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First published online 15 December 2008
doi: 10.1242/dev.027193
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1 Max-Delbrück-Centrum for Molecular Medicine, Robert-Rössle-Strasse
10, 13125 Berlin, Germany.
2 EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Author for correspondence
(cbirch{at}mdc-berlin.de)
Accepted 6 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fate mapping, Neuronal fate, Transcription factor, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
The complex circuitry in which these neurons participate is established during
development and depends on their spatially and temporally ordered appearance.
The anlage of the hindbrain is segmented transiently into rhombomeres, and
rhombomere borders align with the borders of expression of specific Hox genes
(Fienberg et al., 1987;
Wilkinson et al., 1989;
Fraser et al., 1990;
Krumlauf et al., 1993). Along
the dorsoventral axis of the rhombomeres, specific neuronal types appear.
Extensive cell migration and complex morphogenesis make it difficult to follow
the destiny of distinct neuronal types in the hindbrain
(Cobos et al., 2001;
Dauger et al., 2003;
Bloch-Gallego et al., 2005).
Recent progress in genetic fate-mapping techniques that rely on the use of
site-specific recombinases has helped to overcome this impediment
(Branda and Dymecki, 2004;
Joyner and Zervas, 2006).
Combined with targeted mutations, genetic fate mapping can help to define the
molecular determinants that control the development of brainstem nuclei.
One important class of genes that regulates cellular diversity in the
nervous system encodes basic helix-loop-helix (bHLH) transcription factors.
Early work demonstrated that bHLH factors act as generic proneural factors
that function within the Notch pathway to single out neuronal progenitors and
promote their differentiation. Subsequent analysis revealed the important
roles of bHLH factors in the determination of neuronal fates (for reviews, see
Brunet and Ghysen, 1999;
Bertrand et al., 2002;
Ross et al., 2003). The Olig
genes encode a subfamily of bHLH transcription factors. The function of two
family members, Olig1 and Olig2, in the development of
motoneurons and oligodendrocytes has been extensively characterized
(Mizuguchi et al., 2001;
Novitch et al., 2001;
Zhou et al., 2001;
Lu et al., 2002;
Takebayashi et al., 2002a;
Zhou and Anderson, 2002;
Arnett et al., 2004). The third
member, Olig3, is expressed in defined cell populations in the
developing neural tube, among them dorsal progenitors
(Takebayashi et al., 2002b).
Recent work has demonstrated that Olig3 expression in the spinal cord
is controlled by dorsal patterning signals, and that Olig3 is
required to determine neuronal fates in the spinal cord of mice and zebrafish
(Filippi et al., 2005;
Muller et al., 2005;
Zechner et al., 2007).
The dorsal ventricular zone of the medulla and pons generates brainstem
nuclei, including the spinal trigeminal nucleus and the nucleus of the
solitary tract that relay somatosensory and viscerosensory information,
respectively, and precerebellar nuclei that function in motor coordination
(Altman and Bayer, 1980;
Altman and Bayer, 1987).
Neurons that generate these nuclei migrate extensively before they settle, and
arise from dorsal progenitor domains characterized by the expression of bHLH
factors such as Math1 (Atoh1 - Mouse Genome Informatics), Ngn1 (Neurog1),
Mash1 (Ascl1) and Ptf1a (Pattyn et al.,
2000; Qian et al.,
2001; Landsberg et al.,
2005; Wang et al.,
2005; Sieber et al.,
2007; Yamada et al.,
2007). For instance, neurons of the lateral reticular, external
cuneate, pontine and reticulotegmental nuclei arise from Math1+
progenitors at the dorsal lip, and Math1 is required to determine their fate
(Bermingham et al., 2001;
Machold and Fishell, 2005;
Wang et al., 2005). These
neurons express the homeodomain factors Barhl1/2 and Lhx2/9, and depend on
these factors for differentiation (Saito
et al., 1998; Bermingham et
al., 2001; Li et al.,
2004). Mash1+ progenitors locate further ventrally and
appear to give rise to neurons of the nucleus of the solitary tract, as these
are generated in reduced numbers in Mash1 mutant mice
(Dauger et al., 2003;
Pattyn et al., 2006). Neurons
of the nucleus of the solitary tract express the homeodomain factors Phox2b
and Tlx3, and depend on these for differentiation
(Qian et al., 2001;
Dauger et al., 2003). Neurons
of the inferior olivary nucleus arise from a Ptf1a+ ventricular
zone located further ventrally, and Ptf1a is required to determine their fate
(Yamada et al., 2007).
