- versely, ectopic expression of Runx1 in the spinal cord of developing chick embryos suppresses interneuron gene expres-sion and promotes motor neuron differentiation programs.These results identify a role for Runx1 in the establishment of selected motor neuron identities and suggest that maturingmotor neurons must continually suppress interneuron-specificdevelopmental programs.
Results
Runx1
IsExpressedinSpecificPostmitoticMotorNeuronsintheMouseCervical Spinal Cord.
To determine the role of
Runx1
in spinalmotor neuron development, we first characterized its expressionpattern by examining a previously characterized
Runx1
lacZ/
knockin mouse line in which
-galactosidase (
-gal) expressionrecapitulates the expression of endogenous
Runx1
(13, 18, 19). Inembryo day (E)9.5
Runx1
lacZ/
embryos,
-gal expression coin-cided with Runx1 protein, detected by using a previously de-scribed anti-Runx1 antibody (17), in the second branchial arch, where
Runx1
]. The specificity of theanti-Runx1 antibody was demonstrated by the absence of im-munoreactivity in Runx1-deficient embryos (Fig. S1
Runx1
lacZ/
embryos,
-gal expressionbegan at
E9.5 in a small number of ventrolateral cells at levelsC1–C4 (Fig. 1
A
, arrowheads).
-gal
cells did not expressneuronal progenitor markers such as Pax6 or Nkx2.2, nor thegeneral cell proliferation marker Ki67 (Fig. 1
A
–
C
). Moreover, we observed no detectable overlap between the expression of
-gal and the interneuron markers Evx1 (V0 interneurons), En1(V1), and Chx10 (V2) (Fig. 1
D
–
F
), although some occasionaloverlap with Chx10 was observed at later stages (see Fig. 3below). Instead,
-gal
cells expressed the postmitotic motorneuron markers Isl1 and choline acetyltransferase (ChAT) (3, 4,20) (Fig. 1
G
and
H
, arrows). Only a subset of the Isl1
cellsexpressed
-gal,suggestingthat
Runx1
isexpressedinarestrictednumber of spinal motor neurons. The expression of
). These results indicate that
Runx1
expression in the spinal cord is first activated in a subpopulationof postmitotic motor neurons, but not their progenitors, atcervical levels C1–C4.
Runx1
Expression Is Activated in Ventrally Exiting Motor NeuronsAfter the Onset of Differentiation.
To determine the identity of thecervical Isl1
neurons in which
-gal is first expressed in
Runx1
lacZ/
embryos, we compared the expression of
-gal withthat of the Hb9 protein, which is expressed in virtually all motorneurons whose axons exit via the ventral root and innervateskeletal muscles (3, 4). No visible overlap of
-gal and Hb9expression was observed at E9.5 (data not shown) and E10.5, thepeak of motor neuron generation (Fig. 1
J
–
L
). At E9.5, we also
Authorcontributions:N.S.andA.R.O.F.contributedequallytothiswork;N.S.,A.K.,andS.S.designed research; N.S., A.R.O.F., and A.L. performed research; A.L. and A.M. contributednewreagents/analytictools;N.S.,A.R.O.F.,A.K.,andS.S.analyzeddata;andN.S.,A.K.,andS.S. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.
To whom correspondence should be addressed at: Montreal Neurological Institute, 3801rue University, Montreal, QC H3A 2B4, Canada. E-mail: stefano.stifani@mcgill.ca.This article contains supporting information online atwww.pnas.org/cgi/content/full/ 0711299105/DCSupplemental.© 2008 by The National Academy of Sciences of the USA
www.pnas.org
cgi
doi
10.1073
pnas.0711299105 PNAS
April 29, 2008
vol. 105
no. 17
6451–6456
N E U R O S C I E N C E
failed to detect an overlap between the expression of
-gal andthat of Lhx3, a protein whose expression at this stage transientlymarks the majority of ventrally exiting motor neurons (vMNs) as well as their precursors (20) (data not shown). Coexpression of
-gal and Hb9 was first observed in the ventromedial spinal cordat
E11 (Fig. 1
M
–
O
, arrows, and data not shown). At this stage,
Runx1
expression expands ventrally and can be detected at morecaudal levels of the spinal cord (C5–T1) (13). This analysis of
Runx1
lacZ/
embryos suggests that the onset of
Runx1
expressionin vMNs follows the initial activation of
Hb9
expression.In E9.5-E10.5
Runx1
lacZ/
embryos, most, if not all, of the
-gal
cells coexpressed the homeodomain protein Phox2b (Fig.1
P
–
R
), which is selectively expressed in spinal accessory (SAC)motor (nXI) neurons (21). SAC neurons are present at spinallevels C1–C4, express Isl1 but not Hb9, and send their axons outof the spinal cord through lateral exit points located midwayalong the dorsoventral axis of the spinal cord. The exiting axonsassemble into the spinal accessory nerve, which innervatesbranchial arch-derived muscles in the neck (22). Consistent withthese observations, at E15.5 a number of
-gal
cells at levelsC1–C4 were retrogradely labeled from the anterior trapeziusmuscle, a lateral cervical muscle of the neck innervated by thespinal accessory nerve (Fig. 1
S
). Together, these results showthat
Runx1
expression is activated in two distinct spatiotemporalpatterns. It is first expressed in dorsally exiting SAC motorneurons starting at
E9.5 followed by a later expression inselected populations of vMNs after the peak of vMN generationand Hb9 expression (E9.5–E10.5). These results suggest that
Runx1
becomes activated in specific vMNs after their initialdifferentiation and during their developmental maturation.
