© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
RESEARCH ARTICLE
Ascl1 controls the number and distribution of astrocytes and
oligodendrocytes in the gray matter and white matter of the
spinal cord
ABSTRACT
Glia constitute the majority of cells in the mammalian central nervous
system and are crucial for neurological function. However, there is an
incomplete understanding of the molecular control of glial cell
development. We find that the transcription factor Ascl1 (Mash1),
which is best known for its role in neurogenesis, also functions in both
astrocyte and oligodendrocyte lineages arising in the mouse spinal
cord at late embryonic stages. Clonal fate mapping in vivo reveals
heterogeneity in Ascl1-expressing glial progenitors and shows that
Ascl1 defines cells that are restricted to either gray matter (GM) or
white matter (WM) as astrocytes or oligodendrocytes. Conditional
deletion of Ascl1 post-neurogenesis shows that Ascl1 is required
during oligodendrogenesis for generating the correct numbers of WM
but not GM oligodendrocyte precursor cells, whereas during
astrocytogenesis Ascl1 functions in balancing the number of dorsal
GM protoplasmic astrocytes with dorsal WM fibrous astrocytes. Thus,
in addition to its function in neurogenesis, Ascl1 marks glial
progenitors and controls the number and distribution of astrocytes
and oligodendrocytes in the GM and WM of the spinal cord.
KEY WORDS: Astrocyte heterogeneity, Glial specification,
Oligodendrogenesis, Spinal cord gliogenesis
INTRODUCTION
Glia, which include astrocytes and oligodendrocytes, are the most
abundant cell types in the mammalian central nervous system (CNS)
and are crucial for regulating and maintaining neuronal architecture
and function. During development, glial progenitor cells undergo an
extended precursor cell stage, referred to as intermediate astrocyte
precursors (IAPs) or oligodendrocyte precursor cells (OPCs), which
can migrate and continue to proliferate in the surrounding gray matter
(GM) and white matter (WM) prior to terminal differentiation into
mature astrocytes or oligodendrocytes, respectively (Nishiyama
et al., 2009; Ge et al., 2012; Tien et al., 2012). Astrocytes in the GM,
known as protoplasmic astrocytes, are morphologically distinct from
those in the WM, described as fibrous astrocytes (Rowitch, 2004;
Oberheim et al., 2012). Similarly, oligodendrocytes in the GM can
also be distinguished from those in the WM on the basis of their level
of myelin marker expression (Rowitch and Kriegstein, 2010). These
differences suggest that astrocytes and oligodendrocytes in the GM
1
Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390,
2
USA. Division of Molecular Neurobiology, MRC National Institute for Medical
Research, London NW7 1AA, UK.
*Present address: Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
‡
Present address: Institut du Cerveau et de la Moelle é pinière, Inserm U1127,
Université Pierre et Marie Curie-Paris 6, Paris, France.
§
Author for correspondence ( [email protected])
Received 23 October 2013; Accepted 1 August 2014
are developmentally and functionally distinct from their respective
counterparts in the WM. However, how this heterogeneity develops
is not known.
The developing vertebrate spinal cord is segmentally organized
along the dorsal-ventral (DV) axis into distinct progenitor domains
(Jessell, 2000). The pattern of transcription factor expression in the
progenitor domains and the unique spatial organization of the spinal
cord into GM and WM make it an ideal setting to elucidate the
mechanisms of glial heterogeneity (Rowitch and Kriegstein, 2010).
The ventral spinal cord is the site of the earliest waves of IAPs
and OPCs, where, following the completion of neurogenesis,
oligodendrocytes are generated from the pMN domain (Zhou et al.,
2000, 2001; Lu et al., 2002; Zhou and Anderson, 2002), whereas the
surrounding ventral p1-p3 domains give rise to three distinct ventral
WM astrocyte populations (Pringle et al., 2003; Muroyama et al.,
2005; Hochstim et al., 2008). A second wave of gliogenesis arises
from neural progenitors in the intermediate and dorsal domains that
generates oligodendrocytes and astrocytes that reside at similar DV
positions in both the GM and WM (Fogarty et al., 2005; Zhu et al.,
2011; Tsai et al., 2012). Currently, it is unclear which molecular
mechanisms are utilized for specifying IAPs and OPCs in the dorsal
spinal cord, or if IAPs and OPCs from this region are derived from
the same or separate populations of progenitor cells.
One transcription factor that is highly expressed in neural
progenitor cells in the developing dorsal spinal cord is the basic
helix-loop-helix (bHLH) factor Ascl1. Ascl1 mutant mice have been
shown to exhibit significant alterations in differentiation and
specification of both neurons and OPCs (Helms et al., 2005;
Mizuguchi et al., 2006; Wildner et al., 2006; Sugimori et al., 2007,
2008). However, because Ascl1 functions in neurogenesis prior to
gliogenesis, it is unclear whether the disruption in oligodendrocyte
specification and differentiation is a direct effect or secondary to the
fundamental changes to the overall progenitor and/or neuronal
pools. Furthermore, Ascl1 mutant mice die neonatally, thereby
precluding any analysis of the postnatal spinal cord. Thus, the extent
of Ascl1 function in glial cell development remains largely
undefined, particularly in dorsal spinal cord domains where Ascl1
is expressed in progenitor populations that have been shown to also
give rise to astrocytes (Zhu et al., 2011; Tsai et al., 2012).
In this study, we identify the fate of Ascl1-expressing glial
progenitors in the mouse spinal cord and determine if Ascl1 is
required for the development of glial lineages. We confirm
that Ascl1-expressing glial progenitors directly give rise to
oligodendrocytes, and also to dorsally restricted spinal cord
astrocytes. Clonal analysis in vivo reveals that a subset of Ascl1expressing progenitors in the ventricular zone (VZ) is already
lineage restricted toward only the GM or WM, but not to both,
either as astrocytes or oligodendrocytes. We further show that, in
the absence of Ascl1, Ascl1 lineage cells give rise to an increased
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DEVELOPMENT
Tou Yia Vue1, Euiseok J. Kim1, *, Carlos M. Parras2,‡, Francois Guillemot2 and Jane E. Johnson1,§
RESEARCH ARTICLE
number of GM protoplasmic astrocytes and a decreased number of
WM OPCs/oligodendrocytes and WM fibrous astrocytes. Thus,
Ascl1 marks a heterogeneous population of glial progenitor cells
and is crucial for generating a normal distribution of both
astrocytes and oligodendrocytes in the GM and WM of the
spinal cord.
RESULTS
Ascl1 is expressed in progenitor and precursor cells during
gliogenesis in the spinal cord
It was previously reported that Ascl1 is expressed during
oligodendrogenesis in the spinal cord of rat and mouse (Battiste
et al., 2007; Sugimori et al., 2007, 2008). To assess Ascl1
expression during gliogenesis in the spinal cord in more detail,
double immunofluorescence for Ascl1 along with glial lineage
markers was performed from embryonic day (E) 14.5 to E18.5.
At E14.5, Ascl1 is broadly expressed throughout the VZ as well as
in some scattered cells in the GM and WM (Fig. 1B). As
development proceeds, the number of Ascl1-expressing cells
increases in the GM and WM but is diminished in the VZ by E17.5
(supplementary material Fig. S1A,F,K). All Ascl1+ cells in
the VZ, GM and WM are positive for Sox2 (Fig. 1B-C‴;
supplementary material Fig. S1A-D,F-I,K-N), which at these
stages is expressed in glial progenitor and precursor cells
(Hoffmann et al., 2014). A few of the Ascl1+ cells in or near the
VZ are positive for Olig2 or the OPC-specific marker Pdgfrα,
especially in the dorsal spinal cord (Fig. 1D,D′,E,E′), whereas
all Ascl1+ cells in the GM and WM are Olig2+, and most of these
are also Pdgfrα+ (Fig. 1D″,D‴,E″,E‴; supplementary material
Fig. S1E,J). By contrast, all of the Ascl1+ cells in the VZ and most
Ascl1+ cells in the GM and WM from E14.5 to E18.5 are positive
for Nfia (Fig. 1F′-F‴), a transcription factor that is required for the
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
specification and differentiation of IAPs (Deneen et al., 2006; Kang
et al., 2012). However, none of the Nfia+ Ascl1+ cells in the GM and
WM expressed the astrocyte lineage marker ApoE (supplementary
material Fig. S1O) (Bachoo et al., 2004), indicating that Ascl1 is
downregulated in the astrocyte lineage once glial progenitors
transition out of the VZ into IAPs (Fig. 1G,H).
