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First published online 21 June 2006
doi: 10.1242/dev.02441
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1 Department of Human Genetics, University of Chicago, 920 E. 58th Street, CLSC
319 Chicago, IL 60637, USA.
2 Committee on Genetics, University of Chicago, 920 E. 58th Street, CLSC 319
Chicago, IL 60637, USA.
3 Departments of Pathology and Developmental and Cell Biology, University of
California, Irvine, D440 Medical Sciences I, Irvine, CA 92697-4800, USA.
4 Program in Developmental Biology, Howard Hughes Medical Institute, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
* Author for correspondence (e-mail: kmillen{at}genetics.bsd.uchicago.edu)
Accepted 12 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Roof plate, Dorsal midline, Signaling center, Cerebellum, Isthmic organizer, Lmx1a, Bmp, Neuronal specification, Proliferation, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). During
embryonic development it arises from dorsal rhombomere 1 (r1), adjacent to the
fourth ventricle (Chizhikov and Millen,
2003). Normal cerebellar development depends on formation and
function of the isthmic organizer (IsO), a signaling center defining the
cerebellar territory along the anterior-posterior axis of the central nervous
system (CNS) (Liu and Joyner,
2001; Wang and Zoghbi,
2001; Wurst and Bally-Cuif,
2001). As development proceeds, the cerebellar anlage gives rise
to several classes of neurons. Fate mapping studies have shown that Purkinje
cells and other GABAergic neurons, including a subpopulation of deep
cerebellar nuclei (DCN) neurons, basket, stellate and golgi neurons, all arise
from the ventricular zone in overlapping waves commencing at embryonic day
10.5-11.5 (E10.5-E11.5) in the mouse
(Altman and Bayer, 1997;
Goldowitz and Hamre, 1998;
Hoshino et al., 2005). The
rostral rhombic lip is another germinal matrix producing cerebellar
progenitors and represents the dorsal-most neuroectoderm of r1. It is defined
by expression of the transcription factor Math1 (Atoh1 - Mouse Genome
Informatics) (Wang et al.,
2005; Machold and Fishell.,
2005) (reviewed by Wingate,
2005). Math1+ progenitors give rise to superficially
migrating populations of pontomesencephalic neurons, large neurons of the DCN,
and cerebellar granule cells in separate birth cohorts
(Wang et al., 2005;
Machold and Fishell, 2005;
Wingate, 2005). Very few
cell-type-specific markers within the early cerebellar anlage are currently
available, and little is understood regarding the mechanisms driving early
cerebellar anlage patterning.
In many CNS regions, the specification of neural types is controlled by
secreted signals derived from local signaling centers
(Lumsden and Krumlauf, 1996;
Briscoe and Ericson, 2001).
This is best defined in the developing spinal cord where the neural tube is
initially subdivided into gene-expression domains along the dorsal-ventral
axis. Each domain gives rise to distinct subclasses of neurons. In the
developing spinal cord, the roof plate, a specialized group of cells,
differentiates shortly after neural tube closure to form a morphologically
distinct narrow strip of cells along the dorsal midline. Spinal cord roof
plate cells act as a signaling center producing Gdf7 and other Bmp-related
molecules and Wnt molecules to non-autonomously control specification of
numerous classes of adjacent dorsal interneurons
(Lee and Jessell, 1999;
Helms and Johnson, 2003;
Chizhikov and Millen, 2004b;
Chizhikov and Millen, 2005).
Intriguingly, many molecules expressed in the spinal cord roof plate are also
expressed in dorsal r1, raising the possibility that r1 roof plate acts as a
signaling center patterning the adjacent cerebellar anlage and controlling the
development of cerebellar cell types.
