Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
* Author for correspondence (e-mail: kenneth.campbell{at}cchmc.org)
Accepted 19 May 2004
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SUMMARY |
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Key words: DARPP-32 (Ppp1r1b), Pax6, Retinoic acid, Raldh3 (Aldh1a3), Striatum
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Introduction |
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Retinoic acid (RA) is known to be involved in regulating the
differentiation of numerous cell types, including specific neuronal subtypes.
In fact, at limb levels, retinoid signaling is required for the formation of
the lateral, lateral motor column motoneurons
(Sockanathan and Jessell,
1998). Moreover, recent studies have suggested broader
requirements for retinoids at spinal cord levels, both in the specification of
motoneuron identity (Novitch et al.,
2003
) and in the development of motor columns at different
anteroposterior levels (Sockanathan et
al., 2003
). Previous studies have demonstrated that retinoids are
locally produced in the LGE during the period of striatal neurogenesis
(Toresson et al., 1999
;
Mata de Urquiza et al., 1999
).
Since then, the rate-limiting enzyme retinaldehyde dehydrogenase 3 (Raldh3)
has been identified and shown to be expressed in the LGE
(Li et al., 2000
). To date,
this is the only Raldh enzyme known to be expressed in the LGE. Addition of RA
to differentiating LGE cultures increases the number of DARPP-32-expressing
cells, as well as increasing its expression per cell
(Toresson et al., 1999
). This
increase is specific to DARPP-32 expression and is not simply due to an
increase in the number of cells differentiating into neurons in the RA-treated
cultures. As Gsh2 is required for normal LGE development, it seems
likely that retinoid production and/or signaling may be altered in the ventral
telencephalon of these mutants. Accordingly, certain aspects of the
Gsh2 mutant phenotype (i.e. reduced DARPP-32 neurons) could result
from altered retinoid function.
In this study, we have examined the role of Gsh2 in retinoid production and signaling within the ventral telencephalon, and its potential implications for striatal projection neuron differentiation. Our results indicate that Gsh2 is required for normal retinoid production, and that this requirement is distinct from its role in regulating dorsoventral patterning, at least with respect to the repression of Pax6. Furthermore, we have found that exogenous retinoids can improve striatal neuron differentiation in the Gsh2 mutant.
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Materials and methods |
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Immunohistochemistry
Primary antibodies were all produced in rabbits and used at the following
concentrations: Raldh3, 1:5000 (provided by U. Dräger); DARPP-32, 1:1000
(Chemicon); and FoxP1, 1:1000 (provided by E. Morrisey). The secondary
antibodies used were biotinylated swine anti-rabbit antibodies (1:200, DAKO).
The ABC kit (Vector Laboratories) was used to visualize the reaction product
using diaminobenzidine (DAB, Sigma) as the final chromogen.
In situ hybridization
In situ hybridization was performed using digoxigenin-labeled cRNA probes
as previously described (Toresson et al.,
1999). Probes used were: retinoic acid receptor beta
(Rarb; EST-clone, GenBank Accession Number BE854385), Retinoid X
receptor gamma (Rxrg; EST-clone, GenBank Accession Number
AI325376).
Retinoid reporter cell assay
Gsh2+/+, Gsh2+/ and
Gsh2/ embryos were collected from
heterozygous crosses at E12.5 and E16.5. For the E12.5 embryos, the MGE and
LGE were separately dissected. At E16.5, the MGE is no longer morphologically
distinct, so at this stage the LGE and cortical regions were separately
dissected. A small piece of tail from each embryo was used for the genotyping
of Gsh2. Both explants (either MGE, LGE or cortex) from each embryo
were placed on a monolayer of F9 cells stably expressing RARE-lacZ
(Wagner et al., 1992) in a
single well of a 96-well culture dish. The explants and reporter cells were
grown in 150 µl of DMEM supplemented with 15% fetal calf serum (FCS) and
antibiotics. After 24 hours of culture, the explants were removed from each
well and the F9 cells were assayed for ß-galactosidase activity as
described (Wagner et al.,
1992
). X-gal-positive cells were counted in five randomly chosen
fields at 200x magnification for each culture, and the average number of
X-gal-positive cells/well (i.e. per explant pair) was determined for each
embryo.
Retinoic acid treatments
After timed matings of Gsh2 heterozygous parents, pregnant dams
were given all-trans RA (5 mg/kg body weight, Sigma) or vehicle control
(sunflower oil) by oral gavage. The RA was dissolved in DMSO at 50 mg/ml and
diluted in sunflower oil. RA treatments were given twice daily (10-12 hours
apart) starting at E11.5 through E17.5. This period covers the majority of
striatal neurogenesis (Bayer and Altman,
1995), and is after the sensitive period for the teratogenic
effects of RA on embryonic morphology
(Nolen, 1986
;
Simeone et al., 1995
). Embryos
were collected at E18.5, and fixed, genotyped and processed for
immunohistochemistry as described earlier. The DARPP-32 neurons were counted
in each striatal section from either RA-treated (n=4) or oil-treated
(n=4) Gsh2 mutants and a total for each embryo was
determined.
