Nature - USA (2020-01-16)

396 | Nature | Vol 577 | 16 January 2020

Article

before small-diameter neurons, consistent with the historical view^32 ,^33
(Extended Data Fig. 6a, b). Together, these results indicate that cells in
the transcriptionally unspecialized compartment express a broad array
of TFs that become restricted to select subsets of sensory neurons as
development proceeds.

Specification of subtype identity

We next investigated whether broad-to-restricted TFs contribute to
sensory neuron diversification during the transcriptionally unspe-
cialized state, thus broadly influencing the transcriptional matura-
tion of sensory neurons, or whether these TFs primarily influence the
subtypes in which their expression is maintained. We collected DRG
from neonatal (P0–5) pups containing null alleles of either Pou4f2 or
Pou4f3, which are representative broad-to-restricted TFs, and gen-
erated scRNA-seq transcriptomes from Pou4f2KO(Cre)/KO(Cre) mice and
Pou4f2+/+ littermate controls as well Pou4f3−/− mice and Pou4f3+/+ lit-
termate controls. Initial inspection of the scRNA-seq data from both
Pou4f2 and Pou4f3 mutant mice revealed clusters corresponding to
each somatosensory subtype (Fig. 4a, b). We found that cell numbers
were not compromised, as representative ganglia (T7 and T8) from
Pou4f2 or Pou4f3 knockouts had similar numbers of neurons to ganglia
from littermate controls (Extended Data Fig. 7a, b). Notably, genes that
were preferentially expressed in either Pou4f2+ or Pou4f3+ neurons
showed reduced expression in the respective knockouts, compared
to littermate controls, whereas the expression of randomly selected
genes was unchanged (Fig. 4c, d). By contrast, somatosensory neu-
ron subtypes in which expression of Pou4f2 and Pou4f3 was normally
extinguished after E11.5 generally exhibited less marked alterations to
subtype-specific gene expression or subtype-restricted TF expression
in knockout mice when compared to controls (Fig. 4c, d, Extended Data
Fig. 7a, b). Given the reduction in subtype-specific gene expression in
Pou4f2 and Pou4f3 mutants, we also determined the consequences of
Pou4f2 or Pou4f3 ablation on the unique axonal endings associated with
mature somatosensory neuron subtypes. Although the axonal endings

associated with Pou4f2+ subtypes are known to form longitudinal lan-

ceolate endings around hair follicles^1 , the axonal ending morphologies

associated with the Pou4f3+ subtypes were not known. Genetic label-

ling experiments using newly generated Cre lines for each Pou4f3+

subtype revealed that the axons of CGRP-α neurons have free nerve

endings that penetrate the epidermis, whereas those of CGRP-η neurons

form circumferential endings associated with hair follicles (Extended

Data Fig. 8a–d). Longitudinal lanceolate endings and CGRP+ circum-

ferential axonal endings were partially compromised in Pou4f2 and

Pou4f3 knockout mice, respectively (Extended Data Fig. 8e–k). Further-

more, postnatal depletion of Pou4f3 using short hairpin RNA (shRNA)

altered subtype-specific gene expression and function. Together, these

results show that two representative subtype-restricted TFs, Pou4f2

and Pou4f3, control transcriptional maturation of the sensory neuron

subtypes in which they remain expressed.

Extrinsic control of subtype identity

We next investigated whether differential maintenance or extinction of

TFs in emerging subtypes of neurons occurs via a process that is entirely

intrinsic to developing sensory neurons or is guided by extrinsic cues.

The mesenchymal and epidermal environments through which embry-

onic somatosensory axons extend are rich sources of extrinsic signals,

including neuronal growth factors^8. Therefore, we tested whether nerve

growth factor (NGF), an extrinsic cue that is crucial for the growth

and survival of embryonic somatosensory neurons expressing the

NGF receptor NTRK1 (TRKA)^34 —which represent about 80% of adult

DRG—may control the TF selection process. We performed scRNA-seq

on DRG neurons from neonatal mice containing a targeted mutation

in the Ngf gene. This genome-wide analysis of Ngf-dependent gene

expression was carried out on the apoptosis-deficient Bax-knockout

genetic background to circumvent the apoptotic cell death of DRG

neurons associated with developmental loss of NGF^35. Clustering

analysis of the scRNA-seq data revealed that all somatosensory neu-

ron subtypes were present in Bax−/− controls (Fig. 5a), but there were

Pou4f2WT/WT Pou4f2KO/KO

Min

Max

Relative WT

Pou4f2

expression level

CGRP-αSST

MRGPRD+

Cold

thermo.