To address this hypothesis, we first defined several cellular populations of the early cerebellar anlage through gene expression and fate map studies. Using loss- and gain-of-function analysis, we conclude that specification of the cerebellar rhombic lip and its derivative fates is entirely dependent on r1 roof plate. In contrast, roof plate signaling is not absolutely required for specification of more distal cerebellar anlage populations. Rather, roof plate is required for normal positioning of these cells and regulates their numbers by controlling proliferation of the entire cerebellar anlage. Finally, our data indicate that a Bmp/Lmx1a pathway is required for r1 roof plate development and that Bmps are important components of its signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Jackson
Laboratories); Math1lacZ
(Ben-Arie et al., 2000),
Gdf7-DTA (Lee et al.,
2000) with ActB-Cre (Lewandovski et al., 1997) (Jackson
Laboratories) for roof plate ablation; r.p.Lmx1a-Cre (A.G.L., R.
Roberts and K.J.M., unpublished) or Gdf7-Cre
(Lee et al., 2000) with Rosa26
conditional lacZ reporter (129S-Gt(ROSA)26Sortm1Sor/J; Jackson Laboratories)
(Soriano, 1999) for
Gdf7 and Lmx1a fate mapping studies. Genotyping was
previously described (Ben-Arie et al.,
2000; Lee et al.,
2000; Millonig et al.,
2000; Monuki et al.,
2001). For transgenic mice, full-length mouse Lmx1a cDNA
was cloned into pNERV (Panchision et al.,
2001) and mice generated by the University of Chicago Transgenic
Mouse Facility. Founder embryos were collected at E12.5 and E16.5, and
genotyped by PCR (primers are available upon request).
Tissue analysis
Immunohistochemistry was performed as previously described
(Chizhikov and Millen, 2004a),
using the following primary antibodies: rabbit anti-Lmx1a (M. German,
unpublished), anti-Lhx2/9 (Lee et al.,
1998), anti-Ptf1a (H. Edlund, unpublished), mouse anti-Math1,
anti-Lhx1/5, anti-Msx1/2 (DSHB, The University of Iowa, Department of
Biological Sciences) and rat anti-ß-galactosidase (T. Glaser,
unpublished) together with secondary species appropriate antibodies (Jackson
Immunological). In situ hybridization was performed as previously
described (Chizhikov and Millen,
2004a) using mouse Lmx1a, Gdf7, Wnt1, Fgf8, Math1, CyclinD2,
Ptf1a and Lhx5 probes. X-Gal staining, histology, TUNEL assays
and BrdU labeling were performed as previously described
(Currle et al., 2005;
Panchision et al., 2001).
Explants
E8.5 dorsal neural plate and E8.75-E9.0 intermediate neural tube explants
were isolated as described by Alder et al.
(1999). Stage 12-15 chick r1
roof plate was isolated as described by Liem et al.
(Liem et al., 1997) and
co-cultured directly attached to intermediate neural explants. Explants were
cultured (Alder et al., 1999)
with or without noggin and follistatin (R&D Systems). Gdf7
expression was assayed by semi-quantitative RT-PCR as previously described
(Chizhikov and Millen,
2004c).
Measurements and statistical analysis
Serial 10 µm transverse or sagittal sections covering the entire
mid-hindbrain region were collected. Appropriate sections of r1 were
identified based on morphology and En1/2 staining. Quantitative data obtained
from analysis of paramedical sagittal (Lmx1a transgenic and dreher
embryos) and transverse (roof plate ablated embryos) sections are presented.