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) replaces
the Olig3 coding sequence and was fused in frame with the ATG codon
of Olig3. In addition, the neomycin resistance gene (neo)
flanked by FRT sites was inserted. Mutant ES cells were used to
generate a mouse strain, and neo was removed by crossing with FLPe
deleter mice (Farley et al.,
2000). To induce Cre-mediated recombination, tamoxifen
(Sigma-Aldrich; 20 mg/ml in sunflower oil) was administered to pregnant
females at 100 mg/kg (Joyner and Zervas,
2006). The generation of Rosa26R and
Olig3null mice has been described
(Soriano, 1999;
Muller et al., 2005).
Anatomy, immunohistology, in situ hybridization, microscopy and in ovo electroporation
Rhombomeric units were identified using morphological landmarks such as
hindbrain nuclei and exit points of cranial nerves
(Marin and Puelles, 1995;
Cambronero and Puelles, 2000).
Antibodies used were: guinea pig and rabbit anti-Olig3
(Muller et al., 2005), rabbit
anti-Foxd3 (Martyn Goulding, Salk Institute, La Jolla, CA), guinea pig
anti-Foxd3 (Muller et al.,
2005), rabbit anti-Brn3a (Eric Turner, UCSD, La Jolla, CA), guinea
pig anti-Tlx3 and anti-Lbx1 (Muller et
al., 2002), chick anti-β-galactosidase (Abcam, Poole, UK),
mouse anti-Mash1 (BD Biosciences Pharmingen), rabbit anti-Phox2b (Christo
Goridis and Jean-Francois Brunet, Ecole Normale Superieure, Paris, France),
mouse anti-NF68 (Sigma, Munich, Germany), rabbit anti-Pax2 (Zymed, San
Francisco, CA), guinea pig anti-Ptf1a and rabbit anti-Ngn1 (Jane Johnson,
Southwestern Medical Center, Dallas, TX), mouse anti-Lhx1/5 and anti-Pax7
(DSHB, University of Iowa, IA), rabbit anti-Lhx2/9 and anti-Math1 (Tom
Jessell, Columbia University, New York, NY), rabbit anti-Cre (Novagen). Cy2-,
Cy3- and Cy5-conjugated secondary antibodies were obtained from Dianova
(Hamburg, Germany). Fluorescence was visualized by laser-scanning microscopy
(LSM 5 PASCAL, Carl-Zeiss, Germany), using PASCAL software. For the display of
overviews, pictures were merged using Adobe Photoshop.
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), and is available at
http://www.physik.uni-leipzig.de/~waclaw/count/index.html.
Three sections from three different animals each were analyzed.
In situ hybridization and chick in ovo electroporation were performed as
described (Brohmann et al.,
2000; Dubreuil et al.,
2002). Neuron numbers on the electroporated and control sides were
counted on three caudal brainstem sections from three electroporated embryos.
Morphometric analysis was performed on serial hindbrain sections stained by in
situ hybridization with a Barhl1-specific probe.
Barhl1-positive areas were measured on every second section to
determine the volume of external cuneate and lateral reticular nuclei using
ImageJ (W. S. Rasband, NIH, Bethesda, MD).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ding et al., 2005;
Muller et al., 2005). We used
immunohistochemistry to characterize Olig3+ cells on transverse
sections of the hindbrain. At E11.5, we observed Olig3+ cells in a
broad domain that extended from the roof plate ventrally
(Fig. 1A-E, showing the alar
plate). The majority of these Olig3+ cells were located in the
ventricular zone, but Olig3 was also detected in a stripe of cells located in
the mantle zone (Fig. 1A; see
Fig. S1A-D in the supplementary material). This indicates that Olig3 is
expressed by progenitors and postmitotic neurons. Further analysis showed that
Olig3+ cells of the ventricular zone were heterogeneous. In
rhombomere 7, Olig3+ cells close to the roof plate co-expressed
Math1 (Fig. 1B). Ventrally
abutting Olig3+ cells co-expressed Ngn1
(Fig. 1C). Two further
ventrally located domains were defined, one that contained cells co-expressing
Olig3 and Mash1, and a second containing cells that expressed Olig3, Mash1 and
Ptf1a (Fig. 1D,E). Thus, we
defined four distinct ventricular subdomains containing Olig3+
cells (see Fig. 1I for a
summary). The majority of the cells in the dorsal subdomain were
Olig3+, whereas in ventrally located subdomains only a fraction
expressed Olig3 (Fig. 1A-E). In
dorsal ventricular subdomains, Ki67, a marker for proliferation, was
co-expressed with Olig3; in ventrally located subdomains, Ki67+
cells rarely co-expressed Olig3 (Fig.
