Abstract

The molecular mechanisms underlying the formation of hippocampus are unknown in humans. To improve our knowledge of molecules that potentially regulate pyramidal neurogenesis and layering in various hippocampal fields, we investigated the expression of progenitor markers and cell fate molecules from gestational week (GW) 9 to GW 20. At GW 9, the progenitor cell compartment of the hippocampal formation mainly consisted of PAX6+ cells in the ventricular zone. Between GW 9 and 11, a second germinal area, the subventricular zone (SVZ), was formed, as shown by TBR2 labeling. Postmitotic markers (TBR1, CTIP2, SATB2, and CUX1) might reflect the inside-out layering of the plate from GW 11 onwards. TBR1+ neurons appeared in the deep plate, whereas CTIP2+, SATB2+, and CUX1+ neurons occupied the upper layers. From GW 16, differences in layer segregation were observed between the ammonic and subicular plates. Moreover, an ammonic-to-subicular maturation gradient was observed in germinal/postmitotic areas. Taken together, these findings demonstrate for the first time the presence of an SVZ in the hippocampus of human fetuses and laminar differences in transcription factor expression in the pyramidal layer of the human ammonic and subicular plate, and provide new information to further investigate the connectivity of the hippocampal formation.

Introduction

The archicortical hippocampal formation is phylogenetically one of the oldest cortical areas (Kuhlenbeck 1977; Tombol et al. 2000). Animal studies have revealed similarities in the neuroanatomy, development, and functions of the hippocampus among mammals, even though evolutionary differences still exist (Insausti 1993; Seress 2007). Interestingly, as opposed to rodents, the hippocampus of primates develops in the ventral temporal lobe (West 1990), indicating differences in its morphogenetic development. However, little information is available regarding this development.

Several lines of evidence suggest that deep and superficial pyramidal cells of the hippocampal plate differ among species with respect to their morphological and physiological characteristics and connectivity [for review see Slomianka et al. (2011)]. The complex processes of the migration and maturation of ammonic pyramidal cells have been described in rodents and in non-human primates (Rakic and Nowakowski 1981; Altman and Bayer 1990). In these studies, Cornu Ammonis 3 (CA3) neurons were found to be generated before CA1 neurons, although the stratum pyramidale of the CA1 field has been reported to be formed before that of the CA3 field in rats (Altman and Bayer 1990). In humans also, morphological observations and developmental markers have supported the idea of the heterogeneity of hippocampal pyramidal neurons, which show an inside-out maturation gradient (Sidman and Rakic 1973; Kostovic et al. 1989; Arnold and Trojanowski 1996; Abraham et al. 2009). Interestingly, in non-human primates, it has been proposed that cell fate might be determined before the cells reach their final destination within the plate (Nowakowski and Rakic 1981), although the molecular mechanisms underlying the cell fate determination in pyramidal neurons in various hippocampal fields are still unknown.

More recently, the discovery of genes involved in layer and neuronal subtype specification has allowed the study of mechanisms underlying neocortical axonal pathway formation in rodents (Molyneaux et al. 2007; Greig et al. 2013). In the murine neocortex, Pax6-immunoreactive (Pax6+) radial glial cells of the ventricular zone (VZ) have been shown to produce intermediate neuronal progenitor cells, expressing Tbr2, which migrate outwards to form the subventricular zone (SVZ; Rakic and Nowakowski 1981; Haubensak et al. 2004; Noctor et al. 2004; Ochiai et al. 2009). Thus, the sequential expression of several transcription factors, Pax6-Tbr2-Neurod1-Tbr1 (Englund et al. 2005; Bayatti et al. 2008; Roybon et al. 2009; Imamura and Greer 2013), is required for the neuronal commitment of stem cells. Several of these transcription factors have been found to specify neocortical pyramidal cells. Tbr1, a marker of layer VI (Bedogni et al. 2010; Han et al. 2011), and Ctip2, a marker of layer V (Leid et al. 2004), control the formation of corticothalamic and corticospinal projections, respectively (Arlotta et al. 2005; Chen et al. 2005, 2008), whereas Satb2 (Britanova et al. 2005; Szemes et al. 2006) and Cux1 (Nieto et al. 2004), expressed in upper plate neurons, regulate corticocallosal (Alcamo et al. 2008; Britanova et al. 2008) and corticocortical projections (Cubelos et al. 2010), respectively. The expression of several of these genes is known to be conserved in the human neocortex (Saito et al. 2010), but it remains to be determined whether they are similarly regulated during the generation of hippocampal pyramidal cells.

In this study, we investigated the expression pattern of different molecular markers in progenitors (SOX2, PAX6, and TBR2) and postmitotic neurons (TBR1, CTIP2, SATB2, and CUX1) of the human ammonic/subicular/entorhinal fields from gestational week (GW) 9 to GW 20. We investigated the germinal zones of the hippocampal formation and compared them with those described in the human neocortex, using the transient expression of these markers as a valuable tool with which to investigate the development and layering of the archicortical hippocampal formation.

Materials and Methods

Samples

Twelve human fetuses without any neuropathological alterations were collected after legal abortion or spontaneous death (Table 1). All procedures were approved by the ethics committee (Agence de Biomédicine; approval PFS12-0011). The age in GW (or post-ovulatory weeks) of each case was estimated on the basis of anatomy and pregnancy records.

Table 1

Human fetal cases employed in the study

Cases Age in gestational weeks Tissue processing Postmortem delay/fixation time 
Paraffin NA 
Paraffin NA 
10 Paraffin NA 
11 Frozen 2 h/24 h 
11 Frozen 2 h/72 h 
13 Frozen 15 h/15 h 
13 Frozen 15 h/15 h 
13 Frozen NA 
16 Frozen 24 h/48 h 
10 17 Paraffin NA 
11 19 Frozen 24 h/72 h 
12 20 Frozen 10 h/10 h 
Cases Age in gestational weeks Tissue processing Postmortem delay/fixation time 
Paraffin NA 
Paraffin NA 
10 Paraffin NA 
11 Frozen 2 h/24 h 
11 Frozen 2 h/72 h 
13 Frozen 15 h/15 h 
13 Frozen 15 h/15 h 
13 Frozen NA 
16 Frozen 24 h/48 h 
10 17 Paraffin NA 
11 19 Frozen 24 h/72 h 
12 20 Frozen 10 h/10 h 

Immunohistochemistry

For frozen sections, samples were fixed in 4% paraformaldehyde, cryoprotected in 20% sucrose, and stored at −80 °C until use. Fixation time and postmortem delay are reported in Table 1. Samples were cut into 12-µm-thick coronal cryosections, mounted on Super-Frost slides, and maintained at −80 °C.

For paraffin sections (GW 9), samples were dehydrated in an ethanol gradient, cleared in xylene, and embedded in paraffin. The tissue was cut into 5-µm-thick coronal sections. Before labeling, antigen retrieval was performed by incubating in citrate buffer for 1 h at 94 °C (1.8 mM citric acid and 8.2 mM sodium citrate, pH 6).

