A Novel Birthdate-Labeling Method Reveals Segregated Parallel Projections of Mitral and External Tufted Cells in the Main Olfactory System

Birthdate tagging of OB projection neurons. A, The experimental design of the birthdate tag method. The horizontal axis indicates the developmental time course. Individual neurons express CreER only transiently, soon after birth. An injection of TM at a certain developmental time point induces Cre-loxP recombination only in neurons expressing CreER. B, Schematized gene structures of the birthdate tag driver Neurog2^CreER(G2A) and Tau^mGFP-nLacZ reporter used in this figure. C, Proportion of EdU-incorporated cells in birthdate-tagged neurons shown by Cumming estimation plot. TM was given at a fixed time point at E12.5, and EdU was given at the indicated time before the TM injection. The raw values calculated from individual mice and mean ± SD are plotted on the upper axis. On the lower axis, mean differences from the 24-h group are plotted as bootstrap sampling distributions. Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars. D, The number of birthdate-tagged neuron subtypes counted in OB sections prepared from P14 Neurog2^CreER(G2A); Tau^mGFP-nLacZ mice that were given TM at different embryonic stages. The cell numbers are normalized by the layer distance used for cell counting. The color intensity coded columns show the means of tagged-cell numbers at individual TM injection stages. Black bars show the raw values obtained from individual mice (n = 3 for each TM stage). E, F, Immunostaining of MOB sections prepared from P14.5 Neurog2^CreER(G2A); Tau^mGFP-nLacZ mice for nucleus-localized β-gal (E) and membrane-bound GFP (F) reporters. TM was administrated at different embryonic stages as indicated at the top. Arrowheads (E, F) indicate reporter-tagged cell bodies, and arrows (F) indicate the position of reporter-tagged dendrites in the EPL. Asterisks (F) show the reporter-labeled internal plexiform layer that contains intrabulbar association fibers. Scale bar = 100 μm. GRL: granule cell layer. G, Proportion of the neuron subtypes birthdate tagged at different TM stages in the whole OB. The proportions were calculated without normalization by the tissue size, and therefore AOB neurons may look underrepresented compared with D, reflecting the small size of AOB. The diameter of each pie chart is proportional to the number of neurons. The exact numbers of neurons counted are shown in Extended Data Figure 1-1.

Characterization of birthdate-tagged neurons in the MOB. A, B, Distribution of birthdate-tagged MCs (A) and eTCs (B) in different OB sectors. The numbers of MCs and eTCs are counted from the rostral, dorsal, lateral, medial and ventral sectors of MOBs prepared from P14 Neurog2^CreER(G2A); Tau^mGFP-nLacZ mice that were given TM injection at the embryonic stages indicated at the bottom. Black bars indicate the raw values obtained from individual mice (n = 3 for each category), and colored columns represent the means. C–F, Expression of neuron subtype markers in β-gal reporter-labeled (green) neurons that were tagged at TM12.5 (C), TM13.5 (D), TM15.5 (E), and TM17.5 (F) in P14.5 Neurog2^CreER(G2A); Tau^mGFP-nLacZ MOBs. Red staining shows TBR2 (projection neuron marker), cholecystokinin (projection neuron marker), calretinin (periglomerular cell marker), tyrosine hydroxylase (interneuron marker) and calbindin (periglomerular cell marker) from left to right. DAPI (blue) staining is superimposed in all panels. The filled white arrowheads indicate marker-positive birthdate-tagged neurons, and the open arrowheads indicate marker-negative birthdate-tagged neurons. The percentage of marker-positive in β-gal-positive neurons is shown on the right top corner in each panel. The percentage is not shown when no marker-positive cells were found in β-gal-positive birthdate-tagged neurons. The positions of MOB layers are indicated on the right-hand end. Scale bar = 50 μm.

Birthdate tagging of OB projection neurons using OB-specific reporters. The birthdate-tagged projection neuron subtypes were counted in OB sections prepared from P14 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} (A, B) and P14–P15 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP (C, D) mice that had been given TM injection at different embryonic stages. A, C, The color intensity coded columns show the means of tagged cell numbers at individual TM injection stages. Black bars depict the raw values obtained from individual mice (n = 3 for each TM stage). The cell numbers are normalized by the layer distance used for cell counting. B, D, Proportion of neuron subtypes birthdate tagged at different TM stages in the whole OB. The proportions were calculated without normalization by the tissue size, and therefore may look slightly different from the quantifications in A, C. The diameter of each pie chart reflects the number of neurons. The exact numbers of neurons counted are shown in Extended Data Figure 3-1.

