RESEARCH ARTICLE 3399
Development 138, 3399-3408 (2011) doi:10.1242/dev.065482
© 2011. Published by The Company of Biologists Ltd
Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in
ventral midbrain development
Qiaolin Deng1,2, Elisabet Andersson1,*, Eva Hedlund2,*, Zhanna Alekseenko1, Eva Coppola3, Lia Panman2,
James H. Millonig4, Jean-Francois Brunet3, Johan Ericson1,† and Thomas Perlmann1,2,†
SUMMARY
The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at
deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are
important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or
specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into
molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b.
Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves
the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to
originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for
Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons
(RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the
differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the
differentiation of the DN lineage.
INTRODUCTION
Several types of neurons are generated in the ventral midbrain
including DNs, OMNs, RNNs and GABAergic interneurons. They
are all associated with essential neuronal functions and are also
clinically relevant. DNs are important for physiological functions
such as motor control, cognition and reward, and the degeneration of
these cells is a hallmark of Parkinson’s disease (Bjorklund and
Dunnett, 2007; Haber and Fudge, 1997; Marsden, 1994; Van den
Heuvel and Pasterkamp, 2008). A better understanding of DN
development in relation to other neuronal subtypes generated in the
ventral midbrain could facilitate the directed differentiation of stem
cells into DNs for cell replacement therapy for Parkinson’s disease
(Arenas, 2010; Hedlund and Perlmann, 2009; Thomas, 2010).
However, although several signals and transcriptional regulators that
influence the generation of DNs have been identified, it still remains
poorly understood how such factors are mechanistically integrated
into gene regulatory networks that underlie the precise spatiotemporal
specification of cell types in the developing ventral midbrain.
Functionally distinct neuronal subtypes are generated at specific
positions and at defined time points during nervous system
development. Inductive signals play key roles in neuronal
specification by controlling the expression patterns of transcription
factors that, in turn, establish distinct neural progenitor domains via
cross-repressive transcriptional interactions (Briscoe et al., 2000;
1
Karolinska Institutet, Department of Cell and Molecular Biology, von Eulers väg 3,
171 77 Stockholm, Sweden. 2Ludwig Institute for Cancer Research, Nobels väg 3,
Karolinska Institutet, 71 77 Stockholm, Sweden. 3Institut de Biologie de l’Ecole
Normale Supérieure (IBENS), CNRS UMR8197, INSERM U1024, 75005, Paris, France.
4
UMDNJ, Neuroscience and Cell Biology, CABM, 679 Hoes Lane, Piscataway,
NJ 08854, USA.
*These authors contributed equally to this work
Author for correspondence ([email protected]; [email protected])
†
Accepted 10 June 2011
Gunhaga et al., 2003; Muhr et al., 2001; Scholpp et al., 2003).
Moreover, these factors specify neuronal subtype identities as
progenitors leave the cell cycle and differentiate into post-mitotic
neurons (Jessell, 2000). DNs are derived from a pool of neural
progenitors located at the ventral midline of the developing
midbrain. In the midbrain, signaling by Shh (sonic hedgehog),
FGF8 (Fibroblast growth factor 8) and Wnts (Wingless-related
MMTV integration site) promote the generation of DNs (Cajanek
et al., 2009; Hynes and Rosenthal, 1999; Joksimovic et al., 2009b;
McMahon and Bradley, 1990), but these factors also underlie the
induction of other ventrally derived neuronal subtypes, such as
OMNs, RNNs and GABAergic inhibitory interneurons (PerezBalaguer et al., 2009; Prakash et al., 2006; Ye et al., 1998). In
addition, several transcription factors that influence cell patterning
and the differentiation of ventral midbrain neurons have been
identified (Andersson et al., 2006b; Ferri et al., 2007; Hasan et al.,
2010; Kala et al., 2009; Nakatani et al., 2007; Pattyn et al., 1997;
Puelles et al., 2003).
The LIM-homeodomain proteins Lmx1a and Lmx1b are coexpressed in DN progenitors and differentiating DNs and have been
shown to mediate key functions in the initial steps of DN fate
specification (Andersson et al., 2006b; Smidt et al., 2000). Lmx1a is
specifically induced in DN progenitors around embryonic day (E)
9.0 in mice. By contrast, Lmx1b is more broadly expressed in the
ventral midbrain at early developmental stages and becomes
restricted to DN progenitors only at E10.5 when DNs begin to
differentiate (Andersson et al., 2006b). This raises the possibility that
Lmx1b is not only important for developing DNs but may also
influence specification of other cell types generated at more lateral
positions of the ventral midbrain. Moreover, both Lmx1a and Lmx1b
are sufficient to induce DNs when ectopically expressed in the
midbrain (Andersson et al., 2006b; Lin et al., 2009; Nakatani et al.,
2010) or when expressed in differentiating pluripotent stem cells
(Andersson et al., 2006b; Friling et al., 2009; Chen et al., 2009).
DEVELOPMENT
KEY WORDS: Lmx1a/b, Midbrain, Phox2a, Dopamine neuron, Ocular motorneuron, Red nucleus interneuron, Mouse
3400 RESEARCH ARTICLE
MATERIALS AND METHODS
Mouse mutants
All experiments on animals have been approved by the Swedish ethical
council. The DreherJ (referred to as dr in the text) mouse was obtained
from J.H.M.; the Lmx1b-null mouse was obtained from T. Jessell
(Columbia University, NY, USA) and M. Mendelsohn (Columbia
University, NY, USA); and the Phox2a-lacZ mouse has been described by
Jacob et al. (Jacob et al., 2000). All lines were maintained on a C57BL/6J
background. Genotyping was carried out as previously described (Chen et
al., 1998; Millonig et al., 2000). The Lmx1aeGFP/+ mouse was generated by
the Institut Clinique de la Souris, France. The cloning strategy is shown in
Fig. S1A in the supplementary material. In short, the genomic 3⬘ and 5⬘
Lmx1a arms were isolated using PCR, subcloned and verified by
sequencing. The 3 kb 5⬘ arm was cloned into a lox-neo-lox (lNeol) vector
using FseI/PacI. The enhanced green fluorescent protein (eGFP) and the
4.5 kb 3⬘ arm were added using PacI/SgrAI and SbfI/SfiI, respectively.
