Ion channel mRNA distribution and expression in the sinoatrial node and right atrium of dogs and monkeys
Journal of Toxicologic Pathology
SANO, T;YASUNO, H;WATANABE, T;
| DOI: 10.1293/tox.2020-0089
There are limited data on the gene expression profiles of ion channels in the sinoatrial node (SAN) of dogs and monkeys. In this study, the messenger RNA (mRNA) expression profiles of various ion channels in the SAN of naïve dogs and monkeys were examined using RNAscope _in situ _hybridization and compared with those in the surrounding right atrium (RA) of each species. Regional-specific Cav1.3 and HCN4 expression was observed in the SAN of dogs and monkeys. Additionally, HCN1 in dogs was only expressed in the SAN group. The expression profiles of Cav3.1 and Cav3.2 in the SAN and RA were completely different between dogs and monkeys. Dog hearts only expressed Cav3.2; however, Cav3.1 was detected only in monkeys, and the expression score in the SAN was slightly higher than that in the RA. Although Kir3.1 and NCX1 in dogs were equally expressed in both the SAN and RA, the expression scores of these genes in the SAN of monkeys were slightly higher than those in the RA. The Kir3.4 expression score in the SAN of dogs and monkeys was also slightly higher than that in the RA. The mRNA expression scores of Kv11.1/ERG and KvLQT1 were equally observed in both the SAN and RA of dogs and monkeys. HCN2 was not detected in dogs and monkeys. In summary, we used RNAscope to demonstrate the SAN-specific gene expression patterns of ion channels, which may be useful in explaining the effect of pacemaking and/or hemodynamic effects in nonclinical studies.
Bogdanov, V;Soltisz, A;Beard, C;Hernandez Orengo, B;Sakuta, G;Veeraraghavan, R;Davis, J;Gyorke, S;
| DOI: 10.1016/j.bpj.2022.11.1389
Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.
Bando, H;Brinkmeier, ML;Castinetti, F;Fang, Q;Lee, MS;Saveanu, A;Albarel, F;Dupuis, C;Brue, T;Camper, SA;
PMID: 35951005 | DOI: 10.1093/hmg/ddac192
Congenital hypopituitarism is a genetically heterogeneous condition that is part of a spectrum disorder that can include holoprosencephaly. Heterozygous mutations in SIX3 cause variable holoprosencephaly in humans and mice. We identified two children with neonatal hypopituitarism and thin pituitary stalk who were doubly heterozygous for rare, likely deleterious variants in the transcription factors SIX3 and POU1F1. We used genetically engineered mice to understand the disease pathophysiology. Pou1f1 loss of function heterozygotes are unaffected; Six3 heterozygotes have pituitary gland dysmorphology and incompletely ossified palate; and the Six3+/-; Pou1f1+/dw double; heterozygote mice have a pronounced phenotype, including pituitary growth through the palate. The interaction of Pou1f1 and Six3 in mice supports the possibility of digenic pituitary disease in children. Disruption of Six3 expression in the oral ectoderm completely ablated anterior pituitary development, and deletion of Six3 in the neural ectoderm blocked development of the pituitary stalk and both anterior and posterior pituitary lobes. Six3 is required in both oral and neural ectodermal tissues for activation of signaling pathways and transcription factors necessary for pituitary cell fate. These studies clarify the mechanism of SIX3 action in pituitary development and provide support for a digenic basis for hypopituitarism.
Thompson, MJ;Young, CA;Munnamalai, V;Umulis, DM;
PMID: 37237002 | DOI: 10.1038/s41598-023-34725-4
Positional information encoded in signaling molecules is essential for early patterning in the prosensory domain of the developing cochlea. The sensory epithelium, the organ of Corti, contains an exquisite repeating pattern of hair cells and supporting cells. This requires precision in the morphogen signals that set the initial radial compartment boundaries, but this has not been investigated. To measure gradient formation and morphogenetic precision in developing cochlea, we developed a quantitative image analysis procedure measuring SOX2 and pSMAD1/5/9 profiles in mouse embryos at embryonic day (E)12.5, E13.5, and E14.5. Intriguingly, we found that the pSMAD1/5/9 profile forms a linear gradient up to the medial ~ 75% of the PSD from the pSMAD1/5/9 peak in the lateral edge during E12.5 and E13.5. This is a surprising activity readout for a diffusive BMP4 ligand secreted from a tightly constrained lateral region since morphogens typically form exponential or power-law gradient shapes. This is meaningful for gradient interpretation because while linear profiles offer the theoretically highest information content and distributed precision for patterning, a linear morphogen gradient has not yet been observed. Furthermore, this is unique to the cochlear epithelium as the pSMAD1/5/9 gradient is exponential in the surrounding mesenchyme. In addition to the information-optimized linear profile, we found that while pSMAD1/5/9 is stable during this timeframe, an accompanying gradient of SOX2 shifts dynamically. Last, through joint decoding maps of pSMAD1/5/9 and SOX2, we see that there is a high-fidelity mapping between signaling activity and position in the regions that will become Kölliker's organ and the organ of Corti. Mapping is ambiguous in the prosensory domain precursory to the outer sulcus. Altogether, this research provides new insights into the precision of early morphogenetic patterning cues in the radial cochlea prosensory domain.
