Medical Molecular Morphology (2020) 53:168–176 https://doi.org/10.1007/s00795-020-00245-7
ORIGINAL PAPER
Sequential pattern of sublayer formation in the paleocortex and neocortex
Makoto Nasu1 · Kenji Shimamura2 · Shigeyuki Esumi1 · Nobuaki Tamamaki1
Received: 17 December 2019 / Accepted: 13 January 2020 / Published online: 30 January 2020 © The Japanese Society for Clinical Molecular Morphology 2020
Abstract The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfac- tory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have diferent cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neo- cortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The diferences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specifc genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.
Keywords Piriform cortex (paleocortex) · Sublayer · Sequential expression · Ctip2/Bcl11b · Brn1/Pou3f3
Introduction six-layered cortex, which is a mammal-specifc feature, are well established; sequential expression of transcription fac- The piriform cortex (paleocortex) is the olfactory cortex or tors is involved in determining cell identities, and late-born the primary cortex for the sense of smell. It receives the neurons migrate to pass through and take their place outside olfactory input from the mitral and tufted cells in the olfac- earlier-born neurons, i.e., in an inside-out manner [8–12]. tory bulb and is involved in the processing of information Recently, lineage trace analyses demonstrated that the pertaining to odors [1, 2]. Neurons originating from the lat- laminar organization of the piriform cortex is regulated in eral pallium (LP) in the telencephalon migrate laterally or an inside-out manner, while neurons located inside layer II ventrally into the piriform cortex [3, 4]. The dorsally adjoin- are arranged in an outside-in manner [13]. Furthermore, neu- ing dorsal pallium (DP) gives rise to the neocortex, which is rons in the superfcial and deeper layers of layer II, layers IIa responsible for higher mental functions, including memory, and IIb, respectively, exhibit diferent innervation patterns speech, value judgments, and sociality. The adjoining LP to other cortical regions [14, 15]. However, less is known and DP have diferent cytoarchitectures; LP is a three-lay- about the regulatory mechanisms in the piriform cortex. In ered structure, while DP is a six-layered structure [5–7]. particular, the diferences and similarities in the regulatory The regulatory mechanisms underlying the building of the mechanisms between the neocortex and the piriform cortex remain unclear, despite the fact that these two cortices are adjoined. * Makoto Nasu In this study, we examined the expression of some neo- mnas@kumamoto‑u.ac.jp cortex layer-specifc genes in the piriform cortex at embry- 1 Department of Morphological Neural Science, Graduate onic day 16 (E16), postnatal day 0 (P0), and P7, which cor- School of Medical Sciences, Kumamoto University, 1‑1‑1, respond to the late-neurogenesis stage, the post-neurogenesis Honjo, Chuo‑ku, Kumamoto 860‑8556, Japan stage, and the postnatal developmental stage, respectively 2 Department of Brain Morphogenesis, Institute of Molecular [9]. Embryology and Genetics (IMEG), Kumamoto University, 2‑2‑1, Honjo, Chuo‑ku, Kumamoto 860‑0811, Japan
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◂Fig. 1 Sublayers in layer II of the piriform cortex at P7. a Overview (FV-1200, FV-1000; OLYMPUS) and analyzed using the of the telencephalon at P7. The piriform cortex (Pir) adjoins the neo- ImageJ software. cortex (NCx) and the olfactory tubercle (Tu). Their borders are indi- cated by white dotted lines. Pir consists of three layers (I, II and III). NCx consists of six layers (I, II/III, IV, V and VI). b The piriform Image analysis cortex stained with Hoechst 33343. The endopiriform nucleus (EN) is located in the deeper region of the piriform cortex. c Sublayers of Four-colored fuorescent images of the piriform cortex were piriform layer II, IIa and IIb, which are roughly segregated by the cell density. The white dashed lines indicate the layer/sublayer borders. obtained from three individuals at P0 and P7 using a laser- d–f Triple immunostaining for Tbr1 (d), Ctip2 (e) and Brn1 (f). The scanning confocal microscope and split into separate color border between NCx and Pir is indicated by the white dotted