Kim JE Fei L, Yin WC, Coquenlorge S, Rao-Bhatia A, Zhang X, Shi SSW, Lee JH, Hahn NA, Rizvi W, Kim KH, Sung HK, Hui CC, Guo G, Kim TH
PMID: 31953387 | DOI: 10.1038/s41467-019-14058-5
Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.
Nanotube-like processes facilitate material transfer between photoreceptors
Kalargyrou, AA;Basche, M;Hare, A;West, EL;Smith, AJ;Ali, RR;Pearson, RA;
PMID: 34494703 | DOI: 10.15252/embr.202153732
Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo.
Hennessy ML, Corcoran A, Brust RD, Nattie EE, Dymecki S.
PMID: 28073937 | DOI: 10.1523/JNEUROSCI.2316-16.2016
Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the 5-HT system, which have borne out in functional studies, including the modulation of distinct facets of homeostatic control. These functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to co-express other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the transcription factor gene Pet1, thus referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic (CNO-hM4Di) perturbation of Tac1-Pet1 neuron activity resulted in blunting of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2 Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor nuclei. These findings demonstrate that the activity of a Pet1 neuron subtype with potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, likely via motor outputs that maintain airway patency and engage muscles of respiration. These Tac1-Pet1 neurons may complement the activity of Egr2-Pet1 neurons, previously established in respiratory chemoreception, but which do not innervate respiratory motor nuclei.
SIGNIFICANCE STATEMENT:
5-HT neurons modulate outputs as diverse as body temperature, respiration, aggression, and mood. We characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons) which projects to respiratory motor nuclei, and when silenced, blunts the ventilatory response to inhaled carbon dioxide. We employ genetic tools to access this subset of 5-HT neurons to query function, anatomy, and connectivity. Localization of synaptic boutons from Tac1-Pet1 neurons, primarily within motor regions, contrasts with those from previously described Egr2-Pet1 neurons, which are chemosensitive and reside in the raphe magnus and project primarily to chemosensory integration, but not motor, regions of the brainstem.
Wang C, Reyes de Mochel NS, Christenson SA, Cassandras M, Moon R, Brumwell AN, Byrnes LE, Li A, Yokosaki Y, Shan P, Sneddon JB, Jablons D, Lee PJ, Matthay MA, Chapman HA, Peng T.
PMID: 29999500 | DOI: 10.1172/JCI99435
Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.
Yao, Y;Barger, Z;Saffari Doost, M;Tso, CF;Darmohray, D;Silverman, D;Liu, D;Ma, C;Cetin, A;Yao, S;Zeng, H;Dan, Y;
PMID: 36170850 | DOI: 10.1016/j.neuron.2022.08.027
Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.
Kaucka, M;Joven Araus, A;Tesarova, M;Currie, JD;Boström, J;Kavkova, M;Petersen, J;Yao, Z;Bouchnita, A;Hellander, A;Zikmund, T;Elewa, A;Newton, PT;Fei, JF;Chagin, AS;Fried, K;Tanaka, EM;Kaiser, J;Simon, A;Adameyko, I;
PMID: 36376278 | DOI: 10.1038/s41467-022-34266-w
There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.
Molecular Therapy - Nucleic Acids
Kaburagi, H;Nagata, T;Enomoto, M;Hirai, T;Ohyagi, M;Ihara, K;Yoshida-Tanaka, K;Ebihara, S;Asada, K;Yokoyama, H;Okawa, A;Yokota, T;
| DOI: 10.1016/j.omtn.2022.05.006
Neuropathic pain, a heterogeneous condition, affects 7%-10% of the general population. To date, efficacious and safe therapeutic approaches remain limited. Antisense oligonucleotide (ASO) therapy has opened the door to treat spinal muscular atrophy, with many ongoing clinical studies determining its therapeutic utility. ASO therapy for neuropathic pain and peripheral nerve disease requires efficient gene delivery and knockdown in both the dorsal root ganglion (DRG) and sciatic nerve, key tissues for pain signaling. We previously developed a new DNA/RNA heteroduplex oligonucleotide (HDO) technology that achieves highly efficient gene knockdown in the liver. Here, we demonstrated that intravenous injection of HDO, comprising an ASO and its complementary RNA conjugated to α-tocopherol, silences endogenous gene expression more than 2-fold in the DRG, and sciatic nerve with higher potency, efficacy, and broader distribution than ASO alone. Of note, we observed drastic target suppression in all sizes of neuronal DRG populations by in situ hybridization. Our findings establish HDO delivery as an investigative and potentially therapeutic platform for neuropathic pain and peripheral nerve disease.
