1 Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, LPPI, University of California at San Francisco, 401 Parnassus Avenue, San Francisco, CA 94143-0984, USA
2 Loeb Medical Research Institute, University of Ottawa, 1053 Carling Avenue, Ottawa, Ontario K1Y 4E9, Canada
*Author for correspondence (e-mail: jlrr{at}cgl.ucsf.edu)
Accepted 17 October 2001
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SUMMARY |
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Key words: Dlx, Electroporation, Forebrain, GABA, Glutamic acid decarboxylase, Mouse
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INTRODUCTION |
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The molecular mechanisms through which the Dlx genes regulate the development of forebrain GABAergic neurons remain to be elucidated. To complement Dlx loss-of-function analyses, we have implemented a novel electroporation gain-of-function assay in slice cultures of the embryonic mouse forebrain. Using this approach, we demonstrate that Dlx2 and Dlx5, but not Dlx1, can robustly induce cortical cells to express the glutamic acid decarboxylase (Gad) genes. Furthermore, this assay provides evidence for a cross-regulatory cascade of the Dlx genes.
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MATERIALS AND METHODS |
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Telencephalic slices
Preparation and maintenance of slice tissue cultures followed the methods of Anderson et al. (Anderson et al., 1997a).
Electroporation
A tissue vibratome slice (in 4% low melting point agarose in 1x Krebs), with its supporting membrane (Nucleopore Track-Etch membrane, Whatman), was placed onto a 1% agarose block (in 1x Krebs buffer) within a setup of two horizontally oriented platinum electrodes (System CUY-700-1 and CUY-700-2; Protech International, San Antonio, TX) (see Fig. 1 for a drawing of the apparatus). A tiny agarose column, punched with a clipped hypodermic needle (gauge 19 or 21) from a 1% agarose gel in 1x Krebs, was attached to the mobile upper electrode. The agarose blocks and columns were made form a gel that was cast between two glass plates (the layer of agarose should not be thinner than 1.5 mm). A small amount of plasmid solution was applied to the lower end of the agarose column, and then the electrode was lowered to let the solution contact the tissue. The system was powered by a T820 Electro Square Porator (BTX). In empirical trials, we determined that plasmid concentrations of about 1 mg/ml at charging voltages of >100 V (with two pulses of 5 ms each) yielded acceptable numbers of electroporated cells.
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In situ hybridization
In situ hybridization was performed along the lines detailed in Porteus et al. (Porteus et al., 1992).
Immunohistochemistry
Immunohistochemistry was performed on 50 µm free-floating sections. Following pre-incubation for 2 hours in a solution of 2% normal goat serum in PBST (PBS with 0.1% Triton X-100) and 0.1% NaN3, the liquid was changed for a fresh aliquot containing the primary antibody, and the tissue incubated for up to 48 hours at 4°C. Sections were subsequently washed 3x in PBST and the secondary antibody (1:200 goat anti-rabbit IgG, conjugated to either the red fluorescent dye Alexa594 (Molecular Probes), or biotin (Vector)) applied, and incubated for 4 hours at room temperature. The biotinylated antibody was visualized with the ABC kit (Vector), according to the manufacturers instructions. We used the following primary antibodies: anti-distal-less (rabbit polyclonal, 1:400; a kind gift from Dr G. Panganiban, University of Wisconsin-Madison); anti-GAD65 (rabbit polyclonal, 1:2000; Chemicon AB5082); and anti-ß-galactosidase (rabbit polyclonal, 1:2000; 5prime-3prime).
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RESULTS |
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An expression vector encoding green fluorescent protein (GFP) was co-electroporated with the Dlx expression vectors to identify the regions that expressed the transfected plasmids (Fig. 3a,c,e,g), and to assess the efficiency of each electroporation. Electroporation of Dlx1, Dlx2 and Dlx5 expression vectors led to the appearance of ectopic DLX immunoreactivity within 3 hours (not shown); by 7 hours, strong expression (comparable with endogenous levels) was detectable (Fig. 5b). Expression from the Dlx1, Dlx2 and Dlx5 vectors produced roughly equivalent levels of DLX expression, as judged by immunofluorescence with an anti-distal-less antibody (Fig. 3b,d,f). The patterns of GFP and DLX expression were virtually identical (Fig. 3a-f), suggesting extensive co-transfection of the electroporated plasmids. Counting of GFP- and DLX-positive cells showed that >95% of electroporated cells expressed both plasmids.