Runx1
Is Expressed in Selected Types of Ventrally Exiting MotorNeurons and Is Not Required for Their Generation or Survival.
InE13.5
Runx1
lacZ/
embryos, when distinct motor columns arediscernable, two groups of
-gal
motor neurons were observedat the forelimb level (C5–C8) (Fig. 2
A
–
L
). One group wascomposed of motor neurons of the medial component of theaxial muscle-innervating median motor column (MMCm) (20),based on their ventromedial location (Fig. 2
A
, vertical arrows)and the expression of ChAT, Lhx3 (Fig. 2
A
–
F
), and Isl1 (Fig. 2
J
–
L
). The second group consisted of motor neurons of the lateralmotor column (LMC), based on their ventrolateral location andexpression of retinaldehyde dehydrogenase 2 (RALDH2) (Fig.2
G
–
I
, horizontal arrows).
-gal expression was found in sub-populationsofbothmedialLMC(LMCm)motorneurons,whichexpress Isl1 but not Lim1, and project to ventral limb muscles,and lateral LMC (LMCl) motor neurons, which express Lim1and innervate dorsal limb muscles (17, 23) (Fig. 2
G
–
O
).Consistent with these results, a group of
-gal
neurons wasretrogradely labeled from the deltoideus muscle (Fig. S2
-gal
Runx1
is expressed in selectedpopulations of ventrally exiting MMC and LMC motor neurons.
Fig.1.
Runx1
expressioninspinalmotorneurons.(
A–H
)Expressionof
-galandtheindicatedproteinsinthespinalcordofE9.5(
A
–
C
and
G
)orE10.5(
D
–
F
and
H
)
Runx1
lacZ/
embryos.
-gal expression does not overlap with proliferation (
A–C
) or ventral interneuron (
D–F
) markers, but it overlaps with the motor neuronmarkers ChAT and Isl1 (
G
and
H
, arrows). Arrowheads in (
A–C
) point to
-gal
cells. (
I
) Summary of
Runx1
expression (green) in the cervical spinal cord (SC) atE9.5; the location of the progenitor domain, ventral interneuron 0, 1, and 2 domains, and vMN domain are indicated. (
J–O
) Expression of
-gal and Hb9 in thecervical spinal cord of
Runx1
lacZ/
embryos. No overlap is observed at E10.5 (
J
–
L
, arrowheads), whereas
-gal
/Hb9
cells are visible in a ventromedial domainatE11.5(
M
–
O
,arrows;shownathighermagnificationinthe
Inset
).(
P–R
)Coexpressionof
-galandPhox2binthecervicalspinalcordofE9.5
Runx1
lacZ/
embryos.Virtually all
-gal
cells coexpress Phox2b (arrows). (
S
) Analysis of axonal projections in E15.5
Runx1
lacZ/
embryos by using retrograde labeling by rhodamine-dextran (Rho) placed in the anterior portion of the trapezius muscle. Arrows point to examples of
-gal
cells that were retrogradely labeled. (
T
) Schematicdisplayingthelocationoftheventrolateraldomainofthespinalcordanalyzedin
S
.(
U
)Summaryof
Runx1
expressioninthecervicalSCatE11.5;thelateraldomaincontaining Runx1
cells that express Isl1 and Phox2b and project to the anterior trapezius muscle is shown in yellow, and the ventromedial domain containingRunx1
/Hb9
cells is shown in green; LEP, lateral exit point. When shown, dotted lines depict outline of the spinal cord. (
D–S
) Dorsal is to the
Top
and lateralto the
Right
.
6452
www.pnas.org
cgi
doi
10.1073
pnas.0711299105 Stifani
et al.
To characterize the function of Runx1 in motor neurondevelopment, we examined two separate lines of
Runx1
-deficientmice, termed
Runx1
lacZ/rd
and
Tie2
-Cre;
Runx1
Flox:lacZ/Flox:lacZ
,respectively (19, 24). Transheterozygous
Runx1
lacZ/rd
embryosare Runx1-null and die at
E12.5 because of a lack of fetalliver-derivedhematopoiesis(19).Thisembryoniclethalitycanbecircumvented by examining
Tie2
-Cre;
Runx1
Flox:lacZ/Flox:lacZ
em-bryos, in which the expression of Cre recombinase is under thecontrol of the endothelial/hematopoietic-specific
Tie2
promoter(25, 26), resulting in a selective reactivation of
Runx1
in hema-topoietic cells but not in the nervous system. Concomitantly, thisconditional reactivation abolishes
-gal
cells in the spinal cord of both
Runx1
lacZ/rd
and
Tie2
-Cre;
Runx1
Flox:lacZ/Flox:lacZ
embryos was the same as in theircontrol littermates at all stages examined (Fig. 3
A
–
C
). Thisresult shows that Runx1 is not essential for
-
gal
expression andmaintenance nor for the generation and/or survival of theselected spinal SAC, MMC, and LMC neurons in which it isnormally expressed.