1A). Thus, cells in different subdomains of the ventricular zone
appear to express Olig3 in distinct phases of the cell cycle. At later
developmental stages, for instance E12.5, the dorsal Olig3+ domain
was markedly smaller than at earlier stages (data not shown). In rhombomeres
4-6, subdomains containing cells that express Olig3/Math1, Olig3/Mash1 and
Olig3/Mash1/Ptf1a were observed (not shown). In accordance with previous
reports (Landsberg et al.,
2005), a dorsal Ngn1+ ventricular domain in rhombomeres
4-6 was not found (data not shown; see Fig.
1M for a summary).
We used the Olig3CreERT2 allele (see below for details
of the allele) and a lacZ reporter (Rosa26R) for genetic
lineage tracing, and a panel of antibodies to characterize neurons that derive
from Olig3+ cells. Derivatives of cells in which recombination has
occurred inherit the active lacZ allele and express
β-galactosidase (β-Gal). The most dorsal β-Gal+
neuronal population detected, dA1, co-expressed Lhx2/9
(Fig. 1F). Ventral to these
cells, β-Gal+ dA2 and dA4 neurons were observed; dA2 neurons
co-expressed Lhx1/5, whereas dA4 neurons co-expressed Lhx1/5 and Foxd3
(Fig. 1G). In addition, we
detected β-Gal+ dA3 neurons that co-expressed Tlx3
[Fig. 1H; note that
Tlx3+ neurons also express Phox2b and Lmx1b, see
Fig. 7A and Sieber et al.
(Sieber et al., 2007)].
Foxd3+ dA4 neurons were located lateral to the ventricular zone
that contained Olig3+, Mash1+ and Ptf1a+
cells (see Fig. S1E-G in the supplementary material). Others previously
characterized Math1+, Ngn1+, Mash1+ and
Ptf1a+ ventricular subdomains and the neuronal types they generate
(Bermingham et al., 2001;
Qian et al., 2001;
Landsberg et al., 2005;
Machold and Fishell, 2005;
Wang et al., 2005;
Pattyn et al., 2006;
Yamada et al., 2007). Thus,
Olig3+ cells generate four dorsal neuronal subtypes in rhombomere
7, and these appear to arise from ventricular subdomains that contain cells
expressing Olig3/Math1, Olig3/Ngn1, Olig3/Mash1 and Olig3/Mash1/Ptf1a
(summarized in Fig. 1I).
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;
Machold and Fishell, 2005;
Wang et al., 2005). Ventral to
these cells, β-Gal+/Tlx3+ (dA3) neurons were
observed, which co-express Phox2b and Lmx1b and were present in rhombomeres
4-7 but not in more-rostral rhombomeres
(Fig. 1H,K,
Fig. 3C,E and data not shown)
(Pattyn et al., 2000;
Qian et al., 2001;
Dauger et al., 2003;
Sieber et al., 2007). We
identified an additional, small β-Gal+ cell population located
further ventrally that expressed Lhx1/5+ (dA4); dA4 neurons of
rhombomere 7, but not of rhombomeres 4-6, co-expressed Foxd3
(Fig. 1G,L and data not
shown).
Olig3 antagonizes the development of class B neurons
During normal development of rhombomere 7, class A neurons arise in the
dorsal alar plate and Lbx1+ class B neurons in the ventral alar
plate (Fig. 2A,C,E)
(Sieber et al., 2007). We
observed a pronounced dorsal expansion of Lbx1+ neurons in
Olig3 mutant mice in rhombomere 7
(Fig. 2B). Most ectopic
Lbx1+ neurons co-expressed Pax2 and Lhx1/5, and we denote these as
dA2-4* (Fig.