Although at these stages Olig2 and Nfia are dynamically
expressed and are not completely restricted to the oligodendrocyte
or astrocyte lineages, respectively (Cai et al., 2007; Deneen et al.,
2006), these findings indicate that Ascl1 is expressed in glial
progenitor and precursor cells to both the oligodendrocyte and
astrocytes lineages in the spinal cord.
Ascl1 lineage cells include astrocytes and oligodendrocytes
that are found in both the GM and WM of the spinal cord
Previous lineage analysis using bacterial artificial chromosome
(BAC) transgenic mice showed that Ascl1-expressing cells are
restricted to neuronal and oligodendroglial lineages in the
telencephalon and spinal cord, and do not become astrocytes
(Battiste et al., 2007; Parras et al., 2007). By contrast, recent fate
mapping using Ascl1CreERT2 knock-in (KI) mice showed that both
astrocytes and oligodendrocytes are labeled during gliogenesis in
the cerebellum (Sudarov et al., 2011), indicating that the
Ascl1CreERT2 KI allele may be able to label more progenitor types
or an earlier, multipotent progenitor population that was not
detected in the Ascl1 BAC transgenic lines.
To determine whether Ascl1-expressing glial progenitors in the
spinal cord are capable of giving rise to both oligodendrocytes and
astrocytes, Ascl1CreERT2/+ KI mice were crossed with the Cre
recombinase-dependent reporter R26RLSL-tdTOM. Cre recombinase
activity was induced at E14.5 by administering tamoxifen
(2.5 mg/40 g body weight) to gravid females to permanently
DEVELOPMENT
Fig. 1 . Ascl1 is transiently expressed in glial progenitor
and precursor cells in the developing mouse spinal
cord. Immunofluorescence for Ascl1 (green) and glial
lineage markers (red) on thoracic spinal cord sections at
E14.5. (A) Schematic of imaged areas. (B-F‴) Ascl1 (green)
colocalizes (arrows) with Sox2 (B-C‴), Olig2 (D-D‴), Pdgfrα
(E-E‴) and Nfia (F-F‴). Dotted lines indicate the lateral
boundary of the ventricular zone, as determined by the
density of Sox2 expression. (G,H) Schematic of Ascl1
expression in oligodendrocyte and astrocyte lineages in the
post-E14.5 spinal cord relative to other markers used in this
study. GP, glial progenitor; OPC, oligodendrocyte precursor
cell; IAP, intermediate astrocyte precursor; VZ, ventricular
zone (d, dorsal; v, ventral); GM, gray matter; WM, white
matter. Scale bars: 100 μm for B-B″; 25 μm for C-F‴.
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label Ascl1-expressing cells. This stage was specifically chosen
because neurogenesis is largely complete and Ascl1+ glial
progenitors are present in high numbers, especially in the dorsal
spinal cord VZ (Fig. 1). Analysis at E18.5 and postnatal day (P) 14
showed that tdTOM+ cells were widely distributed in both GM
and WM and, as expected, very few if any neurons (NeuN+;
Rbfox3 – Mouse Genome Informatics) were labeled (Fig. 2A,D,
E). Indeed, unlike Ascl1-CreERTM BAC transgenic mice (data not
shown; Battiste et al., 2007), the tdTOM+ cells co-express either
oligodendrocyte [Olig2, Sox10, CC1 (Apc)] (Fig. 2C,E-H) or
astrocyte (Nfia, Gfap) markers (Fig. 2I-L). Quantification at the
level of the thoracic region showed that 112±3.5 tdTOM+ cells
were labeled per spinal cord section at E18.5, and this increased
∼2-fold to 269±17.4 tdTOM+ cells per section by P14 (black line,
Fig. 2M). Notably, Ascl1 lineage OPCs (Olig2+;tdTOM+ and
Sox10+;tdTOM+), despite constituting only ∼20% of the labeled
tdTOM+ cells at E18.5, increased to make up the majority (66.5%)
of the labeled cells by P14 and were found mostly in the WM
(Fig. 2M-O). By contrast, the Ascl1 lineage astrocytes (Nfia+;
tdTOM+) increased to a lesser extent between E18.5 and P14 (red
line, Fig. 2M), were restricted predominantly to the dorsal half of
the spinal cord and were evenly distributed into the GM (22%) and
WM (24.5%) (Fig. 2I-N,P). This dorsal restriction of tdTOM+
astrocytes suggests that they are derived from the dorsal Ascl1+
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
glial progenitors, since astrocytes in the spinal cord are not known
to undergo extensive tangential migration (Zhu et al., 2011; Tsai
et al., 2012).
There was a less than 15% overlap in the tdTOM+ cells marked by
Olig2 or Nfia at the various stages analyzed, which is consistent
with the known overlap of these markers early in the glial lineages.
However, taken together with the later oligodendrocyte- and
astrocyte-specific markers, this long-term fate-mapping analysis
into the postnatal spinal cord illustrates that Ascl1 is expressed in
glial progenitors that are fated to both the oligodendrocyte and
astrocyte lineages.
Individual Ascl1+ glial cells are restricted in distribution to
the GM or WM
To determine if individual Ascl1+ progenitor cells in the spinal cord
could be shown to be bipotential in giving rise to both astrocytes and
oligodendrocytes, a low dose of tamoxifen (0.025 mg/40 g body
weight) was administered to Ascl1CreERT2/+;R26LSL-tdTOM mice at
E14.5 to sparsely label single Ascl1-expressing cells. To verify the
sparse labeling of cells, 50 consecutive sections (totaling 1500 μm
thickness) through the thoracic (N=2) or lumbar (N=2) regions were
examined 12 h post tamoxifen administration at E15.0. tdTOM+
cells were found only in the dorsal spinal cord primarily in or
immediately adjacent to the VZ (64%), and in the GM (36%), but
Fig. 2. Ascl1-expressing glial cells give rise to both astrocytes and oligodendrocytes in the GM and WM of the spinal cord. Immunofluorescence on E18.5
(A-C) or P14 (D-L) thoracic spinal cord (TH-SC) sections of Ascl1CreERT2/+;R26LSL-tdTOM mice treated with tamoxifen at E14.5. Red is tdTOM fluorescence marking
Ascl1 lineage cells. (A-H) tdTOM does not colocalize with neuronal marker NeuN (A,D), which was used here to delineate the GM, but colocalizes with the
oligodendrocyte (OL) markers Olig2, Sox10 and CC1 (B,C,E-H, arrowheads) in the GM and WM. Note that Olig2 and Sox10 are not expressed in tdTOM+
astrocytes (B,E,F, arrows). (I-L) tdTOM also colocalizes with the astrocyte (AS) markers Nfia and Gfap in the GM and WM in the dorsal spinal cord (arrows).
Ventral tdTOM+ cells do not express Nfia and Gfap (arrowheads, L). (M-P) Quantification and schematic summary of the number (mean±s.d.) and distribution of
tdTOM-labeled astrocyte (Nfia+;tdTOM+) and oligodendrocyte (Olig2+;tdTOM+ and Sox10+;tdTOM+) lineage cells per thoracic spinal cord section. Percentage (N)
refers to the percentage of total tdTOM+ cells for each cell type in GM or WM at P14. Dotted lines (I,P) delineate the dorsal-ventral boundary at the level of the
central canal (CC). Number of spinal cords analyzed: N=4 at E18.5 and N=3 at P14. Scale bar: 100 μm for A,D,I; 25 μm for B,C,E-H,J-L.
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not the WM (supplementary material Fig. S2A-G,N). To define the
tdTOM+ cells as clones, a radius of 150 μm (∼20 cell diameters)
was measured for each tdTOM+ cell along the anterior-posterior
(AP) axis, and all labeled cells within this radius were considered a
clone. Out of 16 putative clones identified, 11 consisted of a single
cell (supplementary material Fig. S2A,C). Marker analysis revealed
that these tdTOM+ cells were not positive for Olig2 (supplementary
material Fig. S2D-G) or the OPC marker Pdgfrα (not shown), and
thus were likely to be astrocyte lineage cells. This was distinctly
different from the tdTOM+ cells initially labeled at the higher doses
of tamoxifen (supplementary material Fig. S2H-N), which, as
demonstrated below, contained both astrocyte and oligodendrocyte
lineages.
Screening serial sections through spinal cords at E18.5 showed
that, unlike at E15.0, the sparsely labeled tdTOM+ cells were absent
from the VZ and were found only in the GM or WM. The tdTOM+
cells still consisted of astrocyte (Nfia+, Gfap+) and not
oligodendrocyte (Olig2−) lineage cells (Fig. 3A,E), and were
evenly distributed to the GM and WM in the dorsal spinal cord.
