Nature - USA (2020-01-16)

Nature | Vol 577 | 16 January 2020 | 395

neurons (Extended Data Fig. 5a), like Runx1 and Runx3. These scRNA-seq
findings were verified using double single-molecule RNA fluorescent
in situ hybridization (smRNA-FISH), with Runx1 and Runx3 as well as
Pou4f2 and Pou4f3 serving as test cases. smRNA-FISH measurements
showed that Runx1 and Runx3 as well as Pou4f2 (Aβ rapidly adapting
(RA)-low-threshold mechanoreceptors (LTMRs), Aδ-LTMRs, C-LTMRs)
and Pou4f3 (calcitonin gene-related peptide (CGRP)-αs and CGRP-ηs)
are co-expressed in the majority of E11.5 Avil+ sensory neurons, despite
their mutually exclusive expression patterns in neurons from P0 and
adult ganglia (Extended Data Fig. 5b, c). These observations suggest
that all somatosensory neuron subtypes transit through a postmi-
totic transcriptionally unspecialized state. To further address this, we
genetically labelled descendants in the Avil+ cell compartment at E11.5
by administering a low dose of tamoxifen (0.5 mg) at E11.5 to AvilCreERT2;

Rosa26LSL-tdTomato mice and found tdTomato transcripts to be present in

5–19% of cells in each somatosensory neuron subtype of adult ganglia

by scRNA-seq (Fig. 3a–c). In addition, we labelled all descendants of

one of the ‘broad early’-to-‘subtype restricted late’ TFs, Pou4f2, with

tdTomato using a Pou4f2Cre; Rosa26LSL-tdTomato mouse line, and trans-

duced terminally differentiated Pou4f2+ DRG subtypes in the same

mouse using an adeno-associated virus (AAV) carrying a Cre-dependent

GFP reporter delivered at P14 (Fig. 3d). smRNA-FISH analysis revealed

tdTomato transcripts in more than 90% of DRG sensory neurons in

adult Pou4f2Cre; Rosa26LSL-tdTomato; AAV-CAG:FLEX-GFPP14 I.V mice; by con-

trast, GFP transcripts were restricted to Aβ RA-LTMRs, Aδ-LTMRs and

C-LTMRs (mature Pou4f2+ populations) (Fig. 3e). Notably, a develop-

mental analysis of subtype-specific gene expression revealed that, in

general, large-diameter neurons achieved transcriptional maturity

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N
euron
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SNP
Advillin
tdTGFPomato GFP tdTomato
Calb1 GFP(Aβ RA-LT MR) GFP Calb1 (Aβ RA-LT MR)
Colq GFP(Aδ-LTMR) GFP Colq (Aδ-LTMR)
Th (C-LTMR)
GFP
GFP Th (C-LTMR)
Pou4f2GFP GFP Pou4f2
Aβ RA-LT MR
Aδ-LTMR
C-LTMR
23%
21%
56%
0%Other
0
100
Neu
rons (%)
tdTomato

• GFP


• 
  

  Sensory neurons
  transduced
  at P14
  Descendants of
  sensory neuruncommitted ons
  Pou4f2Cre/WT;
  Rosa26LSL-tdTo mato/WT;
  AAV-CAG:FLEX-GFPP14 I.V
  Pou4f2Cre/WT;
  Rosa26LSL-tdTo mato/WT;
  AAV-CAG:FLEX-GFPP14 I.V
  Pou4f2

  
  


• 
  

  Subtype
  GFP+ neurons only
  92% 99%
  29%
  SST
  MRGPR
  D+^
  Propri
  oceptors
  Aβ RA-L
  TMR
  Aβ eld/SA1
  CGRP-CGRP-αC-LTMR
  ε
  CGRP-
  η
  CGRP-
  γ
  CG
  RP-
  θ
  CGRP-
  ζ
  Cold thr.
  Aδ-LTMR
  Unspecialized
  sensory neuron
  Pou4f2Cre
  Rosa26LSL-tdTo mato AAV-CAG:FLEX-GFP(P14 I.V. )
  Pou4f2-positive
  Pou4f2-negative
  50
  ad
  eAdult DRG (T7/8)
  35 μm
  (E11.5 0.5 mg TAM)
  AvilCreERT2;
  Rosa26LSL-tdTomato
  scRNA-seq
  Unspecialized
  sensory neuron
  (P5)
  SST
  MRGPRD

  
  


• 
  

  Propr
  ioceptor
  Aβ RA-LTM s
  R
  Aβ eld/SA1
  CGRP-C-LTMR
  CGRP-α
  ε
  CGRP-
  η
  CGRP-
  γ
  CGRP-
  θ
  CGRP-
  ζ
  Cold ther.
  Aδ-LTMR
  Unspecialized
  sensory neuron
  tdTomato signal
  (^)
  ClivA
  eEr
  RT^2 ;R
  osa^26
  LSL-tdTomato
  CN
  P
  (^)
  (Cel
  l state)
  (^)
  (^)
  (G
  enetic
  strateg
  y)
  Inferr
  ed trajectory E11.5 DRG
  Pou4f2 Pou4f3
  Unspecialized
  sensory neuron
  Unspecialized
  sensory neuron
  0
  1
  ln(TPT+1)
  0
  1
  ln(TPT+1)
  SST
  MRGPRD+
  Proprioceptors
  Aβ RA-LTMR
  Aβ eld/SA1
  C-LTMR
  CGRP-α
  CGRP-ε
  CGRP-η
  CGRP-γ
  CGRP-θ
  CGRP-ζ
  Cold thermoceptors
  Non-neuronal
  Aδ-LTMR
  P5 DRG (AvilCreERT2;Rosa26LSL-tdTo mato)
  tdTomato
  0%
  (Non-neuronal)
  0
  1
  ln(TPT+1)
  Per cent of cells with >1
  tdTomato tag
  (Subtype)
  b
  c
  7%
  (C-LTMR)
  19%
  (SST)
  6%
  (Cold thermo.)
  0%
  (Non-neuronal)
  19%
  (ε)
  5%
  (α)
  17%
  (MRGPRD)
  0%
  (Non-neuronal)(Proprioceptors)10%
  10%
  (Aβ RA-LTMR)
  16%
  (Aδ-LTMR)
  6%
  (η)
  11%
  (Aβ eld/SA1)
  15%
  (θ) 5%(γ)
  4%
  (ζ)
  Unspecializedneuron
  Terminal
  identity lab
  el
  Fig. 3 | The unspecialized sensory neuron compartment gives rise to most or
  all somatosensory neuron subtypes. a, Developmental trajectory of sensory
  neurons (left) and t-SNE visualization with TF expression overlaid (right).
  b, Schematic description of the AvilCreERT2Rosa26LSL-tdTomato labelling strategy.
  c, t-SNE visualization of AvilCreERT2Rosa26LSL-tdTomato scRNA-seq with cell-type
  identity or tdTomato expression overlaid. d, Schematic representing strategy
  for labelling neurons in Pou4f2Cre/WT mice. e, smRNA-FISH and quantification for
  the indicated transcripts (mean ± s.e.m.). For n values, see Methods.