2B,D,F). In normal development, Lbx1+/Pax2+
neurons of the dorsal spinal cord and hindbrain express glutamic acid
decarboxylase 1 (Gad1), an enzyme essential for GABA synthesis
(Cheng et al., 2005) and these
neurons arise exclusively in the ventral alar plate
(Fig. 2G). In Olig3
mutants, Gad1+ neurons arose ectopically in the dorsal alar plate
(Fig. 2H). We conclude that
dA2-dA4 neurons are not correctly specified in rhombomere 7 of Olig3
mutant mice. At their expense,
Gad1+/Lbx1+/Pax2+/Lhx1/5+ neurons
arose that displayed molecular characteristics of inhibitory neurons. We
identified an additional aberrant neuronal subtype close to the roof plate in
Olig3 mutants; these neurons co-expressed Lbx1 and Lhx2/9, and we
denote them as dA1* (Fig.
2B).
We analyzed genes expressed in the dorsal ventricular zone of rhombomere 7 in Olig3 mutant mice. Compared with control animals, Math1 and Ngn1 expression was reduced in Olig3 mutants (Fig. 2I-L). By contrast, Ptf1a expression was expanded dorsally (Fig. 2M,N). Pax7 and Mash1 were similarly expressed in control and Olig3 mutant mice (Fig. 2O,P and data not shown). We conclude that Olig3 is essential to maintain the correct expression of Math1, Ngn1 and Ptf1a in the dorsal ventricular zone of rhombomere 7.
In rhombomeres 4-6 of Olig3 mutant mice, we also observed a pronounced dorsal expansion of Lbx1+ neurons (Fig. 3A-D). Close to the roof plate, we identified neurons that co-expressed Lhx2/9 and Lbx1 (dA1*, Fig. 3B). Tlx3+/Phox2b+ dA3 neurons were not present, and instead we observed Tlx3+/Lbx1+ (dA3*) neurons, which appeared to intermingle with Lbx1+/Pax2+ and Lhx1/5+/Lbx1- neurons (Fig. 3C-J). These changes were accompanied by a dorsal expansion of Ptf1a (Fig. 3E,F). It should be noted that in the alar plate of rhombomeres 4-6, our panel of markers defined identical neuronal subtypes in control and mutant mice, and we display exemplary data on rhombomere 4. Thus, loss of Olig3 results in expanded expression of Lbx1 and Ptf1a in rhombomeres 4-7. No apparent ectopic Lbx1 expression was observed in rhombomeres 1-3, as assessed by immunohistology and whole-mount in situ hybridization (data not shown). We therefore restricted subsequent analyses to rhombomeres 4-7 and their derivatives.
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We followed various class A neuronal subtypes in Olig3 mutant mice. In control mice, dA4 neurons of rhombomere 7 co-expressed Brn3a (Pou4f1 - Mouse Genome Informatics) and Foxd3 at E11.5 (Fig. 5A). The Brn3a+/Foxd3+ dA4 neurons migrated tangentially, forming a stream that extended at E12.5 into the ventral hindbrain, and they settled close to the midline (Fig. 5B). At E15.5 and E18.5, Foxd3+ neurons were observed close to the ventral midline, and by E18.5 the Brn3a+/Foxd3+ neurons assembled in the characteristic structure of the inferior olivary nucleus (Fig. 5C,D). In Olig3 mutant mice, Brn3a+/Foxd3+ neurons were absent at E11.5, and at E12.5 the ventrally migrating stream of Foxd3+/Brn3a+ neurons was lacking (Fig. 5E,F). At E15.5, Foxd3+ neurons at the ventral midline were absent, and we did not detect Foxd3+/Brn3a+ neurons at the position of the inferior olivary nucleus at E18.5 (Fig. 5G,H). Histological analysis confirmed the absence of the characteristic structure of the inferior olivary nucleus (data not shown; see also Fig. 4C,D and Fig. 5D,H). We conclude that the fate of dA4 neurons is not correctly determined in Olig3 mutant mice, resulting in the absence of climbing fiber neurons of the inferior olivary nucleus.
Foxd3+ climbing fiber neurons of the inferior olivary nucleus
depend on Ptf1a for fate determination
(Yamada et al., 2007). We
therefore assessed a possible interaction between Olig3 and Ptf1a in
overexpression experiments. The hindbrains of chick embryos were
electroporated in ovo at Hamburger-Hamilton stage 16 with an expression
construct that produces mouse Olig3, and the embryos were analyzed 48 hours
later. On the electroporated side, we observed a pronounced decrease in the
numbers of Lbx1+ and dorsal Pax2+ neurons
(Fig. 6A,B; quantification in
Fig. 6D). This was accompanied
by a suppression of Ptf1a (Fig.
