In the embryonic forebrain, the expression pattern of DLX transcription factors is almost identical to that of the glutamic acid decarboxylases (GAD65 and GAD67), enzymes that synthesize the neurotransmitter GABA (Stühmer et al., 2002a
). Ectopic expression of Dlx2
can induce GAD expression in cortical progenitors (Stühmer et al., 2002a
genes are expressed in all migrating GABAergic neurons, and differentiation, migration, and survival are dependant upon Dlx1
function (Anderson et al., 1997a
; Cobos, 2006
; Cobos et al., 2007
; Cobos et al., 2005
). The expression pattern of EGFP in i12b-Cre;Z/EG and URE2-Cre;Z/EG animals during embryonic development and in the adult closely resembles forebrain GABA expression (- and (Katarova et al., 2000
; Stühmer et al., 2002a
). Thus, we hypothesized that I12b-Cre and URE2-Cre activity would label GABAergic neurons. To test this idea, we analyzed co-expression of EGFP and GABA in the somatosensory cortex and hippocampus of I12b-Cre;Z/EG and URE2-Cre;Z/EG mice (). Greater than 95% of GABA+ cells co-expressed EGFP in the somatosensory cortex and hippocampus of I12b-Cre;Z/EG animals (, and data not shown) while approximately 43% of GABA+ cells co-labeled for EGFP in the somatosensory cortex and hippocampus of URE2-Cre;Z/EG animals (, and data not shown). This indicates that nearly all GABAergic neurons in the cortex and hippocampus were captured by lineage-mapping using I12b-Cre, but not with URE2-Cre.
I12b-Cre and URE2-Cre label GABAergic neurons and not glia in the adult brain
We next examined the percentage of EGFP+ cells that co-express GABA. In the cortex and hippocampus, approximately 85% and 75% of EGFP+ cells in I12b-Cre;Z/EG and URE2-Cre;Z/EG mice, respectively, expressed detectable levels of GABA. The lack of 100% overlap probably arises from a well-known limitation in GABA-detection (Pow, 1997
Basal forebrain cholinergic neurons, which express choline acetyltransferase (ChAT), are generated in the MGE, septal area, and anterior POA (Furusho et al., 2006
). To determine if I12b-Cre or URE2-Cre are active in this forebrain population, we double-labeled sections for ChAT and EGFP from adult I12b-Cre;Z/EG and URE2-Cre;Z/EG mice. Strongest ChAT immunoreactivity was detected in the striatum, septal nucleus, substantia innominata, diagonal band, and hypothalamus. In the adult striatum, EGFP was not co-localized with ChAT-expressing neurons in both I12b-Cre;Z/EG and URE2-Cre;Z/EG mice () indicating that I12b and URE2 are not active in striatal cholinergic neurons. Interestingly, a small percentage of basal forebrain cholinergic neurons in the diagonal band, magnocellular preoptic area, substantia innominata, and hypothalamus co-labeled for EGFP and ChAT in i12b-Cre;Z/EG animals (n=3), while no co-localization of EGFP and ChAT was detected in similar regions in URE2-Cre;Z/EG animals (n=3) (Supplemental Figure S4). Thus, I12b, but not URE2, marks a subset of basal forebrain cholinergic neurons.
Progenitors within the mouse MGE and LGE give rise to both GABAergic neurons and oligodendrocytes (He et al., 2001
; Petryniak et al., 2007
; Yung et al., 2002
). Transgenic mice that express Cre in the ventricular zone of the LGE (i.e. Gsh2-Cre) or MGE (i.e. Nkx2.1-Cre) label both GABAergic interneurons and oligodedendrocytes (Fogarty et al., 2007
; Kessaris et al., 2006
; Xu et al., 2008
). We demonstrated that DLX2 is expressed in uncommitted progenitors of the MGE that generate both oligodendrocytes and GABAergic interneurons; continued expression of DLX restricts progenitors to a neuronal fate (Petryniak et al., 2007
). Thus, we predict, unlike other transgenic mice that express CRE in the ganglionic eminences, that I12b-Cre and URE2-Cre would label GABAergic neurons, but not glia, if expression of Cre-recombinase occurs after the neuron-glia fate decision.
To determine whether i12b-Cre and URE2-Cre label oligodendrocytes in addition to GABAergic neurons, we examined the co-expression of EGFP and oligodendrocyte markers OLIG2, OLIG1, and SOX10 in the forebrain of I12b-Cre;Z/EG and URE2-Cre;Z/EG mice (). We detected less than 0.5% of co-expression between all oligodendrocyte-markers and EGFP in both adult and newborn I12b-Cre;Z/EG and URE2-Cre;Z/EG sections ( and data not shown). It has been reported that Dlx2
-lineage cells might give rise to perinatal astrocytes (Marshall and Goldman, 2002
). To determine if I12b-lineage or URE2-lineage cells gave rise to forebrain astrocytes, we analyzed the expression of the astrocytic marker GFAP. Numerous GFAP-expressing cells were detected in the hippocampus and white matter of adult mice, while fewer GFAP+ cells were observed in the neocortex. We did not detect any overlap between EGFP and GFAP in the neonatal or adult hippocampus or neocortex of I12b-Cre;Z/EG or URE2-Cre;Z/EG mice ( and data not shown). Overall this data indicates that I12b-Cre and URE2-Cre are lineage-restricted to neurons within the telencephalon.
Given that DLX2 is expressed in multi-potent progenitors but I12b-Cre and URE2-Cre specifically label neurons but not glia, our data suggests that CRE is expressed after endogenous DLX2 and in progenitors committed to a neuronal fate. To address this directly, we analyzed the expression of DLX2 and CRE in the ganglionic eminence at E12.5, E15.5 and P0 of I12b-Cre and URE2-Cre animals ( and data not shown). Despite functional CRE activity in URE2-Cre animals (-), we did not detect CRE protein by immunolabeling in URE2-Cre forebrain sections at these ages (data not shown), indicating CRE expression is low in URE2-Cre animals. In I12b-Cre forebrain sections, CRE was highly expressed within the ganglionic eminences and tangentially migrating immature interneurons (). Co-labeling showed numerous DLX2+ cells and few CRE+ cells within the VZ, DLX2/CRE co-expression at the VZ/SVZ border, and many more CRE+ than DLX2+ cells within the MZ. Thus, this supports a model that the neural/glial fate switch takes place either in the VZ or in the layer of the SVZ (SVZ1) that is rich in DLX2+ cells, but has few I12b-CRE+ cells.
DLX2 and CRE expression in embryonic and newborn I12b-Cre;Z/EG mice