Ding, CY;Ding, YT;Ji, H;Wang, YY;Zhang, X;Yin, DM;
PMID: 37147705 | DOI: 10.1186/s13578-023-01032-4
Where the gene is expressed determines the function of the gene. Neuregulin 1 (Nrg1) encodes a tropic factor and is genetically linked with several neuropsychiatry diseases such as schizophrenia, bipolar disorder and depression. Nrg1 has broad functions ranging from regulating neurodevelopment to neurotransmission in the nervous system. However, the expression pattern of Nrg1 at the cellular and circuit levels in rodent brain is not full addressed.Here we used CRISPR/Cas9 techniques to generate a knockin mouse line (Nrg1Cre/+) that expresses a P2A-Cre cassette right before the stop codon of Nrg1 gene. Since Cre recombinase and Nrg1 are expressed in the same types of cells in Nrg1Cre/+ mice, the Nrg1 expression pattern can be revealed through the Cre-reporting mice or adeno-associated virus (AAV) that express fluorescent proteins in a Cre-dependent way. Using unbiased stereology and fluorescence imaging, the cellular expression pattern of Nrg1 and axon projections of Nrg1-positive neurons were investigated.In the olfactory bulb (OB), Nrg1 is expressed in GABAergic interneurons including periglomerular (PG) and granule cells. In the cerebral cortex, Nrg1 is mainly expressed in the pyramidal neurons of superficial layers that mediate intercortical communications. In the striatum, Nrg1 is highly expressed in the Drd1-positive medium spiny neurons (MSNs) in the shell of nucleus accumbens (NAc) that project to substantia nigra pars reticulata (SNr). In the hippocampus, Nrg1 is mainly expressed in granule neurons in the dentate gyrus and pyramidal neurons in the subiculum. The Nrg1-expressing neurons in the subiculum project to retrosplenial granular cortex (RSG) and mammillary nucleus (MM). Nrg1 is highly expressed in the median eminence (ME) of hypothalamus and Purkinje cells in the cerebellum.Nrg1 is broadly expressed in mouse brain, mainly in neurons, but has unique expression patterns in different brain regions.
Wang L, Huang J, Moore DC, Zuo C, Wu Q, Xie L, von der Mark K, Yuan X, Chen D, Warman ML, Ehrlich MG, Yang W.
PMID: 28983104 | DOI: 10.1038/s41598-017-12767-9
Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.
Journal of developmental biology
Vonk, AC;Hasel-Kolossa, SC;Lopez, GA;Hudnall, ML;Gamble, DJ;Lozito, TP;
PMID: 35225965 | DOI: 10.3390/jdb10010012
(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.
Brenna Ø, Furnes MW, Munkvold B, Kidd M, Sandvik AK, Gustafsson BI.
PMID: 27044258 | DOI: -
Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.