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The kinetics of Dlx2-mediated GAD65 induction was assessed by analyzing electroporated slices after 7, 19 and 35 hours in culture (Fig. 5). Whereas strong expression of DLX2 could be detected after 7 hours, ectopic expression of GAD65 was not (n=3). GAD65 expression was detectable by 19 hours, and was increased by 35 hours (Fig. 5).
Electroporation of the Dlx2 vector also induced the expression of Gad67. As no antibody specific to GAD67 was available, we studied this induction using in situ RNA hybridization (Fig. 4i-k). Although only 5/28 experiments showed a clear induction of Gad67 by Dlx2, we suggest that this is an underestimate, as most of the negative experiments were performed during early stages of the project, when we were establishing the protocol and often encountered low levels of electroporation. The last three experiments show Gad67 induction by Dlx2. Attempts to compare the efficiencies of GAD65 and GAD67 induction have been hampered by our inability to use the same method to study both of their expression (in situ hybridization for Gad65 has not worked, and we have no antibody specific for GAD67).
Thus, these experiments indicate that Dlx2 and Dlx5 are efficient inducers of a fundamental element of the GABAergic phenotype in cortical cells. However, Dlx2 and Dlx5 failed to clearly induce other markers that are characteristic of basal telencephalic neurons or cortical interneurons such as NPY, nNOS, substance P, enkephalin, OCT6, calretinin or calbindin, although none of these shows as close a temporal or spatial correlation during development to Dlx expression as do the Gad genes. Furthermore, ectopic expression of the DLX proteins did not seem to affect expression of TBR1, a transcription factor that marks most early cortical plate neurons (Hevner et al., 2001) (data not shown).
Dlx genes are regulated by DLX proteins
Previous loss of function studies provided evidence that Dlx1 and Dlx2 together regulate Dlx5 and Dlx6 in most of the forebrain (Anderson et al., 1997a; Zerucha et al., 2000). In addition, an intergenic enhancer for mouse Dlx5 and Dlx6 and zebrafish dlx4 and dlx6 has been shown to be regulated by Dlx1 and Dlx2 in transgenic mice and in tissue culture cells (Zerucha et al., 2000). Here, we used the gain-of-function assay to test whether ectopic expression of Dlx1, Dlx2 and/or Dlx5 is sufficient to induce expression from endogenous Dlx genes and from a co-electroporated Dlx5/6 enhancer/reporter plasmid (mI5/6i-lacZ) (Zerucha et al., 2000).
Electroporation of Dlx2 in the cortex induced Dlx5 RNA, as judged by in situ hybridization (Fig. 4i,j,l). Although the efficiency of this induction was not robust (occurring in only 4/23 cases), we suggest this is an underestimate for the same reasons discussed above regarding the induction of Gad67. Electroporation of Dlx1 did not induce expression of the endogenous Dlx5 gene (data not shown). Induction of Dlx6 expression was not detected following electroporation with either Dlx1, Dlx2 or Dlx5 (data not shown), nor with binary combinations of these expression vectors (n=28).
Next we tested whether the Dlx expression vectors could induce expression from a co-electroporated mI5/6i-lacZ enhancer/reporter plasmid. We performed the electroporations at various rostrocaudal and dorsoventral positions of the E12.5 brain, and assessed the pattern of ß-galactosidase through immunohistochemistry. Control experiments showed that electroporation of the mI5/6i-lacZ plasmid alone resulted in ß-galactosidase expression that was restricted to areas that endogenously express the Dlx genes, such as the ganglionic eminences, septum and ventral thalamus (Fig. 6a-f). Thus, Dlx5/6 enhancer-mediated expression appears to critically depend on the transcription factors that are present in the Dlx+ regions.
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While the Dlx5 expression vector was also a potent inducer of ß-galactosidase expression throughout the brain (Fig. 6n,r; n=15), the Dlx1 expression vector produced a very different result from the Dlx2 and Dlx5 vectors. Dlx1 induced very little ß-galactosidase expression in the cerebral cortex (not shown) and the tectum (Fig. 6p). However, the Dlx1 vector was capable of inducing ß-galactosidase in the dorsal thalamus, the tegmentum (ventral midbrain) and in the hindbrain (Fig. 6t) (n=8).