Homologous recombination was confirmed using Southern blot
hybridization with 5⬘ and 3⬘ external probes. Exon 1, intron 1, exon 2 and
part of intron 2 were removed. The Lmx1a conditional knockout mouse
(Lmx1acko/+) was generated in our laboratory. The cloning strategy is shown
in Fig. S1B in the supplementary material. In short, the genomic 3⬘ and 5⬘
Lmx1a arms were amplified by PCR from RPCI-21 PAC library and
verified by sequencing. Single loxP sites was inserted upstream of exon 1
together with an extra BglII site used for screening. The lNeol cassette was
inserted 288 nucleotides downstream of exon 2, in intron 2, with an extra
ApaLI site for screening. The initial screen was performed by long-range
PCR (internal neo primer and 3⬘ external primer) and the final validation
was carried out using Southern blot hybridization with a 5⬘ and 3⬘ external
probe (see Fig. S1Ba,b in the supplementary material). Chimeric mice were
generated by KCTT, Karolinska Institutet, Sweden. Germline transmission
was verified by Southern blot and PCR. This mouse line was maintained
as a homozygous line (Lmx1acko/cko) and showed a behavior that was
indistinguishable from wild-type mice. To delete exon 1, intron 1, exon 2
and part of intron 2 of the Lmx1a gene, the Lmx1acko/cko mouse line was
crossed with a CMV-Cre mouse line and the resulting mouse line was
termed Lmx1a+/–. This line was maintained as heterozygote and
subsequently intercrossed to generate Lmx1a-null mice (Lmx1a–/–).
Immunohistochemistry and in situ hybridization
The following antibodies were used: mouse Foxa2, Lim1/2, Nkx2.2, Shh and
Nkx6.1 (Developmental Studies Hybridoma Bank); mouse TH and Brn3a
(Chemicon); mouse Ngn2 (a gift from D. Anderson, California Institute of
Technology, CA, USA); and mouse Gata3 (Santa Cruz); rabbit GFP
(Molecular Probes), rabbit Lmx1a (a gift from M. German, UCSF, CA,
USA), rabbit Nurr1 (Santa Cruz), rabbit TH (PelFreeze), rabbit Phox2a
(prepared by J.-F.B.) and rabbit Corin (a gift from M. Parmar, Lund
University, Lund, Sweden); guinea pig Lmx1a, Lmx1b and Pitx3 (prepared
by J.E.), guinea pig Isl1/2 (T. Jessell) and guinea pig Phox2b (prepared by
J.E.); goat -galactosidase (Biogenesis) and goat GFP (Abcam). In situ
hybridization on sections was carried out essentially as previously described
(Briscoe et al., 2000) using probes for Wnt1 (Puelles et al., 2003), Dll1
(Lindsell et al., 1996), Ngn2 (Fode et al., 1998), Hes5 (Akazawa et al., 1992),
Tcf12 (Riken clone AK078415), Drd2 (Geneservice I.M.A.G.E ID 13489)
and Sim1 (Pierani et al., 1999).
Quantification of DNs in Lmx1a and Lmx1b mutant mice
For evaluation of the importance of Lmx1a and Lmx1b for DN generation
in the ventral midbrain, DN markers were counted at E11.5 (Nurr1), E13.5
(Nurr1 and Pitx3) and P0 (TH) in wild-type, dr/dr, Lmx1a-null, Lmx1bnull and/or Lmx1a/b compound mutant mice. At E11.5 and E13.5, all
Nurr1+ and/or Pitx3+ neurons in the ventral midbrain were counted in four
consecutive sections/embryo (four embryos per genotype). At P0, TH+
neurons in the midbrain were counted unilaterally with a subdivision into
two regions, 0-250 M and 250-500 M from the midline. One out of five
sections (12 m sections) were counted across four sections/animal. Four
wild-type and one heterozygous (included in the wild-type group) and five
Lmx1aeGFP/eGFP animals were counted. All counts were performed in a
double-blind fashion.
DNA constructs and in ovo electroporation
cDNAs encoding mouse Lmx1a (Riken; AK044944), Lmx1b (Q. Ma) and
Lmx1adr (mimicking dr mutation with C82Y) in RCASBP were
electroporated (Briscoe et al., 2000) at 1.5 g/l into the midbrain of HH
stage 10-12 chick embryos. Chick embryos were analyzed 84 hours after
electroporation.
cDNAs encoding mouse Lmx1a in CAGG:iresGFP was electroporated
at 0.8 g/l into the midbrain of HH stage 10-12 chick embryos. Chick
embryos were analyzed 48 hours after electroporation.
Optical density measurements
Optical density of Lmx1b and GFP was measured in DNs that were
transfected with CAGG-Lmx1a:iresGFP construct using ImageJ
(http://imagej.nih.gov/ij/). Confocal images used for the optical density
measurements were carefully captured at a setting where all cells displayed
signal intensity below the saturation level. A few extreme high level of
GFP+ cells were excluded from the measurements. The optical density
values were normalized by the medium optical density for each embryos
(n7) and the relationship depicted in a scatter plot.
Statistical analysis
P0 experimental data was analyzed using Student’s t-test and ANOVA.
InStat3 software (GraphPad software) was used for the statistical analyses.
Experimental data from other different embryonic stages was analyzed
using mean±s.e.m. values.
DEVELOPMENT
However, the precise roles for Lmx1a and Lmx1b during
embryogenesis remain poorly characterized and it is unclear to what
degree Lmx1a and Lmx1b mediate redundant or specific activities
in midbrain patterning and neuronal fate determination. DN
development is affected both in Lmx1b-null mutants and in
homozygous dreher (dr/dr) mice carrying an amino acid substitution
of a conserved cysteine residue within the first LIM domain of the
Lmx1a protein (Ono et al., 2007; Smidt et al., 2000). Thus, these two
proteins cannot fully compensate for each other. Importantly,
however, although Lmx1a protein expression is abolished in the
dorsal neural tube in dr/dr embryos, the expression persists in DN
progenitors and differentiating DNs within the ventral midbrain and
it remains possible that the DN phenotype in dr/dr embryos reflects
a partial loss of Lmx1a activity (Millonig et al., 2000) (see also
below). Additionally, few studies have addressed the requirement for
Lmx1b in ventral midbrain development (Smidt et al., 2000) and the
precise role for Lmx1b in cell patterning and neuronal fate
determination therefore remains unresolved.