Quantitative data are expressed as the mean±s.e.m. Statistical
significance was determined by t-test,
*P<0.01.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transcription factors define distinct cellular populations within the early cerebellar anlage
By analyzing the expression of transcription factors which distinguish
neuronal populations in the spinal cord, we defined four cellular populations
(denoted c1-c4) within the E12.5 cerebellar anlage. c1 cells arise adjacent to
the r1 roof plate and express Math1
(Ben-Arie et al., 1996;
Alder et al., 1999). Similar to
E10.5 embryos, only very minimal overlap was detected between Lmx1a and Math1
at E12.5 (Fig. 2A) suggesting
that Math1+ cells remain distinct from Lmx1a+ roof plate
cells (Fig. 2G,H). Beginning
from E10.0, c1 cells and their progeny migrate rostrally and laterally to form
the rostral rhombic lip migratory stream (RLS) and immediately downregulate
Math1 as they exit the rhombic lip territory
(Wang et al., 2005). To
develop early markers of the RLS we used Math1lacZ/+
embryos, in which all RLS cells are transiently labeled by cytoplasmic LacZ
expression. Co-labeling revealed that at E12.5, nuclear Lhx2/9 (Lh2A/B)
expression was initiated immediately as c1 cells became engaged into the RLS
and was detected in all RLS cells, including those in the cerebellar anlage
and pons (Fig. 2B,C). Lhx2/9 is
therefore an early marker of the RLS.
c2 and c4 cells express Lhx1/5, while c3 cells express Lmx1a
(Fig. 2A,D,E). c2 cells are
located directly adjacent to the Math1+ c1 cells and can be
detected along the entire length of r1, except the most anterior domain
(Fig. 2G). At E13.5,
Lhx1/5+ c2 cells initiate expression of the Purkinje cell-specific
marker calbindin as they migrate toward the cerebellar surface (see Fig. S1A
in the supplementary material). At later embryonic stages and in the adult
cerebellum, these cells coexpress Lhx1/5 and calbindin, and acquire typical
morphology of Purkinje cells (see Fig. S1B in the supplementary material). In
the adult, Lhx1/5 is also expressed in GABA+ stellate, basket and
Golgi cells and in small DCN GABAergic neurons (see Fig. S1B in the
supplementary material), all of which originate from Ptf1a-expressing
progenitors (Hoshino et al.,
2005). Comparison of Ptf1a and Lhx1/5 expression at E12.5 revealed
that the Ptf1a expression precisely abuts c2 Lhx1/5 expression. Furthermore, a
small number of cells coexpress both these proteins
(Fig. 2D), strongly suggesting
that Ptf1a+ cells are progenitors of Lhx1/5+ c2 cells.
Therefore, we define Ptf1a+ cells as pc2 (progenitors of c2) cells.
Similar to c2 cells, c4 cells also express Lhx1/5, but are located at a more
ventral position within the E12.5 cerebellar anlage
(Fig. 2A,D). The fate of c4
cells is unknown.
The c3 population consists of Lmx1a+/Msx1/2- cells
(Fig. 2E). These are located
between c2 and c4 cells (Fig.
2A) and are found along the entire r1 except the most anterior
domain (Fig. 2G). c3 cells
initiate Lmx1a expression around E11.5-12.5 and represent the only
group of Lmx1a+ cells within the early cerebellar anlage
(Fig. 2A,E). The location of c3
cells at E12.5 resembles the nuclear transitory zone, a transient
differentiation zone of DCN neurons (Altman
and Bayer, 1985). At E17.5-E18.5, c3 cells become segregated into
several clusters found at the base of the cerebellar anlage (see Fig. S1C in
the supplementary material) corresponding to the positions of the nascent DCN.
Furthermore, at E14.5-E17.5 some of Lmx1a+ cells express
calretinin, a marker for a large population of differentiating DCN neurons
(Jacobowitz and Abbott, 1998)
(see Fig. S1D in the supplementary material). This suggests that c3 cells give
rise to DCN neurons. However, Lmx1a is localized to cells with large nuclei
which do not express GABA. This is in contrast to Lhx1/5 which is expressed in
cells with small nuclei (data not shown). Thus, c2 and c3 cells give rise to
distinct cellular populations. Since RLS cells also give rise to some large
DCN neurons, we assessed Lmx1a expression in Math1LacZ/+
embryos. We found that c3 cells do not originate from the RLS
(Fig. 2F) suggesting that c3
cells give rise to a previously unrecognized population of DCN cells.