Paraffin and frozen sections were permeabilized with 0.1% Triton X-100 in 0.12 M phosphate-buffered saline (PBS-T, 15 min at 25 °C) and treated with blocking solution (10% goat serum in PBS-T, 1 h at 25 °C) to inhibit nonspecific binding. Sections were incubated with primary antibodies at the dilutions reported in Table 2 for about 16 h at 4 °C. Sections were then treated with Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 676-conjugated secondary antibodies (1 : 500 in blocking solution, Invitrogen Molecular Probes) for 1 h at 25 °C. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL, Invitrogen Molecular Probes). Fluoromount-G mounting medium (SouthernBiotech, Birmingham, USA) was used to mount coverslips.

Table 2

Antibodies employed in the study

Antibody Company Species Concentration (IHC/IHC-P) Target References 
Proliferation 
 Ki67 Abcam, ab16667 (clone SP6) Rabbit 1 : 200/– Cell cycle-related nuclear protein Bento et al. (2010
 Ki67 Dako, M7240 (Clone MIB) Mouse 1 : 500/1 : 50 Cell cycle-related nuclear protein Gerdes et al. (1983
 PH3 Millipore 06-570 Rabbit 1 : 1000/– Mitosis marker Yamada et al. (2014
Neuronal progenitors 
 SOX2 Abcam, ab97959 Rabbit 1 : 200/1 : 100 Stem cell self-renewal transcription factor Lundberg et al. (2014
 PAX6 Proteintech, 12323-1-AP Rabbit 1 : 200/1 : 100 Stem cell transcription factor Kreitzer et al. (2013
 PAX6 DSHB Mouse 1 : 10/– Stem cell transcription factor Ericson et al. (1997
 TBR2 Abcam, ab23345 Rabbit 1 : 200/1 : 50 Transcription factors of neurogenic intermediate progenitors Fietz et al. (2010
Postmitotic cells 
 TBR1 Abcam, ab31940 Rabbit 1 : 500/– Transcription factor regulating the maturation of postmitotic cells Bayatti et al. (2008
 CTIP2 Abcam, ab18465 Rat 1 : 500/1 : 200 Transcription factor regulating the maturation of postmitotic cells Johnson et al. (2009
 SATB2 Abcam, ab51502 Mouse 1 : 200/– Transcription factor regulating the maturation of postmitotic cells Ip et al. (2011
 CUX1 Abcam, ab54583 Mouse 1 : 100/1 : 500 Transcription factor regulating the maturation of postmitotic cells Hadjivassiliou et al. (2010
 NeuroD1 Abcam, ab60704 Mouse 1 : 500/– Neuronal differentiation promoting transcription factor Cooper et al. (2010
 NeuN Millipore, MAB377 Mouse 1 : 500/– Neuron-specific nuclear protein Jager et al. (2013
Antibody Company Species Concentration (IHC/IHC-P) Target References 
Proliferation 
 Ki67 Abcam, ab16667 (clone SP6) Rabbit 1 : 200/– Cell cycle-related nuclear protein Bento et al. (2010
 Ki67 Dako, M7240 (Clone MIB) Mouse 1 : 500/1 : 50 Cell cycle-related nuclear protein Gerdes et al. (1983
 PH3 Millipore 06-570 Rabbit 1 : 1000/– Mitosis marker Yamada et al. (2014
Neuronal progenitors 
 SOX2 Abcam, ab97959 Rabbit 1 : 200/1 : 100 Stem cell self-renewal transcription factor Lundberg et al. (2014
 PAX6 Proteintech, 12323-1-AP Rabbit 1 : 200/1 : 100 Stem cell transcription factor Kreitzer et al. (2013
 PAX6 DSHB Mouse 1 : 10/– Stem cell transcription factor Ericson et al. (1997
 TBR2 Abcam, ab23345 Rabbit 1 : 200/1 : 50 Transcription factors of neurogenic intermediate progenitors Fietz et al. (2010
Postmitotic cells 
 TBR1 Abcam, ab31940 Rabbit 1 : 500/– Transcription factor regulating the maturation of postmitotic cells Bayatti et al. (2008
 CTIP2 Abcam, ab18465 Rat 1 : 500/1 : 200 Transcription factor regulating the maturation of postmitotic cells Johnson et al. (2009
 SATB2 Abcam, ab51502 Mouse 1 : 200/– Transcription factor regulating the maturation of postmitotic cells Ip et al. (2011
 CUX1 Abcam, ab54583 Mouse 1 : 100/1 : 500 Transcription factor regulating the maturation of postmitotic cells Hadjivassiliou et al. (2010
 NeuroD1 Abcam, ab60704 Mouse 1 : 500/– Neuronal differentiation promoting transcription factor Cooper et al. (2010
 NeuN Millipore, MAB377 Mouse 1 : 500/– Neuron-specific nuclear protein Jager et al. (2013

IHC, immunohistochemistry; IHC-P, immunohistochemistry-paraffin.

Microscopy

Virtual images in brightfield were created using an Axio Scan.Z1 slide scanner (Zeiss, Germany) and processed with Axio Scan.Z1 ZEN (Zeiss, Germany). Tile scans of each slide were acquired using a Zeiss Axio Observer.Z1 fluorescent microscope with the following excitation/emission frequencies: 359/461 nm for DAPI, 470/509 nm for Alexa Fluor 488, 558/583 nm for Alexa Fluor 555, and 649/670 nm for Alexa Fluor 676, using a Plan-Apochromat ×20/0.8 M27 objective and an AxioCamMR3 camera. Images were processed by the Axiovision Rel. 4.8 software (Zeiss) and contrast-adjusted with Adobe Photoshop CS2 (Adobe Systems, Mountain View, CA, USA).

Confocal analysis was performed with a Leica TCS SP8 confocal scanning system (Leica Microsystems) equipped with 405 nm diode, 488 nm Ar, 561 nm DPSS, and 633 nm HeNe lasers. Eight-bit digital images were collected from a single optical plane using a ×20 HC PL APO CS2 oil-immersion objective (numerical aperture 0.75; Leica) or a ×40 HC PL APO CS2 oil-immersion objective (numerical aperture 1.30; Leica). For each sample, optical sections of 2048 × 2048 pixels were taken at 0.9 µm intervals. Images were processed with the LAS AF.Ink software (Leica). Z-stack images were then analyzed with the ImageJ software (National Institutes of Health, USA). Images were assembled into photomontages using QuarkXPress (Quark, Inc., Denver, CA, USA).

Results

Gestational Week 9–10

The hippocampal primordium was recognizable adjacent to the ventral cortical hem. The following layers were observed, from deep to superficial: the cell-dense VZ, the thin presumptive SVZ/intermediate zone (IZ), and the marginal zone (MZ; Fig. 1A).

Figure 1.

Analysis of progenitors and layer markers in coronal sections of the human hippocampal formation at GW 9. (A) SOX2+ progenitors of the ventral cortical hem. The asterisk indicates the confocal image field in BK. (B) SOX2/Ki67 double-labeling. (CI) PAX6/Ki67/CTIP2 triple labeling. (B) SOX2+ and (C,D,F,I) PAX6+ progenitors show proliferative activity in the VZ and SVZ (arrowheads). (C,E,G,I) PAX6+/CTIP2+ cells are observed in the SVZ, at the border between the VZ and SVZ (arrows), and (H,I) some of which are also co-labeled with Ki67 (arrowheads). (J,K) Few TBR2+ cells show proliferative activity in the SVZ (arrowheads). Scale bar: 200 µm in A; 50 µm in BK. d, dorsal zone; IZ, intermediate zone; l, lateral; m, medial; MZ, marginal; SVZ, subventricular zone; v, ventral; VZ, ventricular zone.

