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, 70 (5), 939-50

Lhx6 and Lhx8 Coordinately Induce Neuronal Expression of Shh That Controls the Generation of Interneuron Progenitors

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Lhx6 and Lhx8 Coordinately Induce Neuronal Expression of Shh That Controls the Generation of Interneuron Progenitors

Pierre Flandin et al. Neuron.

Abstract

Lhx6 and Lhx8 transcription factor coexpression in early-born MGE neurons is required to induce neuronal Shh expression. We provide evidence that these transcription factors regulate expression of a Shh enhancer in MGE neurons. Lhx6 and Lhx8 are also required to prevent Nkx2-1 expression in a subset of pallial interneurons. Shh function in early-born MGE neurons was determined by genetically eliminating Shh expression in the MGE mantle zone (MZ). This mutant had reduced SHH signaling in the overlying progenitor zone, which led to reduced Lhx6, Lhx8, and Nkx2-1 expression in the rostrodorsal MGE and a preferential reduction of late-born somatostatin(+) and parvalbumin(+) cortical interneurons. Thus, Lhx6 and Lhx8 regulate MGE development through autonomous and nonautonomous mechanisms, the latter by promoting Shh expression in MGE neurons, which in turn feeds forward to promote the developmental program of the rostrodorsal MGE.

Figures

Figure 1
Figure 1. Lhx6PLAP/PLAP;Lhx8−/− Double Mutants Fail to Express Shh in Neurons of the E11.5 MGE Mantle Zone (MZ) and Have Reduced Shh Signaling in the Proliferative Zone of the Dorsal MGE
The genotype of the control embryo was Lhx6PLAP/+;Lhx8+/+. Subpallial images of E11.5 three serial coronal hemisections (rostral-most on the left) show the preservation of Lhx6-placental alkaline phosphatase (PLAP) expression (A–C and A′–C′) or in situ RNA hybridization staining for Shh (D–F and D′–F′), Ptc1 (G–I and G′–I′), Nkx6-2 (J–L and J′–L′), and Nkx2-1 (M–O and M′–O′). Arrows in (C) and (C′) show the reduced PLAP+ tangentially migrating cells in the mutant. Arrows in (E) and (E′) indicate the lack of Shh expression in the mantle region of the MGE of the mutant whereas Shh expression in the ventricular zone (VZ) of the ventral MGE and POA is not affected (arrowheads). Arrows in (H) and (H′) indicate the reduction in Ptc1 expression in the VZ of the dorsal MGE of the mutant. Arrows in (L) and (L′) indicate the reduction of Nkx6-2 expression in the dorsal-most MGE (sulcus between LGE and MGE). Arrows in (N) and (N′) indicate lack of Nkx2-1 expression in the dorsal MGE, and arrowheads in (O) and (O′) show ectopic expression of Nkx2-1 in the superficial MZ of the MGE. Abbreviations: LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; dMGE dorsal MGE; vMGE, ventral MGE; MZ, mantle zone; POA, preoptic area. Scale bar: 500 μm. See also Figure S1 and Table S1.
Figure 2
Figure 2. Molecular Defects in the Ventral Telencephalon of the Lhx6PLAP/PLAP;Lhx8−/− Double Mutant at E14.5
The genotype of the control embryo was Lhx6PLAP/+;Lhx8+/+. MGE images of three serial coronal hemisections (rostral-most on the left) show Lhx6-PLAP expression (A–C and A′–C′) and in situ RNA hybridization staining for Nkx2-1 (D–F and D′–F′), Gli1 (G–I and G′–I′), Lmo3 (J–L and J′–L′), and immunofluorescence using an antibody to SOX6 (M–O and M′–O′). Arrows in (D) and (D′) indicate the reduction in Nkx2-1 expression in the SVZ of the MGE of the double mutant. Arrows in (L) and (K′) indicate lack of Lmo3 in the SVZ of the MGE of the double mutant. Arrows in (N) and (N′) indicate maintenance of SOX6 expression in the SVZ of the MGE, and arrowheads in (F′) and (O′) indicate ectopic Nkx2-1+ and SOX6+ cells scattered in the MZ of the caudoventral telencephalon in the double mutant, respectively. Abbreviations: BNST, bed nucleus stria terminalis; GP, globus pallidus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; Se, septum. Scale bar: 500 μm. See also Figure S2 and Table S2.