6C,D). Electroporation of a Ptf1a expression construct had little
effect on the generation of Foxd3+ neurons, and electroporation of
an Olig3 expression construct suppressed the generation of Foxd3+
(dA4) neurons (Fig. 6E,F,H).
However, co-electroporation of Olig3 and Ptf1a expression constructs induced
Foxd3 (Fig. 6G,H). We conclude
that Olig3 and Ptf1a can cooperate to induce Foxd3.
An apparent antagonism exists between Olig3 and Lbx1, and Olig3 might exert
its function by suppressing Lbx1 (Muller
et al., 2005). We therefore investigated whether deficits present
in Olig3 mutants could be rescued in the absence of Lbx1 using
Olig3; Lbx1 double-mutant mice
(Fig. 6I-O). Foxd3+
dA4 neurons were not formed in Olig3 mutants, but their generation
was rescued in Olig3; Lbx1 double-mutant embryos
(Fig. 6I-K). Thus, Olig3 has a
permissive role in the determination of the dA4 fate. However, the generation
of Phox2b+/Tlx3+ dA3 neurons was not rescued, and at
their expense ectopic Foxd3+ neurons appeared
(Fig. 6M-O and data not shown;
summary in Fig. 6P). Lbx1
repression alone cannot therefore explain Olig3 function(s) in the
determination of the dA3 fate.
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; Dauger et al.,
2003). These neurons arose in the dorsal alar plate and migrated
ventrally to settle lateral to vagal motoneurons that express Phox2b but not
Tlx3 (Fig. 7A,B; the arrows
point towards vagal motoneurons and the asterisk indicates the future neurons
of the nucleus of the solitary tract)
(Dauger et al., 2003).
Subsequent morphogenetic movements of the hindbrain resulted in a dorsal
location of the Phox2b+/Tlx3+ neurons, close to the
midline (Fig. 7C)
(Dauger et al., 2003). In
rhombomeres 7 and 4-6 of Olig3 mutant mice,
Phox2b+/Tlx3+ neurons were not formed
(Fig. 7D and
Fig. 3F). At E13.5, we observed
no Phox2b+ neurons at the site of the future nucleus of the
solitary tract in Olig3 mutant mice
(Fig. 7E). At E18.5, we could
not discern Phox2b+/Tlx3+ neurons in the nucleus of the
solitary tract (Fig. 7F). In
addition, the area postrema was not formed
(Fig. 4F). By contrast, vagal
motoneurons (Phox2b+ and Tlx3-) were present at the
expected location at all developmental stages
(Fig. 7, arrows). We conclude
that the fate of Phox2b+/Tlx3+ dA3 neurons was not
correctly determined in Olig3 mutant mice; as a consequence, the
nucleus of the solitary tract and the area postrema were absent.
Lhx2/9+ (dA1) neurons arise from Math1+ cells close
to the roof plate (Fig. 2A,I;
Fig. 8A)
(Bermingham et al., 2001). In
Olig3 mutant mice, we detected Lhx2/9+ cells that arose at
the dorsal lip (Fig. 2B and
Fig. 8B). These neurons
ectopically expressed Lbx1, and we denote them as dA1*
(Fig. 2). Quantification showed
that Lhx2/9+ neurons were generated at reduced numbers in
Olig3 mutant as compared with control mice
(Fig. 8A-C). In normal
development, Lhx2/9+ neurons migrate ventrally in a superficial
migratory stream (Fig. 8A).
During migration and when they settle, Lhx2/9+ neurons of
rhombomere 7 express Barhl1/2 and generate the lateral reticular and external
cuneate nuclei (Saito et al.,
1998; Bermingham et al.,
2001). In situ hybridization demonstrated the presence of
Barhl1+ neurons at the positions of the lateral reticular and
external cuneate nuclei in Olig3 mutant mice, but compared with
control mice the size of these nuclei was reduced
(Fig. 8D-I). We conclude that
Lhx2/9+ dA1* neurons of Olig3 mutant mice
misexpressed Lbx1, but assembled at the sites of the lateral reticular and
external cuneate nuclei. These nuclei were reduced in size, reflecting the
small numbers of Lhx2/9+ dA1* neurons generated in
Olig3 mutants.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) with this study]. Co-electroporation of Olig3 and Ptf1a
expression vectors induced Foxd3, a molecular characteristic of climbing fiber
neurons of the inferior olivary nucleus (see
Fig. 9 for a model of Olig3
function).