To delineate more conclusively the roles of Lmx1a and Lmx1b
during ventral midbrain development, we generated two different
Lmx1a-null mouse strains where the first two exons were removed,
and in one strain replaced with a sequence encoding the enhanced
green fluorescence protein (eGFP). Midbrain patterning and
specification of DNs were compared in dr/dr, Lmx1a- and Lmx1bnull embryos, and in Lmx1a/Lmx1b compound mutants. Analysis
of these transgenic mouse lines revealed both unique and redundant
roles of Lmx1a and Lmx1b in DN generation. Notably, Lmx1a is
more important for neurogenesis in the medial DN domain,
whereas Lmx1b is more important for the generation of lateral
DNs. In addition, our study revealed a broader role for Lmx1b in
midbrain development. Phox2a has previously been shown to
specify OMNs within the ventral midbrain (Hasan et al., 2010;
Nakano et al., 2001; Pattyn et al., 1997). We show here that Lmx1b
is required for the expression of Phox2a, and influences the
temporal specification of OMNs and RNNs in the ventral midbrain.
Thus, Lmx1b and Phox2a are part of a novel gene regulatory
network that controls the spatiotemporal generation of DNs, OMNs
and RNNs during midbrain development.
Development 138 (16)
RESULTS
Generation of Lmx1a-null mice
To determine whether the reduction of DNs observed in dr/dr mice
reflects a partial or complete loss of Lmx1a activity (Ono et al.,
2007), we generated two new Lmx1a mutant mouse strains
(Lmx1aeGFP and Lmx1acko) (see Fig. S1 in the supplementary
material). In Lmx1aeGFP/+ heterozygous mice, exons 1 and 2 in one
Lmx1a gene copy were replaced with a sequence encoding eGFP
(see Fig. S1A in the supplementary material). In heterozygous
Lmx1aeGFP/+ embryos, expression of the eGFP reporter
recapitulated the expression profile of Lmx1a in the ventral
midbrain, as well as in other regions in which Lmx1a is expressed
during embryogenesis (Fig. 1A; data not shown). In heterozygous
Lmx1acko/+ mice, LoxP sites were introduced in one Lmx1a gene
copy allowing conditional deletion of exons 1 and 2 (see Fig. S1B
in the supplementary material). Lmx1a-null mutant embryos
(termed Lmx1a–/–) were generated by using these mice in crosses
with ‘deleter’ mice expressing the Cre-recombinase under the
Fig. 1. dr/dr, Lmx1aeGFP/eGFP and Lmx1a–/– mice show a similar DN
phenotype in the ventral midbrain. (A)In E12.5 Lmx1aeGFP/+
embryos, eGFP is expressed in discrete locations, including the ventral
midbrain (Vm), roof plate and cortical hemisphere (BF, bright field).
Sections of ventral midbrain (bottom) show that eGFP is co-expressed
with Lmx1a. (B)Transverse sections of ventral midbrain at E11.5 show
that Lmx1a protein is undetectable in Lmx1aeGFP/eGFP and Lmx1a–/–, but
is present in dr/dr and wild type. (C)At E13.5, Nurr1+/Pitx3+ DNs were
reduced in dr/dr, Lmx1aeGFP/eGFP and Lmx1a–/– compared with wild type.
(D)Analysis of Nurr1+ DNs at E11.5 and E13.5, and Pitx3+ DNs at
E13.5. Values are presented as mean±s.d., n4. (E)In chick
electroporation, Lmx1awt and Lmx1bwt induce Nurr1+ cells 84 hours
post-transfection, but not the eGFP control or a construct carrying the
dr mutation of Lmx1a (Lmx1adr). Endogenous post-mitotic DNs are
indicated by the yellow dotted line.
RESEARCH ARTICLE 3401
CMV promoter (Su et al., 2002). Importantly, in contrast to wildtype and dr/dr mice, no Lmx1a protein was detected in the ventral
midbrain of either Lmx1aeGFP/eGFP or Lmx1a–/– embryos at E11.5
(Fig. 1B). Thus, Lmx1aeGFP/eGFP and Lmx1a–/– mice represent
Lmx1a-null mutants.
We next examined the generation of DNs in the ventral midbrain
at E11.5 and E13.5 in the different Lmx1a mutant backgrounds. In
both Lmx1aeGFP/eGFP and Lmx1a–/– embryos, the number of DNs
was significantly reduced, as determined by analyzing the coexpression of Nurr1 and Pitx3 at E13.5 (Fig. 1C). Quantification
of Nurr1 and Pitx3 expressing cells showed that the partial loss of
DNs was similar in the different mutant backgrounds (~60% at
E11.5 and ~30-40% at E13.5; Fig. 1D). Thus, these data reveal that
Lmx1a is not absolutely required for the specification of DNs and
also suggest that the dr mutation results in a complete loss of
Lmx1a function. This conclusion is supported by experiments
showing that the ability of Lmx1a to induce ectopic DNs in the
chick midbrain was completely abolished after introducing the dr
mutation in the Lmx1a expression construct (Fig. 1E; data not
shown).
Non-redundant functions of Lmx1a and Lmx1b in
DN fate specification
Lmx1a and Lmx1b are co-expressed in DN progenitors and
differentiating DNs in wild-type embryos at E11.5 (Fig. 2A),
although Lmx1b is more broadly expressed in the ventral midbrain
at early developmental stages (Andersson et al., 2006b) (see
below). Thus, Lmx1b may functionally compensate for the loss of
Lmx1a. The DN progenitor domain in Lmx1aeGFP/eGFP embryos
was similar in size to that of wild-type embryos, as determined by
Lmx1b and Foxa2 expression at E11.5 (Fig. 2A,B,D,E). Despite
the normal size of the DN progenitor domain in Lmx1aeGFP/eGFP
embryos, generation of post-mitotic DNs was limited to lateral
positions and essentially no Nurr1+/TH+ cells could be detected at
the ventral midline of the midbrain at E11.5 (Fig. 2E,H) (Ono et
al., 2007). Moreover, the expression of Lmx1b was significantly
upregulated in the DN progenitor domain of Lmx1aeGFP/eGFP
embryos (Fig. 2B). The upregulated expression was seen in the
entire Lmx1a-postive progenitor domain as determined by double
staining for both Lmx1b and Corin (see Fig. S2 in the
supplementary material). Thus, these results identify a unique
requirement for Lmx1a in the control of DN neurogenesis in the
medial part of the progenitor domain. In addition, these results
show that the elevated expression of Lmx1b cannot compensate for
the loss of Lmx1a function in the medial DN progenitor domain.
