Nature - USA (2020-01-23)

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

Article

we found genes that may regulate cell fate determination at the first
branch point (Extended Data Fig. 7d, e). Microglia, the immune cells
in the CNS, originate from the mesoderm^14. We classified microglia
into 11 subclusters and observed that M9 contained microglia in active
cell cycles from all developing stages (Extended Data Fig. 8a–d). The
immunostaining images also indicated proliferating microglia at GW25
(Extended Data Fig. 8e).

Evolution signatures of developing hippocampus

Although the hippocampus is considered an evolutionarily conserved
part of the brain, transcriptomic correlation coefficient analysis illus-
trated that the developmental timing of the human hippocampus from
GW16 to 20 was similar to that at P0–5 in mice^15 ,^16 (Fig. 4a), suggesting
that the human embryonic hippocampal development occurs earlier
but lasts for longer than in mice. We also found DEGs in the human
hippocampus, some of which are primate-specific, including STX10,
CHMP4A, BEX5, NBPF1 and the long non-coding RNA CASC15 (Fig. 4b,
c). In situ images and ATAC-seq data identified the mRNA localiza-
tion and transcription regulatory sites of these genes (Fig. 4c). Genes
of the neuroblastoma breakpoint family (NBPF) contain a repeated

domain called DUF1220, the copy number of which is related to brain

evolution and complexity^17. Several NBPF family genes are expressed

in hippocampal cells, and the expression of NBPF1 was relatively high

and general in all cell types (Fig. 4c, Extended Data Fig. 9a). NBPF1 with

eight DUF1220 domains exists only in primates, and in particular in spe-

cies that are evolutionally close to humans (Extended Data Fig. 9b, c).

To further investigate its role in hippocampal development, we

transiently expressed NBPF1 in the mouse primordial hippocampal

area at embryonic day 13.5 (E13.5) and observed that these mice had

more PROX1+ cells and an enlarged PROX1+ area at E15.5 and E18.5

when compared with control mice (Fig. 4d–g, Extended Data Fig. 9d–f ).

To understand how NBPF1 regulates hippocampal development, we

collected single GFP+ cells (Extended Data Fig. 9g). LHX2 has been

considered as an essential gene in the hippocampal primordium to

regulate hippocampal neuronal development^18. Single-cell quanti-

tative RT–PCR results indicated that LHX2 expression was higher in

NBPF1–GFP+ cells (Extended Data Fig. 9h). Further analysis of open

chromatin areas close to the PROX1 TSS revealed three potential

sites for LHX2 binding (Extended Data Fig. 9i), indicating a possible

molecular mechanism by which NBPF1 may regulate hippocampal

development via LHX2.

1

10

1

10

1

10

1

10

1

10

1

1

1

10

NR2F2

PROX1

CCK

CALB2

VIP

LHX6

SATB1

SST

Branch CGE MGE

Pseudotime

P 01P 08InN 01InN 02InN 03InN 04InN 05InN 06InN 08InN 07

START

END1 END2

END3

END4

1 kb

NPTX2 chr7 q21.12q22.1

Rep 1

Rep 2

Rep 3

NFIA

30 kb

chr1 p32.2p31.1

Rep 1

Rep 2

Rep 3

DUSP1

1 kb

chr5 q33.3q35.1

Rep 1

Rep 2

Rep 3

Rep 1

Rep 2

Rep 3

chr21

20 kb

KCNJ6 q22.12q22.2

PROX1

EMX2

POU3F2

SULF2

RUNX1T1

KIF5ASOX11

SEMA3ESYT4

KITIGFBP5

FABP5GAP43EMP2

ELAVL2NECAB1EGR1

JUNFOSBFOS

NTMAPP

LMO4SYT1 NRP1 CXCR4

CRYABIGSF21

CLSTN2

SCGNCALB2NFIA

NRGN

RBP1

PROX1

NRP2

RBFOX3 DPF3FGFR1

FLRT3NTF3

EOMESKCNJ6SFRP1NPTX2DUSP1

SEMA5AGRIA2

LRTM1

GW25–27

–log 10 P

Behaviour

Learning

Learning or memory

012

Neuron differentiation

Axon guidance

Axonogenesis

GW20–22

012345

Neurogenesis

Generation of neurons

Neuron differentiation

GW16–18

0246

0.25 K

0.75

ExN 01ExN 04ExN 02ExN 05 ExN 03ExN 06ExN 07

GW27

DG

CA3

CA2

CA1

DG

CA3CA2

CA1

DG

CA3

CA2

CA1

DG

CA3

CA2

CA1

SEMA5A (DG) PID1 (CA1) SULF2 (CA3) NRIP3 (CA3)

GW22 GW25GW18 GW20GW27

GW16

PID1

NRIP3SULF2

SEMA5ASCGN

PROX1MGAT4C

MPPED1

2

0

–2

ExN 01ExN 02ExN 03ExN 04ExN 05ExN 06ExN 07

ExN 01

ExN 02

ExN 03

ExN 04

ExN 05

ExN 06

ExN 07

a

b

c

dh

i

j

kl

e

f

g

Fig. 3 | Dynamics of neurogenesis in the developing human hippocampus.
a, Visualization of seven subtypes of excitatory neuron in the developing
human hippocampus using t-SNE. Sample sizes of clusters: 2, 573, 2,347, 1,838,
1,956, 1,192, 717, 92 cells. b, Heat map showing the expression level and identity
of genes in the excitatory neurons subclasses. Top, distribution of each
subclass by gestational week. c, In situ hybridization of region-specific genes in
DG, CA1 and CA3 at GW27. Scale bar, 600 μm. The experiment was repeated
three times independently with similar results. d, Maturation scores of seven
subtypes of excitatory neuron show that CA1 neurons are more mature than
CA3 and DG neurons. e–g, The enriched gene ontology terms show the cell
properties of the hippocampus at different weeks. Sample sizes: 4,912 cells (e);

4,164 cells (f); 1,639 cells (g). h, The social network Cytoscape graph depicts the

gene network regulation of excitatory neurons. i, j, Motifs of PROX1 (i) and the

normalized ATAC-seq profile of downstream genes of PROX1 (j) in GW25

hippocampus with three independent biological replicates. k, Cell lineage

relationships of progenitors and inhibitory neurons analysed in developing

human hippocampus. Monocle recovered a branched single-cell trajectory

beginning with progenitors and terminating at subgroups of inhibitory

neurons. l, Markers were ordered by Monocle analysis in pseudo-time. Line

with blue shading represents inhibitory neurons derived from CGE; pink

shading represents inhibitory neurons derived from MGE.