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DISCUSSION |
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This gain-of-function study complements the analysis of the Dlx1 and Dlx2 loss-of-function double mutants, which show differentiation and migration defects in the development of most telencephalic GABAergic neurons (Anderson et al., 1997a; Anderson et al., 1997b; Anderson et al., 1999; Anderson et al., 2001; Bulfone et al., 1998; Pleasure et al., 2000). Despite the severe block in GABAergic neuron development in the Dlx1 and Dlx2 mutants, which includes decreased expression of Dlx5 and Dlx6, GAD expression persists, showing that other transcription factors are also capable of regulating GAD expression in the forebrain. Candidates for these genes include Mash1 (Ascl1 Mouse Genome Informatics), and Gsh1 and Gsh2. These transcription factors continue to be expressed in the Dlx1 and Dlx2 mutants (Yun and J. L. R. R., unpublished observations), and gain-of-function evidence suggests that Mash1 is able to induce GABAergic neurons (Fode et al., 2000).
This analysis has begun to dissect the specific roles for the Dlx genes in the development of GABAergic neurons. While they can induce both Gad65 and Gad67 within 20 hours, we did not find induction of other markers of GABAergic projection neurons (e.g. enkephalin and substance P) or of GABAergic interneurons (e.g. calbindin and nNOS) (data not shown). Perhaps additional time is needed to see expression of these genes. Alternatively, cortical cells may not be fully competent to express all genes found in GABAergic neurons, as other transcription factors may be needed in parallel, or in conjunction with, the DLX proteins.
While Dlx2 and Dlx5 were robust inducers of GADs, Dlx1 was not. Based on their amino acid sequences, the Dlx genes fall into two major homology groups: Type A (Dlx2, Dlx3 and Dlx5) and Type B (Dlx1, Dlx6 and Dlx7) (Stock et al., 1996; Liu et al., 1997). These are the first results that suggest biochemical differences in the functions of A and B subtypes. Furthermore, while Dlx2 and Dlx5 efficiently transactivated expression from a co-electroporated Dlx5/6 enhancer/reporter plasmid in every CNS region tested (Fig. 6h,j,l,n,r), Dlx1 appeared more restricted in its ability to activate the Dlx5/6 enhancer. For example, while Dlx1 activated Dlx5/6 enhancer expression in the ventral midbrain (tegmentum) and in the ventral hindbrain (Fig. 6t), we did not observe any effect of Dlx1 on the Dlx5/6 enhancer in the dorsal midbrain (superior colliculus) (Fig. 6p). We also noted that whereas Dlx2 could induce both endogenous Dlx5 and the Dlx5/6 enhancer, it did not effectively induce endogenous Dlx6 expression. This may be explained by the observation that expression from the Dlx5/6 enhancer more closely resembles endogenous Dlx5 than Dlx6 expression. These results suggest that there is a separate Dlx6 enhancer, which may be less sensitive to activation by Dlx2.
Therefore, the results of this and our previous studies suggest both redundant and distinct functions for different members of the Dlx gene family. Dlx1 and Dlx2 are redundant for the control of late-born neurons of the basal telencephalon to efficiently migrate away from the subventricular zone and to express markers of more differentiated neurons (such as DARPP32) (Anderson et al., 1997; Marín et al., 2000) (Yun and J. L. R. R., unpublished). Neither single mutant shows this phenotype. However, we provide evidence that Dlx1 and Dlx2 show different abilities to ectopically induce GAD expression, and to regulate the Dlx5/6 enhancer. Thus, perhaps both Type A (Dlx2 and Dlx5) and Type B (Dlx1 and Dlx6) Dlx genes have redundant functions in regulating aspects of differentiation related to migration, but Type A and Type B Dlx genes may have distinct functions with respect to cross-regulation of Dlx genes and GAD expression. The hypothesis that Type A and B Dlx genes have different functions in vivo, is consistent with the observation that Dlx1 and Dlx2 mutants have distinct maxillary dysmorphologies, despite their similar expression patterns in the first branchial arch (Qiu et al., 1997).
The electroporation method used in these experiments is a novel adaptation of the approach devised for in ovo electroporation of chicken embryos (Funahashi et al., 1999). Slice culture electroporation is spatially more precise and it can be readily applied to diverse species. Given the large number of mouse mutants now available, this opens the possibility of rescuing mutant phenotypes. Furthermore, many developmental and physiological processes are largely unperturbed in slice cultures (e.g. neuronal migrations) (Anderson et al., 1997b; Anderson et al., 2001); thus electroporation that does not restrict the size of the transfected plasmid (unlike most viral vectors) will be an effective method with which to study rapidly the effects of genes on specific processes. Finally, because one can efficiently co-electroporate two or more plasmids into single cells, this approach promises to be an important method to study multi-component processes, such as transcriptional regulation in physiologically relevant cells. This proved to be the case for establishing that Dlx2 and Dlx5 are effective inducers of the Dlx5/6 enhancer.
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ACKNOWLEDGMENTS |
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