|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 13 December 2006
doi: 10.1242/dev.02717
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Biology Graduate Program, University of California, San Diego, La Jolla, CA
92037, USA.
2 Department of Molecular Cell Biology, Max Planck Institute for Biophysical
Chemistry, Gottingen 37077, Germany.
3 Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer
Center, Houston, TX 77030, USA.
4 Laboratory of Mammalian Genes and Development, National Institute of Child
Health and Human Development, Bethesda, MD 20892, USA.
5 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
* Author for correspondence (e-mail: goulding{at}salk.edu)
Accepted 25 October 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Lhx1, Lhx5, Pax2, Inhibitory neurons, Spinal cord, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
), and the glycine transporter, GlyT2, which is responsible
for glycine reuptake and transport across the plasma membrane of glycinergic
neurons (Liu et al., 1993). In
addition, GABAergic neurons express two genes, Gad1 and
Gad2, that encode glutamic acid decarboxylase - the enzyme that
converts glutamate to GABA (Erlander and
Tobin, 1991). During embryogenesis, the expression of these genes
is activated in subsets of differentiating neurons, thus imbuing them with the
necessary cellular machinery for fast inhibitory neurotransmission.
The developmental programs that determine the neurotransmitter status of a
neuron remain largely unknown. Whereas the pattern and levels of a particular
neurotransmitter can be regulated under certain circumstances by neural
activity and target-derived signals during development
(Schotzinger and Landis, 1988;
Borodinsky et al., 2004), a
cells acquisition of a particular neurotransmitter `phenotype' appears to be
closely linked to the gene regulatory events that determine neuronal subtype
identity. In the embryonic spinal cord, developing neurons fall into three
fast neurotransmitter classes: cholinergic neurons, excitatory glutamatergic
neurons and inhibitory neurons that use GABA and or glycine as their primary
transmitters. Motor neurons are primarily cholinergic
(Phelps et al., 1991), as are
a small population of interneurons of unknown function that are located near
the central canal (Barber et al.,
1991). Glutamatergic excitatory interneurons include the
early-born dI1-3, dI5, V2 and V3 neurons, as well as a population of late-born
dorsal interneurons, the so-called dILB neurons. The dI4, dI6, V0
and V1 classes of interneuron that are generated during the first wave of
neurogenesis are inhibitory (Saueressig et
al., 1999; Wenner et al.,
2000; Lanuza et al.,
2004; Glasgow et al.,
2005), as are the late-born dILA neurons that settle in
the dorsal horn (Cheng et al.,
2004; Cheng et al.,
2005; Mizuguchi et al.,
2006).
Studies in the dorsal horn have begun to delineate the transcriptional
mechanisms that control the neurotransmitter phenotype of spinal-interneuron
cell types. Inhibitory neurons in the dorsal spinal cord are derived
exclusively from cells that express the homeodomain transcription factor Lbx1
(Gross et al., 2002;
Muller et al., 2002;
Matise, 2002;
Cheng et al., 2004). These
cells are comprised of two early-born populations - dI4 and dI6 neurons - and
late-born dILA neurons that are generated during the second wave of
neurogenesis, which begins at E12 in the mouse. All three classes of neuron
express the paired-domain transcription factor Pax2 together with the
LIM-homeodomain transcription factors Lhx1 and Lhx5
(Gross et al., 2002;
Muller et al., 2002). A subset
of Lbx1-expressing neurons, dI5 and dILB neurons, also
differentiate as glutamatergic neurons. These cells express the homeodomain
transcription factors Tlx1, Tlx3 and Lmx1b. Tlx1 and Tlx3 function in a
cell-autonomous manner to specify glutamatergic dI5- and
dILB-sensory neurons (Cheng et
al., 2004), in part by over-riding an inhibitory differentiation
program that is Lbx1-dependent (Cheng et
al., 2005). Inactivation of Tlx1 and Tlx3
results in the loss of glutamatergic cell types in the dorsal horn, along with
the concomitant upregulation of Pax2 and GABAergic markers, such as
Viaat. Conversely, Tlx3 overexpression induces a switch from
GABAergic to glutamatergic cell fate (Cheng
et al., 2004; Cheng et al.,
2005; Mizuguchi et al.,
2006). Interestingly, the loss of Lmx1b does not alter the
neurotransmitter status of dI5 and dILB neurons
(Ding et al., 2004).