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Rhombomere 1 roof plate gives rise to choroid plexus and does not significantly contribute to the developing cerebellum
Lineage tracing studies in the mouse have revealed that the roof plate
contributes to several neuronal populations in both the developing spinal cord
and the forebrain (Lee et al.,
2000; Monuki et al.,
2001). In r1, Lmx1a+/Gdf7+
roof plate cells are located directly adjacent to Math1+ c1 cells
suggesting that some rhombic lip cells or other cerebellar cells may be
clonally related to roof plate. To address this possibility we performed
genetic fate mapping studies. We used mice expressing Cre recombinase under
the control of the of Lmx1a promoter (r.p.Lmx1a-Cre; A.G.L.,
R. Roberts and K.J.M., unpublished), which specifically drives expression of
an endogenous gene to the roof plate at early developmental stages, or the
Gdf7 promoter (Gdf7-Cre)
(Lee et al., 2000). These mice
were crossed with ROSA26 cre reporter mice
(Soriano, 1999) to permanently
label roof plate derivatives (Fig.
3A,B). In E12.5 r.p.Lmx1a-Cre;ROSA26 embryos, LacZ
staining was detected in the choroid plexus, with no detectable overlap
between LacZ and c1 marker Math1 (Fig.
3C). At E18.5, very few LacZ+ cells were detected
within the cerebellum (Fig. 3D
and data not shown). This suggests therefore, that the roof plate contributes
cells to the choroid plexus and is not a significant source of cerebellar
cells.
A Bmp/Lmx1a pathway is involved in rhombomere 1 roof plate formation and can repattern the early cerebellar anlage
In the developing spinal cord, overexpression of Lmx1a induces
ectopic roof plate, which, in turn, repatterns the caudal neural tube
(Chizhikov and Millen, 2004a).
To test if Lmx1a has comparable activity in r1, we generated Lmx1a
transgenic mouse embryos overexpressing Lmx1a under control of the
nestin promoter, driving expression to the neuroepithelium from at least E9.5
(Panchision et al., 2001).
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Lmx1a overexpression also induced numerous c1 cells throughout the transgenic cerebellar anlage (4/6 embryos) (Fig. 4L-N). Induction of c1 cells was associated with additional RLS Lhx2/9+ cells (3/6 embryos), with some appearing at ectopic positions in the ventricular zone (Fig. 4O-Q). At E16.5, Lmx1a transgenic embryos (2/3) also had increased numbers of cerebellar granule progenitors in the external granule layer (see Fig. S2A-C in the supplementary material). In contrast, Ptf1a+ pc2, Lhx1/5+ c2 and c4 cells, and Lmx1a+/Msx1/2- c3 cells were greatly reduced in numbers in most (4/6) Lmx1a-transgenic embryos at E12.5 (Fig. 4I-K,R-W). Consistent with this, at E16.5 there was severe reduction or loss of calbindin+ Purkinje cells and GABAergic DCN neurons and Golgi cells in most (2/3) Lmx1a transgenics (see Fig. S2D-G in the supplementary material). These results indicate that Lmx1a is sufficient not only for r1 roof plate development, but it can also repattern the developing cerebellar anlage and substantially alter subsequent cerebellar cell fates.
To identify upstream components of the Lmx1a signaling pathway we
concentrated on Bmps, since Bmp6 and Bmp7 are expressed in
the rostral epidermal ectoderm at the time of r1 roof plate formation
(Furuta et al., 1997;
Dudley and Robertson, 1997).
We isolated the dorsal-most edges of the r1 neural plate together with
adjacent epidermal ectoderm at E8.5, when Lmx1a expression is
initiated (Fig. 5A). When
cultured alone for 24 hours, numerous Lmx1a+ cells and high levels
of Gdf7 expression (Fig.
