Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1h\n M1560V/+\n)

Proceedings of the National Academy of Sciences of the United States of America

2021 Apr 27

Seidel, E;Schewe, J;Zhang, J;Dinh, HA;Forslund, SK;Markó, L;Hellmig, N;Peters, J;Muller, DN;Lifton, RP;Nottoli, T;Stölting, G;Scholl, UI;
PMID: 33879608 | DOI: 10.1073/pnas.2014876118

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1h M1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h -/- ). Adrenal morphology of both Cacna1h M1560V/+ and Cacna1h -/- mice was normal. Cacna1h M1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1h M1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h -/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1h M1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Single-cell roadmap of human gonadal development

Nature

2022 Jul 01

Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Adaptation of the Oxygen Sensing System during Lung Development

Oxidative Medicine and Cellular Longevity

2022 Feb 18

Kirschner, K;Kelterborn, S;Stehr, H;Penzlin, J;Jacobi, C;Endesfelder, S;Sieg, M;Kruppa, J;Dame, C;Sciesielski, L;
| DOI: 10.1155/2022/9714669

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.

The development of dentin microstructure is controlled by the type of adjacent epithelium

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

2021 Nov 16

Lavicky, J;Kolouskova, M;Prochazka, D;Rakultsev, V;Gonzalez-Lopez, M;Steklikova, K;Bartos, M;Vijaykumar, A;Kaiser, J;Porizka, P;Hovorakova, M;Mina, M;Krivanek, J;
PMID: 34783080 | DOI: 10.1002/jbmr.4471

Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry) mouse strain and knockout mice with ectopic enamel (Spry2+/- ;Spry4-/- ) we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of adjacent epithelium. Thus, based on the type of interacting epithelium we introduce more generalized terms for two distinct types of dentins: cementum vs. enamel-facing dentin. In the odontoblasts which produce enamel-facing dentin we identified uniquely expressed genes (Dkk1, Wisp1 and Sall1) which were either absent or downregulated in odontoblasts which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate different effect of two main types of dental epithelium, important for either crown or root formation, on adjacent odontoblasts which give rise to dentin of different quality. This article is protected by

Effect of the spatial-temporal specific theca cell Cyp17 overexpression on the reproductive phenotype of the novel TC17 mouse

Journal of translational medicine

2021 Oct 15

Secchi, C;Belli, M;Harrison, TNH;Swift, J;Ko, C;Duleba, AJ;Stupack, D;Chang, RJ;Shimasaki, S;
PMID: 34654452 | DOI: 10.1186/s12967-021-03103-x

In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women. Androgen excess is observed in women with polycystic ovary syndrome (PCOS) resulting from excess endogenous androgen production, and in transgender males undergoing exogenous testosterone therapy after female sex assignment at birth. However, the molecular and morphological effects of Cyp17 overexpression and androgen excess on folliculogenesis is unknown.In this work, seeking a comprehensive profiling of the local outcomes of the androgen excess in the ovary, we generated a transgenic mouse model (TC17) with doxycycline (Dox)-induced Cyp17 overexpression in a local and temporal manner. TC17 mice were obtained by a combination of the Tet-dependent expression system and the Cre/LoxP gene control system.Ovaries of Dox-treated TC17 mice overexpressed Cyp17 specifically in TCs, inducing high testosterone levels. Surprisingly, TC17 ovarian morphology resembled the human ovarian features of testosterone-treated transgender men (partially impaired folliculogenesis, hypertrophic or luteinized stromal cells, atretic follicles, and collapsed clusters). We additionally assessed TC17 fertility denoting a perturbation of the normal reproductive functions (e.g., low pregnancy rate and numbers of pups per litter). Finally, RNAseq analysis permitted us to identify dysregulated genes (Lhcgr, Fshr, Runx1) and pathways (Extra Cellular Matrix and Steroid Synthesis).Our novel mouse model is a versatile tool to provide innovative insights into study the effects of Cyp17 overexpression and hyperandrogenism in the ovary.