This distribution pattern was similar to that seen at the higher
tamoxifen doses at this stage (GM-AS and WM-AS bars, Fig. 3E),
and suggests that within the astrocyte lineage, both low and high
tamoxifen doses are labeling a similar set of cells. Because IAPs are
known to undergo local proliferation (Ge et al., 2012), the sparsely
labeled tdTOM+ IAPs were defined as clonal if they were found in
the same or neighboring sections within 150 μm of each other, but at
least 300 μm away from other labeled cells along the AP axis.
Strikingly, all tdTOM+ IAPs in a defined clone were found in close
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
proximity to each other within the same or adjacent sections, as
clusters of 2-4 cells in the GM or WM (Fig. 3A,C,D). This spatial
restriction of Ascl1 lineage tdTOM+ astrocytes in the dorsal spinal
cord to just the GM or WM was also observed at P30, although the
number of cells per clone at this stage had doubled (Fig. 3B-D).
These findings suggest that Ascl1+ progenitors in the dorsal VZ, as
labeled at the low tamoxifen dose, are not bipotential but instead are
restricted in fate to the GM or WM as astrocytes.
At the very low dose of tamoxifen administered, no OPCs or
oligodendrocytes were labeled. To increase the probability of
capturing Ascl1+ clones of the oligodendrocyte lineage at E14.5, an
intermediate dose of tamoxifen (0.25 mg/40 g body weight) was
used. At 24 h post tamoxifen administration, a few tdTOM+ cells
were observed per spinal cord section (supplementary material Fig.
S2H), all of which were Nfia+ (supplementary material Fig. S2J).
Occasionally, some of these tdTOM+ cells were also Olig2+
(supplementary material Fig. S2I). The distribution of the tdTOM+
cells in the VZ, GM and WM at the intermediate dose is similar to
that of the high (2.5 mg/40 g body weight) tamoxifen dose
(supplementary material Fig. S2K-N). By E18.5, with the
intermediate tamoxifen dose clusters of 2-4 tdTOM+ OPCs
(Olig2+;tdTOM+) were sparsely labeled in the dorsal WM or GM
(Fig. 3F), along with consistent labeling of astrocytes. These
Olig2+;tdTOM+ clusters account for ∼11% of the total tdTOM+
cells, compared with 24% at the high tamoxifen dose at this time
point (Fig. 3E). Unfortunately, due to the density of cells labeled at
the intermediate dose, it cannot be determined whether the labeled
OPC clusters (Fig. 3F) and surrounding astrocytes (Fig. 3F) arise
Fig. 3. The development of Ascl1 lineage marked clones
is spatially restricted to the GM or WM in the spinal cord.
Immunofluorescence on spinal cord sections of
Ascl1CreERT2/+;R26LSL-tdTOM mice treated with low
(0.025 mg/40 g body weight, A,B) or intermediate (0.25 mg/
40 g body weight, F,G) doses of tamoxifen at E14.5.
(A) Sixty consecutive sections of E18.5 spinal cord
contained only astrocyte labeled clones, separated by
780 μm in this example, and were restricted to WM (sections
20, 21) or GM (section 47). (B) Eighty consecutive sections
of P30 spinal cord contained the progeny of two astrocyte
labeled clones, separated by 1530 μm in this example, and
were restricted to WM (sections 16-22) or GM (sections
73-76). (C,D) Number (mean±s.d.) of cells per GM astrocyte
(red triangle) or WM astrocyte ( purple triangle) clone at
E18.5 and P30 from the lowest tamoxifen doses as in A,B.
(E) Tamoxifen dose curve of E18.5 spinal cord showing the
percentage (mean±s.e.m.) of tdTOM+ AS (Olig2− or Nfia+)
and Olig2+;tdTOM+ cells labeled. (F) Twenty-five
consecutive sections of E18.5 spinal cord showing the
presence of two Ascl1 lineage OPC clusters, separated by
360 μm in this example, that were restricted to WM (sections
4, 6) or GM (section 18). An Olig2−;tdTOM+ cell (astrocyte)
is also present (arrow) but the clonal relationship to the
Olig2+;tdTOM+ cells cannot be determined. (G) P30 thoracic
spinal cord from caudal or rostral thoracic sections
showing spatial restriction of Ascl1 lineage oligodendrocyte
clusters to either WM or GM, but not both. Arrows
indicate IAPs/astrocytes and arrowheads indicate OPCs/
oligodendrocytes. Scale bars: 25 μm for A,B,F,G; 12.5 μm
for insets in A,F.
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from a common or separate Ascl1-expressing cell(s). By P30, the
size of tdTOM+ OPCs/oligodendrocyte clusters had dramatically
increased from 2-4 to 10-50 cells, as assessed by Sox10 expression
(Sox10+;tdTOM+), to occupy large portions (0.5-1 mm) of the
spinal cord along the AP axis. Despite this expansion, however, all
the Sox10+;tdTOM+ cells within a cluster were found together and
restricted only to the dorsal WM or GM (Fig. 3G). This spatial
restriction of tdTOM+ oligodendrocyte lineage clusters at both
E18.5 and P30 to only the WM or GM implies that the labeled
Ascl1+ cells in the oligodendrocyte lineage, similar to what was
seen for the astrocyte lineage, are already restricted to localize in the
GM or WM. These findings do not however exclude the possible
existence of Ascl1-expressing bipotential or multipotential cells that
might not be marked with the low doses of tamoxifen required for
these experiments.
The number and distribution of glial cells in the dorsal spinal
cord are altered in the absence of Ascl1
It was previously shown that Ascl1 mutant mice exhibit defects in
OPC specification and differentiation in the spinal cord (Sugimori
et al., 2007, 2008). However, it was unclear whether these defects
were a direct effect or the result of fundamental changes to the
overall progenitor and/or neuronal pools resulting from the loss of
earlier Ascl1 roles in neurogenesis (Helms et al., 2005; Mizuguchi
et al., 2006; Wildner et al., 2006). Furthermore, because it was
not known that Ascl1-expressing glial progenitors also give rise
to dorsal astrocytes (Figs 2 and 3), the effects on astrocyte
development were not assessed. Finally, Ascl1 null mutants die
shortly after birth, so the consequences for glial cell development
and distribution in the GM and WM, which occur postnatally, could
not be addressed.
To circumvent these limitations, Ascl1 was conditionally
knocked out (Ascl1 CKO) only in Ascl1-expressing cells postneurogenesis at E14.5 in the spinal cord. This was accomplished by
crossing Ascl1CreERT2/+ KI mice with R26RLSL-tdTOM Cre reporter
mice that also carry an Ascl1FL allele, then Cre recombinase was
induced with tamoxifen (2.5 mg/40 mg body weight) at E14.5.
Although the initial function of Ascl1 in glial progenitor cells
cannot be assessed, a unique advantage with this paradigm is that
only those cells that express Ascl1 at the time of tamoxifen
administration will be labeled and affected, while others in the
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
surrounding neural landscape will be spared. The efficient deletion
of Ascl1 was determined at E15.5, 24 h after tamoxifen
administration in control (Ascl1CreERT2/+;R26LSL-tdTOM) or Ascl1
CKO (Ascl1CreERT2/FL;R26LSL-tdTOM) spinal cord. In control,
Ascl1 lineage tdTOM+ cells in the VZ and some in the GM
continued to express Ascl1 (supplementary material Fig. S3B).
However, Ascl1 expression was no longer detected in the
majority of tdTOM+ cells in the Ascl1 CKO (supplementary
material Fig. S3B′). Extending this analysis to E18.5, Ascl1 was
also not expressed in Olig2+;tdTOM+ cells of the Ascl1 CKO
(supplementary material Fig. S3F′-H′), whereas it was in controls
(supplementary material Fig. S3F-H).
To evaluate whether the post-neurogenesis conditional knockout of
Ascl1 had a disruption in spinal cord gliogenesis, the total number and
distribution of tdTOM+ cells in the Ascl1 CKO were compared with
those in control and Ascl1 KO mice (Ascl1CreERT2/null;R26LSL-tdTOM
treated with tamoxifen at E14.5). Analysis at E18.5 showed that over
90% of the tdTom+ cells had migrated out of the VZ in all three
conditions; therefore, Ascl1 lineage cells are not maintained as radial
glial even in the absence of Ascl1 (Fig. 3E). Additionally, the tdTOM+
cells were largely non-neuronal and were mostly restricted to the
dorsal spinal cord. Notably, the Ascl1 CKO and Ascl1 KO had a ∼40%
increase in the number of tdTOM+ cells (Fig. 4A-D). This could be
due in part to the loss of negative feedback on Ascl1 expression, which
is known to occur in this locus (Horton et al., 1999), a confound that is
difficult to eliminate. However, this increase in labeled cells could
also result from additional rounds of division, an interpretation
supported by clonal experiments (see Fig. 8 and related text below).
