Nature - USA (2020-01-23)

2 | Nature | http://www.nature.com

Article

in the PFC, whose progenitors are regulated by HMGA2^7 ,^8. SOX4 and
SOX11, two SOXC transcription factors that are required for neuronal
differentiation during neurogenesis in the adult hippocampus^9 ,^10 , were
relatively highly expressed in the hippocampus (Fig. 1i). GO analysis of
DEGs between ExN from the hippocampus and PFC at GW16 indicate
that hippocampal ExN may undergo synapse organization and axono-
genesis at GW16 (Extended Data Fig. 2d). Comparison of the matu-
ration trajectories of hippocampus and PFC neurons indicated that
hippocampus non-DG ExN were more mature than PFC ExN, whereas
maturation of DG ExN was similar to that of PFC ExN (Fig. 1j). InN of the
PFC and hippocampus generally showed a similar maturation status,
whereas MGE-derived InN were more mature than CGE-derived InN in
the hippocampus (Fig. 1k). Consistent with transcriptome analysis,
immunofluorescence staining for OLIG2 and MBP showed a number of
MBP+ cells in the subfield of the hippocampus, whereas no MBP+ cells
were found in the human PFC at GW16 (Fig. 1l, m, Extended Data Fig. 2e),
suggesting that oligodendrocytes may be involved the maturation of
hippocampal neurons during early development.

Progenitors of the developing hippocampus

To further investigate cellular lineage relationships in the fetal human
hippocampus, we reconstructed five developmental paths by monocle
analysis without microglia and endothelial cells (Fig. 2a). Three major
subgroups of progenitors differentiated to excitatory neuronal, OPC
and oligodendrocyte or astrocyte lineages. The MGE- and CGE-derived
InN were separated in different directions, which is consistent with pre-
vious studies showing that hippocampal InN originate from different

progenitors located in the ganglionic eminence^11. To further reveal the

diversity and molecular properties of human hippocampal progeni-

tors, we used GO analysis of DEGs and marker genes to identify eight

subclusters (Extended Data Fig. 3a–c). EOMES+, MEIS2+ and NEUROD2+

progenitors were in clusters P3 and P4, indicative of ExN generation

(Extended Data Fig. 3a). AQP4, OLIG1/OLIG2 and PDGFRA were highly

expressed in clusters P5, P6 and P7, respectively, indicative of astrocyte

and oligodendrocyte cell fates. Cluster P8 contained a small number

of progenitors that highly expressed DLX1 and DLX2, indicating that

these cells may differentiate as InN (Extended Data Fig. 3a–f ).

To understand how progenitors develop into neuronal and glial cells,

we carried out trajectory analysis (Fig. 2b) and separated three paths

towards neurons, astrocytes and oligodendrocytes. Notably, PAX6+ and

HOPX+ progenitors, which are considered as neurogenic progenitors in

the neocortex^12 , were likely to contribute to both neurogenesis and glio-

genesis in the human hippocampus (Fig. 2b, Extended Data Fig. 3g, h).

We next examined the locations of cells expressing PAX6 or HOPX

by immunofluorescence staining (Fig. 2c–f). At GW11, the primordial

hippocampal area, located adjacent to the cortical hem (CH), was

composed of the dentate neuroepithelium (DNE) and ammonic neu-

roepithelium (ANE). The majority of cells in the DNE and ANE expressed

SOX2, and PAX6+ SOX2+ progenitors of ANE started to migrate (Fig. 2c,

Extended Data Fig. 4a). At the same time, HOPX+ SOX2+ DNE progenitors

also indicated migration potential (Fig. 2d, Extended Data Fig. 4b). As

the hippocampus developed at GW14, a number of PAX6+ progenitors

migrated away from the ventricular zone towards the future DG (PROX1+

region, Fig. 2e, Extended Data Fig. 4c), forming the primary matrix (I)

