Touch receptor end-organ innervation and function requires sensory neuron expression of the transcription factor Meis2

Reviewer #1 (Public Review):

Summary:
This work examined transcription factor Meis2 in the development of mouse and chick DRG neurons, using a combination of techniques, such as the generation of a new conditional mutant strain of Meis2, behavioral assays, in situ hybridization, transcriptomic study, immunohistochemistry, and electrophysiological (ex vivo skin-nerve preparation) recordings. The authors found that Meis2 was selectively expressed in A fiber LTMRs and that its disruption affects the A-LTMRs' end-organ innervation, transcriptome, electrophysiological properties, and light touch-sensation.

Strengths:

1. The authors utilized a well-designed mouse genetics strategy to generate a mouse model where the Meis2 is selectively ablated from pre- and post-mitotic mouse DRG neurons. They used a combination of readouts, such as in situ hybridization, immunhistochemistry, transcriptomic analysis, skin-nerve preparation, electrophysiological recordings, and behavioral assays to determine the role of Meis2 in mouse DRG afferents.

2. They observed a similar preferential expression of Meis2 in large-diameter DRG neurons during development in chicken, suggesting evolutionarily conserved functions of this transcription factor.

3. Conducted severe behavioral assays to probe the reduction of light-touch sensitivity in mouse glabrous and hairy skin. Their behavioral findings support the idea that the function of Meis2 is essential for the development and/or maturation of LTMRs.

4. RNAseq data provide potential molecular pathways through which Meis2 regulates embryonic target-field innervation.

5. Well-performed electrophysiological study using skin-nerve preparation and recordings from saphenous and tibial nerves to investigate physiological deficits of Meis2 mutant sensory afferents.

6. Nice whole-mount IHC of the hair skin, convincingly showing morphological deficits of Meis2 mutant SA- and RA- LTMRs.

Overall, this manuscript is well-written. The experimental design and data quality are good, and the conclusion from the experimental results is logical.

Weaknesses:

1. Although the authors justify this study for the involvement of Meis2 in Autism and Autism associated disorders, no experiments really investigated Autism-like specific behavior in the Meis2 ablated mice.

2. For mechanical force sensing-related behavioral assays, the authors performed VFH and dynamic cotton swabs for the glabrous skin, and sticky tape on the back (hairy skin) for the hairy skin. A few additional experiments involving glabrous skin plantar surfaces, such as stick tape or flow texture discrimination, would make the conclusion stronger.

3. The authors considered von Frey filaments (1 and 1.4 g) as noxious mechanical stimuli (Figure 1E and statement on lines 181-183), which is questionable. Alligator clips or pinpricks are more certain to activate mechanical nociceptors.

4. There are disconnections and inconsistencies among findings from morphological characterization, physiological recordings, and behavior assays. For example, Meis2 mutant SA-LTMRs show a deficiency in Merkel cell innervation in the glabrous skin but not in hairy skin. With no clear justification, the authors pooled recordings of SA-LTMRs from both glabrous and hairy skin and found a significant increase in mean vibration threshold. Will the results be significantly different if the data are analyzed separately? In addition, whole-mount IHC of Meissner's corpuscles showed morphological changes, but electrophysiological recordings didn't find significant alternation of RAI LTMRs. What does the morphological change mean then? Since the authors found that Meis2 mice are less sensitive to a dynamic cotton swab, which is usually considered as an RA-LTMR mediated behavior, is the SAI-LTMR deficit here responsible for this behavior? Connections among results from different methods are not clear, and the inconsistency should be discussed.

Reviewer #2 (Public Review):

Summary:
Desiderio and colleagues investigated the role of the TALE (three amino acid loop extension) homeodomain transcription factor Meis2 during maturation and target innervation of mechanoreceptors and their sensation to touch. They start with a series of careful in situ hybridizations to examine Meis2 transcript expression in mouse and chick DRGs of different embryonic stages. By this approach, they identify Meis2+ neurons as slowly- and rapidly adapting A-beta LTMRs, respectively. Retrograde tracing experiments in newborn mice confirmed that Meis2-expressing sensory neurons project to the skin, while unilateral limb bud ablations in chick embryos in Ovo showed that these neurons require target-derived signals for survival. The authors further generated a conditional knock-out (cKO) mouse model in which Meis2 is selectively lost in Islet1-expressing, postmitotic neurons in the DRG (IsletCre/+::Meis2flox/flox, abbreviated below as cKO). WT and Islet1Cre/+ littermates served as controls. cKO mice did not exhibit any obvious alteration in volume or cellular composition of the DRGs but showed significantly reduced sensitivity to touch stimuli and various innervation defects to different end-organ targets. RNA-sequencing experiments of E18.5 DRGs taken from WT, Islet1Cre/+, and cKO mice reveal extensive gene expression differences between cKO cells and the two controls, including synaptic proteins and components of the GABAergic signaling system. Gene expression also differed considerably between WT and heterozygous Islet1Cre/+ mice while several of the other parameters tested did not. These findings suggest that Islet1 heterozygosity affects gene expression in sensory neurons but not sensory neuron functionality. However, only some of the parameters tested were assessed for all three genotypes. Histological analysis and electrophysiological recordings shed light on the physiological defects resulting from the loss of Meis2. By immunohistochemical approaches, the authors describe distinct innervation defects in glabrous and hairy skin (reduced innervation of Merkel cells by SA1-LTMRs in glabrous but not hairy skin, reduced complexity of A-beta RA1-LTMs innervating Meissner's corpuscles in glabrous skin, reduced branching and innervation of A-betA RA1-LTMRs in hairy skin). Electrophysiological recordings from ex vivo skin nerve preparations found that several, but not all of these histological defects are matched by altered responses to external stimuli, indicating that compensation may play a considerable role in this system.

Strengths:
This is a well-conducted study that combines different experimental approaches to convincingly show that the transcription factor Meis2 plays an important role in the perception of light touch. The authors describe a new mouse model for compromised touch sensation and identify a number of genes whose expression depends on Meis2 in mouse DRGs. Given that dysbalanced MEIS2 expression in humans has been linked to autism and that autism seems to involve an inappropriate response to light touch, the present study makes a novel and important link between this gene and ASD.

Weaknesses:
The authors make use of different experimental approaches to investigate the role of Meis2 in touch sensation, but the results obtained by these techniques could be connected better. For instance, the authors identify several genes involved in synapse formation, synaptic transmission, neuronal projections, or axon and dendrite maturation that are up- or downregulated upon targeted Meis2 deletion, but it is unresolved whether these chances can in any way explain the histological, electrophysiological, or behavioral deficits observed in cKO animals. The use of two different controls (WT and Islet1Cre/+) is unsatisfactory and it is not clear why some parameters were studied in all three genotypes (WT, Islet1Cre/+ and cKO) and others only in WT and cKO. In addition, Meis2 mutant mice apparently are less responsive to touch, whereas in humans, mutation or genomic deletion involving the MEIS2 gene locus is associated with ASD, a condition that, if anything, is associated with an elevated sensitivity to touch. It would be interesting to know how the authors reconcile these two findings. A minor weakness, the first manuscript suffers from some ambiguities and errors, but these can be easily corrected.