Striking parallels between carotid body glomus cell and adrenal chromaffin cell development.

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are 'émigrés' from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the 'émigré' hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.

Positional differences of axon growth rates between sensory neurons encoded by Runx3.

The formation of functional connectivity in the nervous system is governed by axon guidance that instructs nerve growth and branching during development, implying a similarity between neuronal subtypes in terms of nerve extension. We demonstrate the molecular mechanism of another layer of complexity in vertebrates by defining a transcriptional program underlying growth differences between positionally different neurons. The rate of axon extension of the early subset of embryonic dorsal root ganglion sensory neurons is encoded in neurons at different axial levels. This code is determined by a segmental pattern of axial levels of Runx family transcription factor Runx3. Runx3 in turn determines transcription levels of genes encoding cytoskeletal proteins involved in axon extension, including Rock1 and Rock2 which have ongoing activities determining axon growth in early sensory neurons and blocking Rock activity reverses axon extension deficits of Runx3(-/-) neurons. Thus, Runx3 acts to regulate positional differences in axon extension properties apparently without affecting nerve guidance and branching, a principle that could be relevant to other parts of the nervous system.

A local source of FGF initiates development of the unmyelinated lineage of sensory neurons.

The principle by which unmyelinated primary sensory neurons transducing thermal, itch and pain perception are specified in early development is unknown. These classes of sensory neurons diversify from a common population of late-born neurons, which initiate expression of Runt homology domain transcription factor RUNX1 and the nerve growth factor receptor TrkA. Here, we report that signals emanating from within the mouse dorsal root ganglion mediated partly by early-born neurons destined to a myelinated phenotype participate in fating late-born RUNX1(+)/TrkA(+) neurons. Inductive factors include FGFs via activation of FGF receptor 1 (FGFR1). Consistently, FGF2 is sufficient to induce expression of RUNX1, and Fgfr1 conditional mutant mice display deficits in fating of the common population of late-born RUNX1(+)/TrkA(+) neurons that develop into unmyelinated neurons. Thus, the distinct lineages of sensory neurons are acquired in response to increasing FGF levels provided by a rising number of born neurons.

Adult spinal Dmrt3 neurons receive direct somatosensory inputs from ipsi- and contralateral primary afferents and from brainstem motor nuclei.

In the spinal cord, sensory-motor circuits controlling motor activity are situated in the dorso-ventral interface. The neurons identified by the expression of the transcription factor Doublesex and mab-3 related transcription factor 3 (Dmrt3) have previously been associated with the coordination of locomotion in horses (Equus caballus, Linnaeus, 1758), mice (Mus musculus, Linnaeus, 1758), and zebrafish (Danio rerio, F. Hamilton, 1822). Based on earlier studies, we hypothesized that, in mice, these neurons may be positioned to receive sensory and central inputs to relay processed commands to motor neurons. Thus, we investigated the presynaptic inputs to spinal Dmrt3 neurons using monosynaptic retrograde replication-deficient rabies tracing. The analysis showed that lumbar Dmrt3 neurons receive inputs from intrasegmental neurons, and intersegmental neurons from the cervical, thoracic, and sacral segments. Some of these neurons belong to the excitatory V2a interneurons and to plausible Renshaw cells, defined by the expression of Chx10 and calbindin, respectively. We also found that proprioceptive primary sensory neurons of type Ia2, Ia3, and Ib, defined by the expression of calbindin, calretinin, and Brn3c, respectively, provide presynaptic inputs to spinal Dmrt3 neurons. In addition, we demonstrated that Dmrt3 neurons receive inputs from brain areas involved in motor regulation, including the red nucleus, primary sensory-motor cortex, and pontine nuclei. In conclusion, adult spinal Dmrt3 neurons receive inputs from motor-related brain areas as well as proprioceptive primary sensory neurons and have been shown to connect directly to motor neurons. Dmrt3 neurons are thus positioned to provide sensory-motor control and their connectivity is suggestive of the classical reflex pathways present in the spinal cord.

The transcription factor Cux2 marks development of an A-delta sublineage of TrkA sensory neurons.