Pax2-expressing neurons in the hindbrain and spinal cord predominantly
differentiate as inhibitory interneurons
(Maricich and Herrup, 1999;
Gross et al., 2002;
Cheng et al., 2004;
Glasgow et al., 2005;
Mizuguchi et al., 2006;
Wildner et al., 2006). In the
Pax2-mutant cord, there is a marked loss of GABAergic markers in the
dorsal horn, demonstrating that Pax2 functions as an obligatory
regulator of the inhibitory-neurotransmitter program in these cells
(Cheng et al., 2004). These
dorsal inhibitory interneurons, as well as the ventrally-derived V0 and V1
inhibitory interneurons, also express Lhx1 and Lhx5
(Burrill et al., 1997;
Moran-Rivard et al., 2001;
Gross et al., 2002;
Muller et al., 2002) (this
study). This has led to the suggestion that the co-expression of Pax2 and Lhx1
and/or Lhx5 may provide a transcription factor code for inhibitory neurons in
the hindbrain and spinal cord. Although roles for Lhx1 and
Lhx5 have been demonstrated in head, kidney and motor neuron
development (Kobayashi et al.,
2005; Kobayashi et al.,
2004; Zhao et al.,
1999; Kania et al.,
2000), their overlapping expression in spinal interneurons
(Sheng et al., 1997), coupled
with the early embryonic lethal phenotype of the Lhx1 mutant
(Shawlot and Behringer, 1995),
has impeded analyzing their role(s) in spinal-interneuron development.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
) (see
Fig. 3A). Lhx1
conditional-mutant mice (Lhx1loxP) were kindly provided by
R. Behringer (M. D. Anderson Cancer Center, Houston, USA). The Lhx1
coding region is flanked by two loxP sites in the Lhx1loxP
conditional allele (Kwan and Behringer,
2002) (see Fig.
3A). Selective inactivation of Lhx1 in neurons was
achieved by crossing a Nestin-Cre (NesCre)
transgenic line in which Cre-recombinase is under the control of a
nervous-system-specific enhancer present in the second intron of the rat
nestin gene (Lendahl et al.,
1990) into a Lhx1loxP/loxP background. In this
study, conditional Lhx1-mutant mice with the genotype
Lhx1loxP/loxP;NesCre are referred to as
Lhx1-/- mutants. Double mutants with the genotype
Lhx1loxP/loxP;Lhx5-/-;NesCre
are referred to as DKO mice. Lhx5-/-- and
Lhx1-/--mutant mice both die at birth.
Lhx1loxP/loxP homologous strains are, however, healthy and
fertile, and so DKO mice were generated by crossing parental lines
comprised of genetic combinations of
Lhx5+/-;NesCre;Lhx1loxP/+
with Lhx1loxP/loxP;Lhx5+/- mice.
Pax2-/- and Pax8-/- embryos were
provided by A. Mansouri (Mansouri et al.,
1998). Pax5-/- embryos were derived from the
breedings of heterozygous Pax5+/- mice
(Urbanek et al., 1994). The
primers for genotyping all mutant animals are identical to those described in
the aforementioned references.
Immunohistochemistry and in situ hybridization
Mouse embryos were fixed for 1 hour in 4% paraformaldehyde in
phosphate-saline buffer (PBS), cryoprotected in 25% sucrose, embedded in OCT
(Tissue-Tec) and sectioned at 20 µm. Immunohistochemistry was performed on
frozen sections as previously described
(Burrill et al., 1997). The
following antibodies were used in the study: monoclonal anti-Lhx1 and
anti-Lhx5 (4F2-10, Developmental Hybridoma Studies Bank), monoclonal anti-NeuN
(Chemicon International), rat anti-BrdU monoclonal antibody (Harlan),
anti-Lbx1 (Gross et al.,
2002), polyclonal anti-Pax2 (Zymed) and guinea-pig anti-Lmx1b
(gift of T. Jessell, HHMI, Columbia University, NY, USA). Species-specific
antibodies conjugated to Cy2, Cy3 or Cy5 were used (Jackson ImmunoResearch).
In situ hybridizations were performed as previously described
(Goulding et al., 1993). Pax2
immuno-in situ double localization was performed according to Cheng et al.
(Cheng et al., 2004). In situ
hybridizations were preformed using probes specific for mouse Lhx1
(Bertuzzi et al., 1996),
Lhx5 (Zhao et al.,
1999), Viaat and VGluT2, and rat Gad1,
as described previously by Mizuguchi et al.
(Mizuguchi et al., 2006).
|
Histology |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
). Cell
counts were performed on three sections from three cords (i.e. nine sections
each for wild-type and DKO embryos). For each section, cells in a
single dorsal quadrant were counted twice in order to minimize counting
errors. Statistical differences in cell counts between wild-type and mutant
cords were determined using the Student's t-test.
|
BrdU labeling
Pregnant dams were injected intraperitoneally with 50 mg bromodeoxyuridine
(50 µg/ml dissolved in 0.9% saline) per gram of mouse bodyweight at E12.5.
E14.5 embryos were collected and processed for immunohistochemistry sections,
and stained with an antibody to Pax2 followed by anti-BrdU mouse monoclonal
antibody.