Derivatives of Olig3+ progenitor cells
We show here that Olig3 is expressed in the ventricular zone of the dorsal
alar plate of the hindbrain. The expression of the bHLH factors Math1, Ngn1,
Mash1 and Ptf1a further subdivided the Olig3+ progenitor domain. We
used genetic lineage tracing to follow the fate of the Olig3+
cells, which contributed to the nucleus of the solitary tract and to
precerebellar nuclei including the lateral reticular, external cuneate and
inferior olivary nuclei.
Climbing fiber neurons of the inferior olivary nucleus arise in the dorsal
alar plate, and undergo extensive migration before they settle in the ventral
medulla oblongata (Cobos et al.,
2001; Bloch-Gallego et al.,
2005). These neurons derive from Ptf1a+ progenitors and
depend on Ptf1a for determination of their fate
(Yamada et al., 2007). We show
here that Olig3 is also essential to determine the fate of these neurons. The
nucleus of the solitary tract and the area postrema are generated by dA3
viscerosensory relay neurons that co-express Phox2b and Tlx3
(Qian et al., 2001;
Dauger et al., 2003) (this
study), which appear to arise from a ventricular zone containing
Olig3+ and Mash1+ cells in rhombomeres 4-7. In
Olig3 mutant mice, the fate of dA3 viscerosensory relay neurons was
not correctly determined, and the nucleus of the solitary tract and the area
postrema were not formed. Mossy fiber neurons of the lateral reticular,
external cuneate, pontine, reticulotegmental nuclei, and the neurons of the
parabrachial nucleus appear to derive from cells expressing Math1 and Olig3 at
the rhombic lip (Bermingham et al.,
2001; Li et al.,
2004; Wang et al.,
2005; Farago et al.,
2006) (this study). Lhx2/9 neurons arose in Olig3 mutant
mice at reduced numbers, and lateral reticular, external cuneate, pontine,
reticulotegmental and parabrachial nuclei were small. Thus, Lhx2/9+
neurons of Olig3 mutant mice appeared to retain the ability to
migrate and differentiate, despite the fact that they ectopically expressed
Lbx1.
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); dA3,
viscerosensory relay neurons of the nucleus of the solitary tract and the area
postrema (Qian et al., 2001;
Dauger et al., 2003); and dA4,
climbing fiber neurons of the inferior olivary nucleus in rhombomere 7
(Yamada et al., 2007). dA2
neurons appear to arise from a ventricular zone that expresses Olig3 and Ngn1,
which exists in rhombomere 7 but not in other hindbrain segments
(Landsberg et al., 2005). With
the available markers, we have not as yet been able to trace dA2 neurons
during development. The cuneate nucleus is generated by an as yet unidentified
neuronal type that derives from Olig3+ progenitors. Future
experiments, for instance genetic lineage tracing of Ngn1+ cells,
might help to assign the fate of dA2 neurons.
Olig3, Ptf1a and neuronal fate determination
In the dorsal spinal cord and hindbrain, Ptf1a is expressed in a
ventricular zone that gives rise to Lbx1+/Pax2+
inhibitory and dA4 climbing fiber excitatory neurons of the inferior olivary
nucleus, and Ptf1a is essential to determine the fate of both neuronal types
(Glasgow et al., 2005;
Hoshino et al., 2005;
Yamada et al., 2007).
Electroporation of Ptf1a leads to the appearance of supernumerary inhibitory
neurons (Hoshino et al., 2005;
Wildner et al., 2006;
Hori et al., 2008). To
determine an inhibitory neuronal fate, Ptf1a forms a trimeric complex
consisting of Ptf1a, Rbpj and an E-box factor, such as Tcf12
(Hori et al., 2008). Rbpj is
best known as the major transcriptional mediator of Notch signaling, but its
role in the determination of an inhibitory neuronal fate appears to be
independent of Notch (Hori et al.,
2008). It is currently unclear whether a Ptf1a-Rbpj-Tcf12 complex
also functions in the fate determination of climbing fiber neurons.