5B-E) were induced. indicating successful formation of roof plate
in vitro. This induction was significantly blocked by the addition of Bmp
inhibitors noggin (30 nM) and follistatin (130 nM)
(Fig. 5B-E). Therefore, Bmps
act upstream of Lmx1a in the r1 roof plate-inducing signaling
pathway.
Bmps are necessary mediators of roof plate signaling in the developing cerebellar anlage
Previously, Alder et al. (Alder et al.,
1999) determined that Bmps can induce granule cells when added to
naïve r1 neural tissue in vitro, suggesting that they may also be
mediators of r1 roof plate signaling. To determine if roof plate Bmps are
required for its patterning activity, we performed explant experiments.
Specifically, we tested whether roof plate-dependent induction of c1 cells was
blocked by noggin and follistatin using intermediate explants from E8.75-E9.0
mouse mid/hindbrain neural plate or newly formed neural tube
(Fig. 5F). Explants were
cultured for 3 days alone or together with stage 12-15 chicken r1 roof plate
with or without noggin and follistatin. No Math1+ c1 or
Lhx2/9+ RLS cells were detected when intermediate explants were
cultured alone, but significant numbers of both Math1+ and
Lxh2/9+ cells were induced when they were co-cultured together with
chick r1 roof plate (Fig.
5G-N). Addition of noggin (20 nM) and follistatin (85 nM) did not
affect the mature roof plate used in these experiments, as revealed by its
normal Lmx1a (Fig.
5I,M) and Gdf7 expression (data not shown), but
significantly blocked (by 80-85%) induction of both c1 and Lhx2/9+
RLS cells (Fig. 5I,M,J,N). Thus
we conclude that Bmps in r1 roof plate are required for induction of c1 cells
and their derivative RLS cells.
Cerebellar anlage abnormalities in dreher (Lmx1a-/-) mouse embryos
To investigate the effects of roof plate disruption on cerebellar anlage
development, we first analyzed dreher (Lmx1a-/-) embryos.
In the dreher mouse, inactivation of Lmx1a by point mutation
prevents roof plate formation in the developing spinal cord
(Millonig et al., 2000;
Chizhikov and Millen, 2004a).
In dreher embryos, r1 roof plate territory is severely reduced in
size starting from E9.5, but limited marker analysis suggested that r1 roof
plate cells are still present (Millonig et
al., 2000). Here we performed more detailed analysis. Our data
indicate that the anterior roof plate, which expresses low Gdf7
levels and does not express Wnt1, is expanded at the expense of
posterior r1 roof plate in dreher embryos
(Fig. 6A-D,G-H). Gdf7
and Wnt1, however, are still expressed in the reduced posterior r1
roof plate of E10.5 dreher embryos
(Fig. 6A-D). These data
indicate that although Lmx1a is not absolutely required for
development of the roof plate in r1, it is critical for a normal relationship
between anterior and posterior domains of r1 roof plate. Abnormal patterning
of the r1 roof plate did not affect development of the IsO, which was normal
in the dreher embryos, based both on morphological criteria and
retention of isthmic-specific expression of Wnt1, Fgf8, Gbx2 and
Otx2 (Fig. 6C,D and
data not shown). Thus, loss of Lmx1a causes abnormal development of
r1 roof plate but does not interrupt global anterior-posterior patterning of
the mid/hindbrain territory.
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We did not detect any defects in expression of Ptf1a, Lhx1/5 or Lmx1a, marking the pc2, c2 and c3 cell populations (Fig. 6K-R; see Fig. S3 in the supplementary material) at E12.5 in dreher embryos. These data indicate that the relatively mild abnormality in r1 roof plate is not sufficient to grossly disrupt patterning of the E12.5 cerebellar anlage in this mouse model.