Birthdate tagging at TM10.5. A–F, Coronal brain sections prepared from a P22 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} mouse that was given TM injection at E10.5. Images for tdTomato reporter (red) and DAPI (blue). Medial is to the left and dorsal is to the top. The TM10.5 tagging selectively labels AOB projection neurons (A, arrowheads in A’). Their axons run in the deep dorsal side of the LOT (arrowheads in B, C) and project to the AOB-specific targets (D–F). Asterisks indicate the position of the anterolateral edge of the OT. Brain illustration in the middle summarizes TM10.5 axon projections and indicates the levels at which individual sections were prepared. The pie chart shows the proportion of neuron subtypes tagged at this TM stage with this reporter. Representative images from five mice. Scale bar = 100 μm (A’), 500 μm (A–F). BAOT: bed nuclei of the accessory olfactory tract, CoA: cortical amygdala, EC: entorhinal cortex, MeA: medial amygdala, PLCo: posterolateral cortical amygdala, PMCo: posteromedial cortical amygdala.

Birthdate tagging at TM11.5. A–I, Coronal brain sections prepared from a P21 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP mouse that was given TM at E11.5. Images for mGFP reporter (green) and DAPI (blue). A”, D’, E’, Black and white high contrast image converted from the mGFP image. A’, A”, Higher magnifications of the MOB, of which layer positions are indicated on the right-hand end. Arrowheads (A’, A”) indicate cell bodies of birthdate-tagged MCs. Note that the basal dendrites in the lower part of the EPL are labeled with the reporter. The glomerulus (GL) is encircled by a dotted line (A”). The TM11.5 axons project diffusely to all the MOB and AOB targets (B–I) except the small domain in the anterolateral edge of the OT (asterisks in D, D’, E, E’). D’, E’, Position of a cell cluster is encircled by the blue dotted line and marked with the asterisk. The curved dotted line on the left side of the cell cluster in E’ depicts the lateral hook of the OT cell layer. The labeling of the LOT axons looks less intense possibly because the axons are highly myelinated (Inaki et al., 2004). The brain illustration in the middle summarizes TM11.5 axon projections and indicates the levels at which individual sections were prepared. A pie chart shows the proportion of neuron subtypes tagged at this TM stage with this reporter. Representative images from six mice. Scale bars = 100 μm (A’, A”, D’, E’) and 500 μm (A–I). BAOT: bed nuclei of the accessory olfactory tract, CoA: cortical amygdala, EC: entorhinal cortex, GRL: granule cell layer, MeA: medial amygdala, pD: pars dorsalis, pL: pars lateralis, PLCo: posterolateral cortical amygdala, PMCo: posteromedial cortical amygdala, TT: tenia tecta.

Birthdate tagging at TM12.5. A–I, Coronal brain sections prepared from a P20 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP mouse that was given TM at E12.5. Images for mGFP reporter (green) and DAPI (blue). A”, D’, E’, Black and white high contrast image converted from the mGFP image. A’, A”, Higher magnifications of the MOB, of which layer positions are indicated on the right-hand end. Arrowheads (A’, A”) indicate cell bodies of birthdate-tagged MCs. Note that the basal dendrites in the lower part of the EPL are labeled. The glomerulus (GL) is encircled by a dotted line (A”). The TM11.5 axons project diffusely to all the MOB targets (B–I) except the small domain in the anterolateral edge of the OT (asterisks in D, D’, E, E’). D’, E’, Position of a cell cluster is encircled by the blue dotted line and marked with the asterisk. The curved dotted line on the left side to the cell cluster in E’ depicts the lateral hook of the OT cell layer. A brain illustration in the middle summarizes TM12.5 axon projections and indicates the levels at which individual sections were prepared. A pie chart shows the proportion of neuron subtypes tagged at this TM stage with this reporter. Representative images from seven mice. Scale bars = 100 μm (A’, A”, D’, E’) and 500 μm (A–I). BAOT: bed nuclei of the accessory olfactory tract, CoA: cortical amygdala, EC: entorhinal cortex, GRL: granule cell layer, MeA: medial amygdala, pD: pars dorsalis, pL: pars lateralis, PLCo: posterolateral cortical amygdala, PMCo: posteromedial cortical amygdala, TT: tenia tecta.