Invest Ophthalmol Vis Sci.
Chung DD, Frausto RF, Lin BR, Hanser EM, Cohen Z, Aldave AJ.
PMID: 28654985 | DOI: 10.1167/iovs.17-21423
Abstract
PURPOSE:
To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression.
METHODS:
Next-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause).
RESULTS:
Expression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes.
CONCLUSIONS:
Identification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.
Steece-Collier K, Stancati JA, Collier NJ, Sandoval IM, Mercado NM, Sortwell CE, Collier TJ, Manfredsson FP.
PMID: 31002755 | DOI: 10.1002/mds.27695
Abstract
BACKGROUND:
Levodopa-induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well-tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa-induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa-induced dyskinesias.
METHODS:
To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high-potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa-induced dyskinesias in severely parkinsonian rats.
RESULTS:
We demonstrate that vector-mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa-induced dyskinesias, and this antidyskinetic benefit persists long term and with high-dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa-induced dyskinesias. Importantly, motoric responses to low-dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability.
DISCUSSION:
The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa-induced dyskinesia side effect.
Medvedev, R;Turner, D;Gorelik, J;Alvarado, F;Bondarenko, V;Glukhov, A;
| DOI: 10.1016/j.bpj.2022.11.1392
Atrial fibrillation (AF) is commonly observed in patients with hypertension and is associated with pathologically elevated cardiomyocyte stretch. AF triggers have been linked to subcellular Ca2+ abnormalities, while their association with stretch remains elusive. Caveolae are mechanosensitive membrane structures, that play a role in both Ca2+ and cyclic adenosine monophosphate (cAMP) signaling. Therefore, caveolae could provide a mechanistic connection between cardiomyocyte stretch, Ca2+ mishandling, and AF. In isolated mouse atrial myocytes, cell stretch was mimicked by hypotonic swelling, which increased cell width (by ∼30%, p
Nandadasa S, Kraft CM, Wang LW, O'Donnell A, Patel R, Gee HY, Grobe K, Cox TC, Hildebrandt F, Apte SS.
PMID: 30814516 | DOI: 10.1038/s41467-019-08520-7
Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.
EGF and BMPs govern differentiation and patterning in human gastric glands
Wölffling, S;Daddi, A;Imai-Matsushima, A;Fritsche, K;Goosmann, C;Traulsen, J;Lisle, R;Schmid, M;del Mar Reines-Benassar, M;Pfannkuch, L;Brinkmann, V;Bornschein, J;Peter Malfertheiner, ;Ordemann, J;Link, A;Meyer, T;Boccellato, F;
| DOI: 10.1053/j.gastro.2021.04.062
Background & Aims The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organised inside glands and crypts. Regeneration depends on WNT/β-Catenin pathway activation, but to understand homeostasis and its dysregulation in disease we need to identify the signalling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen- and gastric acid- producing cells. Methods We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief and parietal cells. We localised the source of the growth factors in the tissue of origin. Results We show that EGF is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with BMP/NOGGIN signals, EGF controls the differentiation of foveolar cells vs. parietal or chief cells. We also show that EGF is likely to underlie alteration of the gastric mucosa in the pre-cancerous condition atrophic gastritis. Conclusions Use of our recently established mucosoid cultures in combination with analysis of the tissue-of-origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signalling microenvironment in the human gastric glands.