Figure 1.

Analysis of progenitors and layer markers in coronal sections of the human hippocampal formation at GW 9. (A) SOX2+ progenitors of the ventral cortical hem. The asterisk indicates the confocal image field in BK. (B) SOX2/Ki67 double-labeling. (CI) PAX6/Ki67/CTIP2 triple labeling. (B) SOX2+ and (C,D,F,I) PAX6+ progenitors show proliferative activity in the VZ and SVZ (arrowheads). (C,E,G,I) PAX6+/CTIP2+ cells are observed in the SVZ, at the border between the VZ and SVZ (arrows), and (H,I) some of which are also co-labeled with Ki67 (arrowheads). (J,K) Few TBR2+ cells show proliferative activity in the SVZ (arrowheads). Scale bar: 200 µm in A; 50 µm in BK. d, dorsal zone; IZ, intermediate zone; l, lateral; m, medial; MZ, marginal; SVZ, subventricular zone; v, ventral; VZ, ventricular zone.

The localization and subtypes of progenitor cells were analyzed. SOX2+ (Fig. 1A,B) and PAX6+ (Fig. 1C,F,G,I) cells were observed in both the VZ and the IZ. Several of these cells displayed proliferative activity in the deep VZ as they were co-labeled with Ki67 (Fig. 1C,D,F,I). In addition, a few TBR2+ cells were observed above the VZ, delineating the presumptive SVZ in the process of being formed (Fig. 1J,K) and suggesting their early commitment to a neuronal lineage. In this area, some cycling TBR2+/Ki67+ cells were observed (Fig. 1K).

Committed neuronal cells of the IZ were labeled with CTIP2 (Fig. 1J,K). Multiple labeling showed the presence of PAX6+/CTIP2+/Ki67+ triple-labeled cells at the border between the VZ and the SVZ (Fig. 1C–I). CTIP2+/Ki67+/Pax6 cells were not detected (Fig. 1C–I). Taken together, these data suggested that PAX6+/CTIP2+/Ki67+ triple-labeled cells were completing the transition from cycling progenitors to postmitotic neurons.

In summary, at this stage, both PAX6+ and TBR2+ progenitor cells are present in the VZ and SVZ, with the possible production of neuronal cells. In the first compartment, proliferative activity appears more pronounced.

Gestational Week 11

A primordial hippocampal formation was seen to develop on the ventromedial side of the cortical hem (Fig. 2A). In this hippocampal formation, the pyramidal plate was visible between the MZ and the IZ. On the dorsal side, next to the hem, the hippocampal plate displayed a round cell-dense area, the presumptive dentate anlage (Fig. 2A).

Figure 2.

Analysis of progenitors and layer markers in coronal sections of the hippocampal formation at GW 11. (A) DAPI staining. Asterisks indicate the confocal image field in EI and MP. (B) Ki67, (C) PAX6, and (D) TBR2 immunolabeling showing the distribution of cycling and progenitor cells (arrowheads). PAX6+ cells are mainly observed in the VZ. TBR2+ cells are observed in the SVZ, at the border with the VZ. (E) Higher magnification showing the localization of PAX6+ and TBR2+ progenitors. PAX6+/TBR2+ co-labeled cells are present in the SVZ (arrowheads) and IZ. (F) PAX6+/SOX2+ co-labeled cells are visible in the VZ. Several PAX6+/SOX2 cells are observed in the SVZ. (G) Proliferative SOX2+ cells are observed in the VZ. The proliferative activity of (H) PAX6+ and (I) TBR2+ cells was analyzed using Ki67 co-labeling. Cycling PAX6+ progenitors are present in the VZ/SVZ (arrowheads). Cycling TBR2+ cells are observed in the SVZ and IZ (arrowheads). (J) TBR1, (K) CTIP2, and (L) CUX1 immunolabeling showing the distribution of postmitotic cells within the hippocampal plate (arrowheads). (M) PAX6/PH3 and (N) TBR2/PH3 double-labeling showing mitotic cells in the hippocampal VZ and SVZ (arrowheads). (O) Distribution of TBR2+ cells, CTIP2+, and CUX1+ neurons. The density of CTIP2+ cells increases from the SVZ toward the ammonic plate. TBR2/CTIP2 co-labeled cells are present in the SVZ (arrowheads), whereas CTIP2+/CUX1+ neurons are located in the upper hippocampal plate (shown in yellow). (P) Distribution of TBR1+, CTIP2+, and SATB2+ neurons within the hippocampal plate. A few TBR1+ neurons are present in the upper part of the plate. Arrowheads indicate SATB2+ cells. Scale bar: 200 µm in AD and L,J; 25 µm in EI and M and N; 50 µm in O and P. d, dorsal; DA, dentate anlage, HP, hippocampal plate; l, lateral; m, medial; MZ, marginal zone; SVZ, subventricular zone; IZ, intermediate zone; v, ventral; VZ, ventricular zone.

Figure 2.

Analysis of progenitors and layer markers in coronal sections of the hippocampal formation at GW 11. (A) DAPI staining. Asterisks indicate the confocal image field in EI and MP. (B) Ki67, (C) PAX6, and (D) TBR2 immunolabeling showing the distribution of cycling and progenitor cells (arrowheads). PAX6+ cells are mainly observed in the VZ. TBR2+ cells are observed in the SVZ, at the border with the VZ. (E) Higher magnification showing the localization of PAX6+ and TBR2+ progenitors. PAX6+/TBR2+ co-labeled cells are present in the SVZ (arrowheads) and IZ. (F) PAX6+/SOX2+ co-labeled cells are visible in the VZ. Several PAX6+/SOX2 cells are observed in the SVZ. (G) Proliferative SOX2+ cells are observed in the VZ. The proliferative activity of (H) PAX6+ and (I) TBR2+ cells was analyzed using Ki67 co-labeling. Cycling PAX6+ progenitors are present in the VZ/SVZ (arrowheads). Cycling TBR2+ cells are observed in the SVZ and IZ (arrowheads). (J) TBR1, (K) CTIP2, and (L) CUX1 immunolabeling showing the distribution of postmitotic cells within the hippocampal plate (arrowheads). (M) PAX6/PH3 and (N) TBR2/PH3 double-labeling showing mitotic cells in the hippocampal VZ and SVZ (arrowheads). (O) Distribution of TBR2+ cells, CTIP2+, and CUX1+ neurons. The density of CTIP2+ cells increases from the SVZ toward the ammonic plate. TBR2/CTIP2 co-labeled cells are present in the SVZ (arrowheads), whereas CTIP2+/CUX1+ neurons are located in the upper hippocampal plate (shown in yellow). (P) Distribution of TBR1+, CTIP2+, and SATB2+ neurons within the hippocampal plate. A few TBR1+ neurons are present in the upper part of the plate. Arrowheads indicate SATB2+ cells. Scale bar: 200 µm in AD and L,J; 25 µm in EI and M and N; 50 µm in O and P. d, dorsal; DA, dentate anlage, HP, hippocampal plate; l, lateral; m, medial; MZ, marginal zone; SVZ, subventricular zone; IZ, intermediate zone; v, ventral; VZ, ventricular zone.