Another notable phenotype was a shift in the distribution of the
tdTOM+ cells from WM to GM (Fig. 4E-G), with the absolute number
and ratio of Ascl1 lineage oligodendrocytes (Olig2+;tdTOM+ and
Sox10+;tdTOM+) and astrocytes (Nfia+;tdTOM+) in the GM and
WM permanently altered in the Ascl1 CKO into postnatal stages
(supplementary material Fig. S4). These findings implicate a
functional role for Ascl1 during gliogenesis in the spinal cord.
Ascl1 is required to generate the correct number of WM OPCs
in the spinal cord
To understand the role of Ascl1 during oligodendrogenesis in the
spinal cord, the developmental dynamics of Ascl1 lineage tdTOM+
OPCs in the GM and WM were analyzed in the thoracic region over
Fig. 4. The number and distribution of Ascl1 lineage glial
cells into the GM and WM are altered in Ascl1 CKO and
Ascl1 KO spinal cords. Immunofluorescence on thoracic
sections of E18.5 Ascl1CreERT2/+;R26LSL-tdTOM control,
Ascl1CreERT2/FL;R26LSL-tdTOM (Ascl1 CKO) or Ascl1CreERT2/null;
R26LSL-tdTOM (Ascl1 KO) mice treated with tamoxifen at E14.5.
(A-E) NeuN delineates the boundaries between VZ, GM and
WM. The number and distribution of tdTOM+ cells are
increased in the GM of Ascl1 CKO and Ascl1 KO at E18.5.
(F,G) Number of tdTOM+ cells is altered in the GM and WM of
Ascl1 CKO into postnatal stages. Number of spinal cords
analyzed: E18.5, N=4 control, N=3 Ascl1 CKO and Ascl1 KO;
P14, N=3 control and Ascl1 CKO; P30, N=2 control and Ascl1
CKO. *P<0.005 indicates statistical significance using linear
regression analysis. Values in graphs are mean±s.e.m. Scale
bar: 100 μm.
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Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
time in Ascl1CreERT2/+;R26LSL-tdTOM control mice following
tamoxifen (2.5 mg/40 mg body weight) at E14.5. At E18.5, the
number of tdTOM+ cells co-expressing Olig2 (Olig2+;tdTOM+),
which should mark oligodendrocyte lineage cells at this stage, was
distributed evenly into the GM (11.3±3.6) and WM (14.5±3.5)
(Fig. 5A,I,J,K). Fewer tdTOM+ cells at this stage co-expressed
Sox10 (Sox10+;tdTOM+) (Fig. 5O-Q), most likely because Sox10 is
a more mature marker, and Olig2 is also expressed in a small subset
of immature astrocytes (Fig. 6B,C) (Cai et al., 2007).
By P14, the number of Olig2+;tdTOM+ cells per section was
increased ∼4-fold in the GM (47.8±3.8) and 10-fold in the WM
(139.4±8.2) (Fig. 5I,J). A similar number of Sox10+;tdTOM+ cells
was also observed in the GM (36.2±4.3) and WM (134.9±11.5)
(Fig. 5C-G,O,P), consistent with Olig2 and Sox10 both marking
similar sets of cells at this stage. Some Olig2+;tdTOM+ or Sox10+;
tdTOM+ cells in the GM and most in the WM at this stage are also
CC1+ (Fig. 2G,H), illustrating that they have matured into
oligodendrocytes. Interestingly, analysis at P30 revealed that the
number of Sox10+;tdTOM+ cells had increased by 80% in the GM
(65.2±7.8) but decreased by 37% in the WM (84.8±10.9)
compared with that at P14 (Fig. 5O,P). The reason for this
decrease in the WM at P30 is unclear, but it could reflect a faster
rate of WM OPC maturation to oligodendrocyte and/or a turnover
of WM oligodendrocytes within this time frame. Overall, these
findings indicate that the proliferation and differentiation of Ascl1
lineage OPCs in the GM and WM of control spinal cords are highly
dynamic.
3726
The development of the oligodendrocyte lineage in the absence
of Ascl1 was then analyzed. At E18.5, the effect on the GM
oligodendrocyte lineage is complex. In particular, both Olig2+;
tdTOM+ and Sox10+;tdTOM+ cells in the GM were doubled in
number in the Ascl1 CKO compared with control (Fig. 5I,O).
Unexpectedly, the morphology of most of the Olig2+;tdTOM+
cells in the GM of the Ascl1 CKO resembled that of astrocytes
(Fig. 5B′) and were negative for the OPC marker Pdgfrα
(supplementary material Fig. S3C′-E′). This increase in Olig2+;
tdTOM+ astrocyte-like cells was also noted in the GM of Ascl1
KO spinal cords at E18.5 (supplementary material Fig. S3F″-H″).
Marker analysis revealed that, indeed, most of the Olig2+;
tdTOM+ cells in the GM of the Ascl1 CKO co-express astrocyte
markers such as AldoC and ApoE (Fig. 6A-C,A′-C′) (Walther
et al., 1998; Bachoo et al., 2004). However, whether this
phenotype is the result of a transfating of Olig2+ OPCs to GM
astrocytes, or a delay in the downregulation of Olig2 expression
in the astrocyte lineage, could not be determined. But by P14,
these Olig2+;tdTOM+ astrocytes (AldoC+ ApoE+) were no longer
as apparent in the Ascl1 CKO (Fig. 6D,E versus 6D′,E′).
Accordingly, the numbers of Olig2+;tdTOM+ and of Sox10+;
tdTOM+ cells in the GM of the Ascl1 CKO were not significantly
different to the control (Fig. 5I,O). Interestingly, by P30 the
number of Sox10+;tdTOM+ cells in the GM of the Ascl1 CKO had
decreased to 80% of controls (Fig. 5O), suggesting that Ascl1
might be required for continued proliferation or survival of the
GM oligodendrocyte lineage.
DEVELOPMENT
Fig. 5. Number of OPCs and oligodendrocytes is
decreased in WM of Ascl1 CKO spinal cords.
Immunofluorescence on thoracic sections of
Ascl1CreERT2/+;R26LSL-tdTOM control or Ascl1CreERT2/FL;
R26LSL-tdTOM CKO mice treated with tamoxifen at
E14.5. (A-G) Control spinal cords showing very few
Olig2+;tdTOM+ cells at E18.5 (arrowheads, A,B), but
many Olig2+;tdTOM+ (arrowheads, C) and Sox10+;
tdTOM+ (arrowheads, F,G) cells in the WM at P14.
(A′-G′) Ascl1 CKO spinal cords showing Olig2+;
tdTOM+ cells in GM at E18.5 (yellow arrows, A′,B′), and
Olig2+;tdTOM+ (arrowheads, C′,D′) and Sox10+;
tdTOM+ cells (arrowheads, F′,G′) in the WM.
A dramatic decrease in Sox10+;tdTOM+ cells is
detected especially in the dorsal funiculus at P14
(compare yellow cells in F′ and F). (H,H′) Schematic
summary of the oligodendrocyte phenotype of control
and Ascl1 CKO at P14/P30. (I-S) The number and
distribution of Olig2+;tdTOM+ (I-M) and Sox10+;
tdTOM+ (N-S) cells per thoracic section in the GM, WM
and dorsal funiculus of control and Ascl1 CKO. Number
of spinal cords analyzed: E18.5, N=4 control, N=3
Ascl1 CKO; P14, N=3 control and Ascl1 CKO; P30,
N=2 control and Ascl1 CKO. #P<0.05 and *P<0.005
indicate statistical significance using linear regression
analysis. Values in graphs are mean±s.e.m. Scale
bars: 100 μm for A,A′,C-D′; 25 μm for B,B′,E-G′.
RESEARCH ARTICLE
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
Fig. 6. Number of Ascl1 lineage Olig2+ astrocytes is
increased in the GM of Ascl1 CKO spinal cords at E18.5.
Immunofluorescence on thoracic sections of Ascl1CreERT2/+;
R26LSL-tdTOM control or Ascl1CreERT2/FL;R26LSL-tdTOM CKO
mice treated with tamoxifen at E14.5. (A-C) Control spinal
cord showing the Ascl1 lineage from E14.5 includes a few
Olig2+;tdTOM+ cells in the GM that also co-express the
astrocyte markers AldoC and ApoE (arrows, B,C) at E18.5.