Wnt/β-catenin signaling acts cell-autonomously to promote cardiomyocyte regeneration in the zebrafish heart

Developmental biology

2021 Nov 06

Bertozzi, A;Wu, CC;Hans, S;Brand, M;Weidinger, G;
PMID: 34748730 | DOI: 10.1016/j.ydbio.2021.11.001

Zebrafish can achieve scar-free healing of heart injuries, and robustly replace all cardiomyocytes lost to injury via dedifferentiation and proliferation of mature cardiomyocytes. Previous studies suggested that Wnt/β-catenin signaling is active in the injured zebrafish heart, where it induces fibrosis and prevents cardiomyocyte cell cycling. Here, via targeting the destruction complex of the Wnt/β-catenin pathway with pharmacological and genetic tools, we demonstrate that Wnt/β-catenin activity is required for cardiomyocyte proliferation and dedifferentiation, as well as for maturation of the scar during regeneration. Using cardiomyocyte-specific conditional inhibition of the pathway, we show that Wnt/β-catenin signaling acts cell-autonomously to promote cardiomyocyte proliferation. Our results stand in contrast to previous reports and rather support a model in which Wnt/β-catenin signaling plays a positive role during heart regeneration in zebrafish.

The Long and the Small Collide: LncRNAs and Small Heterodimer Partner (SHP) in Liver Disease

Molecular and cellular endocrinology

2021 Mar 26

Wu, J;Nagy, LE;Wang, L;
PMID: 33781837 | DOI: 10.1016/j.mce.2021.111262

Long non-coding RNAs (lncRNAs) are a large and diverse class of RNA molecules that are transcribed but not translated into proteins, with a length of more than 200 nucleotides. LncRNAs are involved in gene expression and regulation. The abnormal expression of lncRNAs is associated with disease pathogenesis. Small heterodimer partner (SHP, NR0B2) is a unique orphan nuclear receptor that plays a pivotal role in many biological processes by acting as a transcriptional repressor. In this review, we present the critical roles of SHP and summarize recent findings demonstrating the regulation between lncRNAs and SHP in liver disease.

A screen of repurposed drugs identifies AMHR2/MISR2 agonists as potential contraceptives

Proceedings of the National Academy of Sciences of the United States of America

2022 Apr 12

Li, Y;Wei, L;Meinsohn, MC;Suliman, R;Chauvin, M;Berstler, J;Hartland, K;Jensen, MM;Sicher, NA;Nagykery, N;Donahoe, PK;Pepin, D;
PMID: 35380904 | DOI: 10.1073/pnas.2122512119

SignificanceThis study aims to identify drugs that activate the Mullerian inhibiting substance pathway to be used for contraception or other applications in women's health. We describe a high-throughput screening pipeline to identify small molecules that activate the Mullerian inhibiting substance type 2 receptor (MISR2) and validate their activity in bioassays. We identify five compounds from a repurposed drug library that specifically induce MISR2 signaling, trigger regression of the Mullerian duct, and inhibit follicle activation. We test these compounds in vivo and show that they can repress folliculogenesis in mice and rats in an Misr2-dependent manner. These drugs may represent a class of ovarian regulators that inhibit preantral follicle activation and growth.

Isolation and Characterization of Human Brown Adipocytes

Methods in molecular biology (Clifton, N.J.)

2022 Feb 15

Scheele, C;Henriksen, TI;Nielsen, S;
PMID: 35167100 | DOI: 10.1007/978-1-0716-2087-8_14

Brown adipose tissue (BAT) is a thermoregulatory fat with energy-consuming properties. The location and heterogeneity of this tissue makes it complicated to sample before and after interventions in humans, and an in vitro model for mechanistic and molecular studies is therefore of great value. We here describe a protocol for isolation of progenitors from the stromal vascular fraction of BAT biopsies obtained surgically from adult humans. We further present how these cells are differentiated in vitro and finally how they are characterized for thermogenic capacity. Methods for characterization described here include norepinephrine-induced thermogenic gene expression using qPCR; norepinephrine-induced mitochondrial uncoupling using the Seahorse XFe96 Analyzer, and norepinephrine-induced expression of UCP1 using the RNAscope Technology.