In Olig3 mutant mice, the Ptf1a expression domain was expanded,
and supernumerary inhibitory neurons appeared. In control mice, the dorsal
Olig3+ domain was substantial in size early on (E11.5), but was
markedly smaller at later developmental stages. Accordingly, the domain that
generated misspecified neurons in Olig3 mutant mice became smaller as
development proceeded. We found that even at E18.5, the number of
Pax2+/Lbx1+ or Gad1+ neurons was increased in
the caudal medulla oblongata of the mutant mice. The physiological consequence
of the increased number of inhibitory neurons is unclear. Homozygous
Olig3 mutant mice become cyanotic and die shortly after birth,
apparently because they are unable to breathe. Breathing is controlled by a
network of neurons in the brainstem
(Feldman et al., 2003;
Dubreuil et al., 2008), and it
is tempting to speculate that an increased inhibition is responsible for the
breathing deficit of Olig3 mutant mice.
Olig3 and Ptf1a are essential to determine the fate of dA4 climbing fiber neurons of the inferior olivary nucleus, which appear to arise from a ventricular domain that contains cells expressing Ptf1a and Olig3 in rhombomere 7. We therefore tested whether the two factors cooperate, and co-electroporated Ptf1a and Olig3 expression vectors in the chick hindbrain. This led to an induction of Foxd3, a molecular characteristic of climbing fiber neurons. We therefore propose that Ptf1a and Olig3 cooperate to determine the fate of the climbing fiber neurons. In such experiments, ectopic Foxd3+ neurons were not observed close to the floor and roof plate, indicating that the two factors act in a context-dependent manner. Our model, a cooperation of Ptf1a and Olig3 during fate determination of climbing fiber neurons, is in apparent contradiction with the observed suppression of Ptf1a by Olig3. However, it should be noted that, in normal development, we found Olig3 to be expressed in Ki67- cells located laterally in the ventricular domain that appears to generate Foxd3+ climbing fiber neurons, indicating that Olig3 is transiently expressed in progenitors that have left the cell cycle and begun to differentiate. Ptf1a is expressed more broadly in this domain, and only a few cells co-expressed Ptf1a and Olig3 (Fig. 1E). Onset of Ptf1a and Olig3 expression thus appear to occur in distinct phases of the cell cycle, and result in a transient co-expression of the two factors in normal development. Distinct time courses of Ptf1a and Olig3 expression could thus explain the data.
Viscerosensory dA3 neurons and climbing fiber dA4 neurons were not
specified in Olig3 mutant mice, and Lbx1+ neurons arose at
their expense. We tested whether Olig3 exerts its role solely by suppressing
Lbx1. If this were the case, the changed fate determination of dA3 and dA4
neurons in Olig3 mutant mice should be reverted by the Lbx1
mutation. Analysis of Olig3; Lbx1 double-mutant mice
demonstrated that this was indeed the case for dA4, but not dA3, neurons.
Thus, Olig3 and Ptf1a together induce a dA4 fate, but the primary role of
Olig3 in this process is the suppression of Lbx1. Ptf1a is known to suppress
Tlx3 (Glasgow et al., 2005;
Mizuguchi et al., 2006;
Hori et al., 2008), and
Phox2b+ viscerosensory neurons are not generated in Tlx3
mutant mice (Qian et al.,
2001). It is tempting to speculate that the determination of the
Foxd3+ dA4 fate depends on the suppression of Lbx1 and Tlx3 by
Olig3 and Ptf1a, respectively.
Olig3 and the molecular mechanisms of neuronal fate determination in the spinal cord and hindbrain
Despite the greater complexity of neuronal types in the hindbrain as
compared with the spinal cord, and the distinct functions of hindbrain and
spinal neurons, neurons with similar molecular characteristics are frequently
generated in longitudinal columns that span the spinal cord and reach into the
hindbrain. This indicates that similarities exist in the mechanisms that
determine neuronal fates in the two units. Olig3 is expressed in the spinal
cord and hindbrain, and our analyses show some similarities in Olig3 function
in these two units. In particular, Olig3 marks the dorsal ventricular zone in
both, and a dorsal expansion of Lbx1 expression was observed in the spinal
cord and hindbrain of Olig3 mutant mice. Furthermore, in the spinal
cord and hindbrain of the chick, ectopic expression of Olig3 suppresses the
emergence of Lbx1+ neurons. Thus, several aspects of Olig3 function
are conserved in the spinal cord and hindbrain [compare
Fig. 9 with Muller et al.]
(Muller et al., 2005).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/295/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
Present address: Bayer Schering Pharma AG, 13353 Berlin, German
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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