Imaging
Antibody, TUNEL and BrdU staining was visualized on a Zeiss LSM 510
confocal microscope. In situ hybridization images were captured on a Zeiss
Axiophot 2 microscope fitted with an Axiocam MRm camera. All figures were
color-corrected and assembled using Photoshop and Canvas software.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
), these
studies did not address the dynamic changes in the expression of Lhx1
and Lhx5 that occur at later developmental times. Previous studies by
Gross et al. (Gross et al.,
2002) using antibodies that recognize both Lhx1 and Lhx5 had
indicated that both proteins are co-expressed in late-born dILA
neurons that populate the dorsal horn. However, although Lhx1 had
been shown to persist dorsally at later stages by in situ hybridization
(Muller et al., 2002), it was
unclear whether Lhx5 was also expressed at later developmental
times. To clarify this issue, we used in situ hybridization to compare the developmental expression profiles of Lhx1 and Lhx5 in the embryonic spinal cord. During the early phase of neurogenesis (E10.5-E11.5), Lhx1 and Lhx5 were found to be co-expressed in multiple spinal-interneuron populations, including three dorsal cell types - the dI2, dI4 and dI6 interneurons (Fig. 1A,B,G,H; also see Fig. S1 and Fig. S2 in the supplementary material). However, from E12.5 onwards, the expression patterns of these two genes began to diverge, leading to complementary patterns of expression in dorsal interneurons at later developmental times (Fig. 1C,I). In the E13.5 dorsal horn, the highest level of Lhx5 transcripts was found medially, decreasing towards the lateral rim of the dorsal cord. Lhx1 was expressed in an inverse gradient, with cells closest to the ventricular zone expressing low levels of Lhx1 transcripts, while cells further away from the ventricular zone expressed higher levels (Fig. 1D,J). Whereas Lhx1 continued to be expressed in a mosaic pattern in the dorsal horn neurons up to birth (Fig. 1E,F, and data not shown), little or no Lhx5 expression was detected in the spinal cord from E14.5 to E17.5 (Fig. 1K,L).
The divergent expression patterns of Lhx1 and Lhx5 in the
late-born dIL cells can be accounted for in two ways. First, the complementary
expression patterns of Lhx1 and Lhx5 in late-born
dILA cells may reflect high-level expression of Lhx1 in
the dILA cells that are born first. These cells would be expected
to accumulate in the more lateral regions of the dorsal horn, whereas
later-born dILA cells that are located more medially might exhibit
high Lhx5-low Lhx1 expression. Alternatively,
differentiating dILA cells may downregulate Lhx5 and
upregulate Lhx1 as they migrate from the subventricular zone into the
dorsal horn. Support for the later possibility comes from the observation that
Lhx5 begins to be downregulated when dILA cells cease
being generated at E13.5 (Gross et al.,
2002). For this reason, we favor a model in which newborn
dILA cells express Lhx5 at high levels, while the more
mature dILA neurons downregulate Lhx5 and upregulate
Lhx1.
Spinal cord development is normal in Lhx1 and Lhx5 single mutants
Our observation that Lhx1 and Lhx5 are co-expressed at
E10.5 and E11.5 suggested to us that Lhx1 and Lhx5 might
function redundantly at these early developmental stages but adopt unique
roles at later times when their expression patterns diverge. Consistent with
this hypothesis, we did not observe any marked differences in the
specification of early-born cells, including inhibitory cells types, in either
of the single mutants (see Fig. S3 in the supplementary material). There were
also no marked changes in the specification or differentiation of late-born
dILA neurons in either of the single mutants. Pax2, which is
selectively expressed in inhibitory dILA and neurons showed a
normal pattern of expression in cords taken from E12.5
Lhx1loxP/loxP and Lhx5-/- single
mutants (Fig. 2A-C). There were
also no marked changes in the expression patterns of either Lbx1 or Lmx1b in
these late-born cells (data not shown). Viaat and Gad1
continued to be expressed in late-born dILA neurons in each of the
single mutants (Fig. 2E,F,H,I),
demonstrating that these cells acquire their normal
inhibitory-neurotransmitter phenotype.
|
|
|
) was used in combination with a Lhx5-null allele
(Zhao et al., 1999) to
generate Lhx1;Lhx5 double mutants. When Lhx1;Lhx5 double
knockout (DKO) mice were generated using a
NestinCre (NesCre) transgene to
selectively inactivate Lhx1 in neural progenitors, we observed a
dramatic abolition of Lhx1 and Lhx5 expression throughout
the spinal cord (Fig. 3).
Nonetheless, some cells that migrate towards the ventral midline continued to
express the Lhx1 protein (approximately 15-20 cells per E11.5
hemicord, Fig. 3D). These cells
are likely to be a subset of V0 or dI6 interneurons.
To further assess the extent of NesCre-mediated
recombination in the embryonic spinal cord, a ROSA26-derived reporter
line that conditionally expresses the lacZ gene
(R26lacZ) was used to identify cells that had undergone
Cre-mediated recombination (Soriano,
1999). When mice carrying the R26-lacZ reporter gene were
crossed with NesCre mice, embryos carrying both alleles
exhibited intense ß-gal staining throughout the nervous system. We
observed diffuse cytoplasmic ß-gal immunofluorescence that overlapped
extensively with the NeuN+ staining at all dorsoventral levels of
the spinal cord, indicating widespread Cre-mediated recombination
(Fig. 3E-G). Greater than 95%
of the cells in the ventricular zone of E11.5 spinal cords were
ß-gal+ (Fig.
3E) and these NeuN+/ß-gal+ neurons were
often in the process of migrating from the ventricular zone
(Fig. 3F,G), demonstrating that
the NesCre transgene effectively inactivates the
Lhx1 gene in most spinal cord progenitors.