In parallel experiments, we analyzed the ventral midbrain in
Lmx1b mutant (Lmx1b–/–) embryos (Chen et al., 1998). Lmx1b is
known to be sufficient to induce Lmx1a expression (Chizhikov and
Millen, 2004; Nakatani et al., 2010), a result that we confirmed by
introducing an Lmx1b expression construct into the chick neural
tube (see Fig. S3 in the supplementary material). However, DN
progenitors expressing Lmx1a and Foxa2 could be detected at the
ventral midline of Lmx1b–/– embryos showing that Lmx1b is not
absolutely required for the induction of Lmx1a expression in this
region (Fig. 2C,F). Moreover, although the size of the DN
progenitor domain in Lmx1b–/– was substantially reduced compared
with wild-type or Lmx1aeGFP/eGFP embryos, a significant number
of medially located neurons expressed Nurr1 and TH in Lmx1b–/–
embryos at E11.5 (Fig. 2F,I). These data raised the possibility that
loss of Lmx1b primarily affects the establishment of the lateral part
of the DN progenitor domain. We therefore analyzed additional
markers that could distinguish between the medial and lateral DN
DEVELOPMENT
Lmx1a/b in midbrain development
3402 RESEARCH ARTICLE
Development 138 (16)
Fig. 2. Characterization of the DN domain in Lmx1aeGFP/eGFP and
Lmx1b–/– mice. Transverse sections of the ventral midbrain at E11.5 and
E13.5, comparing expression patterns in wild type, Lmx1aeGFP/eGFP and
Lmx1b–/–. (A,B)Lmx1b expression remained unchanged, even enhanced,
in DN progenitors in Lmx1aeGFP/eGFP (B) compared with wild type (A). (C)In
Lmx1b–/–, the Lmx1a domain became smaller. (D-I)There was a specific
medial loss of DNs in Lmx1aeGFP/eGFP shown by the lack of Nurr1 (E,H) and
TH (H) which is not seen in wild type (D,G) and Lmx1b–/– (F,I). The medial
region is marked by white bracket. (J-L)Corin expression showed that the
medial domain was maintained in both mutants (K,L). By contrast, the
lateral domain was lost in Lmx1b–/– (L) but not in Lmx1aGFP/GFP (K), as
indicated by the loss of the gap between Corin and Nkx6.1 seen in the
wild type (J). (M-O,S-U) In situ hybridization of Wnt1 and Drd2 in wild
type (M,S), Lmx1aeGFP/eGFP (N,T) and Lmx1b–/– (O,U) showed that Wnt1
and Drd2 expression were lost in Lmx1b–/–, but remained unchanged in
Lmx1aeGFP/eGFP. (P-R)Schematic model of the medial (DNM) and lateral
(DNL) DN domains at E11.5. (V-X)At E13.5, Nurr1+ DNs in Lmx1b–/– failed
to initiate mature DN markers (X) compared with wild type (V) and
Lmx1aeGFP/eGFP (W). (Y)The percentage of Nurr1+ or Pitx3+ DNs of wild
type in different genotypes of Lmx1a/Lmx1b compound mutant mice.
Values are presented as mean±s.d., n4.
Lmx1a ablation delays induction of the Notch
pathway within the medial ventral midbrain
Midbrain DNs are unique in that they are derived from floor-plate
cells. By contrast, floor-plate cells located at more caudal axial
levels do not generate any neurons, indicating that ventral midbrain
floor-plate cells must acquire neurogenic properties during their
development (Joksimovic et al., 2009b; Ono et al., 2007). Lmx1a
is important for the induction of Ngn2 (Ono et al., 2007), a
proneural bHLH protein that promotes the generation of postmitotic DNs (Andersson et al., 2006a; Kele et al., 2006), and may
thus regulate the switch from non-neuronal floor plate cells to
neuronal DN progenitors. Additional genes implicated in the
regulation of neurogenesis were analyzed in Lmx1aeGFP/eGFP
embryos, including components of the Notch signaling pathway.
Interestingly, along with Ngn2, expression of the Notch ligand Dll1
as well as of the bHLH proteins Hes5 and Tcf12 was selectively
lost in the medial part of the DN progenitor domain in these
embryos at E11.5 (Fig. 3A-H). Hes5 operates downstream of Dll1
in the Notch pathway and functions to suppress the expression of
proneural bHLH genes (Ohtsuka et al., 1999). By contrast, Tcf12
DEVELOPMENT
progenitor domains. Wnt1 was selectively expressed in lateral DN
progenitors at E11.5 (Fig. 2M) (McMahon and Bradley, 1990;
Prakash et al., 2006). In addition, the dopamine receptor D2 (Drd2
or D2R) was restricted to lateral DNs at E13.5 (Fig. 2S) (Kim et
al., 2006). Interestingly, both of these lateral markers were almost
completely lost in Lmx1b–/– embryos while remaining largely
unaffected in Lmx1aeGFP/eGFP embryos (Fig. 2N,O,T,U). Moreover,
the expression of Corin, which selectively defines ventral midline
cells, was retained in both Lmx1aeGFP/eGFP and Lmx1b–/– embryos
(Fig. 2J-L). However, lateral DN progenitors residing in between
the expression domains of Corin and Nkx6.1 were abolished in
Lmx1b but not Lmx1a mutants at E11.5 (Fig. 2J-L). Thus, these
data show that the DN progenitor domain can be subdivided into
at least two distinct (medial and lateral) zones, and suggest that
Lmx1a is selectively required for the specification of DNs in the
medial DN progenitor domain, whereas Lmx1b is necessary for the
establishment of the lateral DN progenitor domain (Fig. 2P-R).