Progenitor Cell Types and Proliferative Compartments of the Hippocampal Anlage

The localization and proliferation of SOX2+, PAX6+, and TBR2+ cells were investigated in the ammonic area. SOX2+ cells were mainly detected in the VZ (Fig. 2F,G), whereas several PAX6+ cells were also observed in the SVZ (Fig. 2C,E,F,H). Rare SOX2+ (Fig. 2F,G) and PAX6+ (Fig. 2C,E,F,H) cells could be observed in the IZ. We found that 69 ± 2% of all PAX6+ cells were PAX6/SOX2 co-labeled in the VZ–IZ. Proliferating SOX2+/Ki67+ (Fig. 2G) and PAX6+/Ki67+ (Fig. 2H) progenitor cells were mainly located at the ventricular border.

In the SVZ, TBR2+ cells appeared denser than at the previous stage (Fig. 2E,I and Supplementary Fig. 1H,J), increasing toward the temporal cortex. The production of TBR2+ cells from PAX6+ progenitors was suggested by the presence of TBR2/PAX6 double-labeled cells (Fig. 2E), which represented 8.1 ± 1.5% of the VZ PAX6+ cells and 47 ± 6% of the SVZ TBR2+ cells.

The detection of TBR2/Ki67 double-labeled cells confirmed the germinal property of the SVZ (Fig. 2I). However, Ki67+ cells were still mainly located along the ventricular border in the putative ammonic VZ, appearing less dense in the more superficial SVZ (Fig. 2I). To assess the occurrence of mitoses, we evaluated the frequency of PAX6+/PH3+ and TBR2/PH3+ cells in the VZ and SVZ. PAX6+/PH3+ cells represented 0.86 ± 0.25% of all PAX6+ cells in the VZ (Fig. 2M), and TBR2+/PH3+ cells represented 0.78 ± 0.30% of all TBR2+ cells in the SVZ (Fig. 2N). The frequency of mitotic progenitor cells normalized to the respective total number of cells was indeed not significantly different between the SVZ and the VZ.

Some TBR2+ cells lacking proliferative activity were observed in the MZ (Fig. 2D), and likely represented a population of Cajal–Retzius cells as previously described (Abraham et al. 2004; Hodge et al. 2013).

Taken together, these results show the presence of 2 germinal zones in the hippocampal formation, the VZ and the adjacent SVZ, composed of cycling radial glial cells and intermediate progenitors.

Pyramidal Cells of the Hippocampal Anlage

We identified the presence of different neuronal subpopulations within the plate. In the superficial hippocampal plate, a band of cells labeled with TBR1 (Fig. 2J,P and Supplementary Fig. 1B,E) associated with some CUX1+ cells was observed (Fig. 2L,O and Supplementary Fig. 1I,J), whereas a deeper and thicker band was positive for CTIP2 (Fig. 2K,O,P and Supplementary Fig. 1C,E,H,J). In addition, a few SATB2+ cells were found within the plate, close to the entorhinal cortex (Fig. 2P and Supplementary Fig. 1B,E). Moreover, the presence of TBR2+/CTIP2+ cells (Fig. 2O) in the SVZ suggested production of CTIP2+ cells from TBR2+ cells. In the MZ, a thin band of TBR1+ (Fig. 2J) or CUX1+ (Fig. 2L) cells was observed localized in the same compartment as TBR2+ putative Cajal–Retzius cells (Abraham et al. 2004).

These results suggest that both the VZ and SVZ might contribute to the generation of pyramidal neurons.

Gestational Week 13

The hippocampal formation had enlarged and begun to gyrate into more mature posterior sections (Fig. 3A). The hippocampal plate was increased in thickness, displaying an enlarged dentate anlage located between the well-defined fimbria and the ammonic primordium (Fig. 3A).

Figure 3.

Analysis of progenitor- and cell fate-specific transcription factors in coronal sections of the hippocampal formation at GW 13. (A) Nissl staining. Asterisk indicates the confocal image field in EG. (B) Ki67, (C) PAX6, and (D) TBR2 labeling showing the distribution of cycling and progenitor cells, similar to the previous stage. (E) TBR1, (F) CTIP2, and (G) CUX1 immunolabeling showing the distribution of neuronal subpopulations within the hippocampal plate. (H) Proliferative PAX6+/Ki67+ and (J) TBR2+/Ki67+ progenitors (arrowheads) are localized in the same compartments as observed at GW 11. (I) PAX6+ and TBR2+ progenitors continue to delineate the VZ and SVZ, respectively. Some PAX6+ progenitors are observed in the SVZ. (JM) TBR2/CTIP2/Ki67 multiple labeling of the VZ, SVZ, and IZ. In the SVZ, TBR2+ cells expressing the postmitotic marker CTIP2 are not Ki67+ (arrows). Arrowheads indicate TBR2+/Ki67+ co-labeled cells. Scale bar: 400 µm in AD and EG; 25 µm in HM. d, dorsal; DA, dentate anlage, FI, fimbria; HP, hippocampal plate; IZ, intermediate zone; l, lateral; m, medial; MZ, marginal zone; SVZ, subventricular zone; v, ventral; VZ, ventricular zone.

Figure 3.

Analysis of progenitor- and cell fate-specific transcription factors in coronal sections of the hippocampal formation at GW 13. (A) Nissl staining. Asterisk indicates the confocal image field in EG. (B) Ki67, (C) PAX6, and (D) TBR2 labeling showing the distribution of cycling and progenitor cells, similar to the previous stage. (E) TBR1, (F) CTIP2, and (G) CUX1 immunolabeling showing the distribution of neuronal subpopulations within the hippocampal plate. (H) Proliferative PAX6+/Ki67+ and (J) TBR2+/Ki67+ progenitors (arrowheads) are localized in the same compartments as observed at GW 11. (I) PAX6+ and TBR2+ progenitors continue to delineate the VZ and SVZ, respectively. Some PAX6+ progenitors are observed in the SVZ. (JM) TBR2/CTIP2/Ki67 multiple labeling of the VZ, SVZ, and IZ. In the SVZ, TBR2+ cells expressing the postmitotic marker CTIP2 are not Ki67+ (arrows). Arrowheads indicate TBR2+/Ki67+ co-labeled cells. Scale bar: 400 µm in AD and EG; 25 µm in HM. d, dorsal; DA, dentate anlage, FI, fimbria; HP, hippocampal plate; IZ, intermediate zone; l, lateral; m, medial; MZ, marginal zone; SVZ, subventricular zone; v, ventral; VZ, ventricular zone.

Progenitor Cell Types and Proliferative Compartments of the Ammonic/Subicular Anlage

The presence of 2 proliferating compartments was similar to that observed at GW 11 (Fig. 3B–D), as confirmed by the presence of PAX6+/Ki67+ (Fig. 3H) and TBR2+/Ki67+ double-labeled cells (Fig. 3J), located in the VZ and SVZ. TBR2+/PAX6+ cells were still observed (Fig. 3I), representing 4.5 ± 1.1% of PAX6+ VZ cells and 57 ± 9.8% of TBR2+ SVZ cells.

