Nature - USA (2020-01-23)

Nature | http://www.nature.com | 5

Discussion
We have systematically analysed scRNA-seq and ATAC-seq data to iden-
tify cell type diversities, gene expression trajectories, transcription
regulation networks and signal transduction pathways in the develop-
ing human hippocampus. The hippocampus starts to form from the
hippocampal primordium in response to bone morphogenetic protein
(BMP) and WNT secreted by the CH^18 –^20. An open chromatin area close
to the PROX1 TSS contains the binding motif for LEF1 and TCF4, two
transcription factors that are involved in the WNT signalling pathway
by recruiting the coactivator beta-catenin to enhancer elements of
targeting genes^5 , indicating that WNT signals not only initiate differen-
tiation of the medial pallium to the hippocampus, but also contribute
to subregional patterning of the hippocampus. The adult neural stem
cells located in the subgranular zone give rise to granule cells through-
out adult life in most mammals^21. WNT signalling also helps to regulate
granule cell genesis and neural activity in adult mammals^22 ,^23 , indicating
that the key gene regulation may be conserved in embryonic and adult
neurogenesis in the hippocampal DG.
HOPX has been recently identified as a gene that is expressed by
dentate precursors and contributes to embryonic and postnatal neu-
rogenesis in mice^24. Another unbiased single-cell RNA-seq analysis
has indicated that perinatal, postnatal, and adult neurogenesis in the
mouse DG are fundamentally similar^15. Notably, clonal lineage-tracing
of HOPX+ cells in mice showed that these precursors generate neurons
located in the DG or CA^24. Consistently, we found that at GW11, although
most HOPX+ progenitors are located in the DNE, a subset of HOPX+
progenitors is found in the ANE, indicating that HOPX+ progenitors in
different locations may have different cell fates.
The copy number of the DUF1220 protein domain in the genome is
correlated with the evolutionary proximity of the species to humans as
well as with brain size, cognitive capability, and severity of autism^17 ,^25 –^27.
Major copies of human DUF1220 domains are encoded by the NBPF gene
family. Microarray data from the Allen Brain Atlas suggest that NBPF1

expression decreases when the human brain develops (http://www.

brainspan.org). LHX2 is expressed in the dorsal and medial pallium but

not in the CH, which secretes WNT ligands and functions as an organizer

that is necessary and sufficient to induce the hippocampus^18. Notably,

expression of NBPF1 upregulates LHX2 expression and increases the

number of hippocampal PROX1+ granule cells in the developing mouse

brain. However, the detailed molecular mechanisms of this process

need further investigation.

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availability are available at https://doi.org/10.1038/s41586-019-1917-5.

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0.4 1.0

E16.5

STX10CASC15

BEX5CHMP4A

NBPF1NBPF15

NLGN4X

TUBB5CAMK2A

CALYSLC25A4

HumanMouse

b –2^2

STX10

2

0

CASC15

3

0

NBPF1

2

0

BEX5

3

0

2.5

0

CHMP4A

NBPF110 kb

chr1p36.22p36.11

STX10

1 kb

chr19p13.2p13.13

CHMP4A2 kb

chr14q11.1q12

BEX5 2 kb

chrXq21.31q22.1

Rep 1Rep 2

Rep 3

Rep 1Rep 2

Rep 3

c

E13.5–E15.5 IUE

GFP/PROX1/DAPI

NBPF1-GFP/PROX1/DAPI

1 2

1

2

d GFP/PROX1/DAPIE13.5–E18.5 IUE

NBPF1-GFP/PROX1/DAPI

4

3

3

4

e f

g

P0P5

P18P19

P23GW16

GW18

GW22GW25

GW27

GW20

E16.5

P0P5P18P19P23

GW16GW18GW20GW22GW25GW27

Mouse Human

Mouse

Human

a

**

E13.5–E15.5 IUE

80

60

40

20

(^0) GFPNBPF1–GFP
GFP
+PROX1
+/GFP

• (%)
  **
  E13.5–E18.5 IUE
  80
  100
  60
  40
  20
  (^0) GFPNBPF1–GFP
  GFP
  +PROX1
  +/GFP


• (%)
  Fig. 4 | Specif ic genes expressed in the human developing hippocampus.
  a, Heat map showing correlation of different stages of hippocampus
  development in human and mouse. The developing human hippocampus is
  similar to the developing mouse hippocampus at P0–5. b, Heat map showing
  DEGs in human and mouse hippocampus. c, Expression of human-specific
  genes in t-SNE plots (left). Right, in situ hybridization at GW25; bottom,
  normalized ATAC-seq profile of human-specific genes in hippocampus in GW25
  with three independent biological replicates. Scale bar, 300 μm.
  d, e, Overexpression of NBPF1 promotes DG formation at E13.5, observed at
  E15.5 (d) and E18.5 (e) in mouse. Scale bars, 500 μm (d, left), 100 μm (d, right),
  200 μm (e). f, g, Percentage of PROX1+ cells among GFP+ cells. f, E13.5–E15.5:
  P = 0.0049, two-sided t-test. n = 6, 5 brain slices per experiment; mean ± s.d.
  g, E13.5–E18.5: P = 0.0015. n = 6, 5 brain slices per experiment. IUE, in utero
  electroporation.