Gaitonde, KD;Andrabi, M;Burger, CA;D'Souza, SP;Vemaraju, S;Koritala, BSC;Smith, DF;Lang, RA;
PMID: 37141220 | DOI: 10.1371/journal.pone.0284824
Neurons in the hypothalamic preoptic area (POA) regulate multiple homeostatic processes, including thermoregulation and sleep, by sensing afferent input and modulating sympathetic nervous system output. The POA has an autonomous circadian clock and may also receive circadian signals indirectly from the suprachiasmatic nucleus. We have previously defined a subset of neurons in the POA termed QPLOT neurons that are identified by the expression of molecular markers (Qrfp, Ptger3, LepR, Opn5, Tacr3) that suggest receptivity to multiple stimuli. Because Ptger3, Opn5, and Tacr3 encode G-protein coupled receptors (GPCRs), we hypothesized that elucidating the G-protein signaling in these neurons is essential to understanding the interplay of inputs in the regulation of metabolism. Here, we describe how the stimulatory Gs-alpha subunit (Gnas) in QPLOT neurons regulates metabolism in mice. We analyzed Opn5cre; Gnasfl/fl mice using indirect calorimetry at ambient temperatures of 22°C (a historical standard), 10°C (a cold challenge), and 28°C (thermoneutrality) to assess the ability of QPLOT neurons to regulate metabolism. We observed a marked decrease in nocturnal locomotion of Opn5cre; Gnasfl/fl mice at both 28°C and 22°C, but no overall differences in energy expenditure, respiratory exchange, or food and water consumption. To analyze daily rhythmic patterns of metabolism, we assessed circadian parameters including amplitude, phase, and MESOR. Loss-of-function GNAS in QPLOT neurons resulted in several subtle rhythmic changes in multiple metabolic parameters. We observed that Opn5cre; Gnasfl/fl mice show a higher rhythm-adjusted mean energy expenditure at 22°C and 10°C, and an exaggerated respiratory exchange shift with temperature. At 28°C, Opn5cre; Gnasfl/fl mice have a significant delay in the phase of energy expenditure and respiratory exchange. Rhythmic analysis also showed limited increases in rhythm-adjusted means of food and water intake at 22°C and 28°C. Together, these data advance our understanding of Gαs-signaling in preoptic QPLOT neurons in regulating daily patterns of metabolism.
Orexin receptors 1 and 2 in serotonergic neurons differentially regulate peripheral glucose metabolism in obesity
Xiao, X;Yeghiazaryan, G;Hess, S;Klemm, P;Sieben, A;Kleinridders, A;Morgan, DA;Wunderlich, FT;Rahmouni, K;Kong, D;Scammell, TE;Lowell, BB;Kloppenburg, P;Brüning, JC;Hausen, AC;
PMID: 34475397 | DOI: 10.1038/s41467-021-25380-2
The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.
Endogenous µ-opioid receptor activity in the lateral and capsular subdivisions of the right central nucleus of the amygdala prevents chronic postoperative pain
Journal of neuroscience research
Cooper, AH;Hedden, NS;Corder, G;Lamerand, SR;Donahue, RR;Morales-Medina, JC;Selan, L;Prasoon, P;Taylor, BK;
PMID: 33957003 | DOI: 10.1002/jnr.24846
Tissue injury induces a long-lasting latent sensitization (LS) of spinal nociceptive signaling that is kept in remission by an opposing µ-opioid receptor (MOR) constitutive activity. To test the hypothesis that supraspinal sites become engaged, we induced hindpaw inflammation, waited 3 weeks for mechanical hypersensitivity to resolve, and then injected the opioid receptor inhibitors naltrexone, CTOP or β-funaltrexamine subcutaneously, and/or into the cerebral ventricles. Intracerebroventricular injection of each inhibitor reinstated hypersensitivity and produced somatic signs of withdrawal, indicative of LS and endogenous opioid dependence, respectively. In naïve or sham controls, systemic naloxone (3 mg/kg) produced conditioned place aversion, and systemic naltrexone (3 mg/kg) increased Fos expression in the central nucleus of the amygdala (CeA). In LS animals tested 3 weeks after plantar incision, systemic naltrexone reinstated mechanical hypersensitivity and produced an even greater increase in Fos than in sham controls, particularly in the capsular subdivision of the right CeA. One third of Fos+ profiles co-expressed protein kinase C delta (PKCδ), and 35% of PKCδ neurons co-expressed tdTomato+ in Oprm1Cre ::tdTomato transgenic mice. CeA microinjection of naltrexone (1 µg) reinstated mechanical hypersensitivity only in male mice and did not produce signs of somatic withdrawal. Intra-CeA injection of the MOR-selective inhibitor CTAP (300 ng) reinstated hypersensitivity in both male and female mice. We conclude that MORs in the capsular subdivision of the right CeA prevent the transition from acute to chronic postoperative pain.