Pyramidal Cells of the Ammonic/Subicular Anlage

TBR2/Ki67/CTIP2 multiple labeling (Fig. 3J–M) showed that some non-proliferating TBR2+/Ki67- cells expressed CTIP2 in the SVZ. This suggests that at least some neurons were derived from the SVZ.

Within the hippocampal plate, layer-specific markers showed a significant increase in the number of neurons, which decreased in density from the temporal lobe to the dentate anlage (Fig. 3E–G). In comparison with GW 11, TBR1+ neurons were located deep within the plate and formed a linear band at GW 13 (Fig. 3E and Supplementary Fig. 1L,O), whereas CTIP2+ and CUX1+ neurons were located in the medial and deep plate (Fig. 3F,G and Supplementary Fig. 1MO).

These results are evocative of an inside-out migration of CTIP2+ and CUX1+ neurons, passing through the TBR1+ layer, between GW 11 and 13 as reported in animals (Leid et al. 2004; Nieto et al. 2004; Bedogni et al. 2010).

Within the SVZ, we observed rare TBR1+ cells and several CTIP2+ cells, not detected in the VZ (Fig. 3E,F). However, CUX1+ cells were observed in both the VZ and SVZ (Fig. 3G). Double-labeling showed that CUX1 was expressed in PAX6+ (Supplementary Fig. 2A–C) and TBR2+ cells (Supplementary Fig. 2D–F) in the VZ and SVZ, respectively.

These observations suggest that CUX1+ neurons originate from both VZ and SVZ precursor cells.

Gestational Week 16

The hippocampal formation had rotated, and the limit between the ammonic fields and subicular areas was clearly recognizable. The VZ was thinner in the Ammon's horn area compared with GW 13, but increased in thickness toward the subicular area. The limit between the CA1 and CA2/CA3 compartments was not yet detectable (Fig. 4A). In the presumptive CA1, the superficial layers displayed larger pyramidal cell bodies than the deeper layers (Fig. 6A; Arnold and Trojanowski 1996). The presence of superficial subicular islands allowed the ammonic fields to be distinguished from the subicular complex, although the limit between the subicular complex and the entorhinal plate could not yet be discerned. The lamina dissecans was recognizable in the entorhinal plate (Hevner and Kinney 1996; Fig. 4A).

Figure 4.

Analysis of progenitor markers in coronal sections of the hippocampal formation at GW 16. (A) Asterisks in the VZ/SVZ of CA3 and subiculum indicate the confocal image fields in E, I-K and F, L-N, respectively. (B) PAX6, (C) TBR2, and (D) Ki67 labeling in the hippocampal formation. Ki67+ cells are rarely observed in the VZ and SVZ and difficult to see at low magnification. (E) and (F) show PAX6/Ki67 co-labeling in the VZ of the CA3 and subiculum, respectively. Differences in VZ thickness and cell density are visible between the 2 fields. (G) PAX6/PH3 and (H) TBR2/PH3 double-labeling showing mitotic cells in the hippocampal VZ and SVZ, respectively (arrowheads). (IK) and (LN) show TBR2/CTIP2/Ki67 multiple labeling in the SVZ of the CA3 and subiculum, respectively. Differences in layer thickness and cell density are visible between the 2 fields. Note that TBR2+/Ki67+ cells are not labeled for CTIP2 (arrowheads), whereas TBR2+/Ki67 cells are labeled for CTIP2 (arrows). The dashed line indicates the limit between the VZ and SVZ (EN). Scale bar: 500 µm in AD; 25 µm in EN. d, dorsal; DG, dentate gyrus; l, lateral; LD, lamina dissecans; m, medial; SF, subicular field; v, ventral; VZ, ventricular zone.

Figure 4.

Analysis of progenitor markers in coronal sections of the hippocampal formation at GW 16. (A) Asterisks in the VZ/SVZ of CA3 and subiculum indicate the confocal image fields in E, I-K and F, L-N, respectively. (B) PAX6, (C) TBR2, and (D) Ki67 labeling in the hippocampal formation. Ki67+ cells are rarely observed in the VZ and SVZ and difficult to see at low magnification. (E) and (F) show PAX6/Ki67 co-labeling in the VZ of the CA3 and subiculum, respectively. Differences in VZ thickness and cell density are visible between the 2 fields. (G) PAX6/PH3 and (H) TBR2/PH3 double-labeling showing mitotic cells in the hippocampal VZ and SVZ, respectively (arrowheads). (IK) and (LN) show TBR2/CTIP2/Ki67 multiple labeling in the SVZ of the CA3 and subiculum, respectively. Differences in layer thickness and cell density are visible between the 2 fields. Note that TBR2+/Ki67+ cells are not labeled for CTIP2 (arrowheads), whereas TBR2+/Ki67 cells are labeled for CTIP2 (arrows). The dashed line indicates the limit between the VZ and SVZ (EN). Scale bar: 500 µm in AD; 25 µm in EN. d, dorsal; DG, dentate gyrus; l, lateral; LD, lamina dissecans; m, medial; SF, subicular field; v, ventral; VZ, ventricular zone.

Progenitor Cell Types and Proliferative Compartments of the Ammonic and Subicular Complex/Entorhinal Plate

The PAX6 (Fig. 4B) and TBR2 (Fig. 4C) compartments appeared relatively reduced compared with previous stages, displaying a decreasing in thickness and cell density from the CA3 (Fig. 4E,I–K) area to the subicular/entorhinal area (Fig. 4F,L–N). The VZ thickness of the CA3 and subicular fields was about 40 and 110 µm, respectively. The SVZ thickness of the CA1 and subicular fields was about 50 and 130 µm, respectively. Ki67+/PAX6+ (Fig. 4E,F) and Ki67+/TBR2+ (Fig. 4I,K,L,N) progenitors were found in both the ammonic and subicular germinal layers. We assessed the occurrence of mitosis by evaluating the frequency of PAX6+/PH3+ (Fig. 4G) and TBR2/PH3+ (Fig. 4H) cells in the ammonic VZ and SVZ, respectively. PAX6+/PH3+ cells represented 0.38 ± 0.2% of PAX6+ VZ cells and TBR2+/PH3+ cells represented 0.57 ± 0.38% of TBR2+ SVZ cells, showing that mitotic activity was still present in the 2 compartments, although it tended to decrease compared with GW 11.

In summary, the density of progenitor cells decreases in the ammonic field compared with previous stages, in contrast to the situation in the subicular complex/entorhinal formation.

Pyramidal Neurons of the Ammonic and Subicular/Entorhinal Complex

TBR2/Ki67/CTIP2 multiple labeling (Fig. 4I–K,L–N) showed that some non-proliferating TBR2+/Ki67 cells still expressed CTIP2 in the SVZ. This suggests that CTIP2+ neurons could still be produced in the SVZ.

The expression pattern of layer-specific markers changed drastically compared with earlier stages as the ammonic and subicular plates became discernible (Fig. 5A–D).

Figure 5.

Analysis of postmitotic markers in coronal sections of the hippocampal formation at GW 16. (A) TBR1, (B) CTIP2, (C) SATB2, and (D) CUX1 labeling and (F) overlay showing the distribution of neuronal subpopulations within the hippocampal plate. (E) NeuN+ neurons appear in the CA3/CA2 field. Scale bar: 500 µm.

Figure 5.

