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. 2009 Feb;12(2):141-9.
doi: 10.1038/nn.2241. Epub 2009 Jan 11.

Identification of distinct telencephalic progenitor pools for neuronal diversity in the amygdala

Tsutomu Hirata  1 , Peijun Li, Guillermo M Lanuza, Laura A Cocas, Molly M Huntsman, Joshua G Corbin
Affiliations

Identification of distinct telencephalic progenitor pools for neuronal diversity in the amygdala

Tsutomu Hirata et al. Nat Neurosci. 2009 Feb.

Abstract

The development of the amygdala, a central structure of the limbic system, remains poorly understood. We found that two spatially distinct and early-specified telencephalic progenitor pools marked by the homeodomain transcription factor Dbx1 are major sources of neuronal cell diversity in the mature mouse amygdala. We found that Dbx1-positive cells of the ventral pallium generate the excitatory neurons of the basolateral complex and cortical amygdala nuclei. Moreover, Dbx1-derived cells comprise a previously unknown migratory stream that emanates from the preoptic area (POA), a ventral telencephalic domain adjacent to the diencephalic border. The Dbx1-positive, POA-derived population migrated specifically to the amygdala and, as defined by both immunochemical and electrophysiological criteria, generated a unique subclass of inhibitory neurons in the medial amygdala nucleus. Thus, this POA-derived population represents a previously unknown progenitor pool dedicated to the limbic system.

