Nature - USA (2020-01-23)

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direction but closer to the pial side (Fig. 2e, Extended Data Fig. 4c).
At GW16, the migration of PAX6+ and HOPX+ progenitors continued
and many cells arrived at the hilus and formed an origin hub of DG
cells, called the tertiary matrix (III). Notably, PAX6+ progenitors were
located outside HOPX+ progenitors while migrating (Fig. 2f, Extended
Data Fig. 4d–f ). PAX6+ progenitors were still abundant, and some were
located in the blades of the DG, but only some HOPX+ progenitors were
found in the hilus; the majority of HOPX+ progenitors were in the cornu
ammonis (CA) at GW 22 (Fig. 2f, Extended Data Fig. 4g–i).
We next evaluated the proliferation capacity and cell fate of PAX6+
and HOPX+ progenitors. Both scRNA-seq data and immunostaining
indicate that a subpopulation of PAX6+ and HOPX+ progenitors are
active in the cell cycle even at the mid-gestational stage (Extended Data
Fig. 5a–d). In cell fate assessment, we observed PAX6+ NEUROD1+ cells
in the CA and DG, but PAX6+ GFAP+ cells only in the CA (Fig. 2g, Extended
Data Fig. 5e–g). Similar expression patterns were found in HOXP+ cells
(Fig. 2h, Extended Data Fig. 5h, i). Next, we evaluated the maturation
status of PAX6+ or HOPX+ progenitors and found that NEUROD1+ cells
were more mature than GFAP+ cells, suggesting that they may have been
born earlier (Extended Data Fig. 5j, k). Together, our data suggest that
although the origins and migrating paths of PAX6+ and HOPX+ progeni-
tors differ, they both contribute to neural and glial genesis in a spati-
otemporal manner in the developing human hippocampus (Fig. 2i).

Neurons in developing hippocampus

To further investigate the developmental characteristics of hippocam-
pal neurons, we subclassified all the excitatory neurons into seven
groups by PCA (Fig. 3a, b). Excitatory neurons from CA1, CA3 and DG

were grouped as ExN01–03 (Fig. 3b, Extended Data Fig. 6a). SEMA5A and

PID1 were selected as marker genes for DG and CA1, respectively, while

SULF2 and NRIP3 were considered as CA3 markers (Fig. 3c, Extended

Data Fig. 6b). Consistent with progenitor migration paths, the matura-

tion analysis suggests that CA1 neurons were more mature than CA3 and

DG neurons (Fig. 3d). Excitatory neurons were categorized into three

groups according to their developmental stage, and GO analysis of DEGs

indicates that neurogenesis is the major event at GW16–18, followed

by axonogenesis (GW20–22) and function development (GW25–27)

(Fig. 3e–g). To further analyse the transcriptional regulation of DG

formation, we selected the subclusters of highly variable genes and

clustered them into nine modules by weighted gene coexpression

network analysis (WGCNA) (Extended Data Fig. 6c, d). The green mod-

ule includes PROX1, suggesting that the genes in this module may be

correlated with DG development (Fig. 3h). When we analysed ATAC-seq

data for the hippocampus at GW25, we found PROX1 motifs in ATAC

peaks close to the TSSs of several genes, including KCNJ6, NFIA, DUSP1

and NPTX2, which are also in the green module (Fig. 3i, j). Among these

genes, KCNJ6 (also known as GIRK2) encodes a member of the G-protein-

activated inwardly rectifying K+ channels that is widely abundant in the

brain and has been implicated in learning and memory, reward, motor

coordination, and other functions^13.

Hippocampal inhibitory neurons arise from MGE and CGE precur-

sors. Notably, monocle analysis suggested that the majority of MGE-

derived InN (LHX6+) and CGE-derived InN (NR2F1/2+) were separated

(Fig. 3k, Extended Data Fig. 7a–c). The pseudo-time analysis demon-

strated that InN expressing CCK, CALB2 and VIP accumulated in the

CGE differentiation path, and the majority of SATB1+ and SST+ neurons

were in the MGE path (Fig. 3l, Extended Data Fig. 7b, c). Additionally,

Non-DG ExN

DG ExN

Cajal–Retzius

Progenitor DG InN

Astrocyte

Progenitor

Progenitor

Oligodendrocyte

Non-DG InN

OPC

START

START

START

Primor

dial

hippocampus

Hilus

LMD

V DG

3 3 DG

2

Developing^22

DG

CA

CA CA

Fimbra

Fimbra

Fimbra

HOPXPAX6+

+

GW11 GW14 GW16 GW22

1 1 1 1

VZ

VZ VZ

VZ

CH

DNE DNE

ANE ANE

Non-DG ExNProgenitor

DG ExN

Astrocyte

Astrocyte

Neuron

OPC

Oligodendrocyte

Oligodendrocyte

1.5

–2.0

1.0

–2.0

1.5

–2.0

1.0

–2.0

1.5

–2.0

1.5

–2.0

1.0

–2.0

1.5

–2.0

1.5

–2.0

MKI67 PAX6 HOPX

SOX2 EOMES NEUROD2

OLIG2 GFAP AQP4

START

a

b

i

GW11

DNE

ANE

1

(^12)
2
d
III
III
GW11
GW16
GW22PAX6/SOX2/DAPIHOPX/SOX2/DAPI
PAX6/SOX2/DAPI
HOPX/SOX2/DAPI
DNE
ANE
PROX1/DAPI
II
II II
II
I I
I I
I
I
PROX1
1
(^12)
2
f
GFAP/HOPX/DAPI
NEUROD1/HOPX/DAPI
CA DG
CA DG
11
2
2
11
2
2
GW14PAX6/SOX2/DAPI
HOPX/SOX2/DAPI
PROX1/DAPI
1
1
c
e
GFAP/PAX6/DAPI
NEUROD1/PAX6/DAPI
GW25
1 1CADG
CA DG
2
2
(^11)
2
2
g h
Fig. 2 | Molecular signature of neural progenitor cells of the developing
human hippocampus. a, Cell lineage relationships of all cells analysed except
for microglia and endothelial cells in developing human hippocampus.
Monocle recovered a branched single-cell trajectory beginning with
progenitors and terminating at excitatory neurons, inhibitory neurons,
astrocytes and oligodendrocytes. b, Cell lineage relationships of progenitors,
excitatory neurons, astrocytes and oligodendrocytes in developing human
hippocampus. Known gene expression is shown below. Arrows show the
directions of lineages. c, Immunof luorescence images of PROX1(scale bar,
200 μm), PAX6 and SOX2 at GW11. Scale bars, 500 μm (left), 200 μm (top right),
100 μm (bottom right). I, primary matrix. d, Immunofluorescence images of
HOPX and SOX2 at GW11. Scale bars, 500 μm (left), 200 μm (top right), 100 μm
(bottom right). e, Immunof luorescence images of PROX1 (scale bars, 1,000 μm,
inset 500 μm), PAX6, HOPX and SOX2 in GW14. Scale bar, 200 μm. II, secondary
matrix. f, Immunof luorescence images of PROX1 (scale bar, 1,000 μm), PAX6,
HOPX and SOX2 at GW16 (top) and GW22 (bottom). Scale bar, 500 μm. III,
tertiary matrix. g, h, Immunof luorescence images of PAX6, HOPX, NEUROD1
and GFAP at GW25. Scale bars, 500 μm (left); 100 μm (right). c–h, The
experiments were repeated three times independently with similar results.
i, Schema depicting locations of PAX6+ or HOPX+ progenitors in developing
human hippocampus from GW11 to GW22. Arrows indicate direction of
migration.