line. g–i channels. The fuorescence intensity of three markers (Tbr1, Merged view of d–f. g Tbr1 (green) and Ctip2 (red). h Tbr1 (green) Ctip2 and Brn1) and Hoechst 33342 (nuclear staining) in and Brn1 (red). i Brn1 (green) and Ctip2 (red). j–l Magnifed view of g–i. Hoechst 33342 was used for counterstaining of nuclei (blue). piriform layers IIa and IIb was analyzed by line plot profl- The solid lines roughly indicate the range of each layer. The border ing using the ImageJ software. Two 250 µm-long lines were of layers IIa and IIb was defned by the border of expression of Brn1 arranged at the superfcial and deepest position of piriform LOT Lv and indicated by a crossing bar. lateral olfactory tract, lateral layer II. The number of fuorescence+ neurons was counted ventricle, St striatum. Scale bars, 1000 µm (a); 200 µm (b–l) on each line. The fuorescence intensities per one cell were averaged for each individual. Cut-of values were set to Materials and methods 10% of the peak values. Student’s t tests were conducted to compare the fuorescence intensities and proportions of Tissue preparation marker+ cells among Hoechst 33342+ cells between layers IIa and IIb. P < 0.05 (two-tailed) was considered signifcant Jcl:ICR mice were killed at E16, P0, and P7 (purchased from for all tests. CLEA Japan). All mice were anesthetized with medetomi- dine/midazolam/butorphanol tartrate/phosphate bufered saline (PBS) (fnal dose, 0.3 mg/kg of body weight; 4 mg/kg Results of body weight; and 5 mg/kg of body weight, respectively) and perfused from the left ventricle with iced 4% paraform- Sublayers in layer II of the piriform cortex at P7 aldehyde/PBS (pH 7.2). Brain tissues were extracted and postfxed with 4% paraformaldehyde/PBS (pH 7.2) for 1 h To investigate the differences and similarities in the (E16) or overnight (P0 and P7). Fixed tissues were cryopro- regulatory mechanisms between the neocortex and the tected in 15% sucrose/PBS overnight at 4 °C and embed- piriform cortex (paleocortex) (Fig. 1a, b), we studied the ded in Tissue-Tek O.C.T. compound (Sakura Finetek). All expression of neocortical layer-specific genes. First, we animal experiments were performed in accordance with determined which neocortical layer-specific genes were institutional (Kumamoto University) guidelines and were also expressed in the piriform cortex. We performed approved by the animal care and use committee of Kuma- immunostaining using P7 mice and examined how their moto University. expressions were in each piriform layers and sublayers (Fig. 1c). Tbr1, Ctip2/Bcl11b, and Brn1/Pou3f3 were expressed strongly in the piriform cortex (Fig. 1d–f). Tbr1 Immunostaining and Ctip2 were expressed throughout layer II, whereas Brn1 was mainly expressed in the deeper half of layer II Frozen tissues were coronally sliced at 12 µm. Antigen (Fig. 1g–l), indicating that layer II of the piriform cortex retrieval was achieved through heat treatment (105 °C, can be subcategorized into two sublayers: the superficial 5 min) in 10 mM citrate bufer (pH 6.0). The following layer IIa (Tbr1+ /Ctip2+ /Brn1−) and the deeper layer primary antibodies were used for immunohistochemistry IIb (Tbr1+ /Ctip2+ /Brn1+) using molecular markers. at the indicated dilutions: anti-Brn1/Pou3f3 (goat, 1:200; Tbr1 and Ctip2 were colocalized in a group in the deeper Santa Cruz), anti-Brn2/Pou3f2 (goat, 1:200; Santa Cruz), region of the piriform cortex, which might correspond to anti-Ctip2/Bcl11b (rat monoclonal, 1:3000; Abcam), anti- the endopiriform nucleus (EN) (Fig. 1g). Tbr1, Ctip2, and Foxp2 (goat, 1:100; Santa Cruz), anti-RORb (mouse, 1:500; Brn1 were widely detected in a subset of layer III neurons Perseus), anti-Satb2 (rabbit, 1:1000; Abcam), and anti-Tbr1 with various combinations. Brn2/Pou3f2, Satb2, Foxp2, (rabbit, 1:1000; Abcam). Fluorescence (Alexa Fluor 488, and RORb were sparsely detected in the piriform cortex; 594 and Cy5)-conjugated secondary antibodies were used therefore, they could not be considered representative (donkey, 1:2000; Jackson ImmunoResearch Inc.). Images markers of piriform layers (Fig. 2a–d). Faint expression were acquired using a BZ-X700 fuorescence microscope signals for Brn2/Pou3f2 were detected sparsely in lay- (Keyence) and a laser-scanning confocal microscope ers II and III, whereas Satb2 and Foxp2 were sparsely