Ramlow, L;Falcke, M;Lindner, B;
| DOI: 10.1016/j.bpj.2022.11.1390
Stochastic spiking is a prominent feature of Ca2+ signaling. The main noise source at the cellular level are puffs from inositol-trisphosphate receptor (IP3R) channel clusters in the membrane of the endoplasmic reticulum (ER). While the random cluster activity has been known for decades, a stringent method to derive the puff noise term acting on the cytosolic Ca2+ concentration is still lacking. We adopt a popular description of neural spike generation from neuroscience, the stochastic integrate-and-fire (IF) model, to describe Ca2+ spiking. Our model consists of two components describing i) activity of IP3R clusters and ii) dynamics of the global Ca2+ concentrations in the cytosol and in the ER. Cluster activity is modeled by a Markov chain, capturing the puff. The global Ca2+ concentrations are described by a two-variable IF model driven by the puff current. For the Markov chain we derive expressions for the statistics of interpuff interval, single-puff strength, and puff current assuming constant cytosolic Ca2+, an assumption often well met because the Ca2+ concentrations vary much slower than the cluster activity does. The latter assumption also allows to approximate the driving Ca2+ dependent puff current by a white Gaussian noise. This approximation results in an IF model with nonlinear drift and multiplicative noise. We consider this reduced model in a renewal version and in a version with cumulative refractoriness. Neglecting ER depletion, the stochastic IF model has only one variable and generates a renewal spike train, a point process with statistically independent interspike intervals (ISI). We derive analytical expressions for the mean and coefficient of variation of the ISI and suggest approximations for the ISI density and spike-train power spectrum. Taking into account ER depletion, the two-variable IF model displays cumulative refractoriness as seen in experimental data.
Nutritional regulation of oligodendrocyte differentiation regulates perineuronal net remodeling in the median eminence
Kohnke, S;Buller, S;Nuzzaci, D;Ridley, K;Lam, B;Pivonkova, H;Bentsen, MA;Alonge, KM;Zhao, C;Tadross, J;Holmqvist, S;Shimizo, T;Hathaway, H;Li, H;Macklin, W;Schwartz, MW;Richardson, WD;Yeo, GSH;Franklin, RJM;Karadottir, RT;Rowitch, DH;Blouet, C;
PMID: 34260928 | DOI: 10.1016/j.celrep.2021.109362
The mediobasal hypothalamus (MBH; arcuate nucleus of the hypothalamus [ARH] and median eminence [ME]) is a key nutrient sensing site for the production of the complex homeostatic feedback responses required for the maintenance of energy balance. Here, we show that refeeding after an overnight fast rapidly triggers proliferation and differentiation of oligodendrocyte progenitors, leading to the production of new oligodendrocytes in the ME specifically. During this nutritional paradigm, ME perineuronal nets (PNNs), emerging regulators of ARH metabolic functions, are rapidly remodeled, and this process requires myelin regulatory factor (Myrf) in oligodendrocyte progenitors. In genetically obese ob/ob mice, nutritional regulations of ME oligodendrocyte differentiation and PNN remodeling are blunted, and enzymatic digestion of local PNN increases food intake and weight gain. We conclude that MBH PNNs are required for the maintenance of energy balance in lean mice and are remodeled in the adult ME by the nutritional control of oligodendrocyte differentiation.
The Journal of investigative dermatology
Shim, J;Park, J;Abudureyimu, G;Kim, MH;Shim, JS;Jang, KT;Kwon, EJ;Jang, HS;Yeo, E;Lee, JH;Lee, D;
PMID: 35853486 | DOI: 10.1016/j.jid.2022.06.022
The nail unit and hair follicle are both hard keratin-producing organs that share various biological features. Here we used a Digital Spatial Profiling (DSP) and single cell RNA sequencing (scRNA-seq) to define a spatially resolved expression profile of the human nail unit and hair follicle. Our approach demonstrated the presence of a nail-specific mesenchymal population called onychofibroblasts within the onychodermis. Onychodermis and follicular dermal papilla (DP) both expressed WNT and bone morphogenetic protein (BMP) signaling molecules. In addition, nail matrix epithelium and hair matrix showed very similar expressions profile, including expression of hard keratins and HOXC13, a transcriptional regulator of the hair shaft. Integration of scRNA-seq and DSP data via computational deconvolution methods estimated epithelial and mesenchymal cell abundance in the nail- and hair-specific regions of interest and revealed close transcriptional similarity between these major skin appendages. To analyze the function of BMPs in nail differentiation, we treated cultured human nail matrix keratinocytes (NMKs) with BMP5, which are highly expressed by onychofibroblasts. We observed increased expressions of hard keratin and its regulator genes such as HOXC13. Collectively, our data suggest that onychodermis is the counterpart of DP and that BMP5 in onychofibroblasts play a key role in differentiation of NMKs.