Lhx1 and Lhx5 regulate late aspects of the inhibitory-neuron program in the dorsal horn
The cords of Lhx1;Lhx5 DKO mice were examined at a number of ages
up until birth. Cell-type specific markers were used to analyze the
specification of early-born inhibitory interneurons at E11.5
(Fig. 4). At this stage, Brn3a
and Isl1, which mark early-born excitatory dI1-dI3 and dI5 neurons
(Gross et al., 2002), showed
normal patterns of expression in the DKO cord
(Fig. 4A-D). Furthermore there
was no change in the expression of Lbx1, which marks all Class B neurons,
including inhibitory dI4 and dI6 neurons
(Fig. 4E,F). Pax2, which is
expressed in dI4, dI6, V0 and V1 neurons also exhibited a normal pattern of
distribution (Fig. 4G,H),
indicating that all Pax2-expressing inhibitory-cell types are correctly
specified in the absence of Lhx1 and Lhx5.
At early developmental times (up to E13.5), the loss of Lhx1 and Lhx5 had no obvious effects on the expression of Viaat (Fig. 5A-D); however, a reduction in Viaat and Gad1 mRNA levels in the most dorsal and lateral regions of the developing dorsal horn was noticed at E14.5 (Fig. 5E,F and see Fig. S4 in the supplementary material). This reduction in inhibitory-neurotransmitter gene expression was more pronounced at E17.5, with interneurons in the lateral dorsal horn exhibiting the greatest reduction in Gad1 expression levels (Fig. 5G,H). The loss of Gad1-expressing neurons at E17.5 was confirmed by cell counts, which showed significantly fewer Gad1+ cells in the DKO dorsal horn compared with wild-type dorsal horns (DKO 113±12 s.d. cells versus wild-type 279±33 s.d. cells; P<0.0001).
Interestingly, the expression of VGluT2, a marker of glutamatergic
neurons, was largely unchanged in the DKO dorsal horn
(Fig. 5I,J and see Fig. S4 in
the supplementary material), thereby arguing that dorsal GABAergic neurons do
not activate a glutamatergictransmitter program in response to the loss of
Lhx1 and Lhx5. Lmx1b expression, which marks glutamatergic
dILB neurons, was not upregulated in the cord of DKO
mutants, demonstrating that dILA neurons do not acquire a
dILB fate (see Fig.
6). The DKO phenotype thus resembles that seen in the
Pax2-mutant spinal cord, where the selective loss of inhibitory
markers in the dorsal horn is not accompanied by an upregulation of excitatory
markers such as VGluT2 (Cheng et
al., 2004). Although the loss of Gad1 was most pronounced
in the dorsal horn, some loss of Gad1 was noted in the ventral horn,
suggesting that Lhx1 and Lhx5 also have a similar role in
maintaining inhibitory-gene expression in ventral neurons.
|
To further investigate the nature of the loss of Pax2-expressing
cells in the DKO dorsal horn, we investigated whether a normal
complement of dIL neurons are generated. Spinal cords were pulsed with BrdU at
E12.5, when late-born dILA neurons are in the midst of being born
(Gross et al., 2002), and
these cords were analyzed at E14.5. No difference in BrdU labeling in the
dorsal spinal cord of DKO embryos compared to their wild-type
counterparts was observed (Fig.
7A,B), nor was there any marked change in the distribution of
these BrdU-labeled dIL neurons. Whereas the gross migration of late-born
neurons in the DKO spinal cord appeared to be largely unaffected,
some small differences in their settling patterns were noticed
(Fig. 7C-F).
Cell counts at E17.5 revealed no significant difference in cell numbers in the dorsal horns of wild-type and DKO mice (Fig. 7C-F; wild type 1173±93 s.d. versus DKO 1153±77 s.d., P<0.001). TUNEL assays were also used to assess whether the Pax2-expressing neurons in the dorsal horn undergo premature programmed cell death. There was no increase in apoptotic cell numbers between E14.5 and E17.5 in the DKO cord (see Fig. S5 in the supplementary material), nor was there an increase in activated caspase-3 expression in the DKO cord, thereby arguing that the dIL neurons do not undergo programmed cell death when Lhx1 and Lhx5 are absent. These data demonstrate that the reduction in Gad1 expression at E17.5 in the cord of DKO mutants is unlikely to arise from a loss of dILA neurons. Instead, our data support a model in which Lhx1 and Lhx5 are required to maintain the expression of Pax2 and Gad1 in late-born dILA neurons.
Reciprocal genetic interactions between Lhx1 and Lhx5 with Pax2 in the developing spinal cord
The similarity in the deficits in inhibitory-neurotransmitter gene
expression that occur in Pax2- and Lhx1;Lhx5-mutants led us
to investigate whether there are genetic interactions between these two
classes of genes. Whereas the expression of the Lhx1 and Lhx5 proteins in the
cord of Pax2-/- mutants was initially unchanged at early
developmental times (E11.5-E12.5; Fig.