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Fig. 2 Immunostaining of the piriform cortex and neocortex at P7. to deep orientation of the cortical tissue was shown in the upper to a–d Immunostaining of the piriform cortex. a Immunostaining for bottom orientation. Brn2 (e), Satb2 (f), Foxp2 (g), and RORb (h) Brn2. b Immunostaining for Satb2. c Immunostaining for Foxp2. d were highly expressed in the neocortex. Hoechst 33342 was used for Immunostaining for RORb. The solid lines roughly indicate the range counterstaining of nuclei (blue). Scale bars, 200 µm of each layer. e–g Immunostaining of the neocortex. The superfcial detected in layer III. RORb was undetected at P7. All of piriform cortex at P0 can be divided into layer IIa (Tbr1+ / them were highly expressed in the neocortex at P7; like Ctip2+ /Brn1−) and layer IIb (Tbr1+ /Ctip2dim/Brn1+) Brn2 in upper layers, Satb2 in upper layers and in a subset (Fig. 3d–f). The two sublayers could be diferentiated based of deep layer neurons, Foxp2 in layer VI, and RORb in on a threshold of intensity of Ctip2 expression (Fig. 3g–i). layer IV (Fig. 2e–h). These results suggest that the piri- Thus, we found the diferential expressions of Ctip2 and form cortex contains a wide variety of subpopulations Brn1 between layers IIa and IIb as well as their changed with distinct cell identities. expressions during P0 and P7. To evaluate those features statistically, we examined the fluorescence intensity of three markers and nuclear staining (Hoechst 33342) and the Sublayer‑specifc expression of Ctip2 and Brn1 at P0 proportions of marker positive neurons per nuclear stain- ing+ neurons separately in layers IIa and IIb at P0 and P7 Next, we examined the expression of Tbr1, Ctip2, and Brn1 (Fig. 4a–d). Limited layer IIa neurons expressed Brn1 at at P0, a time point at which neurogenesis in the cerebral cor- P0 (layer IIa, 28.6% ± 5.70%; layer IIb, 86.0% ± 2.93%) tex is complete. Tbr1 was expressed in almost all neurons in and their fuorescence intensities were signifcantly lower the piriform cortex, indicating that this protein is a pan-neu- than Brn1+ layer IIb neurons (Fig. 4e, f). These features ronal marker at this stage (Fig. 3a). We detected two expres- were retained at P7 (layer IIa, 27.1% ± 3.15%; layer IIb, sion patterns for Ctip2 at P0: strong expression in layer IIa 83.6% ± 6.83%) (Fig. 4g, h). The fuorescence intensity of and faint expression in layer IIb (Fig. 3b). Brn1 exhibited Ctip2+ layer IIb neurons was lower than Ctip2+ layer IIa an expression pattern that was similar to that observed at neurons at P0 (Fig. 4f). Consistently, less layer IIb neurons P7; Brn1 was expressed in layer IIb and layer III, but few in tended towards expressing Ctip2 than layer IIa (layer IIa, layer IIa (Fig. 3c). Using molecular markers, layer II of the 82.5% ± 5.99%; layer IIb, 68.3% ± 2.73%, P value < 0.1)
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Fig. 3 Immunostaining of the piriform cortex at P0. a–c Tri- Supra-threshold expression of Tbr1 (g), Ctip2 (h), and Brn1 (i). Hoe- ple immunostaining for Tbr1 (a), Ctip2 (b) and Brn1 (c). Borders chst 33342 was used for counterstaining of nuclei (blue). The solid between the neocortex and piriform cortex are indicated by white dot- lines roughly indicate the range of each layer. The dashed lines indi- ted lines. d–f Merged view of a–c. d Tbr1 (green) and Ctip2 (red). cate the borders between layers I and II and between layers II and III. e Tbr1 (green) and Brn1 (red). f Brn1 (green) and Ctip2 (red). g–i Scale bars, 200 µm
(Fig. 4e). These features of Ctip2 were transient during of layer IIa contains a low density of neurons [14]. We com- development to be undetected at P7 (Fig. 4g, h). The fuo- pared the number of neurons in the dorsal layers IIa and IIb rescence of Tbr1 and nuclear staining was detected equally detected with Hoechst 33342 staining in a line 250 µm long. in two sublayers at P0 and P7. It is known that the dorsal part We found that layer IIa had fewer neurons than layer IIb
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[layer IIa (P0), 17.3 ± 1.53; layer IIb (P0), 23.3 ± 2.31; layer during the postnatal developmental stage (P0–7). Together, IIa (P7), 12.3 ± 0.58; layer IIb (P7), 18.3 ± 3.21] (Fig. 4i, statistical analyses confrmed contrast expression profles of j). Cell density in the piriform layers IIa and IIb declined Ctip2 and Brn1 in two sublayers.
