Fig 1.
Staining patterns for each of the probes used in the present study in the frontal neocortex.
Panel A shows staining for NURR1 labeled cells in the preplate; the deepest region of the cortex. TBR1 (panel B) labeled all projection neurons in the cortex, including those found in layers 2, 3, 5 and 6. Panels c and d depict four markers for deep (5 and 6) layers: NOR1 (Nr4A3), DAARP-32; FOXP2 and CTIP2. BRN2 identified cells in layers 2 and 3 as well as a small number of cells in layer 5 (panel E). The superficial markers used included CUX1 (Layer 2–4, Panel F) and CART (Layer 3, Panel G). Scale bar = 200μm. Numbers in panel A delineate cortical layers. See text for details.
Fig 2.
Nissl section depicting the regions studied.
The low power micrographs in panels A, C and E show the regions from which sections were selected to examine pars principalis of the anterior olfactory nucleus (AONpP, Panel A), anterior piriform cortex (APC) and olfactory tubercle (OT, Panel C) and posterior piriform cortex (PPC, Panel E). The boxed regions in each panel are the zones depicted in Figs 3–6. OB = olfactory bulb, TT = tenia tecta. Panels B, D, and F are higher magnification views of the laminar structure of the areas. All zones have an outer molecular layer (layer 1). The AONpP has one cellular zone (layer 2), while the piriform cortices and OT have two (layers 2 and 3). Scale bar for B, D, and F = 200μm. See text for details.
Table 1.
Antibodies.
Fig 3.
Patterns of neocortical layer markers in the AONpP.
A). TBR1-labelled cells were found throughout Layer 2 of the AONpP as well as in the tenia tecta and mitral cell layer of the OB. B, C) Deep markers were differentially distributed in the region. Layer 2 exhibited dense and evenly-spread CTIP2-positive cells (Fig 3b), while NOR1 was found primarily in the dorsal portion of the structure (Fig 3c, top) Cells expressing the other two marker were rare and found primarily in layer 1: DARRP-32 (note dense staining in the glomerular layer of the OB at left, an area containing large numbers of dopaminergic interneurons, Fig 3c; Liu et al, 2013) and FOXP2 (most often found near the OB, Fig 3b). CTIP2 stained cells were also found in layer 1 but never in cells that expressed one of the other markers. The superficial markers were also differentially distributed. Both BRN2 (Fig 3d) and CUX1 (Fig 3e) were observed primarily in deep cells (except in pars medialis, where CUX1-labeled cells spanned the entire region) with highest densities in the region under the LOT (pars lateralis). All CUX1 cells also expressed BRN2, and over 90% of CUX1 and BRN2 cells also expressed CTIP2. The anti-BRN2 antibody also labeled the LOT (right) and RMS (core of the olfactory peduncle). Scale bar = 200μm. Dorsal to top, lateral to right.
Fig 4.
Patterns of neocortical layer markers in the APC.
A) TBR-1 heavily labeled cells in Layer 2 as well as scattered cells in Layer 3. Of the 4 deep layer markers (B,C), only CTIP2 exhibited dense staining. The other three (FOXP2, NOR1 and DAARP32) labeled sparse number in Layers 1–3. The dense staining for FOXP2 and DAARP32 seen at the bottom of the figures sharply demarcates the APC from the more ventral OT. The other three makers exhibited very different patterns: BRN2 staining was found more in the ventral APC (D), CUX 1 in the deeper portions of both Layer 2 and 3 (E), and CART in the middle of Layer 2(F). Scale bar = 200μm. Dorsal to top, lateral to right.
Fig 5.
Patterns of neocortical layer markers in the PPC.
A) TBR-1 heavily labeled cells in Layer 2 as well as scattered cells in Layer 3. As in the APC many cells in layers 2 and 3 exhibited the deep marker CTIP2 (B). Only widely scattered cells exhibited FOXP2 and DAARP 32 and NOR1 (B,C). The other three makers exhibited very different patterns: CUX 1 staining (E) was strong throughout layers 2 and 3, BRN2 staining much more modest in the same regions, CART was restricted to the middle of layer 2 (F). Scale bar = 200μm. Dorsal to top, lateral to right.
Fig 6.
Patterns of neocortical layer markers in the OT.