8A-D), by E14.5 there was a marked loss of Lhx1 and
Lhx5 expression in the dorsal horn
(Fig. 8E-H, arrows). In view of
previous findings showing that Gad1 expression is lost in the cord of
Pax2-/- mutants (Cheng
et al., 2004), we analyzed in more detail the temporal changes in
Viaat expression that occur when Pax2 is absent. A reduction
in Viaat expression levels in the dorsal spinal cord was seen as
early as E12.5 (Fig. 8K,L),
even though Lhx1 and Lhx5 continued to be expressed in the
cord of Pax2-/- mutants at these times
(Fig. 8B,D). Consequently, the
loss of Viaat expression in the Pax2-/- mutant
cord precedes that of Lhx1 and Lhx5, indicating that the
regulation of Viaat by Pax2 at E12.5 is Lhx1 and/or
Lhx5 independent. At E14.5, the loss of Viaat expression in
the Pax2-/- spinal cord was more apparent
(Fig. 8M,N), which is
consistent with what has previously been reported for Gad1 expression
(Cheng et al., 2004).
|
), which are, in many instances, functionally equivalent
(Bouchard et al., 2000).
Pax2, Pax5 and Pax8 are expressed in the developing
neural tube in overlapping domains (Nornes
et al., 1990; Plachov et al.,
1990; Asano and Gruss,
1992; Schwarz et al.,
1997), with all three proteins being co-expressed with Lhx1 and
Lhx5 in dI4 and dI6 neurons, and in dILA neurons (data not shown).
We therefore investigated whether Pax5 and Pax8 might
continue to be expressed in ventral but not dorsal regions of the
Pax2-/--mutant spinal cord, thus compensating for the loss
of Pax2 expression in neurons that continue to express
inhibitory-neurotransmitter-specific genes, such as Viaat. Sections from Pax2-mutant cords were stained using antibodies that recognize Pax5 and Pax8. In the Pax2-/--mutant cords, we observed a complete absence of Pax5 in the dorsal spinal cord at early stages (E12.5; Fig. 9B) and at E17.5 (Fig. 9E). Pax8 was transiently expressed up to E12.5, albeit at reduced levels (Fig. 9H). However, from E14.5 onwards, Pax8 was also completely absent from the dorsal horn (Fig. 9K, data not shown). Because Pax2, Pax5 and Pax8 are likely to function redundantly, the pronounced loss of Pax5 and Pax8 in the dorsal cord of Pax2-/- mutants could account for the reduced expression of Viaat and Gad1 in this domain. Moreover, the continued expression of Pax5 and Pax8 ventrally at E12.5 may explain the persistence of ventral inhibitory neurons in the Pax2-/- cord (Fig. 8N). There is also a population of GABAergic neurons in the ventral horn that do not express Pax2, Pax5 or Pax8 (G. Lanuza and M.G., unpublished), and these cells may contribute to the residual Viaat expression that is seen in the ventral Pax2-/- cord.
By E17.5, few, if any, neurons in the Pax2-/- spinal cord express Pax5 and Pax8, with only a few ventral neurons continuing to express Pax8 (Fig. 9E,K). This late reduction in Pax5 and Pax8 is consistent with the loss of Viaat and Gad67 expression in ventral neurons that occurs at later times in the Pax2-/- cord. Notably, the Pax5 and Pax8 single-mutant mice do not exhibit any inhibitory-neuron phenotype, nor is there a concomitant loss of Pax2 expression in these animals. Pax5 and Pax8 are therefore epistatic to Pax2 in the dorsal spinal cord.
The observation that Pax2 is required for the continued expression of Pax8 and Pax5 prompted us to examine whether Pax8 and Pax5 are similarly dependent on Lhx1 and/or Lhx5 for their maintenance at E12.5. Although Pax5 and Pax8 expression in the dorsal spinal cord was markedly reduced at E12.5 in the DKO cord (Fig. 9C,I), which is in line with the reduction of Pax2 at this time, expression of both proteins persisted up until E17.5 in some cells scattered throughout the ventral and dorsal horn (Fig. 9F,L). This residual expression of Pax5 and Pax8 in the dorsal horn might explain why some dorsal horn interneurons in the Lhx1;Lhx5 DKO cord continue to express Viaat and Gad1. In summary, our analyses reveal that Lhx1 and Lhx5 play a crucial role in maintaining the expression of not only Pax2, but also that of Pax5 and Pax8 in the dorsal inhibitory neurons. The downregulation of Pax5 and Pax8 in the Pax2-/- cord also suggests that the loss of Pax5 and Pax8 in the Lhx1;Lhx5 DKO is mediated in part by the loss of Pax2.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
|
; Muller et al.,
2002; Cheng et al.,
2004; Glasgow et al.,
2005; Mizuguchi et al.,
2006). Ptf1a and Mash1 are expressed in the
precursors of dI4 and dILA neurons, and they are required for the
initial specification of each cell type. Lbx1 is expressed in
postmitotic Class B neurons, where it functions upstream of Pax2,
Lhx1 and Lhx5 in specifying dI4 and dILA inhibitory
neurons. In analyzing the Lbx1-mutant phenotype, Gross et al.
(Gross et al., 2002) proposed
a model in which Lhx1 and Lhx5 would function in
establishing the identity of dI2 and dI4 neurons. In testing this postulate
with Lhx1;Lhx5 DKO mice, we found no deficits in the initial
specification of dI2 and dI4 neurons (Fig.
4), demonstrating that Lhx1 and Lhx5 do not
confer subtype identity on either of these two dorsal cell types. Although
Lhx1 and Lhx5 are largely dispensable for the specification
of dorsally derived dI4 and dILA neurons, both genes may play roles
in other aspects of dI4 and dILA development. Interestingly, we
observed some loss of ventrally-derived V1 neurons in the DKO cord.