Runx1 Is Important for Persistent Suppression of Interneuron Differ-entiation Programs and Sustained Expression of Motor Neuron Genes.
We traced the fate of motor neurons that normally expressRunx1 by following
-gal expression in
Runx1
mutant embryos.The numbers of
-gal
cells that coexpressed general vMNmarkers such as Isl1 and ChAT were decreased in mutantembryoscomparedwithcontrollittermates(Fig.3
D
and
F
).Theexpression of specific markers of MMC (i.e., Lhx3) or LMC (i.e.,RALDH2)motorneuronswasalsodecreasedin
Runx1
-deficientspinal neurons (Fig. 3
D
and
F
). In
Runx1
mutant embryos of bothgenotypes,wealsonotedthepresenceofincreasednumbersof cells coexpressing
-gal and the exclusive spinal interneuronmarkers Pax2 and Chx10 (3, 27) (Fig. 3
E
and
G
). Moreover, wefound that Pax2, which is not normally expressed in Isl1
spinalmotor neurons (ref. 27; see also Fig. 3
H
–
J
), was coexpressed withIsl1and
-galin
Runx1
mutantmotorneuronsatE11.5(Fig.3
K
–
M
)where
Runx1
-expressing cells correspond to SAC motor neurons (Fig. 1
P
–
R
Runx1
mutant SAC motor neurons at E11.5 (Fig. S4
), suggesting that at least certain Phox2b
SAC neuronsmight coexpress Pax2 in
Runx1
-deficient embryos at this stage.Together, these results strongly suggest that Runx1 inactivationresults in a derepression of interneuron-specific genes in post-mitotic motor neurons.We next tested whether Runx1 was sufficient to cause asuppression of interneuron-specific genes. To this end, weelectroporated a GFP expression plasmid alone or together with a Runx1 expression plasmid into Hamburger and Ham-ilton (HH) (28) stage 14–16 chicken embryo neural tubes. After incubation, the majority of GFP
cells coexpressedRunx1 (Fig. S5). We determined the proportion of GFP
cellsexpressing motor neuron and interneuron markers at HH stage27–28 (Fig. 4
A
–
E
). Compared with control embryos express-ing only GFP, Runx1 caused a significant decrease in theexpression of the interneuron-specific proteins Pax2 andChx10, but it did not affect the expression of Evx1, a V0interneuron marker (Fig. 4
F
). In addition, ectopic Runx1expression led to an increase in the expression of generalmotor neuron markers such as Hb9 and Isl1, as well as themotor- and interneuron markers Lim3 and Lim1/2 (Fig. 4
G
).These effects were observed at both brachial level, where
Runx1
is expressed, and lumbar level, where
Runx1
is notexpressed (17), but occurred only in the ventral, and not dorsal,half of the spinal cord. Importantly, these effects were phe-nocopied by the oncogenic human fusion protein AML1/ETO(hereafter referred to as Runx1/ETO for consistency) (Fig.S6). Runx1/ETO harbors the DNA-binding domain of Runx1fused to the protein eight-twenty one, a potent transcriptional
Fig. 2.
Runx1
expression in MMC and LMC motor neuron subtypes. Expres-sion of
-gal and the indicated marker proteins in the ventral spinal cord ofE13.5
Runx1
lacZ/
embryos at spinal levels C5 (
A–C
), C6–C7 (
D–L
), or C7–C8(
M–O
). In all pictures, dorsal is to the
Top
and lateral to the
Right
.
-galexpression overlaps with ChAT, Lhx3, and Isl1 in MMCm motor neuronslocatedinaventromedial(medial;verticalarrows)domain.These
-gal
cellsdecreaseinnumberinarostral-to-caudaldirection(
cf
.,
A
and
J
).
-galexpres-sion is also observed in ventrolateral (lateral; horizontal arrows) LMC motorneuronsthatexpressChATandRALDH2.MostofthesecellsexpressIsl1atlevelC5-C6 (
J–L
), whereas an overlap with Lim1/2 expression is observed at levelC7–C8(
M–O
).(
O
)DottedlineoutlinesthelateraldomainofLim1/2expressioncontaining
-gal
/Lim1/2
cells; many Lim1/2
cells found at more medialpositions, likely corresponding to V0/V1 interneurons, do not express
-gal;DRG, dorsal root ganglion. In all panels, arrows point to examples of double-labeled cells. Dotted lines, except in
O
, show outline of the spinal cord. (
P
)Summary of
Runx1
expression at levels C5–C8 of the spinal cord (SC) of E13.5
Runx1
lacZ/
embryos;
Runx1
cells mark a medial subdomain of the MMCm(light blue) and specific subdomains of both LMCm and LMCl (yellow).
Stifani
et al.
PNAS
April 29, 2008
vol. 105
no. 17
6453
N E U R O S C I E N C E
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