Analysis of postmitotic markers in coronal sections of the hippocampal formation at GW 16. (A) TBR1, (B) CTIP2, (C) SATB2, and (D) CUX1 labeling and (F) overlay showing the distribution of neuronal subpopulations within the hippocampal plate. (E) NeuN+ neurons appear in the CA3/CA2 field. Scale bar: 500 µm.

The ammonic CA1 field displayed from deep to superficial layers: a very thin band of TBR1+ neurons (Figs 5A,F and 6B), CUX1+ neurons (Figs 5D and 6B,D), intermingled with a few SATB2+ neurons (Figs 5C,F and 6C) and, superficially, CTIP2+ neurons (Figs 5B,F and 6C,D). Some superficial CTIP2+ neurons showed faint CUX1 co-labeling (Fig. 6D). A few NeuN+ neurons were only present in the putative CA3 field located in the superficial layer (Fig. 5E).

Figure 6.

Cell layering in coronal sections of the hippocampal plate at GW 16. (A) Nissl staining of the CA1 field showing a maturation gradient that increases from superficial (upper part of the image) to deep layers. Distribution of (B) TBR1+ and CUX1+, (C) CTIP2+ and SATB2+, and (D) CTIP2+ and CUX1+ neurons in the CA1 field. Note that few TBR1+ and SATB2+ cells are visible in B and C, respectively. TBR1+/CTIP2+/SATB2+ cell localization in the (EG) subicular and (IK) entorhinal plate fields, respectively. (H) and (L) DAPI labeling of the subicular and entorhinal cortical fields, respectively. Different TBR1 (B,E,I) and SATB2 (C,G,K) band thicknesses are evident in the CA1 and subicular/entorhinal plates. Asterisks in IL indicate the lamina dissecans. Scale bar: 50 µm in A and D; and 100 µm in EL.

Figure 6.

Cell layering in coronal sections of the hippocampal plate at GW 16. (A) Nissl staining of the CA1 field showing a maturation gradient that increases from superficial (upper part of the image) to deep layers. Distribution of (B) TBR1+ and CUX1+, (C) CTIP2+ and SATB2+, and (D) CTIP2+ and CUX1+ neurons in the CA1 field. Note that few TBR1+ and SATB2+ cells are visible in B and C, respectively. TBR1+/CTIP2+/SATB2+ cell localization in the (EG) subicular and (IK) entorhinal plate fields, respectively. (H) and (L) DAPI labeling of the subicular and entorhinal cortical fields, respectively. Different TBR1 (B,E,I) and SATB2 (C,G,K) band thicknesses are evident in the CA1 and subicular/entorhinal plates. Asterisks in IL indicate the lamina dissecans. Scale bar: 50 µm in A and D; and 100 µm in EL.

At the border with the subicular complex, the distribution of layer-specific markers changed sharply (Fig. 5A–D,F). The deep layer was composed of TBR1+ (Figs 5A,F and 6E,H,I,L) and CTIP2+ neurons (Figs 5B,F and 6F,H,J,L), whereas the superficial presubicular islands were composed of SATB2+ neurons (Figs 5C,F and 6G,H,K,L). The band of SATB2+ cells increased in thickness from the subicular complex to the entorhinal plate (Fig. 5C), where it split into 2 bands separated by the lamina dissecans (Figs 5C,F and 6K). CUX1+ cells were found throughout the thickness of the subicular complex/entorhinal plate, densely populating the middle-to-upper layers (Fig. 5D).

This analysis of layering by neuronal markers showed clear differences between the ammonic plate on the one hand and the subicular and entorhinal plates on the other.

Gestational Week 19–20

The hippocampal fissure was partly fused at this stage (Fig. 7A). Neuronal maturation was seen by Nissl staining to follow an inside-out gradient throughout the hippocampal formation. In the subicular complex, the lamina dissecans was still observed (Figs 7F–H and 8).

Figure 7.

Analysis of progenitor markers in coronal sections of the hippocampal formation at GW 20. (A) Nissl staining. Asterisks in the VZ/SVZ of the CA1 and subiculum indicate the confocal image fields in BE and IN, respectively. (BE) Analysis of markers in the ammonic VZ. (B) Rare cycling PAX6+/Ki67+ progenitors are visible in the thin ammonic VZ and in the IZ (arrowheads). (C) PAX6+/TBR2+ cells are detected in the SVZ (arrowheads). (D) PAX6+/NeuroD1+ and (E) TBR2+/NeuroD1+ cells are observed in the VZ and in the SVZ (arrowheads). Distribution of (F) PAX6+, (G) TBR2+, and (H) Ki67+ cells in the hippocampal formation. Note that labeled cells are mainly observed in the subicular area at this stage. (IK) PAX6/Ki67 and (LN) TBR2/Ki67 co-labeling in the subicular field showing some double-labeled cells (arrowheads). Larger fields are visible in Supplementary Figure 3AF. Scale bar: 500 µm in A and FH; 25 µm in BE and IL,N. d, dorsal; HF, hippocampal fissure; l, lateral; m, medial; DG, dentate gyrus; SF, subicular field; v, ventral.

Figure 7.

Analysis of progenitor markers in coronal sections of the hippocampal formation at GW 20. (A) Nissl staining. Asterisks in the VZ/SVZ of the CA1 and subiculum indicate the confocal image fields in BE and IN, respectively. (BE) Analysis of markers in the ammonic VZ. (B) Rare cycling PAX6+/Ki67+ progenitors are visible in the thin ammonic VZ and in the IZ (arrowheads). (C) PAX6+/TBR2+ cells are detected in the SVZ (arrowheads). (D) PAX6+/NeuroD1+ and (E) TBR2+/NeuroD1+ cells are observed in the VZ and in the SVZ (arrowheads). Distribution of (F) PAX6+, (G) TBR2+, and (H) Ki67+ cells in the hippocampal formation. Note that labeled cells are mainly observed in the subicular area at this stage. (IK) PAX6/Ki67 and (LN) TBR2/Ki67 co-labeling in the subicular field showing some double-labeled cells (arrowheads). Larger fields are visible in Supplementary Figure 3AF. Scale bar: 500 µm in A and FH; 25 µm in BE and IL,N. d, dorsal; HF, hippocampal fissure; l, lateral; m, medial; DG, dentate gyrus; SF, subicular field; v, ventral.

Figure 8.

Analysis of layering markers in coronal sections of the hippocampal formation at GW 20. Location of (A) TBR1+, (B) CTIP2+, (C) SATB2+, and (D) CUX1+ neurons in the hippocampal formation. Note that TBR1 and SATB2 are poorly expressed in the hippocampus. (E) Overlays of TBR1/CTIP2/SATB2 and (F) TBR1/CTIP2/CUX1 multiple labeling. Scale bar: 500 µm.

Figure 8.

Analysis of layering markers in coronal sections of the hippocampal formation at GW 20. Location of (A) TBR1+, (B) CTIP2+, (C) SATB2+, and (D) CUX1+ neurons in the hippocampal formation. Note that TBR1 and SATB2 are poorly expressed in the hippocampus. (E) Overlays of TBR1/CTIP2/SATB2 and (F) TBR1/CTIP2/CUX1 multiple labeling. Scale bar: 500 µm.