Uehara, K;Koyanagi-Aoi, M;Koide, T;Itoh, T;Aoi, T;
PMID: 35245440 | DOI: 10.1016/j.stemcr.2022.02.002
Human gastric development has not been well studied. The generation of human pluripotent stem cell-derived gastric organoids (hGOs) comprising gastric marker-expressing epithelium without an apparent smooth muscle (SM) structure has been reported. We modified previously reported protocols to generate hGOs with muscularis mucosa (MM) from hiPSCs. Time course analyses revealed that epithelium development occurred prior to MM formation. Sonic hedgehog (SHH) and TGF-β1 were secreted by the epithelium. HH and TGF-β signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-β. TGF-β signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-β signaling in MM formation in recovering gastric ulcers. HH and TGF-β signaling from the epithelium to the stroma and the mechanical properties of the subepithelial environment may influence the emergence of MM in human stomach tissue.
Single-nuclear transcriptomics reveals diversity of proximal tubule cell states in a dynamic response to acute kidney injury
Proceedings of the National Academy of Sciences of the United States of America
Gerhardt, LMS;Liu, J;Koppitch, K;Cippà, PE;McMahon, AP;
PMID: 34183416 | DOI: 10.1073/pnas.2026684118
Acute kidney injury (AKI), commonly caused by ischemia, sepsis, or nephrotoxic insult, is associated with increased mortality and a heightened risk of chronic kidney disease (CKD). AKI results in the dysfunction or death of proximal tubule cells (PTCs), triggering a poorly understood autologous cellular repair program. Defective repair associates with a long-term transition to CKD. We performed a mild-to-moderate ischemia-reperfusion injury (IRI) to model injury responses reflective of kidney injury in a variety of clinical settings, including kidney transplant surgery. Single-nucleus RNA sequencing of genetically labeled injured PTCs at 7-d ("early") and 28-d ("late") time points post-IRI identified specific gene and pathway activity in the injury-repair transition. In particular, we identified Vcam1 +/Ccl2 + PTCs at a late injury stage distinguished by marked activation of NF-κB-, TNF-, and AP-1-signaling pathways. This population of PTCs showed features of a senescence-associated secretory phenotype but did not exhibit G2/M cell cycle arrest, distinct from other reports of maladaptive PTCs following kidney injury. Fate-mapping experiments identified spatially and temporally distinct origins for these cells. At the cortico-medullary boundary (CMB), where injury initiates, the majority of Vcam1 +/Ccl2 + PTCs arose from early replicating PTCs. In contrast, in cortical regions, only a subset of Vcam1 +/Ccl2 + PTCs could be traced to early repairing cells, suggesting late-arising sites of secondary PTC injury. Together, these data indicate even moderate IRI is associated with a lasting injury, which spreads from the CMB to cortical regions. Remaining failed-repair PTCs are likely triggers for chronic disease progression.
Tracing the origin of hair follicle stem cells
Morita, R;Sanzen, N;Sasaki, H;Hayashi, T;Umeda, M;Yoshimura, M;Yamamoto, T;Shibata, T;Abe, T;Kiyonari, H;Furuta, Y;Nikaido, I;Fujiwara, H;
PMID: 34108685 | DOI: 10.1038/s41586-021-03638-5
Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.
Sehgal A, Donaldson DS, Pridans C, Sauter KA, Hume DA, Mabbott NA.
PMID: 29593242 | DOI: 10.1038/s41467-018-03638-6
Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells, but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinallamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium, and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced, whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens.
Abs E, Poorthuis RB, Apelblat D, Muhammad K, Pardi MB, Enke L, Kushinsky D, Pu DL, Eizinger MF, Conzelmann KK, Spiegel I, Letzkus JJ.
PMID: - | DOI: 10.1016/j.neuron.2018.09.001
A wealth of data has elucidated the mechanisms by which sensory inputs are encoded in the neocortex, but how these processes are regulated by the behavioral relevance of sensory information is less understood. Here, we focus on neocortical layer 1 (L1), a key location for processing of such top-down information. Using Neuron-Derived Neurotrophic Factor(NDNF) as a selective marker of L1 interneurons (INs) and in vivo 2-photon calcium imaging, electrophysiology, viral tracing, optogenetics, and associative memory, we find that L1 NDNF-INs mediate a prolonged form of inhibition in distal pyramidal neuron dendrites that correlates with the strength of the memory trace. Conversely, inhibition from Martinotti cells remains unchanged after conditioning but in turn tightly controls sensory responses in NDNF-INs. These results define a genetically addressable form of dendritic inhibition that is highly experience dependent and indicate that in addition to disinhibition, salient stimuli are encoded at elevated levels of distal dendritic inhibition.