Birthdate tagging at TM13.5. Coronal brain sections prepared from P21 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP (A–H) and P21 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} (I–P) mice that were given TM at E13.5. Images for mGFP reporter (A–H) and tdTomato reporter (I–P) counterstained with DAPI. A”, D’, E’, I”, L’, M’, Black and white high contrast image converted from the reporter image. A’, A”, I’, I”, High magnifications of the MOB, of which layer positions are indicated on the right-hand end. Arrows (A, A’, I, I’) indicate the internal plexiform layer that contains intrabulbar association fibers. Arrowheads (A’, A”, I’, I”) indicate cell bodies of birthdate-tagged imTCs and eTCs. Note that the basal dendrites in the upper part of the EPL are labeled. The glomerulus (GL) is encircled by a dotted line (A”, I”). The TM13.5 axons project to only anterior part of the olfactory target areas (B–H, J–P). Arrowheads (E, F, M, N) indicate axons projecting the lateral part of the OT. Arrowheads (G, O) indicate labeled axons reaching to the caudal end of the LOT. Asterisks (D, D’, E, E’, L, L’, M, M’) indicate the small domain in the anterolateral edge of the OT. D’, E’, L’, M’, Position of a cell cluster is encircled by the blue dotted line and marked with the asterisk. The curved dotted line on the left side of the cell cluster in E’, M’ depicts the lateral hook of the OT cell layer. At this TM injection stage, scattered cells in the PC express reporters (E, F, L–P). The brain illustration in the middle summarizes TM13.5 axon projections and indicates the levels at which individual sections were prepared. The pie chart shows the proportion of neuron subtypes tagged at this TM stage in each reporter line. Representative images from eight (A–F) and seven (G–L) mice. Scale bars = 100 μm (A’, A”, D’, E’, I’, I”, L’, M’) and 500 μm (A–P). CoA: cortical amygdala, GRL: granule cell layer, pD: pars dorsalis, pL: pars lateralis, TT: tenia tecta.

Birthdate tagging at TM15.5. Coronal brain sections prepared from P22 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP (A–F) and P21 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} (G–L) mice that were given TM at E15.5. Images for mGFP reporter (A–F) and tdTomato reporter (G–L) counterstained with DAPI. A”, D’, E’, G”, J’, K’, Black and white high contrast image converted from the reporter image. A’, A”, G’, G”, High magnifications of the MOB, of which layer positions are indicated on the right-hand end. Arrows (A, A’, G, G’) indicate the internal plexiform layer that contains intrabulbar association fibers. Arrowheads (A’, A”, G’, G”) indicate cell bodies of birthdate-tagged eTCs, whose basal dendrites horizontally extend in the upper part of the EPL. The glomerulus (GL) is encircled by a dotted line (A”, G”). The TM15.5 axons only project to the pE (B, H) and the small domain in the anterolateral edge of the OT (asterisks in D, D’, E, E’, J, J’, K, K’). This domain contains a cell cluster, which is encircled by the yellow dotted line and marked with the asterisk (D’, E’, J’, K’). The curved dotted line on the left of the cell cluster in E’, K’ depicts the lateral hook of the OT cell layer. The brain illustration summarizes TM15.5 axon projections and indicates the levels at which individual sections were prepared. The pie chart shows the proportion of neuron subtypes tagged at this TM stage in each reporter line. Representative images from five (A–F) and nine (G–L) mice. Scale bars = 100 μm (A’, A”, D’, E’, G’, G”, J’, K’) and 500 μm (A–L). GRL: granule cell layer, pD: pars dorsalis, pL: pars lateralis, TT: tenia tecta.