Figure 3
Figure 3. Cortical Interneuron Defect in the Lhx6PLAP/PLAP;Lhx8−/− Double Mutant at E14.5 and E18.5
The genotype of the control embryo was Lhx6PLAP/+;Lhx8+/+. E14.5 cortex and LGE stained for Lhx6-PLAP activity (A and A′), SOX6 (B and B′), and Nkx2-1 (C and C′). Note the severe reduction of Lhx6-PLAP+ and SOX6+ cells in the MZ (arrow), whereas the SVZ is less affected (A and A′; B and B′). Also note the ectopic Nkx2-1+ cells in the ventrolateral cortex and external capsule (EC; arrow in C), and the increase numbers in the striatum (arrowhead in C′). E18.5 neocortex and hippocampus stained for Lhx6-PLAP activity (D and D′; F and F′), or immunostaining for NKX2-1 (E and E′) and SOX6 (G and G′). Note the severe reduction of PLAP staining in most of the neocortex (except the SVZ), but its preservation in the hippocampus (D and D′); the regions with preserved PLAP expression coincide with regions containing ectopic NKX2-1+ cells (E and E′). (F) and (F′) illustrate an ectopic collection (E) of Lhx6-PLAP expressing cells in SVZ of the MGE of the double mutant at E18.5, suggesting a defect in cell migration. Abbreviations: CP, cortical plate; EC, external capsule; E, ectopia; GP, globus pallidus; H, hippocampus; MZ, marginal zone; St, striatum; SVZ, subventricular zone. Scale bar: 200 μm. See also Figure S3 and Table S2.
Figure 4
Figure 4. Molecular Defects in the MGE of the Dlx1/2-cre;ShhF/− Mutant at E11.5
The genotype of the control embryo was ShhF/−. MGE images of three serial coronal hemisections (rostral-most on the left) show in situ RNA hybridization staining for Shh exon 2 (A–C and A′–C′), Shh (640 bp covering most of the coding region) (D–F and D′–F′), Ptc1 (G–I and G′–I′), Gli1 (J–L and J′–L′), and Nkx6-2 (M–O and M′–O′), and NKX2-1 immunostaining (P–R and P′–R′). Note the loss of Shh exon2 expression in the MGE MZ (arrows in B and B′), but its maintenance in the MGE VZ (arrowheads in C and B′), and the reduced expression of Ptc1, Gli1, and Nkx6-2 in the VZ of the dorsal MGE (arrows in H and G′, K and J′, and N and M′); NKX2-1 expression was only reduced in a small dorsal MGE region (arrows in Q and Q′). Abbreviations: LGE, lateral ganglionic eminence; MGE, ventral medial ganglionic eminence; dMGE, dorsal MGE. Scale bar: 400 μm. See also Figure S4 and Table S1.
Figure 5
Figure 5. Molecular Defects in the MGE of Dlx1/2-cre;ShhF/− Mutant at E14.0
The genotype of the control embryo was ShhF/−. MGE images of two serial coronal hemisections (rostral-most on the left) show in situ RNA hybridization staining for Lhx6 (A and B; A′ and B′) and Lhx8 (C and D; C′ and D′), and NKX2-1 immunostaining (E and F; E′ and F′). Note the reduction in Lhx6, Lhx8, and NKX2-1 expression in the VZ, SVZ, and MZ of the rostrodorsal MGE and ventral septum of the mutant (arrows). Abbreviations: rdMGE, rostrodorsal medial ganglionic eminence; Se, septum; St, striatum. Scale bar: 200 μm. See also Figure S5 and Table S3.
Figure 6
Figure 6. Molecular Defects in the Rostral Basal Ganglia (Striatal and Septal Regions) of Dlx1/2-cre;ShhF/− Mutant at E18.5
The genotype of the control embryo was ShhF/−. Two serial coronal hemisections (rostral-most on the left) show in situ RNA hybridization staining for Nkx2-1 (A and B; A′ and B′), Lhx6 (C and D; C′ and D′) and Lhx8 (E and F; E′ and F′). Note the reduction in Nkx2-1, Lhx6, and Lhx8 expression in the VZ, SVZ and MZ of the rostrodorsal MGE (arrow) and ventrolateral septum of the mutant. Increased apoptosis (activated Caspase-3) in the Dlx1/2-cre;ShhF/− mutant compare to the control (G and H; G′ and H′); boxed regions are 2-fold higher magnification. Abbreviations: AC, anterior commissure; DB, diagonal band; Se, septum; St, striatum. Scale bar: 500 μm. See also Figure S6 and Table S3.