The exact function of Lhx1 and Lhx5 in these cells is not
clear and needs to be investigated further.
Lhx1, Lhx5 and Pax2 coordinately regulate GABAergic-interneuron development
Although Lhx1 and Lhx5 do not regulate the initial choice
between inhibitory dILA and excitatory dILB cell fates
in the dorsal horn (Fig. 4),
one or other gene is needed for dILA neurons to maintain their
differentiated inhibitory phenotype, and for the full induction of Pax2 in
newborn dILA neurons. The observation that some spinal neurons
continue to express inhibitory-neurotransmitter markers when Lhx1 and
Lhx5 are inactivated argues that both genes are not obligatory
determinants for inhibitory neurotransmission, and that they are thus unlikely
to directly control the transcription of inhibitory-neurotransmitter-specific
genes, such as Viaat, Gad1, Gad2 and GlyT2. This conclusion
is also consistent with the gradual loss of Viaat and Gad1
transcripts that occurs in the cord of Lhx1;Lhx5 DKO mutants
(Fig. 5).
Our study did not precisely define the time period when Lhx1 and Lhx5 are required for inhibitory-neuron differentiation; however, the reduced expression of Pax2 at E12.5 in the DKO cord suggests that there may be a critical period up to E12.5 when either Lhx1 or Lhx5 is needed to consolidate Pax2 expression and the inhibitory program. Further support for the idea that the Lhx genes are required at early rather than later times comes from the observation that Lhx1 expression after E13.5 is apparently not necessary for continued Pax2 expression, or for the maintenance of Viaat and Gad1, because all three inhibitory markers continue to be expressed in the Lhx1-mutant cord after E13.5.
In spite of the strong similarities in the spinal cord phenotypes of the Lhx1;Lhx5 DKO and Pax2-/- mutants, there are differences. Although these dissimilarities most likely reflect temporal differences in Pax2 expression in the DKO versus Pax2-/- cord, it is nonetheless possible that Lhx1, Lhx5 and Pax2 have distinct roles in GABAergicneuron development. For instance, Lhx1 and Lhx5 might regulate inhibitory markers at later developmental times in a manner that is independent of its role in maintaining Pax2. Alternatively, the transient expression of Pax2 that occurs at E10.5-E11.5 in the Lhx1;Lhx5 DKO cord might be sufficient for the initiation of Viaat and Gad1 expression, and for its persistence in some neurons even after Pax2 is downregulated, thus accounting for any differences in Viaat and/or Gad1 expression between the two mutants.
Conservation of the Pax2-Pax5-Pax8 gene cassette in the spinal cord
Our studies also implicate Pax5 and Pax8 in the
regulation of inhibitory-neurotransmitter cell identity in the spinal cord,
because Pax5 and Pax8 are expressed together with
Pax2 in many spinal inhibitory neurons. Studies in the kidney and
midbrain and/or hindbrain have provided evidence that Pax2, Pax5 and
Pax8 are functionally redundant in many contexts
(Bouchard et al., 2002;
Kobayashi et al., 2005). In
the CNS, Pax2 and Pax5 have been shown to be functionally
equivalent in the development of the mid- to hind-brain boundary
(Bouchard et al., 2000), and
Pax5 and Pax8 are epistatic to Pax2 at the
midbrain-hindbrain junction (Pfeffer et
al., 1998). Our results reveal that Pax5 and
Pax8 expression in dorsal inhibitory neurons also depends on
Pax2, with the loss of GABAergic cells in the DKO- and
Pax2-mutant cords being closely correlated with the reduction in
Pax5 and Pax8 expression
(Fig. 9). By contrast,
Pax5 and Pax8 are less dependent on Pax2 in the
ventral spinal cord (Fig. 9).
Consequently, the differential effects that losing Pax2 has on the
expression of Pax5 and Pax8 in dorsal versus ventral neurons
may be the major reason why inhibitory-neurotransmitter gene expression is
preferentially depleted in dorsal interneurons. In summary, the close
correlation between neurons that continue to express Viaat and
Gad1, and those cells in which Pax5 and Pax8 protein expression
perdures in the Pax2- and DKO-mutant cords, provides further
evidence that Pax2, Pax5 and Pax8 may function redundantly
to regulate inhibitory-neurotransmitter gene expression in the developing
spinal cord.
|
; Mizuguchi et
al., 2006; Wildner et al.,
2006). This program is not universal, as forebrain inhibitory
interneurons do not express Ptf1a, Pax2, Pax5 or Pax8.
Instead, it is the Dlx genes, together with Mash1, that
regulate the development of these GABAergic neurons
(Yun et al., 2002).
Furthermore, we have recently identified a population of inhibitory
interneurons in the ventral spinal cord that do not express Pax2,
Pax5 or Pax8 (G. Lanuza and M.G., unpublished). Taken together,
these findings suggest that multiple developmental programs in the developing
nervous system can specify an inhibitory-neurotransmitter fate. How these
divergent transcriptional programs activate the genes required for fast
inhibitory neurotransmission remains to be determined. Inhibitory-neuron
determinants, such as Ptf1a and Pax2, rather than directly
controlling genes such as Viaat and Gad1, could activate a
set of `core factors' that regulate their expression. Alternatively, these
`neurotransmitter' genes could contain multiple cis-regulatory elements that
are recognized by the different combinations of cell-type-specific
transcription factors such that their expression is activated in a
context-dependent manner.