(A′-C′) Number of Olig2+;tdTOM+ astrocytes (AldoC+
ApoE+) is dramatically increased in the GM of Ascl1 CKO
spinal cord at E18.5 (arrows, A′-C′). (D-E′) Olig2+;ApoE+;
tdTOM+ astrocytes are no longer as apparent by P14 in the
Ascl1 CKO compared with controls. Scale bar: 100 μm for A,
A′,D,D′; 25 μm for B-C′,E,E′.
Ascl1 is required to balance the number of GM and WM
astrocytes in the dorsal spinal cord
Tracing the lineage of Ascl1-expressing cells at E14.5 in the
Ascl1CreER/+;R26LSL-tdTOM control mice revealed that they give rise
equally to both protoplasmic and fibrous astrocytes in the dorsal
spinal cord (Fig. 2I-K,N and Fig. 7A-I). To determine if Ascl1 plays
a role in the development of these two dorsal astrocyte lineages,
spinal cords of Ascl1 CKO and Ascl1 KO (2.5 mg tamoxifen/40 mg
body weight) were examined in comparison to controls. At E18.5,
the number and percentage of Nfia+;tdTOM+ cells were significantly
increased in the GM and decreased in the WM of the Ascl1 CKO and
Ascl1 KO relative to control (Fig. 7A versus 7A′,K-M), suggesting
that Ascl1 normally functions to suppress the generation of GM
astrocytes. This phenotype is unlikely to be due to a delay in
migration or maturation of these astrocytes because it persists into
postnatal stages through P30 of the Ascl1 CKO (Fig. 7D,H versus
7D′,H′,K-O). More importantly, at these postnatal stages the
increased GM Nfia+;tdTOM+ cells were able to migrate into
the dorsal horn and begin to exhibit a complex morphology
resembling that of protoplasmic astrocytes (Fig. 7E,E′,I,I′). Indeed,
there were twice as many tdTOM+ cells with a protoplasmic
astrocyte morphology in the Ascl1 CKO than in the control (Fig. 7P).
To gain insight into how the imbalance of GM protoplasmic and
WM fibrous astrocytes arises in the absence of Ascl1, a low dose of
tamoxifen (0.025 mg/40 g body weight) was administered to Ascl1
CKO mice at E14.5. Unlike in the control, in which GM-only and
WM-only astrocyte clones were noted at E18.5 (Fig. 3), in the Ascl1
CKO mixed clones, which contained tdTOM+ cells in both the GM
and WM, were also observed in addition to GM-only and WM-only
clones (Fig. 8A-D). The GM-only and WM-only clones in the Ascl1
CKO were positive for astrocyte markers (Sox2, ApoE, tdTOM)
(Fig. 8A,C) and contained a similar number of tdTOM+ cells per
clone as in the control (Fig. 8E). By contrast, the mixed clones
contained twice as many tdTOM+ cells per clone, suggesting that an
additional division occurred between E14.5 and E18.5 in the
absence of Ascl1. Thus, additional cell divisions could account for
the overall increase in tdTOM+ cells seen in Ascl1 CKO and Ascl1
KO (Fig. 4A-D). The mixed clones can be found on the same or
immediately adjacent sections (Fig. 8B, mixed clone 1), or
distributed across multiple nearby sections (Fig. 8D, mixed
clone 2). Marker analysis showed that these mixed clones express
astrocyte markers (Nfia, Gfap), but also contain cells that co-express
Olig2 and ApoE (insets, Fig. 8D). Interestingly, extensive analysis
through large portions (∼4 mm) of the Ascl1 CKO spinal cords
revealed that there was a greater occurrence of GM-only and mixed
clones than WM-only clones. This difference in occurrence resulted
in an altered distribution of tdTOM+ cells, with more cells settling in
the GM (70%) than WM (30%) in the Ascl1 CKO as compared with
control littermates (52% GM, 48% WM) (Fig. 8F), and is
reminiscent of that seen at the population level with the high
tamoxifen dose (73% GM, 27% WM) (Fig. 7O).
In summary, these findings suggest that, at both the population
and clonal level, Ascl1 is playing a role in governing the balance of
GM protoplasmic and WM fibrous astrocytes generated in the
dorsal spinal cord.
DISCUSSION
Ascl1 defines cells that are fated to astrocyte lineages in the
dorsal spinal cord
An emerging concept over the past few years is that astrocyte
specification and heterogeneity are pre-patterned and segmentally
defined by transcription factor codes (for a review see Molofsky
et al., 2012). This concept is demonstrated in the ventral spinal
cord, where homeodomain (Pax6, Nkx6.1) and bHLH (Scl1)
transcription factors mark progenitor cells that give rise to different
spatially defined ventral astrocyte populations (Hochstim et al.,
2008; Muroyama et al., 2005). Lineage analysis of progenitor cells
in the dP6-p0 domains demonstrate that they also give rise to both
GM protoplasmic and WM fibrous astrocytes that are found at
similar intermediate regions of the spinal cord (Fogarty et al., 2005;
Tsai et al., 2012). Not surprisingly, lineage analysis of Ascl1+ glial
progenitors, which predominantly comprise those of the dP3-dP5
domains, reveal that they contribute to only dorsal GM protoplasmic
3727
DEVELOPMENT
By contrast, in the WM there was a persistent and significant
decrease in the number of Olig2+;tdTOM+ and Sox10+;tdTOM+
cells in the Ascl1 CKO from E18.5 to P30 (compare Fig. 5A-G with
5A′-G′,J,P), suggesting that the proliferation or expansion of WM
OPCs is disrupted. This decrease was especially severe in the dorsal
funiculus of the Ascl1 CKO at P14, in which there was almost a
complete absence of Olig2+;tdTOM+ and Sox10+;tdTOM+ cells in
contrast to the control (Fig. 5F versus 5F′,M,N). This preferential
loss in the WM resulted in an apparent proportional shift in the
distribution of Ascl1 lineage OPC/oligodendrocytes from the WM
to the GM in the spinal cord (Fig. 5K,L,Q-S).
Collectively, these findings demonstrate a differential requirement
for Ascl1 for OPCs in the GM and WM, and, together with the
results from the lineage tracing using the intermediate tamoxifen
dose (Fig. 3F,G), imply that GM and WM oligodendrocytes arise
from distinct Ascl1 lineage cells (see Fig. 9A,B).
RESEARCH ARTICLE
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
Fig. 7. The balance of GM and WM
astrocytes is altered in the spinal cord of
Ascl1 CKO mice. Immunofluorescence
on thoracic spinal cord sections of
Ascl1CreERT2/+;R26LSL-tdTOM control and
Ascl1CreERT2/FL;R26LSL-tdTOM CKO mice
treated with tamoxifen at E14.5. (A-I) E18.5
control showing that Nfia+;tdTOM+ cells are
Gfap– in GM (arrows, B) and Gfap+ in WM
(arrows, C). By P14 and P30, most Nfia+;
tdTOM+ cells in the GM exhibited a complex
morphology resembling that of protoplasmic
astrocytes (arrows, E,I). Note that Nfia and
Gfap are not expressed in tdTOM+ OL
(arrowheads, G,I). (A′-I′) Ascl1 CKO
showing an increase in the number of Nfia+;
tdTOM+ cells in the dorsal GM at E18.5
(A′-C′), which exhibited protoplasmic
morphology at P14 (D′-G′) and P30 (H′,I′).
(J,J′) Schematic summary comparing the
astrocyte phenotype of control and
Ascl1 CKO spinal cords at P14/P30.
(K-O) Number and distribution of Nfia+;
tdTOM+ cells per section in the GM and WM
of control, Ascl1 CKO and Ascl1 KO
spinal cords at E18.5, P14 and P30.
(P) Percentage of tdTOM+ cells with
protoplasmic morphology among Nfia+;
tdTOM+ cells at P14. Number of spinal cords
analyzed: E18.5, N=3 control, N=2 Ascl1
CKO and Ascl1 KO; P14, N=3 control and
Ascl1 CKO; P30, N=2 control and Ascl1
CKO. #P<0.05 and *P<0.005 indicate
statistical significance using linear
regression analysis. Values in graphs are
mean±s.e.m. Scale bars: 100 μm for A,A′,D,
D′,H,H′; 25 μm for B-C′,E-G′,I,I′.
Ascl1 balances the number of GM protoplasmic and WM
fibrous astrocytes generated
The presence of two classes of morphologically distinct astrocytes,
namely protoplasmic and fibrous, was first noted in the GM and
WM, respectively, more than 100 years ago (Oberheim et al., 2012).