Nevertheless, it is noteworthy that differentiation of medially
derived DNs is also affected by the loss of Lmx1b, as the majority
of Nurr1+ DNs detected in Lmx1b mutants fail to initiate expression
of late DN markers including TH and Pitx3 (Fig. 2X; data not
shown) (Smidt et al., 2000).
Our comparison of Lmx1a and Lmx1b single mutants revealed
unique roles for these two proteins in DN progenitors. To explore
whether Lmx1a and Lmx1b also mediate redundant activities, we
analyzed the generation of DNs in different Lmx1a/Lmx1b
compound mutant backgrounds at E13.5. Embryos heterozygous
both for Lmx1a and Lmx1b exhibit a ~20-25% reduction in the
number of Nurr1- and Pitx3-expressing cells compared with wildtype and single Lmx1a or Lmx1b heterozygous embryos (Fig. 2Y).
In addition, a dramatic reduction in the number of DNs was evident
when Lmx1a-null mutant embryos carried one mutant Lmx1b allele
(~70-75% loss of Nurr1- and Pitx3-expressing cells compared with
a ~30-40% reduction in Lmx1a homozygous embryos) (Fig. 2Y).
Owing to the early lethality, presumably reflecting early
developmental functions of Lmx1a and Lmx1b, we were unable to
obtain E13.5 embryos that were null for both genes. However, the
Lmx1a gene dose-dependent reduction of DNs strongly indicates
that the combined and partly redundant activities of Lmx1a and
Lmx1b are essential for specification of all DNs in the ventral
midbrain.
Lmx1a/b in midbrain development
RESEARCH ARTICLE 3403
Fig. 3. Lmx1aeGFP/eGFP mice showed defects in
neurogenesis in the ventral medial region.
(A-H)Transverse sections of wild-type, Lmx1aeGFP/eGFP
and Lmx1b–/– embryos at E11.5 and E12.5. In situ
hybridization of Ngn2, Tcf12, Dll1 and Hes5 RNAs at
E11.5 showed a specific medial loss in Lmx1aeGFP/eGFP
(B,D,F,H) compared with wild type (A,C,E,G). (I-N)At
E11.5, in Lmx1b–/–, the expression of Ngn2 and Shh
(K,N) was similar to the wild type (I,L), in contrast to
Lmx1aeGFP/eGFP (J,M). (O-T)At E12.5, the expression
pattern of Hes5, Ngn2 and Shh was restored in the
Lmx1aeGFP/eGFP. Red brackets indicate the ventral medial
region; broken red lines indicate the DN domain.
Lmx1b controls the generation of OMNs and RNNs
located lateral to midbrain DNs
Studies of Lmx1a and Lmx1b in midbrain development have
primarily focused on the generation of DNs. However, in contrast
to Lmx1a, Lmx1b is broadly expressed in the ventral midbrain at
early developmental stages (Andersson et al., 2006b). We,
therefore, examined the expression of Lmx1b relative to other cell
fate-determining transcription factors expressed in midbrain
progenitors and examined how the generation of additional
neuronal populations was affected in Lmx1a and Lmx1b mutants.
Based on the expression of transcription factors, at least three
molecularly distinct progenitor domains can be defined in the
ventral-most midbrain at E10.5. First, Lmx1a+ cells define DN
progenitors at the ventral midline. Second, OMNs are generated
immediately lateral to the Lmx1a+/Lmx1b+ DN progenitor domain
from cells that express Phox2a, Sim1 and Nkx6.1 (Fig. 5C,D; see
also Fig. 6A,D,G,J). RNNs are also generated from this region,
although the precise spatiotemporal origin of these neurons remains
Fig. 4. Postnatal Lmx1aeGFP/eGFP mice show a significant loss of
VTA DNs. DNs in the VTA of wild-type (n5) and Lmx1aeGFP/eGFP (n5)
mice were quantified at P0. (A)The VTA of 500m is shown in the
picture and divided into regions of 0-250m and 250-500m from the
ventral midline (broken red line indicating 250m) and all TH neurons
within these subregions were subsequently counted unilaterally. (B)A
~20% reduction in the number of TH+ neurons within the 250-500m
region of the VTA was detected (0-250m: wild type, 124.3±12.7;
Lmx1aeGFP/eGFP, 111.6±12.2; 250-500m: wild type, 184.8±9.3;
Lmx1aeGFP/eGFP, 147.9±9.2) (P0.01 at 250-500m, analyzed by
Student’s t-test).
DEVELOPMENT
functions to promote transcription of neuronal genes by
heterodimerizing with proneural bHLH proteins (Neuman et al.,
1993). Thus, Lmx1a appears to promote the expression of several
genes implicated in the Notch signaling pathway, presumably
facilitating Notch signaling and establishing neuronal potential in
floor plate cells at the ventral midline.
The induction of Ngn2 and concomitant loss of Shh expression
that occurs in midline cells at around E10.5-E11.5 are indicative of
a conversion of floor-plate cells into DN progenitors (Fig. 3I,L)
(Andersson et al., 2006b; Joksimovic et al., 2009b). These
regulatory events occurred normally in Lmx1b–/– mice supporting
that loss of Lmx1b primarily affects lateral DN progenitors, which
are not derived from floor-plate cells (Fig. 3K,N). By contrast, loss
of Ngn2 expression accompanied by maintained Shh expression in
the midline was detected in Lmx1aeGFP/eGFP embryos at E11.5 (Fig.
3J,M). However, from E12.5 and onwards, Shh became suppressed
and Ngn2, as well as Notch signaling-related genes, became
activated in these embryos, which is consistent with the previously
observed partial restoration of DNs in dr/dr mice over time (Fig.
3O-T) (Ono et al., 2007). Thus, the initial loss of DNs in
Lmx1aeGFP/eGFP embryos most probably reflects a prolonged
maintenance of floor-plate properties and delayed neurogenesis at
the ventral midline. Interestingly, however, a modest but significant
loss of DNs persists in early postnatal Lmx1aeGFP/eGFP mice within
the ventral tegmental area (VTA) (~20% reduction within the
specified region) demonstrating that the medial delay in
neurogenesis cannot be fully compensated for at later
developmental stages (Fig. 4A,B).