The observation that the anterior limit of Ptf1a, Lhx1/5 and Lmx1a was unchanged in dreher embryos was surprising. In wild-type embryos, these genes are expressed only within the posterior r1 domain and not the anterior `negative' domain (labeled as `neg' in Fig. 6K-R). This negative domain normally correlates with the morphological extent of the anterior roof plate (labeled as `a' in Fig. 6). In dreher embryos these two domains no longer coincide (Fig. 6Q,R). Two hypotheses can explain this. First, anterior roof plate is sufficient to support formation of pc2, c2 and c3 cells when expanded into caudal r1. Alternatively, the development of pc2, c2 and c3 cells may be independent of r1 roof plate signals.
Cerebellar anlage abnormalities following roof plate ablation by Gdf7-mediated diphtheria toxin expression
To investigate the effect of complete loss of the roof plate on early
cerebellar anlage development, we moved to a genetic ablation mouse model
using a mouse line that conditionally expresses the diphtheria toxin A subunit
(DTA) from the Gdf7 locus beginning at E9.0
(Fig. 7A)
(Lee et al., 2000). Expression
from this conditional Gdf7 allele faithfully recapitulates expression
of endogenous Gdf7 (Lee et al.,
2000; Currle et al.,
2005) (data not shown).
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To confirm specificity and determine timing of r1 roof plate ablation, we performed TUNEL labeling at E8.5-E12.5. At E8.5, no differences in apoptosis or neural plate morphology were detected between wild-type and ablated embryos (data not shown). In E9.5 roof plate-ablated embryos, apoptosis was specifically elevated in the most dorsal edges of r1, exactly where Gdf7 is normally expressed (see Fig. S4A,B in the supplementary material). At E10.5-E12.5, no significant differences in apoptosis were detected between wild-type and ablated embryos (see Fig. S4C,D in the supplementary material; data not shown), indicating the temporal and spatial specificity of ablation. Further, no dorsal expression of Lmx1a, Gdf7 or Wnt1 was detected in the mutant embryos (Fig. 7B-E and data not shown) revealing complete loss of the r1 roof plate cells. Despite the open neural tube, the IsO was developed normally in ablated embryos, as assayed by Wnt1 and Fgf8 expression (Fig. 7D,E), indicating that roof plate is not required for maintenance of the IsO.
There was complete loss of Math1+ c1 cells in both E10.5 (Fig. 7F,G) and E12.5 (Fig. 8G,H) roof plate-ablated embryos. Also, no Lhx2/9+ RLS cells were detected in the cerebellar anlage or pons of roof plate-ablated embryos at E12.5 (Fig. 8M,N). These data indicate that loss of r1 roof plate prevents formation of both c1 cells and their derivative Lhx2/9+ RLS cells.
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Reduced numbers of pc2, c2 and c3 cells
(Fig. 8I,L) and the overall
reduced size of the cerebellar anlage (Fig.
7L) suggested abnormal proliferation in ablated embryos. This was
confirmed by BrdU labeling at E10.5 (Fig.
7M,N). The decrease in proliferation is unlikely to be mediated by
loss of c1 cells in ablated embryos, since no gross proliferation
abnormalities were detected in E10.5 Math1LacZ/LacZ
embryos in which development of c1 cells is severely disrupted (data not
shown). Instead, decreased proliferation may be associated with loss of dorsal
expression of the well-known mitogen Wnt1 in roof plate ablated
embryos (Fig. 7D,E). In the
developing spinal cord, Wnt1 is sufficient to increase cellular proliferation
by activating transcription of members of the cyclin D family
(Megason and McMahon, 2002).