These two classes of astrocytes utilize different mechanisms for
calcium wave propagation in the cortex (Haas et al., 2006), and may
be functionally distinct in their ability to communicate and regulate
neuronal homeostasis and functions. However, how GM
protoplasmic and WM fibrous astrocytes are generated from glial
progenitor cells during development in the CNS is unknown. Here,
Ascl1+ glial progenitors labeled at E14.5 in the dorsal spinal cord
contribute equally to both types of astrocytes (Figs 2 and 7). The fact
that both GM and WM astrocytes were similarly labeled suggested
that Ascl1 could potentially regulate an asymmetric division of
astrocyte progenitor cells to generate these two astrocyte cell types.
However, by administering low doses of tamoxifen to sparsely label
individual Ascl1-expressing cells, the resulting clones were clusters
of astrocytes restricted to either the GM or WM but not to both
(Fig. 3A,B). This implies that GM protoplasmic and WM fibrous
3728
astrocytes in the dorsal spinal cord are not the result of asymmetric
division from a common Ascl1+ astrocyte progenitor cell, but are
produced from two separate Ascl1+ progenitors (Fig. 9A). A similar
heterogeneity in astrocyte progenitors was recently reported from
lineage-tracing studies in the cortex, where protoplasmic and fibrous
astrocyte clones were distinctly labeled by the combinatorial
expression of fluorescent proteins (Garcia-Marques and LopezMascaraque, 2013). However, neither study excludes the possibility
of earlier Ascl1-negative or Ascl1low bipotential progenitors that
were not labeled in these experiments. Indeed, the presence of
mixed clones in the Ascl1 CKO spinal cords supports the existence
of a GM/WM bipotential progenitor cell. Nevertheless, examining
the Ascl1 astrocyte lineage in the Ascl1 CKO as clones or as a
population demonstrated that Ascl1 is necessary for the correct
distribution of astrocyte types in the dorsal spinal cord, and, in its
absence, there is an increase in the number of GM astrocytes along
with a loss of WM astrocytes (Figs 7-9).
Requirement for Ascl1 in the generation of OPCs in the WM of
the spinal cord
It has been known for some time that Ascl1 is expressed in
progenitors to the oligodendrocyte lineage in the brain (Parras et al.,
2004, 2007; Kim et al., 2007) and spinal cord (Battiste et al., 2007;
Sugimori et al., 2007, 2008). Furthermore, the number of OPCs is
significantly decreased in the spinal cord of germline null Ascl1
mice at the onset of oligodendrogenesis, and this is followed by a
lack or delayed expression of more mature oligodendrocyte markers
in the ventral WM at E18.5 (Sugimori et al., 2007, 2008). However,
because the progenitor and neuronal pools are significantly altered
DEVELOPMENT
and WM fibrous astrocytes, and this spatial restriction is retained
into the adult spinal cord. This dorsal restriction of astrocytes
deriving from dorsal glial progenitor cells has also been noted for
Msx3-Cre and Pax3-Cre lineage cells in the dP1-dP6 domains (Zhu
et al., 2011; Tsai et al., 2012). Collectively, findings in this study
further support the concept that the expression of homeodomain and
bHLH factors spatially defines the progenitor origin of regionally
distinct astrocytes along the DV axis of the spinal cord.
RESEARCH ARTICLE
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
Fig. 8. The proliferation and distribution of astrocyte
clones in the GM and WM are altered in Ascl1 CKO
spinal cords. Immunofluorescence on spinal cord
sections of Ascl1CreERT2/FL;R26LSL-tdTOM mice treated with
a low dose of tamoxifen (0.025 mg/40 g body weight) at
E14.5. (A,C) Consecutive sections showing the presence
of WM-only (A) or GM-only (C) labeled astrocyte clones
found at the center of two separate 780 μm spinal cord
portions. (B,D) Consecutive sections showing the
presence of two mixed (GM and WM) astrocyte-labeled
clones that are found separately in a 780 μm or 1350 μm
spinal cord segment. Note that the number of cells in the
mixed clones is increased compared with GM-only or WMonly clones. Dotted lines delineate the GM/WM boundary.
Arrows indicate labeled AS noted in both low- and highmagnification images. (E) Number (mean±s.e.m.) of cells
per AS clone. The number of each type of clone is also
shown. (F) Distribution of sparsely labeled tdTOM+ AS cells
in the VZ, GM and WM between control and Ascl1 CKO
littermates. *P<0.005, Student’s t-test; n.s, not significant.
Scale bars: 100 μm for low-magnification images; 25 μm
for high-magnification insets.
loss of OPCs/oligodendrocytes was especially apparent in the dorsal
funiculus (Fig. 5F′), a domain where many of the OPCs are likely to
be derived from dorsally located Ascl1+ cells (Fig. 3C,D) (Zhu et al.,
2011; Tsai et al., 2012). This phenotype could reflect a role for Ascl1
in the specification of glial progenitors into the oligodendrocyte
lineage, OPC proliferation, and/or OPC migration from the GM to
the WM. In contrast to the consistent decrease in the WM
oligodendrocyte lineage, the number of GM oligodendrocyte
lineage cells, as assessed by Sox10 expression, was initially
increased in the Ascl1 CKO, but eventually decreased back to
slightly below normal numbers at postnatal stages (Fig. 5O). This
indicates differences in the way the GM and WM OPCs respond to
Fig. 9. Summary of Ascl1 expression and function
during gliogenesis in the spinal cord. (A) Ascl1 is
expressed in progenitors to four different glial lineages. The
number of arrows at precursor cell stage indicates the
degree of proliferation for each lineage, as inferred between
E18.5 and P30. (B) Ascl1 CKO showed an increase in the
GM-AS lineage and decreases in the WM-AS and WM-OL
lineages. In the absence of Ascl1, progenitors that normally
give rise to WM-AS also give rise to mixed WM/GM
progeny. Ascl1 regulates the development of these lineages
in the spinal cord at the progenitor and precursor cell stages.
3729
DEVELOPMENT
in the Ascl1 mutant prior to gliogenesis as a result of its earlier role
in neurogenesis (Torii et al., 1999; Helms et al., 2005; Mizuguchi
et al., 2006; Wildner et al., 2006), a direct role for Ascl1 in the
specification and differentiation of OPCs in the spinal cord has not
been completely addressed. In the neonatal brain, however,
conditional deletion of Ascl1 in SVZ stem/progenitor cells
showed that Ascl1 is required for OPC specification, and OPCspecific deletion of Ascl1 resulted in an increase in proliferation and
a decrease in differentiation of OPCs during remyelination
(Nakatani et al., 2013).
We have shown that oligodendrogenesis in the spinal cord is
dramatically reduced in the WM of Ascl1 CKO mice (Fig. 5). This
RESEARCH ARTICLE
MATERIALS AND METHODS
Mouse strains, tamoxifen administration and tissue preparation
Protocols for the generation and genotyping of mouse strains are as
previously described: Ascl1null (Guillemot et al., 1993); Ascl1CreERT2
[Ascl1tm1.1(Cre/ERT2)Jejo/J] (Kim et al., 2011); Ascl1FL (Pacary et al., 2011);
and the Cre reporter line R26LSL-tdTom [Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J]
(Madisen et al., 2010). All procedures on animals followed NIH guidelines
and were approved by the UT Southwestern Institutional Animal Care and
Use Committee.
The appearance of a vaginal plug was considered E0.5 and the day of
birth was noted as P0. Tamoxifen (Sigma, T55648) administration was
accomplished by intraperitoneal injection of gravid females at 2.5 mg
tamoxifen/40 g body weight, except for clonal analysis, where doses of
0.025 mg to 0.25 mg tamoxifen/40 g body weight were used. Spinal cords
of postnatal animals were fixed via perfusion with 4% paraformaldehyde in
PBS, immersion fixed overnight, washed in PBS and submerged in 30%
sucrose. All tissues were embedded in O.C.T. (Sakura Finetek) for
cryosectioning at 30 μm.
Immunofluorescence
Spinal cord sections were incubated with primary antibody (supplementary
material Table S1) in 1% goat (or donkey) serum/0.3% Triton X-100×/PBS
overnight, followed by secondary antibody conjugated with Alexa Fluor
488, 568 or 647 (Molecular Probes).
