Periodic inhibition of Erk activity drives sequential somite segmentation

Nature

2023 Jan 01

Simsek, MF;Chandel, AS;Saparov, D;Zinani, OQH;Clason, N;Özbudak, EM;
PMID: 36517597 | DOI: 10.1038/s41586-022-05527-x

Sequential segmentation creates modular body plans of diverse metazoan embryos1-4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6-8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

Hedgehog signaling regulates Wolffian duct development through the primary cilium

Biology of reproduction

2022 Dec 15

Alves, MBR;Girardet, L;Augière, C;Moon, KH;Lavoie-Ouellet, C;Bernet, A;Soulet, D;Calvo, E;Teves, ME;Beauparlant, CJ;Droit, A;Bastien, A;Robert, C;Bok, J;Hinton, BT;Belleannée, C;
PMID: 36525341 | DOI: 10.1093/biolre/ioac210

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). While mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; Ift88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix (ECM), mesenchymal-epithelial transition, canonical Hh, and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Cd59 and inflammation regulate Schwann cell development

eLife

2022 Jun 24

Wiltbank, AT;Steinson, ER;Criswell, SJ;Piller, M;Kucenas, S;
PMID: 35748863 | DOI: 10.7554/eLife.76640

Efficient neurotransmission is essential for organism survival and is enhanced by myelination. However, the genes that regulate myelin and myelinating glial cell development have not been fully characterized. Data from our lab and others demonstrates that cd59, which encodes for a small GPI-anchored glycoprotein, is highly expressed in developing zebrafish, rodent, and human oligodendrocytes (OLs) and Schwann cells (SCs), and that patients with CD59 dysfunction develop neurological dysfunction during early childhood. Yet, the function of Cd59 in the developing nervous system is currently undefined. In this study, we demonstrate that cd59 is expressed in a subset of developing SCs. Using cd59 mutant zebrafish, we show that developing SCs proliferate excessively and nerves may have reduced myelin volume, altered myelin ultrastructure, and perturbed node of Ranvier assembly. Finally, we demonstrate that complement activity is elevated in cd59 mutants and that inhibiting inflammation restores SC proliferation, myelin volume, and nodes of Ranvier to wildtype levels. Together, this work identifies Cd59 and developmental inflammation as key players in myelinating glial cell development, highlighting the collaboration between glia and the innate immune system to ensure normal neural development.

Serotonin limits generation of chromaffin cells during adrenal organ development

Nature communications

2022 May 25

Kameneva, P;Melnikova, VI;Kastriti, ME;Kurtova, A;Kryukov, E;Murtazina, A;Faure, L;Poverennaya, I;Artemov, AV;Kalinina, TS;Kudryashov, NV;Bader, M;Skoda, J;Chlapek, P;Curylova, L;Sourada, L;Neradil, J;Tesarova, M;Pasqualetti, M;Gaspar, P;Yakushov, VD;Sheftel, BI;Zikmund, T;Kaiser, J;Fried, K;Alenina, N;Voronezhskaya, EE;Adameyko, I;
PMID: 35614045 | DOI: 10.1038/s41467-022-30438-w

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.

Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis

Developmental cell

2022 May 02

van Bruggen, D;Pohl, F;Langseth, CM;Kukanja, P;Lee, H;Albiach, AM;Kabbe, M;Meijer, M;Linnarsson, S;Hilscher, MM;Nilsson, M;Sundström, E;Castelo-Branco, G;
PMID: 35523173 | DOI: 10.1016/j.devcel.2022.04.016

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

An oxygen-adaptive interaction between SNHG12 and occludin maintains blood-brain barrier integrity

Cell reports

2022 Apr 12

Li, Y;Wei, JY;Liu, H;Wang, KJ;Jin, SN;Su, ZK;Wang, HJ;Shi, JX;Li, B;Shang, DS;Fang, WG;Qin, XX;Zhao, WD;Chen, YH;
PMID: 35417709 | DOI: 10.1016/j.celrep.2022.110656

Tight junctions (TJs) of brain microvascular endothelial cells (BMECs) play a pivotal role in maintaining the blood-brain barrier (BBB) integrity; however, precise regulation of TJs stability in response to physiological and pathological stimuli remains elusive. Here, using RNA immunoprecipitation with next-generation sequencing (RIP-seq) and functional characterization, we identify SNHG12, a long non-coding RNA (lncRNA), as being critical for maintaining the BBB integrity by directly interacting with TJ protein occludin. The interaction between SNHG12 and occludin is oxygen adaptive and could block Itch (an E3 ubiquitin ligase)-mediated ubiquitination and degradation of occludin in human BMECs. Genetic ablation of endothelial Snhg12 in mice results in occludin reduction and BBB leakage and significantly aggravates hypoxia-induced BBB disruption. The detrimental effects of hypoxia on BBB could be alleviated by exogenous SNHG12 overexpression in brain endothelium. Together, we identify a direct TJ modulator lncRNA SNHG12 that is critical for the BBB integrity maintenance and oxygen adaption.