Changes in Mitochondrial Size and Morphology in the RPE and Photoreceptors of the Developing and Ageing Zebrafish

Cells

2022 Nov 09

Burgoyne, T;Toms, M;Way, C;Tracey-White, D;Futter, CE;Moosajee, M;
PMID: 36428971 | DOI: 10.3390/cells11223542

Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. Investigation of temporal gene expression of several mitochondrial-related markers indicated fission as the dominant mechanism contributing to the changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction.

Maternal high-fat diet in mice induces cerebrovascular, microglial and long-term behavioural alterations in offspring

Communications biology

2022 Jan 11

Bordeleau, M;Comin, CH;Fernández de Cossío, L;Lacabanne, C;Freitas-Andrade, M;González Ibáñez, F;Raman-Nair, J;Wakem, M;Chakravarty, M;Costa, LDF;Lacoste, B;Tremblay, MÈ;
PMID: 35017640 | DOI: 10.1038/s42003-021-02947-9

Various environmental exposures during pregnancy, like maternal diet, can compromise, at critical periods of development, the neurovascular maturation of the offspring. Foetal exposure to maternal high-fat diet (mHFD), common to Western societies, has been shown to disturb neurovascular development in neonates and long-term permeability of the neurovasculature. Nevertheless, the effects of mHFD on the offspring's cerebrovascular health remains largely elusive. Here, we sought to address this knowledge gap by using a translational mouse model of mHFD exposure. Three-dimensional and ultrastructure analysis of the neurovascular unit (vasculature and parenchymal cells) in mHFD-exposed offspring revealed major alterations of the neurovascular organization and metabolism. These alterations were accompanied by changes in the expression of genes involved in metabolism and immunity, indicating that neurovascular changes may result from abnormal brain metabolism and immune regulation. In addition, mHFD-exposed offspring showed persisting behavioural alterations reminiscent of neurodevelopmental disorders, specifically an increase in stereotyped and repetitive behaviours into adulthood.

Local coordination between intracortical bone remodeling and vascular development in human juvenile bone

Bone

2023 May 05

Andreasen, CM;El-Masri, BM;MacDonald, B;Laursen, KS;Nielsen, MH;Thomsen, JS;Delaisse, JM;Andersen, TL;
PMID: 37150243 | DOI: 10.1016/j.bone.2023.116787

Although failure to establish a vascular network has been associated with many skeletal disorders, little is known about what drives development of vasculature in the intracortical bone compartments. Here, we show that intracortical bone resorption events are coordinated with development of the vasculature. We investigated the prevalence of vascular structures at different remodeling stages as well as their 3D organization using proximal femoral cortical bone from 5 girls and 6 boys (aged 6-15 years). A 2D analysis revealed that non-quiescent intracortical pores contained more vascular structures than quiescent pores (p < 0.0001). Type 2 pores, i.e., remodeling of existing pores, had a higher density of vascular structures than type 1 pores, i.e., de novo created pores (p < 0.05). Furthermore, pores at the eroded-formative remodeling stage, had more vascular structures than pores at any other remodeling stage (p < 0.05). A 3D reconstruction of an intracortical remodeling event showed that osteoclasts in the advancing tip of the cutting cone as well as preosteoclasts in the lumen expressed vascular endothelial growth factor-A (VEGFA), while VEGFA-receptors 1 and 2 mainly were expressed in endothelial cells in the adjacent vasculature. Consequently, we propose that the progression of the vascular network in intracortical remodeling events is driven by osteoclasts expressing VEGFA. Moreover, the vasculature is continuously reconfigured according to the demands of the remodeling events at the surrounding bone surfaces.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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