3404 RESEARCH ARTICLE
Development 138 (16)
Fig. 5. Characterization of lateral domains in the ventral midbrain. (A)Transverse sections of ventral midbrain at E9.5, E10.5 and E12.5. At
E9.5, Lmx1b is co-expressed with Phox2a+ progenitors lateral of the DN progenitor domain. (B)Nkx2.2 is co-expressed with Phox2a. (C,D)At E10.5,
Lmx1b (C), Phox2a (C,D) and Nkx2.2 (D) expression are segregated into three defined domains. (E-J)Isl1/2+ OMNs are detected from E9.5 (E,H);
Nurr1+ DNs are detected from E10.5 (F,I); Brn3a+ RNNs are not detected at these time points (G,J). (K-M,Q-S) Lmx1b–/– mutants showed a loss of
OMNs (Q,R), an increase of RNNs (R) and largely unaffected GABAergic interneurons (S) when compared with wild type (K-M). (N-P)Lmx1aeGFP/eGFP
mutants did not show any effects on the lateral populations.
Because the analysis showed that Lmx1b operates upstream of
Phox2a in the specification of OMN fate, we speculated that the
temporal mis-specification of RNNs in Lmx1b mutants reflected the
downregulation of Phox2a. To directly test this idea, Phox2a mutant
embryos (Jacob et al., 2000) were analyzed. Interestingly, although
spatial patterning appeared normal (Fig. 6C,F,I,L), loss of Phox2a
resulted in a similar phenotype to that observed in Lmx1b mutants:
the loss of OMNs and a striking overproduction of Lim1/2+ and
Brn3a+ RNNs at E11.25 (Fig. 6O,R). The coding exons are replaced
by the lacZ reporter gene in this Phox2a mutant allele, which allows
short-term tracing of Phox2a-expressing cells. Many Brn3a+ and
Lim1/2+ cells co-expressed -gal in Phox2alacZ/lacZ embryos at E11.5
while lacZ expression appeared exclusively confined to the Isl1/2+
OMN lineage in Phox2alacZ/+ control embryos (Fig. 6S-X). Thus,
cells fated to generate OMNs differentiate into RNNs in the absence
of Phox2a function. Taken together, these data establish novel roles
for Lmx1b and Phox2a in controlling the sequential generation of
OMNs and RNNs from a common progenitor pool in the ventral
midbrain. However, it is notable that Lmx1b may also function partly
independently of Phox2a in this process as the loss of Lmx1b, but
not of Phox2a, increased Sim1 expression and induced ectopic
production of RNNs also in the DN progenitor domain at the ventral
midline (Fig. 6K,L,N,O,Q,R).
DISCUSSION
As Lmx1a and Lmx1b can activate one another when their expression
is forced in progenitors or differentiating DNs, it has remained
unclear to what extent Lmx1a and Lmx1b mediate redundant and/or
unique activities during ventral midbrain development (Andersson
et al., 2006b; Chung et al., 2009; Nakatani et al., 2010). In addition,
the role of Lmx1b in ventral midbrain patterning has remained
poorly understood (Guo et al., 2007; Smidt et al., 2000). Thus, our
current analysis of Lmx1a and Lmx1b mutant mouse embryos
provide important new insight into unique and redundant activities
for Lmx1a and Lmx1b in DN fate specification, and have revealed
entirely unexpected roles of Lmx1b and Phox2a in the temporal
regulation of OMN and RNN generation.
DEVELOPMENT
uncertain (Prakash et al., 2009). Third, progenitors located lateral
to the Phox2a+ OMN progenitors express Nkx2.2 and differentiate
into Gata2/3+ GABAergic neurons (Fig. 5D,M) (Kala et al., 2009;
Nakatani et al., 2007). At E9.5, many lateral Lmx1b+ cells coexpressed Phox2a and Nkx2.2, and the expression of these genes
did not segregate into distinct expression domains until E10.5 (Fig.
5A-D). As Isl1/2+ OMNs, but not DNs and RNNs, have been
generated already by E9.5, these data strongly suggest that at least
early born OMNs originate from progenitors that express Phox2a,
Lmx1b and Nkx2.2 (Fig. 5E-J). Analysis of neuronal subtypespecific markers at E12.5 indicated that the generation of OMNs,
RNNs and GABAergic neurons was unaffected by the loss of
Lmx1a (Fig. 5K-P). By contrast, OMNs were almost completely
lost in Lmx1b mutants at E12.5 and this phenotype was
accompanied by a significant increase in the number of Brn3a+
RNNs (Fig. 5Q,R). As in controls, these cells were located
in between Nurr1+ DNs and Gata3+ GABAergic neurons (Fig.
5M,S).
Additional markers were analyzed at E10.5-E11.5 (Fig. 6). This
analysis showed that the patterned expression of Lmx1a, Nkx6.1
and Nkx2.2 remained largely unaffected in Lmx1b mutants at E10.5
(Fig. 6B,E,H). However, Phox2a expression was almost completely
lost in Lmx1b mutants at E10.5, whereas the expression of Sim1
was upregulated and had further extended ectopically into the
Lmx1a+ progenitor domain at the ventral midline (Fig. 6E,K).
Thus, these data strongly suggest that OMNs and RNNs are
derived from the same progenitor domain but at different time
points, and that Lmx1b is necessary for the generation of OMNs
and for the suppression of RNNs at early developmental stages.
Notably, and in support of this idea, an extensive overproduction
of Brn3a+ and Lim1/2+ RNNs was observed in Lmx1b mutants
already by E11.25, a stage when only few RNNs could be detected
in wild-type controls (Fig. 6M,N,P,Q). Scattered Lim1/2+ and
Brn3a+ cells were also found intermingled with Lmx1a+ and
Nurr1+ cells in Lmx1b mutants, indicating that Lmx1b also
functions to suppress the generation of RNNs within the DN
progenitor domain (Fig. 6N,Q).