Quantification and statistical analyses
For quantification, cell numbers and distribution in the spinal cord were
counted manually using ImageJ (NIH) software on sections from the
thoracic region (∼T2-T9). NeuN or Gfap staining was used to demarcate
the boundary between GM and WM for each section. TDP43 (Tardbp –
Mouse Genome Informatics), a ubiquitously expressed nuclear protein
(Sephton et al., 2010), was used as a pan-nuclei marker, and
colocalization of TDP43 with tdTomato (tdTOM) was used to
determine the total number of tdTOM+ cells per section. For each
marker, ∼5-9 sections per spinal cord were counted for marker+;tdTOM+
cells in the VZ, GM and WM. A 10-15% overlap between the
oligodendrocyte (Olig2, Sox10) and astrocyte (Nfia) markers with
respect to the total number of glial cells labeled by tdTOM was noted,
indicating that these markers are mostly specific but not exclusive for
their respective lineages.
To assess the differences in cell number and distribution of marker+;
tdTOM+ cells between control, Ascl1 CKO and Ascl1 KO mice, we used a
linear regression technique to model the observed cell number as a linear
combination of fixed effect (genotype) and random effect (animal ID). The
animal ID is included as the random effect because multiple spinal cord
sections were counted per animal, and multiple animals were counted per
genotype. We then performed the likelihood ratio test to determine the
statistical significance (chi-square, degree of freedom and P-value) using R
lmer function to conduct the linear mixed model fit by maximum likelihood.
The output value of this analysis is interpreted as estimates from a traditional
least squares linear regression, where the estimate for the control is the
3730
reference (or Y intercept), and the estimate for the Ascl1 CKO or Ascl1 KO is
the value of the slope of the line from the control. These estimated output
values, which are approximately the means, are reported for each genotype.
A P<0.05 indicates statistical significance that genotype (or the loss of
Ascl1) accounts for the change in cell numbers and percentage cell
distributions observed.
Acknowledgements
We acknowledge Lauren Tyra and Rachel Yuengert for excellent technical services
with mouse genotyping and husbandry. We thank Dr Ben Deneen for Nfia
antibodies; Dr Gang Yu for TDP43 antibodies; Dr K. C. Tung for help with statistical
analyses; Drs Helen Lai and Richard Lu for critical reading of the manuscript; and all
members of the J.E.J. laboratory for many helpful discussions throughout this study.
Competing interests
The authors declare no competing financial interests.
Author contributions
E.J.K. initiated the project with J.E.J., performed early experiments and generated
CreER
FLOX
KI mice. C.M.P. and F.G. generated the Ascl1
mice. T.Y.V.
the Ascl1
designed and performed all the experiments and data analysis, and prepared the
manuscript with J.E.J. All authors provided scientific insight and edited the
manuscript.
Funding
This work was supported by Public Health Service grants from the National Institutes
of Health [R01 NS032817 to J.E.J. and F32CA168330 to T.Y.V.] and a Grant-in-Aid
from the Medical Research Council [U117570528 to F.G.]. Deposited in PMC for
release after 6 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.105270/-/DC1
References
Bachoo, R. M., Kim, R. S., Ligon, K. L., Maher, E. A., Brennan, C., Billings, N.,
Chan, S., Li, C., Rowitch, D. H., Wong, W. H. et al. (2004). Molecular diversity of
astrocytes with implications for neurological disorders. Proc. Natl. Acad. Sci. USA
101, 8384-8389.
Battiste, J., Helms, A. W., Kim, E. J., Savage, T. K., Lagace, D. C., Mandyam,
C. D., Eisch, A. J., Miyoshi, G. and Johnson, J. E. (2007). Ascl1 defines
sequentially generated lineage-restricted neuronal and oligodendrocyte precursor
cells in the spinal cord. Development 134, 285-293.
Borromeo, M. D., Meredith, D. M., Castro, D. S., Chang, J. C., Tung, K.-C.,
Guillemot, F. and Johnson, J. E. (2014). A transcription factor network
specifying inhibitory versus excitatory neurons in the dorsal spinal cord.
Development 141, 2803-2812, 3102.
Cai, J., Chen, Y., Cai, W.-H., Hurlock, E. C., Wu, H., Kernie, S. G., Parada, L. F.
and Lu, Q. R. (2007). A crucial role for Olig2 in white matter astrocyte
development. Development 134, 1887-1899.
Castro, D. S., Martynoga, B., Parras, C., Ramesh, V., Pacary, E., Johnston, C.,
Drechsel, D., Lebel-Potter, M., Garcia, L. G., Hunt, C. et al. (2011). A novel
function of the proneural factor Ascl1 in progenitor proliferation identified by
genome-wide characterization of its targets. Genes Dev. 25, 930-945.
Deneen, B., Ho, R., Lukaszewicz, A., Hochstim, C. J., Gronostajski, R. M. and
Anderson, D. J. (2006). The transcription factor NFIA controls the onset of
gliogenesis in the developing spinal cord. Neuron 52, 953-968.
Fogarty, M., Richardson, W. D. and Kessaris, N. (2005). A subset of
oligodendrocytes generated from radial glia in the dorsal spinal cord.
Development 132, 1951-1959.
Garcia-Marques, J. and Lopez-Mascaraque, L. (2013). Clonal identity determines
astrocyte cortical heterogeneity. Cereb. Cortex 23, 1463-1472.
Ge, W.-P., Miyawaki, A., Gage, F. H., Jan, Y. N. and Jan, L. Y. (2012). Local
generation of glia is a major astrocyte source in postnatal cortex. Nature 484,
376-380.
Guillemot, F., Lo, L.-C., Johnson, J. E., Auerbach, A., Anderson, D. J. and
Joyner, A. L. (1993). Mammalian achaete-scute homolog 1 is required for the
early development of olfactory and autonomic neurons. Cell 75, 463-476.
Haas, B., Schipke, C. G., Peters, O., Sö hl, G., Willecke, K. and Kettenmann, H.
(2006). Activity-dependent ATP-waves in the mouse neocortex are independent
from astrocytic calcium waves. Cereb. Cortex 16, 237-246.
Helms, A. W., Battiste, J., Henke, R. M., Nakada, Y., Simplicio, N., Guillemot, F.
and Johnson, J. E. (2005). Sequential roles for Mash1 and Ngn2 in the
generation of dorsal spinal cord interneurons. Development 132, 2709-2719.
DEVELOPMENT
the loss of Ascl1, and supports the concept of heterogeneity between
these populations of cells as seen in multiple contexts. For example,
the proliferation and differentiation efficiency of adult cortical WM
and GM OPCs were shown to be intrinsically distinct when they
were transplanted heterotopically to the GM or WM in the cortex
(Viganò et al., 2013). Furthermore, WM OPCs can proliferate in
response to the growth factor PDGF but GM OPCs cannot (Hill
et al., 2013). Given that the function of Ascl1 in the CNS is highly
dynamic and context dependent (Helms et al., 2005; Mizuguchi
et al., 2006; Wildner et al., 2006; Jacob et al., 2013), and that Ascl1
has been shown to regulate a complex set of downstream targets
(Castro et al., 2011; Borromeo et al., 2014), it is possible that Ascl1
in the oligodendrocyte lineages directly contributes to the
differences in their intrinsic properties in the GM and WM.
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
Hill, R. A., Patel, K. D., Medved, J., Reiss, A. M. and Nishiyama, A. (2013). NG2
cells in white matter but not gray matter proliferate in response to PDGF.
J. Neurosci. 33, 14558-14566.
Hochstim, C., Deneen, B., Lukaszewicz, A., Zhou, Q. and Anderson, D. J.
(2008). Identification of positionally distinct astrocyte subtypes whose identities
are specified by a homeodomain code. Cell 133, 510-522.
Hoffmann, S. A., Hos, D., Kuspert, M., Lang, R. A., Lovell-Badge, R., Wegner, M.
and Reiprich, S. (2014). Stem cell factor Sox2 and its close relative Sox3 have
differentiation functions in oligodendrocytes. Development 141, 39-50.
Horton, S., Meredith, A., Richardson, J. A. and Johnson, J. E. (1999). Correct
coordination of neuronal differentiation events in ventral forebrain requires the
bHLH factor MASH1. Mol. Cell. Neurosci. 14, 355-369.
Jacob, J., Kong, J., Moore, S., Milton, C., Sasai, N., Gonzalez-Quevedo, R.,
Terriente, J., Imayoshi, I., Kageyama, R., Wilkinson, D. G. et al. (2013).
Retinoid acid specifies neuronal identity through graded expression of Ascl1. Curr.
Biol. 23, 412-418.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and
transcriptional codes. Nat. Rev. Genet. 1, 20-29.
