Studies on peripheral nervous system development and function in mice lacking the neurturin receptor GFRα2

The Glial cell line-derived neurotrophic factor (GDNF) family ligands, which include GDNF, neurturin (NRTN), persephin (PSPN) and artemin (ARTN), signal through a glycosyl phosphatidyl inositol (GPI)-linked cognate-receptor (GFRα1-4) and the transmembrane receptor tyrosine kinase receptor RET. The members of the GDNF family play a particularly important role in the development of the peripheral nervous system (PNS). In the autonomic nervous system, GDNF and NRTN regulate important steps in the development of the enteric and parasympathetic nervous systems from migration and proliferation to soma size and target innervation, whereas ARTN takes part in the early phases of sympathetic nervous system development. In the sensory system, GFRα2 ¬ the co-receptor of NRTN has been shown to mediate trophic signaling for nonpeptidergic nociceptive neurons, and is also required for their innervation of the glabrous epidermis. However, several aspects of the role of GFRα2-signaling in normal PNS development and function remain poorly understood. Therefore, the aims of this study were to elucidate (1) the role of GFRα2-signaling in the development of parasympathetic neurons; (2) the role of GFRα2-signaling in two classes of somatosensory mechanoreceptor neurons and their target innervation; and (3) the role of GFRα2-signaling in the cholinergic innervation of the gastric mucosa and the role of this innervation in gastric secretion. We discovered that programmed cell death (PCD) is a normal part of parasympathetic neuron development in mice. GFRα2-signaling was found to regulate parasympathetic neuron survival in pancreatic and submandibular ganglia during late embryonic development; lack of GFRα2-mediated signaling resulted in the loss of intrapancreatic neurons through PCD. In argreement with previous studies, apoptosis in the ENS was found to be rare, and was not increased in the absence of GFRα2, implying that the normal number of enteric neurons is not determined by PCD. In the dorsal root ganglia (DRGs), we found that GFRα2 regulates the cell size, but not the peripheral innervation of hair follicles in both the large early-RET RA Aβ-class low threshold mechanoreceptors (LTMRs) and in the small C-LTMRs. In contrast, GFRα2 was found to regulate both the cell size and the epidermal innervation in the small Mrgprd+ C-nociceptors. We also found evidence that the RA Aβ-LTMRs downregulate GFRα2-expression at some point after birth, suggesting a possible switch in neurotrophic signaling pathways. In the enteric nervous system, we demonstrated that GFRα2-signaling via NRTN is required for cholinergic innervation of the gastric mucosa. Interestingly, this innervation was found to be unnecessary for maintaining the gastric mucosa and for gastrin secretion and basal acid secretion. Even though vagally-stimulated secretion is lost in the GFRα2-KO mice, their ability to secrete acid in response to direct parietal cell stimulation remains in the absence of gastric mucosal innervation.GDNF-perheen hermokasvutekijöihin kuuluvat GDNF, neurturiini (NRTN), persefiini (PSPN) ja artemiini (ARTN). Nämä useista hermosolujen kohdekudoksista erittyvät proteiinit signaloivat GFRα1-4 -apureseptorista ja RET-tyrosiinikinaasireseptorista muodostuvan kompleksin kautta ja osallistuvat muun muassa ääreishermoston kehitykseen ja toimintaan. Autonomisen hermoston kehityksessä GDNF ja NRTN säätelevät tärkeitä vaiheita enteeristen ja parasympaattisten hermosolujen vaeltamisessa ja kohdekudoksen hermotuksessa. Artemiini taas säätelee osaltaan sympaattisen hermoston kehityksen alkuvaiheita. Ääreishermoston sensorisissa solmukkeissa NRTN/GFRα2-signaloinnin on osoitettu välittävän kasvua ja ylläpitoa vieviä viestejä tietylle populaatiolle pieniä kipuhermosoluja, säädellen samalla niiden ihon pinnalle ulottuvan hermotuksen määrää. GFRα2-signaloinnin roolissa ääreishermoston kehityksessä on kuitenkin useita avonaisia kysymyksiä. Tämän väitöskirjan tavoitteena oli selvittää GFRα2-reseptorin merkitystä: (1) parasympaattisten hermosolujen kehityksessä, (2) kahden mekanosensorisen hermosolutyypin kehityksessä ja (3) mahan limakalvon kolinergisen hermotuksen kehityksessä ja tämän hermotuksen osuudessa mahan limakalvon eritystoimintaan. Tutkimuksissa havaitsimme, että parasympaattisten hermosolujen ohjelmoitu solukuolema on osa normaalia yksilönkehitystä. GFRα2-signalointi näytti säätelevän parasympaattisten hermosolujen solukuolemaa, josta todisteena oli solukuoleman huomattava lisääntyminen GFRα2-poistogeenisillä hiirillä haiman ja sylkirauhasten parasympaattisissa hermosoluissa yksilönkehityksen loppupuolella. Kehittyvien enteeristen hermosolujen joukossa vastaavaa solukuolemaa ei ollut havaittavissa. Tuntoaistimuksia välittävissä spinaalisolmukkeissa havaitsimme GFRα2-signaloinnin osallistuvan kahden mekanosensorisen hermosolutyypin solukeskuksen koon säätelyyn. Toisin kuin GFRα2-reseptoria ilmentäville kipuhermosoluille, joiden vapaat hermopäätteet vaativat kehittyäkseen NRTN/GFRα2-signalointia, GFRα2 ei ollut tarpeellinen mekanoreseptorien muodostamien hermopäätteiden kehittymisessä ihon karvatuppien ympärille. Enteerisessä hermostossa osoitimme NRTN/GFRα2-signaloinnin olevan tarpeellinen mahan limakalvon kolinergisen hermotuksen kehittymiselle. Tärkein havaintomme oli, että ehyt hermotus ei ollut tarpeellinen limakalvon rakenteen ylläpidolle, hapon basaaliselle eritykselle eikä gastriinin eritykselle. Vaikka vagushermostimulaation kautta tapahtuva haponeritys puuttui GFRα2-poistogeenisiltä hiiriltä, niiden kyky erittää happoa suoraan parietaalisoluja stimuloitaessa oli normaali