Progenitor Cell Types and Proliferative Compartments of the Ammonic and Subicular/Entorhinal Complex

The gradient observed in germinal layers at GW 16 was still visible at this stage (Fig. 7F–H). In the ammonic VZ/SVZ, proliferative activity of PAX6+ progenitor cells, as seen by Ki67+ labeling (Fig. 7B), was greatly reduced. In parallel, TBR2+ cells were rarely observed in the SVZ (Fig. 7CE), suggesting a reduced production of intermediate progenitors. The transition from PAX6+ to TBR2+ cells was still occurring, as suggested by PAX6/TBR2 co-labeled cells (Fig. 7C), which represented 43 ± 4.3% of all TBR2+ SVZ cells. The expression of NeuroD1 in some PAX6+ cells of the VZ (Fig. 7D) and in TBR2+ cells of the SVZ (Fig. 7E) suggested that they were differentiating into neuronal cells.

Conversely, in the subicular/entorhinal fields, numerous PAX6+/Ki67+ (Fig. 7I–K and Supplementary Fig. 3A–C) and TBR2+/Ki67+ (Fig. 7L–N and Supplementary Fig. 3D–F) progenitor cells remained in the VZ and SVZ/IZ, respectively.

Moreover, as observed in previous stages, a few CTIP2+/TBR2+ cells were seen in the SVZ (Supplementary Fig. 3G,I,J), whereas a few CUX1+ neurons were detected in both the VZ and SVZ (Supplementary Fig. 3K–N).

Taken together, these data confirm a decrease in the germinal compartments and reduced neurogenesis in the ammonic field when compared with the subicular/entorhinal areas.

Pyramidal Neurons of the Ammonic and Subicular/Entorhinal Complex

In the ammonic plate, the expression of markers in different layers extended to the different ammonic fields. From deep to superficial layers: A few TBR1+ neurons formed a very thin deep band similar to that observed at GW 16 (Figs 8A,E,F and 9C); more superficially, numerous CTIP2+ neurons predominated in the deep plate (Figs 8B,E,F and 9D,E), mixed with numerous CUX1+ neurons (Figs 8D,F and 9C,E), which were preferentially located superficially. A few SATB2+ neurons were dispersed within the middle-to-deep plate (Figs 8C,E and 9D). CTIP2+ neurons now extended near the border of CA3/CA2 (Fig. 8B,E,F). A similar gradient was observed for CUX1 labeling, which extended toward the CA3 field (Fig. 8D,F). Within the middle-to-deep CA1 field, some CTIP2+/CUX1+ neurons were still detected (Fig. 9E). NeuN-labeling increased in the CA3 field, and extended toward the CA1 field (Fig. 9A). NeuN staining decreased from the deep to the superficial plate (Fig. 9A), in accordance with the inside-out gradient of neuronal maturation previously reported (Arnold and Trojanowski 1996).

Figure 9.

Cell layering in coronal sections of the hippocampal plate at GW 20. (A) The distribution of NeuN+ neurons predominates in the deep layers of the ammonic plate. The asterisks indicate the picture field in (BE). (B) Nissl staining of the CA1 field. Distribution of (C) TBR1+ and CUX1+, (D) CTIP2+ and SATB2+, and (E) CTIP2+ and CUX1+ neurons in the CA1 field. Note that a few TBR1+ and SATB2+ cells are visible in C and D, respectively. (FH) Triple labeling showing the localization of TBR1+/CTIP2+/SATB2+ neurons in the subicular field (I: DAPI staining). (JL) Triple labeling showing the localization of TBR1+/CTIP2+/CUX1+ neurons in the entorhinal plate field. Different TBR1 (C,F,J) and SATB2 (D,H) band thicknesses are evident in the CA1 and subicular complex/entorhinal plate (M: DAPI staining). Scale bar: 500 µm in A; 50 µm in BE; 100 µm in FM.

Figure 9.

Cell layering in coronal sections of the hippocampal plate at GW 20. (A) The distribution of NeuN+ neurons predominates in the deep layers of the ammonic plate. The asterisks indicate the picture field in (BE). (B) Nissl staining of the CA1 field. Distribution of (C) TBR1+ and CUX1+, (D) CTIP2+ and SATB2+, and (E) CTIP2+ and CUX1+ neurons in the CA1 field. Note that a few TBR1+ and SATB2+ cells are visible in C and D, respectively. (FH) Triple labeling showing the localization of TBR1+/CTIP2+/SATB2+ neurons in the subicular field (I: DAPI staining). (JL) Triple labeling showing the localization of TBR1+/CTIP2+/CUX1+ neurons in the entorhinal plate field. Different TBR1 (C,F,J) and SATB2 (D,H) band thicknesses are evident in the CA1 and subicular complex/entorhinal plate (M: DAPI staining). Scale bar: 500 µm in A; 50 µm in BE; 100 µm in FM.

In the subicular complex/entorhinal plate, the thickness of the TBR1+ (Figs 8A,E,F and 9F,I,J,M; Supplementary Fig. 4A,E,I) and CTIP2+ (Figs 8B,E,F and 9G,I,K,M; Supplementary Fig. 4B,F,J) cell bands was increased. In the subicular complex, TBR1+ neurons clearly predominated in the deep layer (Figs 8A,E,F and 9F,I,J,M), whereas CTIP2+ neurons were still migrating and continued to segregate from the TBR1+ band, occupying a more superficial position (Figs 8B,E,F and 9G,I,K,M; Supplementary Fig. 4B,F). The SATB2+ band was better delineated, as it was more restricted to the superficial plate, including the presubicular islands, and seemed to be migrating (Figs 8C,E and 9H,I; Supplementary Fig. 4C). In the entorhinal cortex, SATB2+ neurons formed 2 bands, localized superficially and in a more intermediate position, respectively (Fig. 8C,E). CUX1+ neurons were still present throughout the thickness of the subicular and entorhinal plates (Figs 8D,F and 9L,M; Supplementary Fig. 4G,K). Mature NeuN+ neurons were more numerous in the subicular/cortical entorhinal plate, confirming an ammonic-to-subicular gradient of expression (Fig. 9A).

In summary, these observations reveal the maturation processes of the different neuronal subpopulations in the ammonic and subicular/entorhinal plates. The staining pattern of neuronal markers still suggested the establishment of a laminating plate (Leid et al. 2004; Nieto et al. 2004; Britanova et al. 2008; Bedogni et al. 2010), and extended from CA3/CA2 to the rest of the hippocampal formation.

Discussion

This is the first study characterizing progenitor cells and hippocampal plate layering of the ammonic and subicular/entorhinal fields during human development. We have shown here that the hippocampal plate is composed of heterogeneous pyramidal cell populations that suggest an “inside-out” lamination pattern at mid-gestation, similar to that shown by experimental studies in the neocortex. Moreover, we have provided evidence of regional gradients in pyramidal cell differentiation.