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Figures

Figure 1
Figure 1. Expression of Dbx1 and knockin approach
Expression of Dbx1 (a,b) and Foxg1 (c,d) in wild-type embryos, and Cre (e,f) in Dbx1+/CreERT2 knock-in embryos, at E11.5 is shown at rostral and caudal levels in coronal sections. Dbx1 is expressed in the septum (a, arrow), the ventral pallium (b, arrowhead) and the preoptic area (b, double arrowhead). Foxg1 expression delineates the telencephalon from the diencephalon (c, d). Dotted lines in b,d indicate the telencephalic-diencephalic border. Cre expression recapitulates Dbx1 expression in the septum (e, arrow), the ventral pallium (f, arrowhead) and the preoptic area (f, double arrowhead). Schematic of the knockin approach in which an IRES-CreERT2 cassette was inserted into the 3’ UTR of the 4th exon of Dbx1 is shown in (g). Abbr: CGE; caudal ganglionic eminence, CTX; cerebral cortex, HIP; hippocampus, HYP; hypothalamus, MGE; medial ganglionic eminence SEP; septum (Note: Embryonic anatomy is based on37). Scale bar; 250 µm
Figure 2
Figure 2. Dbx1-derived cells in the post natal amygdala
β-galactosidase staining of coronal sections from Dbx1+/CreERT2;R26RLacZ brains at P21 reveals numerous recombined LacZ+ cells in the amygdala. Distribution of recombined cells from tamoxifen (TM) administration at E9.5 (b,g,l), E10.5 (c,h,m), E11.5 (d,i n) or E12.5 (e, j o) are shown. Boxed area on schematics (a, f, k) indicate the region of the amygdala shown in each panel. E9.5 TM delivery results in recombined cells in the cortical and medial nuclei (b,g,l, arrowheads), but not the lateral and basolateral nuclei. In addition to the cortical and medial nuclei, TM delivery at E10.5 (c,h,m) or E11.5 (d,i,n) results in recombined cells in the lateral and basolateral nuclei. TM delivery at E12.5 results in recombined cells in the lateral and basolateral nuclei only (e,j,o). Abbr: BLA; basolateral nucleus, Co; cortical nucleus, HYP; hypothalamus, LA; lateral nucleus, MePD; medial nucleus posterior dorsal, MePV; medial nucleus posterior ventral. Scale bar; 500 µm
Figure 3
Figure 3. Dbx1-derived cells in the developing basal telencephalon
β-galactosidase staining of coronal sections of E13.5 Dbx1+/LacZ knock-in (a-c) and Dbx1+/CreERT2;R26RLacZ embryos (d-f) reveals putative migrating cells to the region of the developing amygdala. LacZ+ cells are shown along the both LCS (a-f, single arrow, inset in d shows a higher magnification of LacZ+ cells of the LCS) and the putative POA-amygdala migratory stream (PAS) (b,c,e,f, double arrows). Foxg1 expression in corresponding sections in Dbx1+/LacZ knock-in embryos (g-i) indicates that Dbx1-derived cells distributed along PAS are located within the telencephalon. Note the relative paucity of LacZ+ cells in the ganglionic eminences and the developing cerebral cortex. Abbr: LGE; lateral ganglionic eminence, POA; preoptic area, POH; preopto-hypothalamic region, TH; thalamus. Scale bar; 250 µm
Figure 4
Figure 4. Distribution of YFP+ recombined cells at embryonic stages
Tamoxifen was administered to Dbx1+/CreERT2;R26RYFP embryos at E9.5 (a-c) or E10.5 (d-i) and analyzed at E12.5 as shown. E9.5 TM administration results in YFP+ recombined cells (green) primarily in the POA (asterisk), with only a few YFP+ recombined cells observed along the LCS as shown in a coronal section at low power magnification (a). Higher power magnification of areas highlighted by arrows in (a) shows leading processes of recombined cells (arrows,b, c). Numerous recombined cells are observed in the VZ of the POA (c, dotted line marks the edge of the tissue and the location of the lateral ventricle “v”). As shown in a low power coronal section (d), E10.5 TM administration (d, g) results in recombined cells along both the PAS migratory stream (arrow, f) and LCS (arrow, e). Higher power magnification shows Pax6 (red) negative, recombined cells along the LCS with migratory profiles directed toward the basal telencephalon (arrow, e). Recombined PAS cells also display migratory profiles directed toward the developing amygdala (arrow, f). The PAS migratory stream is most evident in horizontal sections (g, asterisk marks the POA), with higher power magnification of YFP+ cells also revealing recombined cells with leading processes oriented toward the developing amygdala (arrows, h, i). Abbr: AMY; developing amygdala, M-HB; midbrain-hindbrain region, PTH; prethalamus. Scale bar in (a) for (a, b, g); 250 µm. Scale bar in (i) for (b, c, e, f, h, i); 20 µm
Figure 5
Figure 5. Migration from the PSB and POA
DiI crystals (red) were placed in either the PSB in coronal slices (a, b, n=2) or in the POA (f, g, n=4) in horizontal slices at E13.5 in Dbx1+/CreERT2;R26RYFP embryos treated with tamoxifen at E10.5. After two days in vitro (DIV), DiI labeled cells (red) from both the PSB and the POA migrate to the basal telencephalon (b, g). Boxed regions show that migratory streams from both the PSB (arrows, c-e) and POA (arrows, h-j) comprise Dbx1-derived cells as revealed by co-expression of DiI (red) and YFP (green). DiI and YFP co-expressing cells are also shown from another brain slice (arrows, k-m). β-galactosidase stained horizontal sections from E12.5 Dlx2+/tauLacZ embryos (n) and Lhx2 mRNA expression (o) also mark the PAS migratory route (double arrows, asterisk marks the POA in (o)). Scale bar in (a) for (a, b, f, g); 250 µm. Scale bar in (c) for (c-e, h-m); 50 µm. Scale bar in (n) for (n, o); 250 µm.
Figure 6
Figure 6. Dbx1-derived cells express regional cell fate markers during embryogenesis
The expression of Tbr1 (a-g) and caldindin (h-n) in Dbx1+/CreERT2;R26RYFP embryos, and Dlx2 (o-r) in Dbx1+/CreERT2;R26RYFP; Dlx2tauLacZembryos at E12.5 in which TM was administered at E10.5 is shown. As shown in a coronal section (a), expression of Tbr1 (red) is detected in 90.2% (46/51) of YFP+ recombined cells (green) along the LCS (boxed region “b” and arrows in b-d show double labeled cells). In contrast, Tbr1 is not detected in any (0/33) YFP+ recombined cells along the POA-amygdala migratory stream (PAS) (boxed region “e” and arrows in e-g show lack of double labeling). As shown in a horizontal section (h), no (0/88) YFP+ recombined cells (green) of the LCS co-express calbindin (red) (boxed region “i” and arrows in i-k show lack of double labeling). However, 41.4% (46/111) of YFP+ recombined cells of the PAS (green) co-express calbindin (red) (boxed region “i” and arrows in l-n show double labeled cells). As shown in a coronal section (o), 80.8% (21/26) of YFP+ recombined cells of the PAS (green) also co-express Dlx2 (red) (boxed region “p” and arrows in p-r show double labeled cells). Asterisks in h and o mark the POA. Scale bar in (o) for (a, h, o); 250 µm. Scale bar in (p) for (b-g, i-n, p-r); 20 µm
Figure 7
Figure 7. Dbx1+ progenitors generate amygdala excitatory and inhibitory neurons
The cellular phenotype of recombined cells in the amygdala was examined at P21 in Dbx1+/CreERT2;R26RYFP mice that were administered tamoxifen at E10.5. All (32/32) YFP+ recombined cells (green) in the basolateral complex (a,d) and 95.3% (41/43) of recombined cells in the cortical nucleus (b,e) are positive for Tbr1 (red) as shown at low and high power magnification (arrows show double labeled cells). In contrast, no (0/41) YFP+ recombined cells (green, arrow) in the medial nucleus express Tbr1 (arrowhead) (c,f). Higher power magnification of the medial nucleus reveals YFP+ recombined cells co-expressing nNOS (arrows, g-i), calbindin (arrows, j-l), or calretinin (arrows, m-o). Percentages of YFP+ cells co-expressing each inhibitory marker in the medial nucleus is shown in (p), with 78.6% (114/145) of recombined YFP+ cells in the medial nucleus are positive for nNOS, 50.8% (30/59) are positive for calbindin, and 19.1% (18/94) are positive for calretinin. Scale bar in (a) for (a-c); 250 µm. Scale bar in (d) for (d-o); 20 µm.
Figure 8
Figure 8. Electrophysiological properties of Dbx1-derived medial amygdala neurons
DIC (a), fluorescent (b) and overlay (c) images of a typical YFP+ recombined medial amygdala neuron (arrows) with attached recording electrode. Live coronal slice indicating region of physiological recordings (d, asterisk). Firing patterns of a typical medial amygdala YFP+ recombined cell at threshold and sub-threshold (e) and supra-threshold (f) firing. Injected level of current for each trace is indicted below. Confocal image of biocytin filled cell (g) represented in d and e following physiological characterization. Note the fusiform-like dendritic arborization and the presence of sparsely spiny dendrites. Confocal image of biocytin filled cell conjugated to texas red following whole cell patch clamp recording is shown in (h). The current clamp trace of this cell is located in the upper right corner (scale bar: 200 ms horizontal, 20 mV vertical.) Fluorescent immunostaining for nNOS (green) reveals that this cell is also nNOS+ (yellow) (i).
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