Different Requirements for GFR alpha 2-Signaling in Three Populations of Cutaneous Sensory Neurons

Peer reviewe

Dynamic MAPK/ERK Activity Sustains Nephron Progenitors through Niche Regulation and Primes Precursors for Differentiation

The in vivo niche and basic cellular properties of nephron progenitors are poorly described. Here we studied the cellular organization and function of the MAPK/ERK pathway in nephron progenitors. Live-imaging of ERK activity by a Forster resonance energy transfer biosensor revealed a dynamic activation pattern in progenitors, whereas differentiating precursors exhibited sustained activity. Genetic experiments demonstrate that MAPK/ERK activity controls the thickness, coherence, and integrity of the nephron progenitor niche. Molecularly, MAPK/ERK activity regulates niche organization and communication with extracellular matrix through PAX2 and ITGA8, and is needed for CITED1 expression denoting undifferentiated status. MAPK/ERK activation in nephron precursors propels differentiation by priming cells for distal and proximal fates induced by the Wnt and Notch pathways. Thus, our results demonstrate a mechanism through which MAPK/ERK activity controls both progenitor maintenance and differentiation by regulating a distinct set of targets, which maintain the biomechanical milieu of tissue-residing progenitors and prime precursors for nephrogenesis.Peer reviewe

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney

Background: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.Peer reviewe

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF

Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.Peer reviewe

Dissection of progenitor compartments resolves developmental trajectories in B-lymphopoiesis

To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19(+) progenitor compartment.Peer reviewe

C- and Aβ-LTMR innervation of hair follicles in GFRα2-KO mouse back skin remains unchanged.

<p>(A–C) The images show two small caliber hair follicles with TH⁺ and TrkB⁺ LLEs. Immunoreactivity for the markers is localized in different populations of endings (C). The asterisks in (B) mark the two separate hair shafts. (D, E) The images demonstrate TH⁺ sensory innervation around small caliber hair follicles in wild-type (D) and KO (E) back skin. The arrowheads indicate obliquely cut hair follicles with TH⁺ LLEs. The small arrows mark hair follicles with few or no visible endings. Sebaceous glands (sg) around the follicles are indicated. No changes are obvious in the morphology of TH⁺ LLEs around GFRα2-KO hair follicles. (I, left side) The percentage of small hair follicles that are innervated by TH⁺ LLEs is similar between the genotypes (WT 87.8±6.4%, KO 90.6±1.1%; three animals/genotype, 60–80 small hair follicles/animal, U-test). (F, G) The morphology of NFH⁺ LLEs (arrowheads) appears similar between the genotypes. Double arrowheads point to nerve structures innervating the touch dome (located outside of the image). (I, right side) NFH⁺ LLEs remain in KO back skin (WT 88.7±0.8%, KO 81.6±3.6%; innervated follicles/total follicles, three animals/genotype, 60–80 small hair follicles/animal, U-test). Scale bars: 10 µm.</p

An Atlas of Vagal Sensory Neurons and Their Molecular Specialization

Summary: Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems. : Visceral sensory neurons are necessary for the control of organ functions, but knowledge on the complexity of neuron types involved is missing. Kupari et al. molecularly identify jugular and nodose ganglion neurons and find a large diversity of neuron types that are consistent with the numerous sensory functions of the vagus nerve. Keywords: single cell RNA-sequencing, vagus nerve, sensory neurons, viscerosensory, somatosensory, transcriptome, jugular ganglion, nodose ganglion, mechanoreceptor, nocicepto

Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control

Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney

Background: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.N