and secondary matrix (II). HOPX+ progenitors also migrated in the same

AQP4ASLC OLIG2GAPDH

1

RELNGAD1SPAR

C

NEURODPTPRC

2

P 1P 2P 3

P 4

P 5

P 8

P 7

CR

P 6

ExN 4

ExN 5

ExN 6

ExN 7ExN 3ExN 2

InN 1

InN 2

InN 3

InN 4

InN 5

ExN 1

InN 6InN 8

OPC 1

OPC 2

OPC 3

OPC 4OPC 6

OPC 5

AC 1AC 5AC 2

AC 3AC 4

M 1

M 11M 2

M 3

M 4

M 5

M 6

M 7M 8

M 9

M 10

EC

InN 7

0.3 (^41) N (^12) CC (^2) H
0.9
InN maturation
HP CGE-derivedInN
00 .. 39 HP MGE-derived InNPFC InN d
In
0.250.75 HP DG ExN
PFC ExN
HP non-DG ExN
ExN maturation
2
1
0
Relative expression–1
in PFC
Relative expression
in HP
2
1
0
–1
OPHN1
CDK2AP1TOXPLXNA3
SOX11SOX4
YWHAE
BTG1MARCKSL1
RTN4
PFC HP
HP endothelial
PFC microglia
HP microglia
HP microglia
PFC OPCHP OPC
HP oligodendrocyte
PFC astrocyte
HP astrocyte
HP astrocyte
HP Cajal–Retzius
PFC InN
HP MGE-derived InN
HP CGE-derived InN
PFC ExN
HP non-DG ExN
HP DG ExN
PFC progenitor
HP progenitor
Non-DG ExN
Progenitor CGE-derived InNMGE-derived InN
DG ExN
Astrocyte
Cajal–RetziusOPC
Microglia Oligodendrocyte
Endothelial cell
ASCL1 NEUROD2 GAD1 PROX1 MEIS2 LHX6
NR2F2 AQP4 OLIG2MBP PTPRC SPARC
3
0
2.5 1 20 40 2
(^300)
30 30 20 20 30 03
GW16 HP GW16 PFC
OLIG2/MBP/DAPI
1
12
2
10 kb
PROX1
chr1q32.2q42.1
Rep 1
Rep 2Rep 3
LEF1/TCF4
p31.1cchr1hr1 10 kb
TCF4
LEF1
GW25CA DG
MEIS2/PROX1/DAPI
i
j
k
l m
h
f g
e
d
c
a b
Fig. 1 | Molecular diversity of single cells from the developing human
hippocampus. a–c, Visualization of eleven major classes using t-SNE in 3D (a)
and 2D (b) visualization. c, Expression of known markers. HP, hippocampus;
PFC, prefrontal cortex. Dots, individual cells; grey, no expression; red, relative
expression (log-normalized gene expression). d, Immunostaining of MEIS2 and
PROX1. Scale bar, 500 μm. e, Normalized ATAC-seq profiles of PROX1 in GW25
hippocampus show the activation of PROX1. Amplified view (pink) shows
predicted LEF1 and TCF4 binding sites. f, LEF1 and TCF4 binding motifs are
identified in the ATAC-seq peaks close to the PROX1 TSS. g, Hierarchical
clustering analysis of 47 subclasses. AC, astrocyte; P, progenitor; CR, Cajal–
Retzius cell; M, microglia; EC, endothelial cell. n = 134, 141, 95, 275, 58, 300, 397,
159, 204, 483, 101, 74, 670, 1,019, 1,765, 2,334, 793, 1,073, 909, 3,189, 2,347, 92,
1,838, 717, 1,956, 730, 1,192, 2, 573, 54, 259, 84, 84, 44, 489, 465, 246, 68, 229, 638,
540, 131, 103, 139, 257, 227, 459 and 282 cells, top to bottom. h, Abstracted graph
shows the connections on the transcriptome between different cell types in the
developing human hippocampus and PFC. i, Scatterplot of all genes for
correlation with conserved differentiation network across PFC and
hippocampus. Blue plot shows genes related to PFC; red plot shows genes
related to hippocampus. j, k, Maturation scores of excitatory neurons (j) and
inhibitory neurons (k) in PFC and hippocampus. l, m, Immunostaining for
oligodendrocyte markers at GW16 in human hippocampus and prefrontal
cortex. Scale bars, 500 μm (l, left); 100 μm (l, right, m). The experiment was
repeated three times independently with similar results.