A) TBR-1 (A), BRN2 (D) and CART (E) were only found scattered in the very deepest regions of the OT. All four of the deep laminar markers heavily labeled the region. Both CTIP2 and FOXP2 cells were broadly present in Layer 2 and scattered in Layer 3 (Fig 6b). On the medial side most FOXP2 cells coexpressed CTIP2 but the percentage of cells with both markers was reduced laterally. DARRP-32 cells were dense on the lateral side near the APC and in deep regions of the OT, while NOR1 cells were found in Layer 2 in the medial OT (Fig 6c). Scale bar = 200μm. Dorsal to top, lateral to right.
Fig 7.
Patterns of staining in the frontal cerebral cortex of pups receiving the mitotic marker EDU on either E12 (A) or E16 (B).
Note the age-related differences in EDU (red) staining: at E12 EDU is found primarily deep in the cortex (bottom of panel), while at E16 it is found in superficial layers (top). In the E12 tissue (A) substantial numbers of cells expressing the deep marker CTIP2 co-labelled with EDU (pink) while only a few of the cells labelled with CUX also have the maker (yellow). E16 injections (B) labeled cells in layer 3 expressing the superficial marker CART as wells as more superficial cells. The marker labels all proliferating cells, including glial and perivascular cells. Scale bar = 200μm.
Fig 8.
Patterns of staining in the AONpP of pups that receiving the mitotic marker EDU either on E12 (A-D) or E16 (E-H).
Panels B, D, F and H provide higher power views of the cellular regions of the figures to their left. A gradient of EDU labeling was observed with more cells in the superficial half of layer 2 labeled at E12 and more in the deep portion at E16. In the E12 samples both CUX1/CTIP2 (light blue; CUX1/CTIP2+EDU are depicted in white) and CTIP2 alone cells (CTIP2+EDU depicted as pink) were found co-labeled with EDU (Fig 8A and 8B) and a small number of BRN2/EDU-positive cells (yellow) were also observed (Fig 8C and 8D), mostly in the superficial portion of layer 2. By E16, a) EDU was rarely found in CTIP2 single-labeled cells (Fig 8E and 8F) and b) BRN2/EDU cells were much more abundant (Fig 8G and 8H). Taken together, the findings suggest that the pattern of cell birth is consistent with that seen in the cerebral cortex in that cells with cortical “deep markers” were labelled early while those with “superficial” markers were produced on E16. Scale bar = 200μm.
Fig 9.
Patterns of staining in the APC of pups that receiving the mitotic marker EDU either on E12 (A,B) or E16 (C,D).
Panels B and D provide higher power views of the cellular regions of the figures to their left. E12 injections labeled many cells in Layer 3 and in the deep portion of Layer 2. CUX1/CTIP2 (light blue; CUX1/CTIP2+EDU are depicted in white) and CTIP2+EDU (pink) were observed. Few labelled cells were observed in layers 2 and 3 after E16 EDU treatment. Scale bar = 200μm.
Fig 10.
Patterns of staining in the PPC of mice that receiving the mitotic marker EDU either on E12 (A,B) or E16 (C,D).
Panels B and D provide higher power views of the cellular regions of the figures to their left. The E12 injection labeled many CTIP2 cells (CTIP2+EDU depicted as pink) in layer 3 and in the deep portion of Layer 2. Relatively few CUX1 +EDU (yellow) or CUX1/CTIP2+EDU (white) were observed. Scattered labelled cells were seen in layer 2 after EDU injection on E16; most were CUX1+EDU (yellow) positive. Numbers and lines on the left margin of A and C delineate cortical layers. Scale bar = 200μm.
Fig 11.
Retrograde tracer (BDA, red) injections into the OB labelled cell bodies expressing each of the deep and superficial markers tested.
This figure depicts examples for each structure projects to the OB. A, B: AONpP sections exhibiting double labeled cells (yellow) containing BDA and BRN2 (A) and NOR1 (B). Piriform cortices: double labeling for CART (APC: C, PPC: D, yellow), CUX1 (APC: E, PPC:F yellow) and CTIP2 (APC: E, PPC:F pink). White cells in panels E and F are triple labeled for BDA, CUX1 and CTIP2. Scale bars = 200μm.
Table 2.
Regional distribution of neocortical markers in olfactory cortex.