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◂Fig. 4 Line plot profle analysis at P0 and P7. a, b Plot profl- layer II, the superfcial layer IIa and the deeper layer IIb, of ing of layer IIa. Representative image of plot profling of Hoechst the paleocortex by Brn1 at the postnatal developmental stage 33342+ neurons (a) and the plot of fuorescence intensities (b). The red line indicates the line 250 µm long for plot profling of neurons (P7). Although Ctip2 was expressed throughout layer II at at the superfcial position of layer II. c, d Plot profling of layer IIb. P7, contrastive expressions of Ctip2 and Brn1 in two sublay- Representative image of plot profling of Hoechst 33342+ neurons (c) ers were observed at the post-neurogenesis stage (P0); strong and the plot of fuorescence intensities (d). The red line indicates the expression of Ctip2 in layer IIa and specifc expression of line 250 µm long for plot profling of neurons at the deepest position Brn1 in layer IIb. The migration of the Ctip2+ neurons was of layer II. e, f Plot profling at P0. e The proportions (%) of marker (Tbr1, Ctip2, and Brn1)+ cells among Hoechst 33342+ cells. f Aver- completed at the late-neurogenesis stage (E16), whereas that aged fuorescence intensity of marker+ cells. g, h Plot profling at P7. of the Brn1+ neurons was still engaged in lateral migration g The proportions (%) of marker+ cells among Hoechst 33342+ cells. at E16, indicating that layer IIa was achieved by E16 and h Averaged fuorescence intensity of marker+ cells. i, j The number of Hoechst 33342+ neurons on the line 250 µm long in the layers layer IIb was done between E16 and P0. IIa and IIb at P0 (i) and P7 (j). Values expressed as mean ± stand- Sublaminae of layer II have been described based on the ard error of the mean (SEM) (e–h) and as mean ± standard deviation diferent cell density at frst and their neural connectivity, (SD) (i, j). The white dashed lines indicate the layer/sublayer borders. cell morphology, and electrophysiology have been studied *P < 0.05, **P < 0.01, ***P < 0.001 thus far [14–17]. Layer IIa consists of sparse semilunar cells with few basal dendrites, which tend to output to other olfac- Temporal regulation of Ctip2‑ and Brn1‑expressing tory cortex and to receive weak associational input from neurons them. Layer IIb consists of dense superfcial pyramidal cells with bitufted dendritic morphology, which output and input In the neocortex, Ctip2 and Brn1 were exclusively expressed both associational and commissural fbers. For deep inves- in a layer-specifc manner and the onset of their expression tigations of cell features, it is required to label individual was regulated in a time-specifc manner; Ctip2 in the frst and neurons separately from other cell types [18]. In consistent Brn1 in the next. To investigate whether the sublayer-specifc with our data, the piriform cortex contains a wide variety of expression of Ctip2 and Brn1 in the piriform cortex was neuronal subpopulations with distinct cell identities, which achieved via temporal regulation, we investigated the expres- are not likely to be segregated exclusively [13, 15]. It will sion of Ctip2 and Brn1 in the piriform cortex at an earlier stage be required to determine the relationships between neuronal (E16). Both Ctip2 and Brn1 were strongly expressed in the functions and cell identities using molecular markers. Our neocortex in a layer-specifc manner at this stage: Ctip2 was fndings contributed to add new marker information avail- detected in the deeper layer (layer V) and Brn1 was expressed able for studying subpopulations in the piriform cortex in the upper layers (II/III) and in neurons under migration separately. On the other hand, our data showed that marker toward the outside (Fig. 5a). In the piriform cortex at E16, expression changed during development; Ctip2 strongly Ctip2 was widely detected in the entire layer II, which was expresses layer IIa neurons at P0; however, Ctip2 widely still narrow, and in a lump in the sub-piriform position, which expresses in the entire layer II at P7. Lineage tracing tech- might correspond to the endopiriform nucleus, indicating that nology, such as an inducible Cre-mediated recombination the expression profle of Ctip2 at E16 was similar to those at system, will be helpful for delineating cell properties, which P0 and P7 (Fig. 5b). Conversely, scattered expression of Brn1 will be a future study. was observed in the piriform cortex; however, an abundance The neocortical