Figure 7
Figure 7. Molecular Defects in the Neocortex of Dlx1/2-cre;ShhF/− Mutant at E14.0, E18.5, and P24
The genotype of the control embryo was ShhF/−. Coronal hemisections show in situ RNA hybridization staining for Lhx6 at E14.0 (A and A′) and E18.5 (B and B′). Coronal sections from 24-day-old mice were stained using PV (C and C′), SOM (D and D′), CR (E and E′), and NPY (F and F′) antibodies. The graph in (G) summarizes the reduction in PV+, SOM+, and CR+ cells in the superficial (Sup) and deep layers of the somatosensory cortex. NPY+ cells were not reduced. Data are the mean ± SEM. *p < 0.05; **p < 0.01 (paired Student's t test; see Experimental Procedures). Abbreviations: CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; Sup, superficial; St, striatum; SVZ, subventricular zone. Scale bar: 100 μm for (A)–(B′) and 400 μm for (C)–(F′). See also Table S3.
Figure 8
Figure 8. LHX6 and LHX8 Can Bind to and Regulate the Shh Enhancer (SBE3)
(A) Sequence of SBE3. The predicted LHX-binding sequence is shown in red and its core region (TAATCA) is underlined. A box shows the region called probe A used in EMSA assays (below). (B) Electrophoretic mobility shift assay showing that LHX6 and LHX8 bind to the SBE3 Shh enhancer. DNA binding assay, using LHX6- or LHX8-containing nuclear extracts, results in a probe A-protein complex (arrows; lanes 2 and 7), compared to control extract (lanes 1 and 6; two “LHX site A nonspecific” bands; asterisks). When 200-fold mole excess unbiotinylated wild-type (WT) probe A (WT competitor [Comp]) was added to the binding assay, the DNA/protein complexes were nearly eliminated (lanes 3 and 8). On the other hand, biotinylated mutant (mut) probe A (LHX site A core sequence changed from TAATCA to TTTTTT) prevented the formation of the LHX-specific complex (arrows) (lanes 4 and 9). Addition of anti-FLAG antibody to the 3× FLAG-tagged LHX8/WT probe A binding reaction resulted in a supershift of the LHX8-probe A complex (arrowhead; lane 5). (C) Titration of wild-type competitor A (lanes 3, 4, 5) and mutant competitor A (lanes 6, 7, 8) in LHX8 EMSA competition assays. Nuclear extracts from control (lane 1) and LHX8 transfected (lanes 2–8) cells. LHX8-probe A complex (arrow) formation was differentially reduced using 200-fold, 50-fold, and 10-fold mole excess of either WT or mutant unbiotinylated probe A. Ten-fold excess unbiotinylated WT probe A (lane 5) resulted in a similar reduction to 200-fold unbiotinylated mutant probe A (lane 6). (D) Lhx6 and Lhx8 can activate transcription from the SBE3 Shh enhancer. Lanes 1–4: dissociated E12.5 MGE cells cotransfected with a WT Shh enhancer (SBE3-wtA) driving mCherry expression with expression vectors that drive expression of Lhx6, Lhx8, or a 1:1 mixture of Lhx6&8. Each transfection condition was analyzed by immunofluorescence for expression of mCherry. All three conditions induced a 3- to 5-fold increase in mCherry expression (red asterisks: statistical significance compare to control). Lanes 5–8: MGE cells cotransfected with the mutant SBE3 Shh enhancer driving mCherry (SBE3-mutA; see above in A); in this case Lhx6, Lhx8, and Lhx6&8 resulted in a slight (not statistically significant) increase in mCherry. Mutation of the LHX site in SBE3 resulted in ~2.5 reduced Lhx6- and Lhx6&8-mediated expression (black asterisks). Data are the mean ± SEM. n = 4; *p < 0.05 (two-tailed unpaired Student t test). (E) Schema summarizing results described in the paper, and hypothesized regulatory cascades (see Discussion). Abbreviations: Cx, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantle zone; POA, preoptic area.

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