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
Rodriguez, LA;Kim, SH;Page, SC;Nguyen, CV;Pattie, EA;Hallock, HL;Valerino, J;Maynard, KR;Jaffe, AE;Martinowich, K;
PMID: 36369482 | DOI: 10.1038/s41386-022-01487-y
The lateral septum (LS) is a basal forebrain GABAergic region that is implicated in social novelty. However, the neural circuits and cell signaling pathways that converge on the LS to mediate social behaviors aren't well understood. Multiple lines of evidence suggest that signaling of brain-derived neurotrophic factor (BDNF) through its receptor TrkB plays important roles in social behavior. BDNF is not locally produced in LS, but we demonstrate that nearly all LS GABAergic neurons express TrkB. Local TrkB knock-down in LS neurons decreased social novelty recognition and reduced recruitment of neural activity in LS neurons in response to social novelty. Since BDNF is not synthesized in LS, we investigated which inputs to LS could serve as potential BDNF sources for controlling social novelty recognition. We demonstrate that selectively ablating inputs to LS from the basolateral amygdala (BLA), but not from ventral CA1 (vCA1), impairs social novelty recognition. Moreover, depleting BDNF selectively in BLA-LS projection neurons phenocopied the decrease in social novelty recognition caused by either local LS TrkB knockdown or ablation of BLA-LS inputs. These data support the hypothesis that BLA-LS projection neurons serve as a critical source of BDNF for activating TrkB signaling in LS neurons to control social novelty recognition.
Morphological and neurochemical characterization of glycinergic neurons in laminae I-IV of the mouse spinal dorsal horn
The Journal of comparative neurology
Miranda, CO;Hegedüs, K;Wildner, H;Zeilhofer, HU;Antal, M;
PMID: 34382691 | DOI: 10.1002/cne.25232
A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.
Gertler, TS;Cherian, S;DeKeyser, JM;Kearney, JA;George, AL;
PMID: 35346832 | DOI: 10.1016/j.nbd.2022.105713
KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.
Chen, HJ;Barske, L;Talbot, JC;Dinwoodie, OM;Roberts, RR;Farmer, DT;Jimenez, C;Merrill, AE;Tucker, AS;Crump, JG;
PMID: 36905926 | DOI: 10.1016/j.devcel.2023.02.011
Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.
Miura Y, Ota S, Peterlin M, McDevitt G, Kanazawa S.
| DOI: 10.1002/jbm4.10132
Specific MHC class II genes result in a high susceptibility to rheumatoid arthritis (RA), with co‐stimulatory molecules working together with MHC class II during the progression of the disease. To elucidate the involvement of the B7.1 co‐stimulatory molecule in RA, we analyzed the phenotype of B7.1 transgenic (named D1BC) mice and the sequential differentiation of synovial fibroblasts (SFs) by studying the expression of chondrogenic and osteogenic lineage markers together with lineage tracing experiment using B7.1 transgene in vivo. The B7.1 transgene was driven by a collagen type II (CII) promoter and enhancer in the D1BC mouse. A low‐dose of bovine CII (bCII) was used to induce chronic articular inflammation with interstitial pneumonitis. Joint damage was analyzed by histopathological examination and computed tomography. B7.1 was expressed in articular cartilage and SFs of D1BC mice. Chronic inflammatory arthritis in bCII‐D1BC mouse shared common features with those found in patients with RA, such as pannus formation, bone destruction, osteoporosis, and joint ankylosis. A subpopulation of SFs (Runx2+, Sox9+, Col10a1+, Osx+ and CX‐) in the pannus was classified as osteochondrogenic lineage rather than mesenchymal stromal lineage. These cells underwent differentiation into osteogenic lineage via hypertrophic chondrocytes at the end of the chronic phase. The ectopic expression of B7.1 in chondrocytes and SFs leads to an increased susceptibility to chronic inflammatory arthritis and subsequent new bone formation, reminiscent of ankylosis. The regulation of cartilage remodeling in pannus tissue is an important consideration in the treatment of RA.