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Results |
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Retinaldehyde dehydrogenase 3 (Raldh3), the gene encoding a
rate-limiting enzyme in retinoid production, has previously been shown to be
restricted to the LGE during telencephalic development
(Li et al., 2000). Recent
studies have confirmed that Raldh3 protein expression closely matches that of
the endogenous gene expression (Wagner et
al., 2002
). At E12.5, Raldh3 is expressed in the subventricular
zone of the ventromedial LGE, and in the sulcus between the LGE and MGE
(Fig. 1A). This expression
domain is considerably truncated in Gsh2 mutants, leaving mostly
low-level expression in and around the LGE/MGE sulcus
(Fig. 1B). The Gsh2
mutant LGE exhibits a partial recovery at later stages of neurogenesis
(Corbin et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
), which has
been shown to depend on a compensatory response by the family member
Gsh1 (Toresson and Campbell,
2001
; Yun et al.,
2003
). Despite this, only a modest recovery of Raldh3 expression
is observed in the Gsh2 mutant LGE at E16.5
(Fig. 1D), as compared to wild
type (Fig. 1C). These findings
are consistent with Raldh3 gene expression results in Gsh2
mutants (Toresson and Campbell,
2001
) (data not shown).
|
LGE explants were dissected from E12.5 and E16.5 Gsh2+/+, Gsh2+/ and Gsh2/ embryos. As controls, MGE (E12.5) and cortex (E16.5) were dissected from the same embryos. Co-culture of LGE explants from the Gsh2+/+ and Gsh2+/ embryos with the F9 reporter cells resulted in many X-gal-positive cells (Fig. 2A,B). However, the reporter cells co-cultured with E12.5 Gsh2/ LGE explants displayed very little X-gal staining (Fig. 2C), indicating that these explants produced significantly less RA than the wild types or heterozygotes (Fig. 2D). There were no significant differences in the co-cultures with MGE explants from all three genotypes, which contained low levels of retinoids, similar to the mutant LGEs. At E16.5, Gsh2/ LGE explants still induce a significantly lower level of lacZ gene expression than the Gsh2+/+ and Gsh2+/ LGE explants (Fig. 2E), indicating a continued reduction in retinoid production. It should be noted, however, that there was more variability in the response to the Gsh2/ LGEs at E16.5. While some of the mutant explants activated the lacZ gene to levels near the range of the wild types and heterozygotes, others were severely deficient. As was the case with the MGE explants, cortical explants only contain low levels of retinoids, which do not differ between genotype. Overall, the RA reporter cell data are consistent with the findings of Raldh3 expression in Gsh2 mutants, and demonstrate that retinoid production in these mutants is markedly reduced. Moreover, these findings indicate that Raldh3 expression itself is a good indicator of retinoid production in the embryonic mouse ventral telencephalon.
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Discussion |
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Although our results show that Gsh2 is required for the normal
expression of Raldh3 in the LGE, and thus normal levels of retinoids within
this structure, the mechanism by which Gsh2 is required for this
retinoid synthesizing enzyme to be correctly expressed remains unclear. It
seems unlikely that this is direct because Raldh3 expression in the LGE is
much more restricted than that of Gsh2. Gsh2 is expressed in the
ventricular zone of the entire LGE, with its highest levels observed in the
dorsal half (Toresson et al.,
2000; Yun et al.,
2001
), whereas Raldh3 is only found in the subventricular zone of
the rostral LGE, where it is largely confined to the ventral portion
(Fig. 1)
(Li et al., 2000
) (data not
shown). Thus it seems more likely that Gsh2 plays a permissive role
in the expression of this enzyme, allowing for other factors to regulate its
precise expression domain within the LGE. It is possible that Raldh3
expression in the LGE is induced by retinoid producing mesenchymal cells that
have previously been shown to signal to the ventrolateral telencephalon prior
to (and concurrent with) the appearance of the LGE
(LaMantia et al., 1993
). This
process may require Gsh2 expression in the receptive cells in order
to establish and/or maintain Raldh3 expression within the LGE.
An important aspect of the Gsh2 mutant phenotype in the
telencephalon is the compensation by its family member Gsh1
(Toresson and Campbell, 2001;
Yun et al., 2003
). As a result
of this compensation, the molecular identity (i.e. Mash1 and Dlx gene
expression) of the Gsh2 mutant LGE improves at late stages of
embryogenesis (e.g. E16.5). The present results show that Raldh3 expression in
the Gsh2 mutant LGE does not significantly improve, indicating that
Gsh1 cannot fully compensate for Gsh2 in the regulation of
this gene. However, this does not exclude a role for Gsh1 in
regulating Raldh3 expression because this gene appears to be
completely absent in Gsh1/2 double mutants
(Toresson and Campbell, 2001
).