Fig. 6. Lmx1b is involved in the patterning and generation of
lateral neuronal subtypes in the ventral midbrain. (A-L)Transverse
sections of ventral midbrain at E10.5, E11.25 and E11.5. At E10.5,
three progenitor domains could be identified in both Lmx1b–/– and
Phox2alacZ/lacZ, as defined by combined expression of Lmx1a, Lmx1b,
Phox2a, Nkx6.1, Nkx2.2 and Sim1 (broken lines). In Lmx1b–/–, Phox2a+
progenitors were lost (E) and the Sim1+ domain was expanded into the
DN domain (K). By contrast, in Phox2alacZ/lacZ, the Sim1+ domain
remained unchanged (L). (M,P)At E11.25, Isl1/2+ OMNs (broken circle),
Brn3a+ RNNs and Lim1/2+ cells are expressed dorsal to the DN domain
(broken line) in wild type. (N,Q)In Lmx1b–/–, the majority of Isl1/2+
OMNs was lost, whereas the number of Brn3a+ RNNs and Lim1/2+ cells
was increased and encroached into the DN domain. (O,R)Isl1/2+ OMNs
were lost in Phox2alacZ/lacZ accompanied by an increase of Brn3a+ RNNs
and Lim1+ cells within their normal domain. (S-U)-Gal staining of
E11.5 Phox2alacZ/+ showed that the Isl1/2+ OMNs (S), but not Brn3a+
RNNs (T) and Lim1/2+ cells (U), are derived from Phox2a+ progenitors.
(V-X)In Phox2alacZ/lacZ, -gal+/Brn3a+ (W) and -gal+/Lim1/2+ (X) cells
could be detected, but not Isl1/2+ cells (V).
Our analysis of Lmx1a- and Lmx1b-null mutant mice confirms
that Lmx1a is not absolutely required for the specification of DNs.
However, ablation of Lmx1a and Lmx1b alleles within the same
embryos strongly indicated that the combined Lmx1a/b activities
is essential for generation of all DNs. Moreover, this study also
RESEARCH ARTICLE 3405
revealed specific requirements for Lmx1a and Lmx1b in medial
and lateral DN progenitors, respectively. Importantly, these data
establish that the DN progenitor domain is not a homogenous
population of cells, but can be subdivided into distinct medial and
lateral subpopulations that can be distinguished based on
expression of subtype-specific markers and on their differential
dependency on Lmx1a and Lmx1b for their early development
(Fig. 7). It is notable that Lmx1a previously have been implicated
as a key regulator of Wnt1 expression in the midbrain (Chung et
al., 2009). However, we found that the expression of Wnt1 was
largely unaffected in the midbrain of Lmx1a mutants, but was
almost completely abolished in Lmx1b mutants. Consistent with the
early role for Lmx1b in establishing the isthmus at the
midbrain/hindbrain boundary (Guo et al., 2007), our loss-offunction analysis also indicates that Lmx1b, in contrast to Lmx1a,
is a crucial regulator of Wnt1 expression in midbrain DN
progenitors at later developmental stages.
The floor plate is restricted to the extreme ventral midline at
most axial levels of the neural tube, but has been proposed to be
broader and encompass the entire DN progenitor domain in the
midbrain (Andersson et al., 2006b; Ono et al., 2007; Placzek and
Briscoe, 2005). This conclusion is primarily based on studies
showing that floor-plate cells and DN progenitors express several
common genes, including several of those identified as floor-plate
markers in the spinal cord (Ericson et al., 1995; Sasaki and Hogan,
1994). However, the molecular differences between medial and
lateral DN progenitors identified here, and the finding that Wnt1+
cells are largely unaffected in Lmx1a mutants, suggest that only
medially derived DNs originate from bona fide floor plate cells.
Like most other progenitors in the neural tube, laterally derived
DNs are likely to be derived from progenitors that have already
acquired their neurogenic potential at early developmental stages
(Lek et al., 2010; Placzek and Briscoe, 2005). In contrast to
Lmx1b, Lmx1a has a unique role in mediating the conversion of
floor-plate cells into neurogenic DN progenitors, and consistent
with a previous analysis of dr/dr mice, Ngn2 expression is initially
diminished in the medial DN progenitor domain in Lmx1aeGFP/eGFP
mice (Ono et al., 2007). However, in addition to Ngn2, several
additional genes implicated in the Notch signaling pathway are
initially lost at the midline of Lmx1aeGFP/eGFP and the
downregulation of Shh expression was delayed. It is therefore
conceivable that Lmx1a not only triggers cell cycle exit and
neuronal differentiation simply by activating Ngn2, but also
facilitates the establishment of functional Notch signaling and
lateral inhibition at the ventral midline, which thereby provides
neuronal potential to floor-plate cells (Fig. 7). However, additional
activities, including Wnt signaling (Prakash et al., 2006), are also
likely to contribute to this process as the requirement for Lmx1a in
midline cells is limited to early developmental stages and
neurogenesis recovers over time in the midbrain of Lmx1a mutants.
Functionally distinct DNs are topologically organized in defined
areas of the midbrain. DNs of the VTA, which are involved in
motivational and cognitive functions, occupy the ventral midline,
whereas cells of the substantia nigra pars compacta (SNpc), which
are implicated in motor control, are located in more lateral
positions (Bjorklund and Dunnett, 2007; Haber and Fudge, 1997;
Van den Heuvel and Pasterkamp, 2008). It remains unclear how
distinct DN subtypes are established during development, but it is
conceivable that the molecularly distinct medial and lateral DN
progenitors contribute to discrete DN subtype populations. We
found that the Drd2 is a marker of lateral DNs at E13.5 and that
these cells are missing in Lmx1b-null, but spared in Lmx1a-null,
DEVELOPMENT
Lmx1a/b in midbrain development
Fig. 7. Model outlining specific roles for Lmx1a, Lmx1b and
Phox2a in the spatiotemporal control of neuronal fate
determination in the midbrain. Lmx1a and Lmx1b are co-expressed
in DN progenitors in the ventral midbrain, and mediate both specific
and redundant activities in DN specification. The DN progenitor
domain can be subdivided into medial (DNM) and lateral (DNL)
subpopulations of cells. There is specific requirement for Lmx1a in
regulating the onset of neurogenesis in medial cells, and this process
involves establishment of Notch signaling and a concomitant
conversion of non-neuronal floor-plate (FP) into neuronal DN
progenitors. Lmx1b has little effect on midline neurogenesis but is
instead required for the establishment of lateral DN progenitors, and
for suppressing Sim1 expression and RNN fate within the DN
progenitor domain. This spatial patterning activity of Lmx1b is
probably essential at early time points when Lmx1b is broadly
expressed in the ventral midbrain but Lmx1a expression is restricted to
floor-plate cells. In addition to its role in the DN differentiation lineage,
Lmx1b also controls the sequential generation of OMNs and RNNs by
ensuring the activation of Phox2a in progenitors located immediately
lateral to DN progenitors. Phox2a and Sim1 are co-expressed within
this progenitor pool, and Phox2a is required to promote OMN fate and
suppress the generation of RNNs at early developmental stages. Sim1
is likely to contribute to the specification of RNNs within this
progenitor pool, but such activity must be subordinated to Phox2a and
therefore not revealed until the expression of Phox2a is
downregulated.