Although the initial expression of inhibitory-neurotransmitter-specific
genes, such as Viaat and Gad1, are closely linked to the
initial acquisition of particular cell fates, this study demonstrates that
their continued expression in these neurons is dependent upon transcription
factors such as Lhx1 and Lhx5 that act to consolidate the inhibitory
differentiation program. Interestingly, it appears that the loss of
Pax2 and/or Lhx1 and Lhx5 does not result in a
cell-fate switch by these cells (Cheng et
al., 2004) (this study). Rather, presumptive `inhibitory' neurons
simply downregulate many of the genes that are necessary for fast inhibitory
neurotransmission. These findings are consistent with a model in which the
initial choice of neurotransmitter phenotype is closely tied to
neuronal-specification events, and it suggests that for certain neuronal
subtypes, the choice of neurotransmitter phenotype, once made, is irrevocable.
Nonetheless, some neurons are able to change their neurotransmitter expression
in response to changes in neural activity
(Borodinsky et al., 2004) or
target-derived signals (Schotzinger and
Landis, 1988), which argues that plasticity exists in the
developmental programs that control the neurotransmitter status of a
neuron.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/2/357/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSHistologyRESULTSDISCUSSIONREFERENCES |
|---|
Asano, M. and Gruss, P. (1992). Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development. Mech. Dev. 39,29 -39.[CrossRef][Medline]
Barber, R. P., Phelps, P. E. and Vaughn, J. E. (1991). Generation patterns of immunocytochemically identified cholinergic neurons at autonomic levels of the rat spinal cord. J. Comp. Neurol. 311,509 -519.[CrossRef][Medline]
Bertuzzi, S., Sheng, H. Z., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Taira, M., Dawid, I. B. and Westphal, H. (1996). Molecular cloning, structure, and chromosomal localization of the mouse LIM/homeobox gene Lhx5. Genomics 36,234 -239.[CrossRef][Medline]
Borodinsky, L., Root, C. M., Cronin, J. A., Sann, S. B., Gu, X. and Spitzer, N. C. (2004). Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429,523 -530.[CrossRef][Medline]
Bouchard, M., Pfeffer, P. and Busslinger, M. (2000). Functional equivalence of the transcription factors Pax2 and Pax5 in mouse development. Development 127,3703 -3713.[Abstract]
Bouchard, M., Souabni, A., Mandler, M., Neubuser, A. and
Busslinger, M. (2002). Nephric lineage specification by Pax2
and Pax8. Genes Dev. 16,2958
-2970.
Burrill, J. D., Moran, L., Goulding, M. D. and Saueressig, H. (1997). PAX2 is expressed in multiple spinal cord interneurons, including a population of EN1+ interneurons that require PAX6 for their development. Development 124,4493 -4503.[Abstract]
Cheng, L., Arata, A., Mizuguchi, R., Qian, Y., Karunaratne, A., Gray, P. A., Arata, S., Shirasawa, S., Bouchard, M., Luo, P. et al. (2004). Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7,510 -517.[CrossRef][Medline]
Cheng, L., Samad, O., Xu, Y., Mizuguchi, R., Luo, P., Shirasawa, S., Goulding, M. and Ma, Q. (2005). Lbx1 and Tlx3 are opposing switches in determining GABAergic versus glutamatergic transmitter phenotypes. Nat. Neurosci. 8,1510 -1515.[CrossRef][Medline]
Ding, Y. Q., Yin, J., Kania, A., Zhao, Z. Q., Johnson, R. L. and
Chen, Z. F. (2004). Lmx1b controls the differentiation and
migration of the superficial dorsal horn neurons of the spinal cord.
Development 131,3693
-3703.
Erlander, M. G. and Tobin, A. J. (1991). The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem. Res. 16,215 -226.[CrossRef][Medline]
Glasgow, S. M., Henke, R. M., Macdonald, R. J., Wright, C. V.
and Johnson, J. E. (2005). Ptf1a determines GABAergic over
glutamatergic neuronal cell fate in the spinal dorsal horn.
Development 132,5461
-5469.
Goulding, M., Sterrer, S., Fleming, J., Balling, R., Nadeau, J., Moore, K. J., Brown, S. D., Steel, K. P. and Gruss, P. (1993). Analysis of the Pax-3 gene in the mouse mutant splotch. Genomics 17,355 -363.[CrossRef][Medline]
Gross, M. K., Dottori, M. and Goulding, M. (2002). Lbx1 specifies somatosensory association interneurons in the dorsal spinal cord. Neuron 34,535 -549.[CrossRef][Medline]
Kania, A., Johnson, R. L. and Jessell, T. M. (2000). Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb. Cell 102,161 -173.[CrossRef][Medline]
Kobayashi, A., Shawlot, W., Kania, A. and Behringer, R. R.
(2004). Requirement of Lim1 for female reproductive tract
development. Development
131,539
-549.