Kang, P., Lee, H. K., Glasgow, S. M., Finley, M., Donti, T., Gaber, Z. B.,
Graham, B. H., Foster, A. E., Novitch, B. G., Gronostajski, R. M. et al. (2012).
Sox9 and NFIA coordinate a transcriptional regulatory cascade during the
initiation of gliogenesis. Neuron 74, 79-94.
Kim, E. J., Leung, C. T., Reed, R. R. and Johnson, J. E. (2007). In vivo analysis of
Ascl1 defined progenitors reveals distinct developmental dynamics during adult
neurogenesis and gliogenesis. J. Neurosci. 27, 12764-12774.
Kim, E. J., Battiste, J., Nakagawa, Y. and Johnson, J. E. (2008). Ascl1 (Mash1)
lineage cells contribute to discrete cell populations in CNS architecture. Mol. Cell.
Neurosci. 38, 595-606.
Kim, E. J., Ables, J. L., Dickel, L. K., Eisch, A. J. and Johnson, J. E. (2011). Ascl1
(Mash1) defines cells with long-term neurogenic potential in subgranular and
subventricular zones in adult mouse brain. PLoS ONE 6, e18472.
Lu, Q. R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C. D. and Rowitch, D. H.
(2002). Common developmental requirement for Olig function indicates a motor
neuron/oligodendrocyte connection. Cell 109, 75-86.
Madisen, L., Zwingman, T. A., Sunkin, S. M., Oh, S. W., Zariwala, H. A., Gu, H.,
Ng, L. L., Palmiter, R. D., Hawrylycz, M. J., Jones, A. R. et al. (2010). A robust
and high-throughput Cre reporting and characterization system for the whole
mouse brain. Nat. Neurosci. 13, 133-140.
Mizuguchi, R., Kriks, S., Cordes, R., Gossler, A., Ma, Q. and Goulding, M.
(2006). Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal
sensory interneurons. Nat. Neurosci. 9, 770-778.
Molofsky, A. V., Krenick, R., Ullian, E., Tsai, H.-H., Deneen, B., Richardson,
W. D., Barres, B. A. and Rowitch, D. H. (2012). Astrocytes and disease: a
neurodevelopmental perspective. Genes Dev. 26, 891-907.
Muroyama, Y., Fujiwara, Y., Orkin, S. H. and Rowitch, D. H. (2005). Specification
of astrocytes by bHLH protein SCL in a restricted region of the neural tube. Nature
438, 360-363.
Nakatani, H., Martin, E., Hassani, H., Clavairoly, A., Maire, C. L., Viadieu, A.,
Kerninon, C., Delmasure, A., Frah, M., Weber, M. et al. (2013). Ascl1/Mash1
Promotes Brain Oligodendrogenesis during Myelination and Remyelination.
J. Neurosci. 33, 9752-9768.
Nishiyama, A., Komitova, M., Suzuki, R. and Zhu, X. (2009). Polydendrocytes (NG2
cells): multifunctional cells with lineage plasticity. Nat. Rev. Neurosci. 10, 9-22.
Oberheim, N. A., Goldman, S. A. and Nedergaard, M. (2012). Heterogeneity of
astrocytic form and function. Methods Mol. Biol. 814, 23-45.
Pacary, E., Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel-Potter, M., Parras, C.,
Bell, D. M., Ridley, A. J., Parsons, M. et al. (2011). Proneural transcription factors
regulate different steps of cortical neuron migration through Rnd-mediated inhibition
of RhoA signaling. Neuron 69, 1069-1084.
Parras, C. M., Galli, R., Britz, O., Soares, S., Galichet, C., Battiste, J., Johnson,
J. E., Nakafuku, M., Vescovi, A. and Guillemot, F. (2004). Mash1 specifies
neurons and oligodendrocytes in the postnatal brain. EMBO J. 23, 4495-4505.
Development (2014) 141, 3721-3731 doi:10.1242/dev.105270
Parras, C. M., Hunt, C., Sugimori, M., Nakafuku, M., Rowitch, D. and
Guillemot, F. (2007). The proneural gene Mash1 specifies an early
population of telencephalic oligodendrocytes. J. Neurosci. 27, 4233-4242.
Pringle, N. P., Yu, W.-P., Howell, M., Colvin, J. S., Ornitz, D. M. and Richardson,
W. D. (2003). Fgfr3 expression by astrocytes and their precursors: evidence that
astrocytes and oligodendrocytes originate in distinct neuroepithelial domains.
Development 130, 93-102.
Rowitch, D. H. (2004). Glial specification in the vertebrate neural tube. Nat. Rev.
Neurosci. 5, 409-419.
Rowitch, D. H. and Kriegstein, A. R. (2010). Developmental genetics of vertebrate
glial-cell specification. Nature 468, 214-222.
Sephton, C. F., Good, S. K., Atkin, S., Dewey, C. M., Mayer, P., Herz, J. and Yu,
G. (2010). TDP-43 is a developmentally regulated protein essential for early
embryonic development. J. Biol. Chem. 285, 6826-6834.
Sudarov, A., Turnbull, R. K., Kim, E. J., Lebel-Potter, M., Guillemot, F. and
Joyner, A. L. (2011). Ascl1 genetics reveals insights into cerebellum local circuit
assembly. J. Neurosci. 31, 11055-11069.
Sugimori, M., Nagao, M., Bertrand, N., Parras, C. M., Guillemot, F. and
Nakafuku, M. (2007). Combinatorial actions of patterning and HLH transcription
factors in the spatiotemporal control of neurogenesis and gliogenesis in the
developing spinal cord. Development 134, 1617-1629.
Sugimori, M., Nagao, M., Parras, C. M., Nakatani, H., Lebel, M., Guillemot, F. and
Nakafuku, M. (2008). Ascl1 is required for oligodendrocyte development in the
spinal cord. Development 135, 1271-1281.
Tien, A.-C., Tsai, H.-H., Molofsky, A. V., McMahon, M., Foo, L. C., Kaul, A.,
Dougherty, J. D., Heintz, N., Gutmann, D. H., Barres, B. A. et al. (2012).
Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF
signalling in mammalian spinal cord. Development 139, 2477-2487.
Torii, M., Matsuzaki, F., Osumi, N., Kaibuchi, K., Nakamura, S., Casarosa, S.,
Guillemot, F. and Nakafuku, M. (1999). Transcription factors Mash-1 and Prox-1
delineate early steps in differentiation of neural stem cells in the developing central
nervous system. Development 126, 443-456.
Tsai, H.-H., Li, H., Fuentealba, L. C., Molofsky, A. V., Taveira-Marques, R.,
Zhuang, H., Tenney, A., Murnen, A. T., Fancy, S. P. J., Merkle, F. et al. (2012).
Regional astrocyte allocation regulates CNS synaptogenesis and repair. Science
337, 358-362.
Viganò, F., Mö bius, W., Gö tz, M. and Dimou, L. (2013). Transplantation reveals
regional differences in oligodendrocyte differentiation in the adult brain. Nat.
Neurosci. 16, 1370-1372.
Walther, E. U., Dichgans, M., Maricich, S. M., Romito, R. R., Yang, F., Dziennis, S.,
Zackson, S., Hawkes, R. and Herrup, K. (1998). Genomic sequences of aldolase
C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic
mice. Proc. Natl. Acad. Sci. USA 95, 2615-2620.
Wichterle, H., Lieberam, I., Porter, J. A. and Jessell, T. M. (2002). Directed
differentiation of embryonic stem cells into motor neurons. Cell 110, 385-397.
Wildner, H., Mü ller, T., Cho, S.-H., Brö hl, D., Cepko, C. L., Guillemot, F. and
Birchmeier, C. (2006). dILA neurons in the dorsal spinal cord are the product of
terminal and non-terminal asymmetric progenitor cell divisions, and require
Mash1 for their development. Development 133, 2105-2113.
Zhou, Q. and Anderson, D. J. (2002). The bHLH transcription factors OLIG2 and
OLIG1 couple neuronal and glial subtype specification. Cell 109, 61-73.
Zhou, Q., Wang, S. and Anderson, D. J. (2000). Identification of a novel family of
oligodendrocyte lineage-specific basic helix-loop-helix transcription factors.
Neuron 25, 331-343.
Zhou, Q., Choi, G. and Anderson, D. J. (2001). The bHLH transcription factor Olig2
promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31,
791-807.
Zhu, Q., Whittemore, S. R., Devries, W. H., Zhao, X., Kuypers, N. J. and Qiu, M.
(2011). Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal
myelination during development and remyelination following focal demyelination.
Glia 59, 1612-1621.
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