We noted that cyclin D2 is normally highly expressed in the early cerebellar
anlage (Fig. 7H,O). Roof plate
ablation was associated with loss of cyclin D2 expression in this region
(Fig. 7I,P), suggesting that r1
roof plate at least partially activates cerebellar proliferation by regulating
cyclin D2 expression.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The r1 roof plate and mechanisms of its formation
There are considerable discrepancies in the literature regarding the
definition of roof plate in r1. Here we have defined the roof plate, based on
gene expression and lineage analysis, as the
Lmx1a+/Gdf7+ dorsal domain throughout
the anterior/posterior extent of r1 at E10.5. In anterior r1, both
Lmx1a and Gdf7 are restricted to a narrow medial domain
(Fig. 1J). In posterior r1,
they are expressed in both the expanded medial single cell layer and in the
edges of the adjacent neuroepithelium, where the
Lmx1a+/Gdf7+ domain significantly
overlaps with the Wnt1+ territory
(Fig. 1B-E,J,K). Despite the
neurepithelial morphology and ongoing proliferation in this edge domain, it
does not express Math1, a marker of the rhombic lip, and does not contribute
significantly to the adjacent cerebellum. Rather, fate mapping data indicate
that Lmx1a+/Gdf7+ cells (this study)
(Currle et al., 2005;
Landsberg et al., 2005) and
early Wnt1+ cells
(Zervas et al., 2004)
contribute to the choroid plexus.
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In the developing spinal cord, Lmx1a is absolutely necessary for
roof plate development (Millonig et al.,
2000). Loss of Lmx1a in r1, however, resulted in an
interesting roof plate developmental defect instead of its complete loss, as
predicted from our spinal cord studies
(Millonig et al., 2000). In
particular, the anterior roof plate was expanded at the expense of the
posterior roof plate in dreher r1. Lmx1a is therefore
required to establish the normal relationship between anterior and posterior
r1 roof plate. Chick-quail transplants have shown that the r1 anterior and
posterior roof plates have distinct origins. The anterior roof plate is
derived from a small dorsomedial population of isthmus-derived cells, which
migrate caudally, stopping at the boundary between anterior and posterior r1
(Louvi et al., 2003;
Alexandre and Wassef, 2003).
Other genes known to influence the extent of r1 anterior roof plate include
Wnt1 and Otx2 (Louvi et
al., 2003), both of which disrupt global isthmic patterning when
mutant (Liu and Joyner, 2001).
Loss of Lmx1a, however, does not influence the positioning of the
isthmus or expression of general markers of the mid/hindbrain territory such
as Otx2 and Gbx2. It is possible that the low Lmx1a
expression levels in the anterior roof plate are cell-autonomously required to
stop excessive caudal migration of this midline population. Alternatively,
high Lmx1a levels in the posterior roof plate may normally limit the
extent of the anterior roof plate. Further experiments are required to
distinguish these mechanisms. Regardless, the presence of residual r1 roof
plate in dreher embryos indicates that other genes can partially
compensate for loss of Lmx1a, revealing redundancy in roof
plate-inducing mechanisms in r1.
Roof plate signaling controls specification of the adjacent rhombic lip and its derivative cells, and influences proliferation throughout the entire cerebellar anlage
To understand the role of r1 roof plate in cerebellar patterning, we first
developed early markers for cerebellar neuronal progenitors and newly
differentiated neurons. By analyzing the expression patterns of additional
transcription factors, we determined that the E12.5 cerebellar anlage is
divided into several discrete populations (c1-c4), providing the first
comprehensive molecular map of the entire early cerebellar anlage. To test the
role of the r1 roof plate on specification of these cellular types, we
performed gain- and loss-of-function experiments in mice. Induction of ectopic
roof plate by Lmx1a expression was associated with significant
repatterning of the developing cerebellar anlage. This included induction of
c1 cells and their derivative RLS cells (including granule cell progenitors of
the external granule layer), and loss of c2-c4 cells and their derivative
Purkinje cells and other GABAergic neurons. Loss of c2 and c4 cells can be
explained by cell-autonomous conversion of their ventricular zone progenitors
into ectopic roof plate cells by exogenous Lmx1a. In contrast, the appearance
of excessive c1 cells adjacent to the ectopic Lmx1a-induced roof
plate indicates the non-autonomous patterning activity of the ectopic roof