Kobayashi, A., Kwan, K. M., Carroll, T. J., McMahon, A. P.,
Mendelsohn, C. L. and Behringer, R. R. (2005). Distinct and
sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for
tubular morphogenesis during kidney development.
Development 132,2809
-2823.
Kwan, K. M. and Behringer, R. R. (2002). Conditional inactivation of Lim1 function. Genesis 32,118 -120.[CrossRef][Medline]
Lanuza, G. M., Gosgnach, S., Pierani, A., Jessell, T. and Goulding, M. (2004). Genetic identification of neurons that coordinate left-right motor activity during locomotion. Neuron 42,375 -386.[CrossRef][Medline]
Lendahl, U., Zimmerman, L. B. and McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. Cell 60,585 -595.[CrossRef][Medline]
Liu, Q. R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H. and
Nelson, N. (1993). Cloning and expression of a spinal cord-
and brain-specific glycine transporter with novel structural features.
J. Biol. Chem. 268,22802
-22808.
Mansouri, A., Chowdhury, K. and Gruss, P. (1998). Follicular cells of the thyroid gland require Pax8 gene function. Nat. Genet. 19, 87-90.[CrossRef][Medline]
Maricich, S. M. and Herrup, K. (1999). Pax-2 expression defines a subset of GABAergic interneurons and their precursors in the developing murine cerebellum. J. Neurobiol. 41,281 -294.[CrossRef][Medline]
Matise, M. (2002). A dorsal elaboration in the spinal cord. Neuron 34,491 -493.[CrossRef][Medline]
McIntire, S. L., Reimer, R. J., Schuske, K., Edwards, R. H. and Jorgensen, E. M. (1997). Identification and characterization of the vesicular GABA transporter. Nature 389,870 -876.[CrossRef][Medline]
Mizuguchi, R., Kriks, S., Cordes, R., Gossler, A., Ma, Q. and Goulding, M. (2006). Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons. Nat. Neurosci. 9,770 -778.[CrossRef][Medline]
Moran-Rivard, L., Kagawa, T., Saueressig, H., Gross, M. K., Burrill, J. and Goulding, M. (2001). Evx1 is a postmitotic determinant of V0 interneuron identity in the spinal cord. Neuron 29,385 -399.[CrossRef][Medline]
Muller, T., Brohmann, H., Pierani, A., Heppenstall, P. A., Lewin, G. R., Jessell, T. M. and Birchmeier, C. (2002). The homeodomain factor Lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord. Neuron 34,551 -562.[CrossRef][Medline]
Nornes, H. O., Dressler, G. R., Knapik, E. W., Deutsch, U. and
Gruss, P. (1990). Spatially and temporally restricted
expression of Pax2 during murine neurogenesis.
Development 109,797
-809.
Pfeffer, P. L., Gerster, T., Lun, K., Brand, M. and Busslinger, M. (1998). Characterization of three novel members of the zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development 125,3063 -3074.[Abstract]
Phelps, P. E., Barber, R. P. and Vaughn, J. E. (1991). Embryonic development of choline acetyltransferase in thoracic spinal motor neurons: somatic and autonomic neurons may be derived from a common cellular group. J. Comp. Neurol. 307, 77-86.[CrossRef][Medline]
Plachov, D., Chowdhury, K., Walther, C., Simon, D., Guenet, J.
L. and Gruss, P. (1990). Pax8, a murine paired gene expressed
in the developing excretory system and thyroid gland.
Development 110,643
-651.
Saueressig, H., Burrill, J. and Goulding, M. (1999). Engrailed-1 and netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons. Development 126,4201 -4212.[Abstract]
Schotzinger, R. J. and Landis, S. C. (1988). Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo. Nature 335,637 -639.[CrossRef][Medline]
Schwarz, M., Alvarez-Bolado, G., Urbanek, P., Busslinger, M. and
Gruss, P. (1997). Conserved biological function between Pax2
and Pax5 in midbrain and cerebellum development: evidence from targeted
mutations. Proc. Natl. Acad. Sci. USA
94,14518
-14523.
Shawlot, W. and Behringer, R. R. (1995). Requirement for Lim1 in headorganizer function. Nature 374,425 -430.[CrossRef][Medline]
Sheng, H. Z., Bertuzzi, S., Chiang, C., Shawlot, W., Taira, M., Dawid, I. and Westphal, H. (1997). Expression of murine Lhx5 suggests a role in specifying the forebrain. Dev. Dyn. 208,266 -277.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F. and Busslinger, M. (1994). Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79,901 -912.[CrossRef][Medline]
Walther, C., Guenet, J.-L., Simon, D., Deutsch, U., Jostes, B., Goulding, M., Plachov, D., Balling, R. and Gruss, P. (1991). The murine Pax genes: a multigene family of paired-box containing genes. Genomics 11,424 -434.[Medline]
Wenner, P., O'Donovan, M. and Matise, M. P.
(2000). Topographical and physiological characterization of the
interneurons that express engrailed-1 in the embryonic chick spinal cord.
J. Neurophysiol. 84,2651
-2657.
Wildner, H., Muller, T., Cho, S.-H., Brohl, D., Cepko, C. L., G