In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency

Molecular Therapy - Methods & Clinical Development

2022 Mar 01

Huichalaf, C;Perfitt, T;Kuperman, A;Gooch, R;Kovi, R;Brenneman, K;Chen, X;Hirenallur-Shanthappa, D;Ma, T;Assaf, B;Pardo, I;Franks, T;Monarski, L;Cheng, T;Le, K;Su, C;Somanathan, S;Whiteley, L;Bulawa, C;Pregel, M;Martelli, A;
| DOI: 10.1016/j.omtm.2022.02.002

Friedreich's ataxia is a rare disorder resulting from deficiency of frataxin, a mitochondrial protein implicated in the synthesis of iron-sulfur clusters. Preclinical studies in mice have shown that gene therapy is a promising approach to treat individuals with Friedreich's ataxia. However, a recent report provided evidence that AAVrh10-mediated overexpression of frataxin could lead to cardiotoxicity associated with mitochondrial dysfunction. While evaluating an AAV9-based frataxin gene therapy using a chicken β-actin promoter, we showed that toxic overexpression of frataxin could be reached in mouse liver and heart with doses between 1 × 1013 and 1 × 1014 vg/kg. In a mouse model of cardiac disease, these doses only corrected cardiac dysfunction partially and transiently and led to adverse findings associated with iron-sulfur cluster deficiency in liver. We demonstrated that toxicity required frataxin's primary function by using a frataxin construct bearing the N146K mutation, which impairs binding to the iron-sulfur cluster core complex. At the lowest tested dose, we observed moderate liver toxicity that was accompanied by progressive loss of transgene expression and liver regeneration. Together, our data provide insights into the toxicity of frataxin overexpression that should be considered in the development of a gene therapy approach for Friedreich's ataxia.

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides

Nucleic acids research

2022 Jan 11

Relizani, K;Echevarría, L;Zarrouki, F;Gastaldi, C;Dambrune, C;Aupy, P;Haeberli, A;Komisarski, M;Tensorer, T;Larcher, T;Svinartchouk, F;Vaillend, C;Garcia, L;Goyenvalle, A;
PMID: 34893881 | DOI: 10.1093/nar/gkab1199

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

Glutaminase 2 Knockdown Reduces Hyperammonemia and Associated Lethality of Urea Cycle Disorder Mouse Model

Journal of inherited metabolic disease

2022 Jan 06

Mao, X;Chen, H;Lin, A;Kim, S;Burczynski, ME;Na, E;Halasz, G;Sleeman, MW;Murphy, AJ;Okamoto, H;Cheng, X;
PMID: 34988999 | DOI: 10.1002/jimd.12474

Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle. This article is protected by

Clinical and Pathological Characteristics of Hyaline-Vascular Type Unicentric Castleman Disease: A 20-Year Retrospective Analysis

Diagnostics (Basel, Switzerland)

2021 Oct 28

Nishimura, MF;Nishimura, Y;Nishikori, A;Maekawa, Y;Maehama, K;Yoshino, T;Sato, Y;
PMID: 34829355 | DOI: 10.3390/diagnostics11112008

The first case of hyaline vascular type of unicentric Castleman disease (HV-UCD) was reported more than six decades ago. Since patients with HV-UCD are often asymptomatic and this condition is generally discovered incidentally on imaging tests, most of the previous reports were of mediastinal origin detected by chest radiography. In recent years, improved access to imaging modalities has provided new insights in the diagnosis of this condition. In this study, we reviewed the detailed clinical and pathological findings of 38 HV-UCD cases (20 males and 18 females, mean age: 42.8 years). The most common site involved was the abdominal cavity (34.2%), followed by mediastinum (23.7%) and retroperitoneum (15.8%). In the abdominal cavity, mesenteric origin was the most common. The mean size of masses was 4.8 cm. Pathologically, thick hyalinized collagen fibers surrounding large blood vessels and calcification were observed (81.6% and 23.7%, respectively). Multinucleated giant cells resembling Warthin-Finkeldey cell were also observed in occasional cases (23.7%). This is a unique paper that summarizes detailed clinical and pathological findings of a large series of a rare disease. The clinical information presented in this paper is more plausible than previous views and is useful for accurate diagnosis and understanding of the disease.

Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy

Nature communications

2021 Dec 08

Saad, NY;Al-Kharsan, M;Garwick-Coppens, SE;Chermahini, GA;Harper, MA;Palo, A;Boudreau, RL;Harper, SQ;
PMID: 34880230 | DOI: 10.1038/s41467-021-27430-1

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating myopathy caused by de-repression of the DUX4 gene in skeletal muscles. Effective therapies will likely involve DUX4 inhibition. RNA interference (RNAi) is one powerful approach to inhibit DUX4, and we previously described a RNAi gene therapy to achieve DUX4 silencing in FSHD cells and mice using engineered microRNAs. Here we report a strategy to direct RNAi against DUX4 using the natural microRNA miR-675, which is derived from the lncRNA H19. Human miR-675 inhibits DUX4 expression and associated outcomes in FSHD cell models. In addition, miR-675 delivery using gene therapy protects muscles from DUX4-associated death in mice. Finally, we show that three known miR-675-upregulating small molecules inhibit DUX4 and DUX4-activated FSHD biomarkers in FSHD patient-derived myotubes. To our knowledge, this is the first study demonstrating the use of small molecules to suppress a dominant disease gene using an RNAi mechanism.

Cell position within human pluripotent stem cell colonies determines apical specialization via an actin cytoskeleton-based mechanism

Stem cell reports

2021 Nov 27

Kim, Y;Jang, H;Seo, K;Kim, JH;Lee, B;Cho, HM;Kim, HJ;Yang, E;Kim, H;Gim, JA;Park, Y;Ryu, JR;Sun, W;
PMID: 34919810 | DOI: 10.1016/j.stemcr.2021.11.005

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.

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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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