Segregated projections of TM15.5 axons to the target cell cluster in the anterolateral OT. A–D, Coronal brain sections prepared from a P22 Neurog2^CreER(G2A); Cdhr1^tTA; ROSA26-TRE^mGFP mouse that was given TM at E15.5. Images for mGFP reporter (green), FluoroMyelin (red) and DAPI (blue). FluoroMyelin marks myelinated OB axons running through the LOT. The TM15.5 axons (arrowheads) ventrally segregate from other OB axons in the LOT (A, B) and terminate at a small cell cluster in the anterolateral edge of the OT (arrowheads and asterisk in C). In a posterior section (D), the cell cluster fuses with the lateral hook of the OT dense cell layer (arrowheads). Representative images from five mice. Scale bars = 500 μm (A–D). pD: pars dorsalis, pL: pars lateralis.

Birthdate tagging at TM17.5. A–D, Coronal brain sections prepared from a P21 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} (A–D) mouse that was given TM at E17.5. Images for tdTomato reporter and DAPI. D’, Black and white high contrast image converted from the tdTomato reporter image. A’, A”, High magnifications of the MOB, of which layer positions are indicated on the right-hand end. Arrowheads (A’, A”) indicate cell bodies of birthdate-tagged eTCs that barely have basal dendrites. The glomerulus (GL) is encircled by a dotted line (A”). A small number of TM17.5 axons project in the ventral surface of the LOT (arrowheads in B–D). Asterisks (D, D’) show the anterolateral edge of the OT. The cell cluster encircled by the blue dotted line (D’) is not penetrated by TM17.5 axons (arrowheads). The brain illustration summarizes TM17.5 axon projections and indicates the levels at which individual sections were prepared. The pie chart shows the proportion of neuron subtypes tagged at this TM stage in this reporter line. Representative images from seven mice. Scale bars = 100 μm (A’, A”, D’) and 500 μm (A–D). GRL: granule cell layer, pD: pars dorsalis, pL: pars lateralis, TT: tenia tecta.

Histochemical characteristics of the target cell cluster of TM15.5 axons. A–E, Five serial coronal sections (20 μm thick), from anterior (A) to posterior (E), prepared from a P21 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} mouse that was given TM at E15.5. Arrowheads show the cell cluster targeted by TM15.5 axons. The TM15 axons expressing tdTomato (A, C, E) and synaptophysin-GFP (C) reporters densely project to the target cell cluster stained with DAPI (A”–E”). The target cell cluster expresses DARPP (A’, E’), dopamine receptors D1R (B’, D), but not D2R (D’). Dopaminergic axons expressing tyrosine hydroxylase (C’) project over this cell cluster. Representative images from three mice. F, Enlarged image of neurons in the cell cluster, which express DARPP (green) and are projected by the tdTomato-expressing TM15.5 axons (red). Scale bars = 500 μm (A–E) and 10 μm (F).

Afferent projections from aiCAP, the target cell cluster of TM15.5 axons. A, Rhodamine-dextran focally injected into the aiCAP (arrowheads) of a P14.5 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} mouse that was given TM at E15.5. B, B’, Enlarged image of the aiCAP (arrowheads) labeled with dextran and DAPI. C, In a posterior section, the dextran-labeled axons make a small fascicle (arrow) coursing posteriorly through the GAD67-expressing plexus (arrowheads) in the OT polymorph layer. D, D’, High magnification of the dextran-labeled fibers (D), which surround neurons (yellow arrowheads), sparsely embedded in the fascicle (D’). Representative images from nine mice in which the aiCAP was successfully labeled with dextran. Scale bars = 500 μm (A, C), 100 μm (B, B’), and 50 μm (D, D’).

Topographic and non-topographic maps formed by TM15.5 axons. A–C, Coronal sections prepared from a Neurog2^CreER(G2A); Cdhr1^tTA mouse that was given TM15.5 and then injected with AAV2-TRE^TurboFP635 and VSVG-TRE^GFP viruses in the MOB. Medial is to the left and dorsal is to the top. The eTCs on the medial and lateral sides of the MOB are specifically labeled with red and green reporter proteins, respectively (A). The axons make topographic projections to the pE (B) but not the aiCAP (arrowheads in C). Representative images from four mice with successful focal labeling. Scale bar = 200 μm.