In any case, our results suggest that in the Gsh2 mutant,
Gsh1 is not as efficient in regulating Raldh3 expression as it is in
restoring Dlx and Mash1 gene expression at later embryonic stages
(Corbin et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
). This
reduced efficacy may simply be due to timing, as the Gsh1
compensation does not occur until around E14
(Toresson and Campbell, 2001
;
Yun et al., 2001
;
Yun et al., 2003
). It may be
that for normal Raldh3 expression in the LGE, Gsh gene expression is
required at early stages (i.e. between E10.5 and E14). This timeframe
corresponds well with the period during which RA-producing mesenchymal cells
are signaling to the ventrolateral telencephalon (i.e. the presumptive LGE)
(LaMantia et al., 1993
).
The previously established role for Gsh2 in the developing
telencephalon was to establish the correct dorsoventral identity of
telencephalic progenitor cells (Corbin et
al., 2000; Toresson et al.,
2000
; Yun et al.,
2001
). The Gsh2 mutant LGE cells ectopically express
dorsal telencephalic genes (i.e. Pax6) and lose their expression of
ventral regulators (e.g. Dlx and Mash1 genes). Removal of
Pax6 from the Gsh2 mutant background (i.e.
Pax6;Gsh2 double homozygous mutants) improves the molecular identity
of LGE cells (i.e. Dlx and Mash1 gene expression) and results in a
larger striatum, as compared with Gsh2 mutants
(Toresson et al., 2000
) (data
presented here). However, the present results show that Pax6;Gsh2
double mutants do not improve all aspects of the Gsh2 mutant
phenotype. In particular, Raldh3 expression remains reduced in the
Pax6;Gsh2 double mutant LGE, showing no improvement over that
observed in the Gsh2 mutant LGE. Therefore, the role that
Gsh2 plays in regulating retinoid production within the LGE is novel
and independent of its role in repressing Pax6 gene expression (and
thus maintaining the correct dorsoventral identity in LGE cells). Furthermore,
the number of DARPP-32 neurons in the Pax6;Gsh2 double mutant
striatum continues to be severely reduced, similar to that seen in the
Gsh2 mutant striatum, suggesting that the observed retinoid
deficiency underlies this differentiation defect.
The fact that Gsh2 mutants maintain expression of the RA receptor
heterodimer RARß and RXR suggests that the mutant striatum has the
ability to respond to retinoids. It has previously been shown that RA promotes
the expression of DARPP-32 in wild-type LGE cultures
(Toresson et al., 1999
).
Accordingly, exogenous retinoids also increase the number of DARPP-32-positive
striatal neurons in vivo, improving striatal differentiation in the
Gsh2 mutants. These results show that the retinoid signaling
machinery is functional in Gsh2 mutants but that it is not actively
used because of the reduction in retinoids. As expected, exogenous RA had no
effect on the size of the Gsh2 mutant striatum, which remains
severely truncated, typical of the Gsh2 mutant striatum. In our
experimental paradigm, we provided RA to the Gsh2 mutant striatum
during the period of striatal neurogenesis. It is possible, however, that
retinoids may play an earlier role (i.e. prior to E11) in LGE/striatal
development (e.g. in regulating the size of the striatal progenitor pool),
which would not be affected by our treatments. Nevertheless, the present
findings strongly support the notion that the decreased retinoid production in
the Gsh2 mutant LGE is responsible for the disproportionate reduction
of DARPP-32-positive striatal neurons seen in these mutants. Taken together,
our results indicate that Gsh2 is required both for the repression of
Pax6 in LGE cells (so that the correct number of striatal progenitors
are maintained throughout striatogenesis) and for the production of retinoids
within the LGE (which are needed for the correct differentiation of at least a
portion of the striatal projection neurons).
The LGE of Gsh1/2 double mutants does not express detectable
levels of Raldh3 (Toresson and
Campbell, 2001). Moreover, these double mutant embryos completely
lack DARPP-32-expressing neurons in their striatum. This suggests that a
complete retinoid deficiency may exist in the Gsh1/2 double mutants.
Unlike the case in Gsh2 mutants, the Gsh1/2 double mutants
do not show a recovery of Dlx and Mash1 gene expression within the
LGE (Toresson and Campbell,
2001
; Yun et al.,
2003
). Thus the specification of LGE precursor cells in
Gsh1/2 double mutants remains abnormal. For this reason, it seems
unlikely that exogenous retinoids could improve striatal projection neuron
differentiation in these double mutants.
Our findings add further support to our previous suggestion that retinoids
regulate striatal neuron differentiation
(Toresson et al., 1999).
Moreover, our results show that Gsh2 is required for the correct
expression of the retinoid synthesizing enzyme Raldh3, and thus for normal
retinoid production in the ventral telencephalon. However, it remains unclear
what aspect(s) of striatal neuron differentiation retinoids promote. Although
DARPP-32 is a marker of all striatal projection neurons in the mature striatum
(Ouimet et al., 1984
;
Anderson and Reiner, 1991
), at
birth striatal neurons expressing this protein are largely confined to the
patch compartment (Foster et al.,
1987
). Because the Gsh2 mutants and the
Pax6;Gsh2 double mutants die at birth, analysis of the mature
striatum has, thus far, not been possible. Future studies will be focused on
identifying the specific role of retinoids during striatal projection neuron
differentiation in both wild types and Gsh2 mutants.
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ACKNOWLEDGMENTS |
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