neuroepithelium generates the layer- of Brn1+ neurons were detected in the deepest position of the specifc neurons in a sequential manner and arrange them piriform cortex (indicated by arrows in Fig. 5c) (Fig. 5c, d). in an inside-out pattern; deep layer (DL) neurons (layer VI These cells were neurons that had migrated laterally from their and V) are born during the early phase and upper layer (UL) region of origin, i.e., the ventricular zone of the pallium, to the neurons (layer IV, III, and II) are born later. Our data showed piriform cortex. This indicates that Brn1+ neurons were born that the laminar arrangement of the paleocortex layer II was after Ctip2+ neurons and still engaged in lateral migration at built in an outside-in manner, unlike the inside-out structure E16 to form piriform layer IIb between E16 and P0. detected in the neocortex, which was consistent with the These observations demonstrate that Ctip2- and Brn1- results of a previous study [13]. These indicated that the expressing neurons are spatiotemporally regulated. sequential pattern of sublayer formation in the paleocortex was similar to that of layer formation in the neocortex, whilst their spatial patterns were diferent. The sequential genera- Discussion tion of layer-specifc neurons in the neocortex is intrinsically programmed, as sequential generation of neocortical neu- In this study, we carried out expression analyses of the piri- rons can be seen even in primary culture of isolated cortical form cortex (paleocortex) at three developmental stages progenitors [11]. It will be an important question to inves- (E16, P0 and P7). Two sublayers can be distinguished in tigate whether sequential generation of sublayer neurons in
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Fig. 5 Sequential expression of Brn1 and Ctip2, as assessed using immunostaining at E16. a Immunostaining of the neocortex (NCx). Brn1 (green) and Ctip2 (red) were highly expressed in the upper layers (II/III) and deep layer (V), respectively. b–d Immunostain- ing of the piriform cortex for Brn1 (b) and Ctip2 (c). d Merged view of Brn1 (green) and Ctip2 (red). Hoechst 33342 was used for counterstaining of nuclei (blue). The white dotted line indicates the border between the neocortex (NCx) and piriform cortex (Pir). The solid line indicates the range of layer II. The arrows indicate the migrating neurons at the deeper position. St striatum. Scale bars, 200 µm
the paleocortex is also intrinsically programmed or regulated neocortex, the three-layered mammalian paleocortex (piri- in a non-cell autonomous way. form cortex), the three-layered reptilian cortex, and the avian The neocortex plays a critical role in high-order functions nuclear-structured pallium share similar temporal regula- such as memory, speech, value judgements, and sociality. tory machinery for the pallial neuron subtypes. Accord- These characteristic functions might be based on a mam- ingly, although the relationships among those pallia are mal-specifc, six-layered structure. Recently, a single-cell still unknown, deep investigations of the three-layered pale- transcriptome analysis implied that the three-layered reptil- ocortex will lead us to know how the six-layered neocortex ian cortex could be divided into layers IIa and IIb, which was evolved from the ancestral pallium, which might have a were correlated with the neocortical DL and UL, respec- three-layered structure, and how neuronal functions, which tively [19]. Another group reported that avian DL and UL are characteristic to mammals, were achieved. neurons are not layered; rather, they segregate into distinct cortical areas, while the neurogenetic program that occurs sequentially to produce DL and UL neurons is conserved among mammals and birds [20]. The sequential order of Conclusions DL-like layer IIa neurons and UL-like layer IIb neurons in the reptilian cortex has not been investigated; however, These fndings suggest that two sublayers can be distin- the laminar position of the reptilian layer II was similar to guished in layer II, layers IIa and IIb, of the paleocortex by that of the mammalian paleocortex. Although there may be Ctip2/Bcl11b and Brn1/Pou3f3 and the formation of neurons some diferences in the regulatory mechanisms underlying of layers IIa and IIb was temporally regulated. The sequen- cell migration or arrangement, the six-layered mammalian tial order of their expression in layer II of the paleocortex,
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