plate. Previously it has been shown that Bmps can induce granule cells when
added to naïve r1 neural tissue in vitro
(Alder et al., 1999). These
studies, however, could not conclusively distinguish whether Bmps directly
induce c1 cells or they first induce roof plate structures, which, in turn,
produce other, Bmp-unrelated signals, to induce c1 cells and their derivative
granule cells. Our explant experiments indicate, however, that Bmps are direct
mediators of roof plate signaling in rhombomere 1, since roof plate-dependent
induction of Math1+ c1 cells and derivative Lhx2/9+ RLS
cells is blocked by the Bmp inhibitors noggin and follistatin. These data are
in good agreement with the results of a recent genetic study that showed that
Bmp receptors 1a and 1b are required for development of cerebellar granule
cells (Qin et al., 2006). At
the same time however, the Bmp inhibitors used in our explant experiments
significantly blocked, yet did not completely prevent, the appearance of c1
and Lhx2/9+ RLS cells. This suggests that other roof plate-derived
secreted factors may also be involved in the development of c1 cells. It is
also possible that the repatterning of the cerebellar anlage observed in our
Lmx1a transgenic embryos is associated with activation of expression
of not only Gdf7, detected in the current study but also other
factors. Since Lmx1a activates expression of Wnt1 in the developing
spinal cord (Chizhikov and Millen,
2004a), members of the Wnt family represent good candidates for
this activity.
The dependence of c1 induction on roof plate signaling was further demonstrated by analysis of dreher and roof plate-ablated embryos. In the dreher embryos, which display relatively mild roof plate abnormalities, c1 cells were lost but only in the most anterior domain of r1, further emphasizing differences in patterning mechanisms operating in anterior and posterior r1 dorsal domains. In roof plate-ablated embryos, all c1 cells and their RLS derivatives were completely lost in both the anterior and posterior r1, leading to our conclusion that the roof plate is absolutely required for their specification in vivo.
Surprisingly pc2, c2 and c3 cells were still generated in both dreher and roof plate-ablated embryos. These cellular fates are therefore specified independently of roof plate signals. Although this mechanism remains unresolved, absence of c2 and c3 cells from the most anterior region of r1 in wild-type embryos suggests that IsO signaling negatively influences their development.
Although c2 cells and c3 cells appeared at the appropriate time in roof plate-ablated embryos, they were generated in significantly reduced numbers and were displaced. Reduction of these cells was associated with loss of cyclin D2 expression and decreased proliferation of the cerebellar anlage, indicating that the roof plate controls the total number of c2, c3, and probably other cerebellar cells, at least partially by regulating proliferation of the early cerebellar anlage by activating cyclin D2 expression. Dorsal Wnt1 expression may normally provide this signal. The displacement of c3 cells argues that that roof plate is also required for the normal positioning of this population. This phenotype may be a direct result of loss of roof plate signaling, or may be mediated by loss of the c1-derived RLS cells in roof plate-ablated embryos.
We conclude that the r1 roof plate is an important signaling center
required for the specification of directly adjacent c1 cells and their
derivative RLS cells. r1 roof plate is not required for initial specification
of more distant early cerebellar populations, such as the c2 and c3 cells
defined in this study. The roof plate however does regulate the numbers of
these distant populations, at least partially, by regulating proliferation of
cerebellar progenitors, and controls positions of some of these cells within
the developing cerebellar anlage. This contrasts with the situation in the
developing spinal cord, where the roof plate is absolutely required for
development of the immediately adjacent (dI1) population and two more distant
populations (dI2 and dI3) of dorsal interneurons
(Lee et al., 2000). Taken
together with significant gene expression differences between anterior and
posterior r1, our data argue that r1 dorsal cell fates rely on more complex
mechanisms than those in posterior CNS axial levels. Our comprehensive gene
expression map will be useful for deciphering these mechanisms.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/15/2793/DC1
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ACKNOWLEDGMENTS |
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