CGRP-θ

C-LTMR

CGRP-CGRP-γ ε

Aδ-LTMR

ProprioceptorsAβ RA-LTMR

Aβ eld/SA1

CGRP-ζ

CGRP-η

SST

MRGPRD+

Cold

thermo.

CGRP-α

CGRP-ε

CGRP-γ

CGRP-CGRP-ηζ

Aβ eld/SA1

Aβ RA-LTMR

Aδ-LTMR

C-LTMR

CGRP-θ

Proprioceptors

−4

0

4

Pou4f3HighPou4f3Low

CGRP-

η

CGRP-

α

CGRP-

θ

C-L

TMR

Decr

ease

Incr

ease

Randomized genes

Aβ

eld/SA1

CGRP-

γ

ProprioceptorsCGRP-

ζ

CGRP-

ε

Aδ

-LTMR

MRGPRDCold thermo.Aβ

RA-L

TMR

−4 SST

0

4

Pou4f3HighPou4f3Low

CGRP-

η

CGRP-

α

CGRP-

θ

C-L

TMR

Decr

ease

Incr

ease

Cell-type-specic genes

**

Aβ

eld/SA1

CGRP-

γ

ProprioceptorsCGRP-

ζ

CGRP-

ε

Aδ

-LTMR

MRGPRDCold thermo.Aβ

RA-L

TMR

SST

−4

0

4

log

( 2

)

Pou4f2-WT

TPT

Pou4f2HighPou4f2Low

Aδ

-LTMR

C-L

TMR

CGRP-

ε

SST

Decr

ease

Incr

ease

Cell-type-specic genes

**

Aβ

RA-L

TMR

Cold thermo.MRGPRDAβ

eld/SA1

ProprioceptorsCGRP-

γ

CGRP-

η

CGRP-

ζ

CGRP-

α

CGRP-

θ

Pou4f2-KO

TPT *

−4

0

4

Pou4f2HighPou4f2Low

Aδ

-LTMR

C-L

TMR

CGRP-

ε

SST

Decr

ease

Incr

ease

Randomized genes

Aβ

RA-L

TMR

Cold thermo.MRGPRDAβ

eld/SA1

ProprioceptorsCGRP-

γ

CGRP-

η

CGRP-

ζ

CGRP-

α

CGRP-

θ

bd

a Pou4f3WT/WT Pou4f3KO/KO c

SST

MRGPRD+

Proprioceptors

Aβ eld/SA1

Cold

thermo.

C-LTMR CGRP-θ

CGRP-ζ

CGRP-ε

CGRP-γ Aδ-LTMR

Aβ RA-LTMR

CGRP-α

CGRP-η

MRGPRD+

Proprioceptors

Cold

therm.

CGRP-θ

C-LTMR

CGRP-ε

CGRP-γ

Aδ-LTMR

Aβ RA-LTMR

SSTCGRP-ηCGRP-α

Aβ eld/SA1CGRP-ζ

log

( 2

)

Pou4f3-WT

TPT

Pou4f3-KO

TPT

Min

Max

Relative WT

Pou4f3

expression level

Fig. 4 | Pou4f 2 and Pou4f3 regulate select somatosensory neuron subtype
maturation. a, t-SNE visualizations of scRNA-seq data for neurons generated
from Pou4f3WT/WT and Pou4f3KO/KO littermates. b, t-SNE visualizations of scRNA-
seq data for neurons generated from Pou4f2WT/WT and Pou4f2KO/KO littermates.
c, Fold-change distribution of cell-type-specific genes when comparing
Pou4f3KO/KO and Pou4f3WT/WT littermate control samples. d, Fold-change

distribution of cell-type-specific genes when comparing Pou4f2KO/KO and

Pou4f2WT/WT littermates. c, d, Boxes represent IQR, whiskers represent

minimum and maximum values, and notches represent the 95% confidence

interval of the median. *P < 0.01, two-sided Wilcoxon rank-sum test with

Bonferroni correction. For n values, see Methods.