Nature - USA (2020-01-16)

394 | Nature | Vol 577 | 16 January 2020

Article

unspecialized, specifically with respect to subtype-specific genes.
These observations led us to consider whether a ‘transcriptionally
unspecialized state’ serves as the starting point for somatosensory
subtype diversification.
To investigate this idea, we compared scRNA-seq transcriptomes
generated from sensory neurons between E11.5 and adulthood. Pro-
spective identities of sensory neurons at each developmental stage
were assigned on the basis of transcriptional similarity using canoni-
cal correlation analysis^27 (Fig. 1a, Extended Data Fig. 1f ) and a graph-
based strategy for locally embedding consecutive time points based
on the transcriptional variation they share. We constructed single-
cell k-nearest neighbour graphs for each time point (ti) with nodes
representing cells and edges linking neighbours. These graphs were
then joined by identifying neighbouring cells in adjacent time points
using a coordinate system learned from the subsequent time point
(ti + 1; see Methods). The resulting graph forms a branching network
that can be visualized using a force-directed layout. This representa-
tion spans all developmental stages and provides a consolidated view
of the transcriptional maturation of each principal somatosensory
neuron subtype from E11.5 to adulthood (Fig. 2a).
We next tested whether this graph-based representation of devel-
opmental gene expression profiles of sensory neuron subtypes reca-
pitulates known developmental relationships. We inspected the gene
expression patterns of the transcription factors (TFs) Runx1 and Runx3,
which have been implicated in the development of select unmyelinated
(C-fibre) neuron subtypes and proprioceptors, respectively^28 –^30. Runx1
expression was prominent in unmyelinated sensory neuron subtypes,
whereas Runx3 expression was restricted to mature proprioceptors of
adult ganglia, as previously described^28 ,^29 (Fig. 2b). Furthermore, the

graph-based representation accurately depicts the developmental

switch from Ntrk1+ to Ret+ that occurs in subsets of non-peptidergic

C-fibre neurons^30 (Fig. 2b). To facilitate exploration of this dataset

by the community, we created an HTML-based interactive interface

to enable visualization of the expression pattern of any gene at each

developmental time point, from E11.5 to adulthood, for each of the

somatosensory neuron subtypes (interactive somatosensory neuron

browser: https://kleintools.hms.harvard.edu/tools/springViewer_1_6_

dev.html?datasets/Sharma2019/all).

TFs in sensory neuron development

One observation from our initial analysis of the graph-based repre-

sentation of developmental transcriptomes of sensory neurons is that

the TFs Runx1 and Runx3, which have been implicated in the develop-

ment of sensory neuron subtypes, are broadly co-expressed in nascent

E11.5 Avil+ sensory neurons, in contrast to their mutually exclusive

expression patterns in terminally differentiated subtypes of adult DRG

(Fig. 2b). This is consistent with the finding that RUNX1 and RUNX3

proteins are colocalized in embryonic DRG^31. This observation led us

to consider whether other TFs that are subtype-restricted in adult gan-

glia may be co-expressed in nascent, transcriptionally unspecialized

sensory neurons. To address this possibility, we identified TFs other

than Runx1 and Runx3 that are expressed in select subtypes of soma-

tosensory neurons from mature ganglia by inspecting 1,152 neuronally

expressed TFs, and found that 23 are expressed in distinct subsets

of adult somatosensory neurons (Fig. 2c). Strikingly, the scRNA-seq

data revealed that several TFs expressed in select subtypes of sensory

neurons of mature DRG are co-expressed in newborn E11.5 sensory

a

b

Adult

P5

P0

E15.5

E12.5

E11.5

c

SST

MRGPRD

+^

Propr

ioceptors

Aβ RA-

LT MR

Aβ eld/SA1

CGRP-α C-LTMR

CGRP-

ε

CGRP-

η

CGRP-

γ

CGRP-

θ

CGRP-

ζ

Cold thermo.

Aδ-LTMR

SST

MRGPRD+

Proprioceptors

Aβ RA-LTMR

Aβ eld/SA1

C-LTMR

CGRP-α

CGRP-ε

CGRP-η

CGRP-γ

CGRP-θ

CGRP-ζ

Cold thermo.

USN

Aδ-LTMR

Unspecialized

sensory neuron

SST

MRGPRD

+^

Prop

Aβ rioceptors

RA-LT MR

Aβ eld/SA1

C-LTMR

CGRP-α

CGRP-

ε

CGRP-

η CGRP-

γ

CGRP

• θ

CGRP-

ζ

Aδ-LTMR Cold thermo.

Runx1

SST

MRGPRD

+

Prop

A rioceptors

β RA-

LTMR

Aβ eld/SA1

C-LTMR

CGRP-α

CGRP

• ε

CGRP-

η CGRP-

γ

CGRP-

θ

CGRP-

β

Aδ-LTMR Cold thermo.

Runx3

Ntrk1

MRGPRD

+^

C-LTMR

Pvalb

Prop

riocept

ors

C-LTMR

Aδ-LT MR

Th Mrgprd

MRGPRD

+

Ret

MRGPRD

+ C-LTMR

0

2

ln(TPT+1)

Bcl11bBcl11a

Bhlha9Bex1

Btbd11Casz1

Pou6f2Plagl1

Pou4f2Pou4f3

Runx1Runx3

Shox2To x

Zfp521To x3

Onecut2

Chd5Etv1

Foxp2Hopx

Hoxd1Klhl5

CGRP-

η

CGRP-

α

CGRP-

θ

C-L

TMR

Aβ

eld/SA1

CGRP-

γ

CGRP-Proprioceptors

ζ

CGRP-

ε

Aδ

-LTMR

Aβ MRGPRDCold thermo.

RA-L

TMR

SST

Individual cells

TFs

restricted TFsSubtype-

OtherTFs

0

2

ln(TPT+1)

E11.5 USN

Fig. 2 | Transcriptional development of subtypes of DRG neurons. a, Force-
directed layout of DRG sensory neurons overlaid with time point or cell-type
information. b, Force-directed layout of DRG development overlaid with

expression of indicated genes. c, Heat map of subtype-restricted TFs in each

somatosensory neuron subtype of adult ganglia. For n values, see Methods.