mutants. These findings indicate that lateral DNs detected at this
stage are generated from lateral DN progenitors. Thus, although
additional lineage-tracing experiments are necessary, the distinct
medial and lateral progenitor domains defined in this study,
together with previous cell-tracing experiments showing that
‘young’ floor-plate cells primarily generate VTA DNs, begin to
suggest a lineage relationship between DN progenitor populations
with defined molecular properties (Joksimovic et al., 2009a). In this
context, it is interesting that we observed a small but significant
decrease in the number of VTA neurons that persists in newborn
Lmx1a-null mice.
Importantly, our analysis shows that Lmx1b is not only devoted
to DN specification in the midbrain as the loss of Lmx1b also
affects the generation of OMNs and RNNs located lateral to DNs.
There is a dramatic reduction of Phox2a expression and a
concomitant loss of OMNs in the ventral midbrain of Lmx1b-null
mutants, suggesting that Lmx1b operates upstream of Phox2a in
OMN fate specification (Fig. 7). The loss of OMNs does not
simply reflect a failure to establish defined ventral progenitor
domains, as determined by the expression pattern of Lmx1a, Sim1
and Nkx2.2 in Lmx1b-null mutants. Instead, OMNs and RNNs
originate from the same ventral progenitor pool, but at sequential
developmental stages, and Lmx1b controls this process by
promoting the generation of OMNs and suppressing RNNs at early
time points. Moreover, lacZ-lineage analysis in Phox2a mutant
Development 138 (16)
cells provided direct evidence that these neuronal subtypes have a
common origin. The premature induction and extensive
overproduction of RNNs observed in Phox2a mutants also indicate
that a primary role of Lmx1b in this process is to trigger Phox2a
expression, which subsequently specifies OMNs while suppressing
RNN fate within the OMN domain (Fig. 7) (Hasan et al., 2010).
It remains unclear how Lmx1b mediates this activity, but the
broad expression pattern of Lmx1b at an early developmental stage
raises the possibility that Lmx1b directly induces Phox2a
transcription in OMN progenitors at early stages of midbrain
development. However, as Lmx1b is also required for Wnt1
expression in DN progenitors, we cannot exclude that a non-cell
autonomous role for Lmx1b contributes to the induction of Phox2a.
It is notable that Phox2b, which is highly related to Phox2a, can act
as a temporal cell fate determinant by promoting the generation of
visceral motoneurons and suppressing serotonergic (5HT) neurons
in the ventral hindbrain (Pattyn et al., 1997; Pattyn et al., 2003). A
unifying role of these two factors may thus be to promote early
born and suppress late-born neurons in the ventral neural tube, an
idea that raises the interesting possibility that Phox2a and Phox2b
also control temporal cell fate decisions at other sites in the
developing CNS and/or peripheral nervous system where these
transcription factors are known to be expressed (Pattyn et al., 2000;
Pattyn et al., 2003).
Furthermore, our data indicate that Lmx1b can suppress RNN
fate in a Phox2a-independent manner as ectopic RNNs are also
generated within the DN progenitor domain in Lmx1b-mutant
embryos. In normal development, Sim1 and Phox2a are coexpressed in OMN progenitors; however, Phox2a expression is
almost abolished in Lmx1b mutants, whereas Sim1 expression is
ectopically upregulated in presumptive DN progenitors at the
ventral midline. As the expression of Sim1 is unaffected in
Phox2a mutants, and the premature generation of RNNs in these
embryos is limited to the OMN progenitor domain, it seems
plausible that Sim1 functions as a determinant of RNNs and that
Lmx1b maintains the integrity of DN progenitors by suppressing
Sim1 in ventral midline progenitors (Fig. 7). Lmx1b would thus
promote DN fate and concomitantly limit the probability that DN
progenitors adopt an aberrant RNN differentiation program.
However, in more lateral OMN progenitors where Sim1 is
normally expressed, it can be predicted that the cell fatedetermining activity of Phox2a dominates over Sim1. In fact,
such a relationship between Phox2a and Sim1 activities would
explain the sequential generation of OMNs and RNNs in the
OMN progenitor domain, and also the premature generation of
RNNs after genetic ablation of Phox2a or after the loss of Phox2a
expression in Lmx1b mutants (Fig. 7). Thus, taken together, our
genetic analysis indicates that the function of Lmx1a is devoted
to the DN lineage, whereas Lmx1b has a more broad function and
influences the specification of multiple neuronal subtypes in the
ventral midbrain. In particular, Lmx1b, Phox2a and possibly
Sim1 appear to be integral parts of a novel gene regulatory
circuitry that controls both temporal and spatial aspects of
neuronal fate determination in the developing midbrain (Fig. 7).
Acknowledgements
We thank T. Jessell and M. Mendelsohn for providing Lmx1b mutant mice, and
Jose Dias for valuable discussions. This work was supported by the Knut och
Alice Wallenberg Foundation, by the EU 7th Framework Programme, by
NeuroStemcell (222943) (to J.E. and T.P.), by mdDANEURODEV (to T.P.), by
Vetenskapsrådet via support to the Linné Center for Developmental Biology
and Regenerative Medicine, by Vetenskapsrådet (to J.E. and T.P.), by Göran
Gustafssons Stiftelse, and by the Swedish Foundation for Strategic Research
and research funds of the Karolinska Insititutet (to J.E.).
DEVELOPMENT
3406 RESEARCH ARTICLE
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.065482/-/DC1
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