The developmental process and unique molecular identity between the many different types of dorsal root ganglion (DRG) sensory neurons generated during embryogenesis provide the cellular basis for the distinct perceptual modalities of somatosensation. The mechanisms leading to the generation of different types of nociceptive sensory neurons remain only partly understood. Here, we show that the transcription factor Cux2 is a novel marker of sensory neuron subpopulations of three main sublineages as defined by the expression of neurotrophic factor receptors TrkA, TrkB and TrkC. In particular, it is expressed in a subpopulation of early TrkA(+) neurons that arise during the early, Ngn1-independent initiated neurogenesis in the DRG. Postnatally, Cux2 marks a specific subtype of A-delta nociceptors as seen by expression of TrkA and NF200 but absence of TrpV1. Analysis of Cux2 mutant mice shows that Cux2 is not required for specification of Trk(+) neuronal subpopulations. However, Cux2 mutant mice are hypersensitive to mechanical, but not to heat or cold stimuli, consistent with a requirement in the process of specification of the mechanoreceptive neuron circuit. Hence, our results show that Cux2 is expressed and may participate in development of a specific subtype of myelinated TrkA(+) nociceptors.

Essential role of Ret for defining non-peptidergic nociceptor phenotypes and functions in the adult mouse.

Transduction of pain following noxious stimuli is mediated by the activation of specialized ion channels and receptors expressed by nociceptive sensory neurons. A common early nociceptive sublineage expressing the nerve growth factor receptor TrkA diversifies into peptidergic and non-peptidergic nociceptors around birth. In this process, peptidergic neurons maintain TrkA expression, while non-peptidergic neurons downregulate TrkA and upregulate the common glial-derived neurotrophic factor family ligand receptor Ret and bind the isolectin B4 (IB4). Although Ret can have profound impacts on the molecular and physiological properties of nociceptive neurons, its role is not fully understood. Here we have deleted Ret in small- and medium-size sensory neurons, bypassing the early lethality of the full Ret knockout. We identify that Ret is expressed in two distinct populations of small-medium sized non-peptidergic neurons, an IB4(+) and an IB4(-) population. In these neurons, Ret is a critical regulator of several ion channels and receptors, including Nav1.8, Nav1.9, ASIC2a, P2X3, TrpC3, TrpM8, TrpA1, delta opioid receptor, MrgD, MrgA1 and MrgB4. Ret-deficient mice fail to respond to mustard oil-induced neurogenic inflammation, have elevated basal responses and a failure to terminate injury-induced sensitization to cold stimuli, hypersensitivity to basal but not injury-induced mechanical stimuli, while heat sensation is largely intact. We propose that elevated pain responses could be contributed by GPR35, which is dysregulated in adult Ret-deficient mice. Our results show that Ret is critical for expression of several molecular substrates participating in the detection and transduction of sensory stimuli, resulting in altered physiology following Ret deficiency.

Spinal cord interneurons expressing the gastrin-releasing peptide receptor convey itch through VGLUT2-mediated signaling.

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox;Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch.

Dynamic expression of the TRPM subgroup of ion channels in developing mouse sensory neurons.

Despite the significance of transient receptor potential (TRP) channels in sensory physiology, little is known of the expression and developmental regulation of the TRPM (melastatin) subgroup in sensory neurons. In order to find out if the eight TRPM subgroup members (TRPM1-TRPM8) have a possible role in the sensory nervous system, we characterized the developmental regulation of their expression in mouse dorsal root ganglion (DRG) from embryonic (E) day 12 to adulthood. Transcripts for all channels except for TRPM1 were detected in lumbar and thoracic DRG and in nodose ganglion (NG) with distinguishable expression patterns from E12 until adult. For most channels, the expression increased from E14 to adult with the exception of TRPM5, which displayed transient high levels during embryonic and early postnatal stages. Cellular localization of TRPM8 mRNA was found only in a limited subset of very small diameter neurons distinct in size from other populations. These neurons did not bind isolectin B4 (IB4) and expressed neither the neuropeptide calcitonin gene-related peptide (CGRP) nor neurofilament (NF)200. This suggests that TRPM8(+) thermoreceptive sensory neurons fall into a separate group of very small sized neurons distinct from peptidergic and IB4(+) subtypes of sensory neurons. Our results, showing the expression and dynamic regulation of TRPM channels during development, indicate that many TRPM subfamily members could participate during nervous system development and in the adult by determining distinct physiological properties of sensory neurons.