Temporal and Regional Gradients in Germinal Areas of the Hippocampal Anlage

Our results show that PAX6+ radial glial cells and TBR2+ intermediate progenitors are already present in the VZ and SVZ of the hippocampal formation, respectively, at GW 9. Moreover, the presence of scattered PAX6+ cells in the IZ of the human hippocampus suggests the presence of a primordial outer SVZ (OSVZ). This germinal area could be limited in extent, compared with that of the neocortical OSVZ (Hansen et al. 2010), and reminiscent of the radial glia-like progenitor cells described in the mouse neocortex (Wang et al. 2011). Thus, developmental similarities seem to exist between this archicortex and the neocortex. However, we have shown that the SVZ of the human hippocampal formation is thinner and lacks the distinct OSVZ described in the developing human neocortex [(personal data, see also PAX6 and TBR2 gene expression at: http://www.brain-map.org/) and (Hansen et al. 2010)].

Similar to the process described in rodent neocortical development (Englund et al. 2005; Bayatti et al. 2008; Hodge and Hevner 2008), our results suggest the possible production of TBR2+ cells from PAX6+ radial glia, some of which still undergo proliferation. Thus, pyramidal neurons might also be generated by indirect neurogenesis through TBR2+ progenitors, similar to the situation described in the human neocortex at early stages of development (GW 11; Masood et al. 1990; Zecevic et al. 2005; Mo et al. 2007; Hansen et al. 2010). However, our observations also suggest that the proliferative capacity of hippocampal TBR2+ progenitor cells is rather low (judging by the occurrence of TBR2+/Ki67+ cells) when compared with that described in the mouse and human neocortex. This may be related to the relatively thin hippocampal SVZ in comparison with the neocortical SVZ (personal data, Arnold et al. 2008; Hansen et al. 2010; Mairet-Coello et al. 2012; Malik et al. 2013).

The density and proliferative activity of radial glial cells and intermediate progenitors in the VZ and SVZ decline from GW 16 onwards, suggesting that most ammonic neurons might be generated before this time point. Conversely, proliferating layers of the subicular/entorhinal complex continue to be well delineated at GW 20. Thus, the hippocampal anlage appears to be one of the first cortices (archicortex) to develop in humans, as also suggested by previous observations (Kostovic et al. 1989). The prolonged proliferative activity detected in the subicular/entorhinal complex might be linked to the greater number of layers and increased cytoarchitectural complexity of these areas.

Temporal, Regional, and Laminar Gradients in the Pyramidal Layer of the Hippocampal Anlage

Within the hippocampal plate, the first neuronal population to be specified is that of TBR1+ cells, located deep in the plate at GW 13. When the limit between the CA1 area and the subicular complex becomes apparent (GW 16), different layering patterns are visible in the 2 compartments. Within the ammonic fields, we found 2 bands: A thin deep layer populated by TBR1+ cells and a thick superficial layer mainly populated by CTIP2+ and/or CUX1+ cells. Moreover, our data show a radial increase in NeuN-labeling at mid-gestation, in agreement with an inside-out neuronal maturation gradient (Arnold and Trojanowski 1996; Abraham et al. 2009). These data support previous findings suggesting the presence of different pyramidal cell populations along the radial axis. A bilayered structure has been suggested based on neurochemical and functional studies of the CA1 and CA2 of several mammalian species, including humans [Ramon y Cajal 1893; Lorente de Nó 1934, for review, see Slomianka et al. (2011)].

The cytoarchitecture of the hippocampal pyramidal layer is more complex in the human subicular and entorhinal complex. Up to 7 layers have been identified in the presubiculum (Ding 2013), whereas 6 layers have been described in the entorhinal cortex (Krimer et al. 1997; Nieuwenhuys et al. 2007).

In our study, in the subicular and entorhinal complex, 4 markers appeared at mid-gestation: TBR1, CTIP2, SATB2, and CUX1, with an inside-out layering pattern similar to that reported in animal studies (Leid et al. 2004; Nieto et al. 2004; Britanova et al. 2008; Bedogni et al. 2010) and in the human neocortex (Saito et al. 2010). The identification of laminar differences in molecular expression in Ammon's horn is significant in supporting previous studies based on other methods (Ramon y Cajal 1893; Lorente de Nó 1934, for review Slomianka et al. 2011). Thus, these layer-specific markers might reflect the greater cytoarchitectural and layering complexity of these compartments in comparison with the ammonic field. However, the role of these transcription factors in the hippocampal pyramidal layer remains to be elucidated.

Our data also show that NeuN-labeling first appears in the CA3 field at GW 16, extending to the CA1 and subiculum by GW 20. Interestingly, CA3 neurons have been found to be generated before CA1 neurons in rats and non-human primates (Nowakowski and Rakic 1981; Altman and Bayer 1990; Lopez-Gallardo and Prada 2001). However, the stratum pyramidale of the CA1 field has been reported to be formed before that of the CA3 field in rodents and humans (Altman and Bayer 1990; Arnold and Trojanowski 1996). Thus, our data confirm that the maturation of CA3 neurons represents a more complex process than that of CA1 cells.

Layer-Specific Markers and Connectivity of the Hippocampal Formation

The neuroanatomy, connectivity, and functions of the hippocampus have mainly been studied in animals, revealing some similarities across mammalian species. However, there are also differences between species, and very few studies on human development have been carried out. Animal studies suggest that the intrinsic connections of the hippocampal formation constitute a unidirectional circuit (Insausti and Amaral 2012). However, the molecular mechanisms that regulate the formation of these hippocampal connections are not known.

Layer-specific markers have previously been shown to regulate neuronal projections in the neocortex [Leid et al. 2004; Nieto et al. 2004; Arlotta et al. 2005; Chen et al. 2005, 2008; Szemes et al. 2006; Alcamo et al. 2008; Britanova et al. 2008; Bedogni et al. 2010; Cubelos et al. 2010, for review, see Greig et al. (2013)]. In this study, we show that some of these cortical markers are expressed in the hippocampal formation, creating new opportunities for the study of the role of such transcription factors in the development of archicortical structures and connectivity. For example, projections to the hippocampus and subiculum originate from neurons in layers 2 and 3 of the entorhinal cortex, where we observe SATB2 and CUX1 labeling. Moreover, reciprocal projections to the entorhinal cortex originate from the CA1, where we observe CTIP2 and CUX1 expression, as well as the subiculum (Hevner and Kinney 1996). Conversely, the low levels of TBR1 and SATB2 expression in human ammonic fields supports data from animal studies showing the paucity of thalamic projections and the absence of callosal projections from the CA1 (Cenquizca and Swanson 2007; Nielsen et al. 2014). Thus, a link between layer-specific transcription factors and hippocampal neuronal specification/connectivity can be inferred, leading to novel and exciting hypotheses concerning the development of the human hippocampal formation (Leid et al. 2004; Nieto et al. 2004; Arlotta et al. 2005; Chen et al. 2005, 2008; Szemes et al. 2006; Alcamo et al. 2008; Britanova et al. 2008; Bedogni et al. 2010; Cubelos et al. 2010).

In conclusion, the pattern of expression of layer- and stage-specific markers during human brain development has allowed us to identify various layers and neuronal subtypes in subfields of the human hippocampal formation. This knowledge opens up new opportunities for the exploration of hippocampal connectivity and functions.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org.

Funding

This study was supported by EU grant HEALTH-2011-2.2.2-2/Develage (H.A.-B.), the Inserm (P.G.), Paris Diderot University (P.G.), the Roger de Spoelberch Foundation (P.G.), and the Princess Grace of Monaco Foundation (P.G.).

Notes

Conflict of Interest: None declared.

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